Nitric Oxide in Health and Disease | Leaders in Pharmaceutical Business Intelligence Group, LLC, Doing Business As LPBI Group, Newton, MA

Archive for the ‘Nitric Oxide in Health and Disease’ Category

Functional Correlates of Signaling Pathways

Posted in Academic Publishing, Biochemical pathways, Biological Networks, Ca2+ triggered activation, Calcium Signaling, Calmodulin, Calmodulin Kinase and Contraction, Cardiomyopathy, Chemical Biology and its relations to Metabolic Disease, Curation, Cytoskeleton, Experimental validation, Explanatory, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Genome Biology, Historical relevance, Human Immune System in Health and in Disease, Innovations in Neurophysiology & Neuropsychology, Ionic Transporters Na+, Metabolism, Metabolomics, Mg++, Molecular Genetics & Pharmaceutical, Na-K transport, Neurohumoral Transmission, Neurological Diseases, Nitric Oxide in Health and Disease, Nutrition, Nutrition and Phytochemistry, Pharmaceutical Analytics, Population Health Management, Proteomics, Synaptic vesicle, Technology Advance Assessment of, tagged enzymes, pathways, Proteins, signaling, therapeutics on November 4, 2014| Leave a Comment »

Functional Correlates of Signaling Pathways

Author and Curator: Larry H. Bernstein, MD, FCAP

We here move on to a number of specific, key published work on signaling, and look at the possible therapeutic applications to disease states.

Scripps Research Professor Wolfram Ruf and colleagues have identified a key connection between

the signaling pathways and the immune system spiraling out of control involving
the coagulation system and vascular endothelium that,
if disrupted may be a target for sepsis. (Science Daily, Feb 29, 2008).

It may be caused by a bacterial infection that enters the bloodstream, but

we now recognize the same cascade not triggered by bacterial invasion.

The acute respiratory distress syndrome (ARDS) has been defined as

a severe form of acute lung injury featuring
pulmonary inflammation and increased capillary leak.

ARDS is associated with a high mortality rate and accounts for 100,000 deaths annually in the United States. ARDS may arise in a number of clinical situations, especially in patients with sepsis. A well-described pathophysiological model of ARDS is one form of

the acute lung inflammation mediated by

neutrophils,
cytokines, and
oxidant stress.

Neutrophils are major effect cells at the frontier of

innate immune responses, and they play
a critical role in host defense against invading microorganisms.

The tissue injury appears to be related to

proteases and toxic reactive oxygen radicals
released from activated neutrophils.

In addition, neutrophils can produce cytokines and chemokines that

enhance the acute inflammatory response.

Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is

mediated by a group of chemoattractants,
especially CXC chemokines.

Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP).

Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control

http://pharmaceuticalintelligence.com/2012/10/13/sepsis-multi-organ-dysfunction-syndrome-and-septic-shock-a-conundrum-of-signaling-pathways-cascading-out-of-control/

Integrins and extracellular matrix in mechanotransduction

ligand binding of integrins

Integrins are a family of cell surface receptors which

mediate cell–matrix and cell–cell adhesions.

Among other functions they provide an important

mechanical link between the cells external and intracellular environments while

the adhesions that they form also have critical roles in cellular signal-transduction.

Cell–matrix contacts occur at zones in the cell surface where

adhesion receptors cluster and when activated

the receptors bind to ligands in the extracellular matrix.

The extracellular matrix surrounds the cells of tissues and forms the

structural support of tissue which is particularly important in connective tissues.

Cells attach to the extracellular matrix through

specific cell-surface receptors and molecules

including integrins and transmembrane proteoglycans.

The integrin family of αβ heterodimeric receptors act as

cell adhesion molecules

connecting the ECM to the actin cytoskeleton.

The actin cytoskeleton is involved in the regulation of

1.cell motility,

2.cell polarity,

3.cell growth, and

4.cell survival.

The combination of αβ subunits determines

binding specificity and

signaling properties.

Both α and β integrin subunits contain two separate tails, which

penetrate the plasma membrane and possess small cytoplasmic domains which facilitate

the signaling functions of the receptor.

There is some evidence that the β subunit is the principal site for

binding of cytoskeletal and signaling molecules,

whereas the α subunit has a regulatory role. The integrin tails

link the ECM to the actin cytoskeleton within the cell and with cytoplasmic proteins,

such as talin, tensin, and filamin. The extracellular domains of integrin receptors bind the ECM ligands.

binding of integrins depends on ECM divalent cations ch19

integrin coupled to F-actin via linker

http://www.nature.com/nrm/journal/vaop/ncurrent/images/nrm3896-f4.jpg

Schematic of the ‘focal adhesion clutch’ on stiff (a) versus soft (b) extracellular matrix (ECM). In all cases, integrins are coupled to F-actin via linker proteins (for example, talin and vinculin). The linker proteins move backwards (as indicated by the small arrows) as F-actin also moves backwards, under pushing forces from actin polymerization and/or pulling forces from myosin II activity. This mechanism transfers force from actin to integrins, which pull on the ECM. A stiff ECM (a) resists this force so that the bound integrins remain immobile. A compliant matrix (b) deforms under this force (as indicated by the compressed ECM labelled as deformed matrix) so that the bound integrins can also move backwards. Their movement reduces the net loading rate on all the force-bearing elements, which results in altered cellular responses

The ECM is a complex mixture of matrix molecules, including –

glycoproteins, collagens, laminins, glycosaminoglycans, proteoglycans,
and nonmatrix proteins, – including growth factors

The integrin receptor formed from the binding of α and β subunits is

shaped like a globular head supported by two rod-like legs (Figure 1).

Most of the contact between the two subunits occurs in the head region, with

the intracellular tails of the subunits forming the legs of the receptor.

Integrin recognition of ligands is not constitutive but

is regulated by alteration of integrin affinity for ligand binding.

For integrin binding to ligands to occur

the integrin must be primed and activated, both of which involve
conformational changes to the receptor.

Linking integrin conformation to function

Figure Integrin binding to extracellular matrix (ECM). Conformational changes to integrin structure and clustering of subunits which allow enhanced function of the receptor.

Integrins work alongside other proteins such as

cadherins,

immunoglobulin superfamily

cell adhesion molecules,

selectins, and

syndecans

to mediate

cell–cell and

cell–matrix interactions and communication.

Activation of adhesion receptors triggers the formation of matrix contacts in which

bound matrix components,

adhesion receptors,

and associated intracellular cytoskeletal and signaling molecules

form large functional, localized multiprotein complexes.

Cell–matrix contacts are important in a variety of different cell and

tissue properties including

1.embryonic development,

2.inflammatory responses,

3.wound healing,

4.and adult tissue homeostasis.

Integrin extracellular binding activity is regulated from inside the cell and binding to the ECM induces signals that are transmitted into the cell. This bidirectional signaling requires

dynamic,

spatially, and

temporally regulated formation and

disassembly of multiprotein complexes that

form around the short cytoplasmic tails of integrins.

Ligand binding to integrin family members leads to clustering of integrin molecules in the plasma membrane and recruitment of actin filaments and intracellular signaling molecules to the cytoplasmic domain of the integrins. This forms focal adhesion complexes which are able to maintain

not only adhesion to the ECM

but are involved in complex signaling pathways

which include establishing

1.cell polarity,

2.directed cell migration, and

3.maintaining cell growth and survival.

Initial activation through integrin adhesion to matrix recruits up to around 50 diverse signaling molecules

to assemble the focal adhesion complex

which is capable of responding to environmental stimuli efficiently.

Mapping of the integrin

adhesome binding and signaling interactions

a network of 156 components linked together which can be modified by 690 interactions.

Genetic programming occurs with the binding of integrins to the ECM

Signal transduction pathway activation arising from integrin-ECM binding results in

changes in gene expression of cells and
leads to alterations in cell and tissue function.

Various different effects can arise depending on the

1.cell type,

2.matrix composition, and

3.integrins activated

It has been suggested that integrin-type I collagen interaction is necessary for

the phosphorylation and activation of osteoblast-specific transcription factors
present in committed osteoprogenitor cells.

During mechanical loading/stimulation of chondrocytes there is an

influx of ions across the cell membrane resulting from
activation of mechanosensitive ion channels
which can be inhibited by subunit-specific anti-integrin blocking antibodies or RGD peptides.

Using these strategies it was identified that

α5β1 integrin is a major mechanoreceptor in articular chondrocyte
responses to mechanical loading/stimulation.

Osteoarthritic chondrocytes show a depolarization response to 0.33 Hz stimulation

in contrast to the hyperpolarization response of normal chondrocytes.

The mechanotransduction pathway in chondrocytes derived from normal and osteoarthritic cartilage

both involve recognition of the mechanical stimulus
by integrin receptors resulting in
the activation of integrin signaling pathways
leading to the generation of a cytokine loop.

Normal and osteoarthritic chondrocytes show differences

at multiple stages of the mechanotransduction cascade.

Signaling pathways activated in chondrocytes

http://dx.doi.org/10.1016/j.matbio.2014.08.007

Chondrocyte integrins are important mediators of cell–matrix interactions in cartilage

by regulating the response of the cells to signals from the ECM that

control cell proliferation,
survival,
differentiation,
matrix remodeling.

Integrins participate in development and maintenance of the tissue but also

in pathological processes related to matrix destruction, where
they likely play a role in the progression of OA.

Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels

Cells exhibited four types of mechanical responses:

(1) an immediate viscoelastic response;

(2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces;

(3) later adaptive cell stiffening with sustained (>15 second) static stresses; and

(4) a large-scale repositioning response with prolonged (>1 minute) stress.

Importantly, these adaptation responses differed biochemically.

The immediate and early responses were affected by

chemically dissipating cytoskeletal prestress (isometric tension), whereas

the later adaptive response was not.

The repositioning response was prevented by

inhibiting tension through interference with Rho signaling,

similar to the case of the immediate and early responses, but it was also prevented by

blocking mechanosensitive ion channels or

by inhibiting Src tyrosine kinases.

All adaptive responses were suppressed by cooling cells to 4°C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.

Microtubule-Stimulated ADP Release, ATP Binding, and Force Generation In Transport Kinesins

All three classes of molecular motor proteins are now known to be

large protein families with diverse cellular functions.

Both the kinesin family and the myosin family have been defined and their proteins grouped into subfamilies. Finally, the elusive cytoplasmic version of dynein was identified and a multigene family of flagellar and cytoplasmic dyneins defined. Members of a given motor protein family share

significant homology in their motor domains with the defining member,
kinesin, dynein or myosin; but they also contain
unique protein domains that are specialized for interaction with different cargoes.

This large number of motor proteins may reflect

the number of cellular functions that require force generation or movement,
ranging from mitosis to morphogenesis to transport of vesicles.

Kinesins are a large family of microtubule (MT)-based motors that play important roles in many cellular activities including

mitosis,

motility, and

intracellular transport

Their involvement in a range of pathological processes

also highlights their significance as therapeutic targets and
the importance of understanding the molecular basis of their function

They are defined by their motor domains that contain both

the microtubule (MT) and
ATP binding sites.

Three ATP binding motifs—

the P-loop,
switch I,
switch II–

are highly conserved among

kinesins,
myosin motors, and

small GTPases.

They share a conserved mode of MT binding such that

MT binding,
ATP binding, and
hydrolysis

are functionally coupled for efficient MT-based work.

The interior of a cell is a hive of activity, filled with

proteins and other items moving from one location to another.

A network of filaments called microtubules forms tracks

along which so-called motor proteins carry these items.

Kinesins are one group of motor proteins, and a typical kinesin protein has

one end (called the ‘motor domain’) that can attach itself to the microtubules.

The other end links to the cargo being carried, and a ‘neck’ connects the two. When two of these proteins work together,

flexible regions of the neck allow the two motor domains to move past one another,
which enable the kinesin to essentially walk along a microtubule in a stepwise manner.

Although the two kinesins have been thought to move along the microtubule tracks in different ways, Atherton et al. find that the core mechanism used by their motor domains is the same.

When a motor domain binds to the microtubule, its shape changes,

first stimulating release of the breakdown products of ATP from the previous cycle.

This release makes room for a new ATP molecule to bind. The structural changes caused by ATP binding

produce larger changes in the flexible neck region that
enable individual motor domains within a kinesin pair to
co-ordinate their movement and move in a consistent direction.

The major and largely invariant point of contact between kinesin motor domains and the MT is helix-α4,

which lies at the tubulin intradimer interface.

The conformational changes in functionally important regions of each motor domain are described,

starting with the nucleotide-binding site,
from which all other conformational changes emanate.

The nucleotide-binding site (Figure 2) has three major elements:

(1) the P-loop (brown) is visible in all our reconstructions;

(2) loop9 (yellow, contains switch I) undergoes major conformational changes through the ATPase cycle; and

(3) loop11 (red, contains switch II) that connects strand-β7 to helix-α4, the conformation and flexibility of which is

determined by MT binding and motor nucleotide state.

Movement and extension of helix-α6 controls neck linker docking

the N-terminus of helix-α6 is closely associated with elements of the nucleotide binding site suggesting that

its conformation alters in response to different nucleotide states.

Further,

because the orientation of helix-α6 with respect to helix-α4 controls neck linker docking and
because helix-α4 is held against the MT during the ATPase cycle,
- conformational changes in helix-α6 control movement of the neck linker.

Mechanical amplification and force generation involves conformational changes across the motor domain

A key conformational change in the motor domain following Mg-ATP binding is

peeling of the central β-sheet from the C-terminus of helix-α4 increasing their separation;
this is required to accommodate rotation of helix-α6 and consequent neck linker docking

ATP binding draws loop11 and loop9 closer together; causing

(1) tilting of most of the motor domain not contacting the MT towards the nucleotide-binding site,

(2) rotation, translation, and extension of helix-α6 which we propose contributes to force generation, and

(3) allows neck linker docking and biases movement of the 2nd head towards the MT plus end.

In both motors, microtubule binding promotes

ordered conformations of conserved loops that

stimulate ADP release,

enhance microtubule affinity and

prime the catalytic site for ATP binding.

ATP binding causes only small shifts of these nucleotide-coordinating loops but induces

large conformational changes elsewhere that

allow force generation and

neck linker docking towards the microtubule plus end.

The study presents evidence provide evidence for a conserved ATP-driven

mechanism for kinesins and

reveals the critical mechanistic contribution of the microtubule interface.

Phosphorylation at endothelial cell–cell junctions: Implications for VE-cadherin function

This review summarizes the role of VE-cadherin phosphorylation in the regulation of endothelial cell–cell junctions and highlights how this affects vascular permeability and leukocyte extravasation.

The vascular endothelium is the inner lining of blood vessels and

forms a physical barrier between the vessel lumen and surrounding tissue;

controlling the extravasation of fluids,

plasma proteins and leukocytes.

Changes in the permeability of the endothelium are tightly regulated. Under basal physiological conditions, there is a continuous transfer of substances across the capillary beds. In addition the endothelium can mediate inducible,

transient hyperpermeability

in response to stimulation with inflammatory mediators,

which takes place primarily in post-capillary venules

However, when severe, inflammation may result in dysfunction of the endothelial barrier

in various parts of the vascular tree, including large veins, arterioles and capillaries.

Dysregulated permeability is observed in various pathological conditions, such as

tumor-induced angiogenesis,
cerebrovascular accident and
atherosclerosis.

Two fundamentally different pathways regulate endothelial permeability,

the transcellular and
paracellular pathways.

Solutes and cells can pass through the body of endothelial cells via the transcellular pathway, which includes

vesicular transport systems,
fenestrae, and
biochemical transporters.

The paracellular route is controlled by

the coordinated opening and closing of endothelial junctions and
thereby regulates traffic across the intercellular spaces between endothelial cells.

Endothelial cells are connected by

tight, gap and

adherens junctions,

of which the latter, and particularly the adherens junction component,

vascular endothelial (VE)-cadherin,

are of central importance for the initiation and stabilization of cell–cell contacts.

Although multiple adhesion molecules are localized at endothelial junctions,

blocking the adhesive function of VE-cadherin using antibodies
is sufficient to disrupt endothelial junctions and
to increase endothelial monolayer permeability both in vitro and in vivo.

Like other cadherins, VE-cadherin mediates adhesion via

homophilic, calcium-dependent interactions.

This cell–cell adhesion

is strengthened by binding of cytoplasmic proteins, the catenins,

to the C-terminus of VE-cadherin.

VE-cadherin can directly bind

β-catenin and plakoglobin, which
both associate with the actin binding protein α-catenin.

Initially, α-catenin was thought to directly anchor cadherins to the actin cytoskeleton, but recently it became clear that

α-catenin cannot bind to both β-catenin and actin simultaneously.

Numerous lines of evidence indicate that p120-catenin

promotes VE-cadherin surface expression and stability at the plasma membrane.

Different models are proposed that describe how

p120-catenin regulates cadherin membrane dynamics, including the hypothesis
that p120-catenin functions as a ‘cap’ that prevents the interaction of VE-cadherin
with the endocytic membrane trafficking machinery.

In addition, p120-catenin might regulate VE-cadherin internalization

through interactions with small GTPases.

Cytoplasmic p120-catenin, which is not bound to VE-cadherin, has been shown to

decrease RhoA activity,

elevate active Rac1 and Cdc42, and thereby is thought

to regulate actin cytoskeleton organization and membrane trafficking.

The intact cadherin-catenin complex is required for proper functioning of the adherens junction.

Several mechanisms may be involved in the

regulation of the organization and function of the cadherin–catenin complex, including

endocytosis of the complex,
VE-cadherin cleavage and
actin cytoskeleton reorganization.

The remainder of this review primarily focuses on the

role of tyrosine phosphorylation in the control of VE-cadherin-mediated cell–cell adhesion.

Regulation of the adhesive function of VE-cadherin by tyrosine phosphorylation

It is a widely accepted concept that tyrosine phosphorylation of

components of the VE–cadherin-catenin complex
Correlates with the weakening of cell–cell adhesion.

A general idea has emerged that

tyrosine phosphorylation of the VE-cadherin complex

leads to the uncoupling of VE-cadherin from the actin cytoskeleton

through dissociation of catenins from the cadherin.

However, tyrosine phosphorylation of VE-cadherin

is required for efficient transmigration of leukocytes.

This suggests that VE-cadherin-mediated cell–cell contacts

1.are not just pushed open by the migrating leukocytes, but play

2.a more active role in the transmigration process.

A schematic overview of leukocyte adhesion-induced signals leading to VE-cadherin phosphorylation

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin.

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs

N-glycosylation of E-cadherin has been shown to inhibit cell–cell adhesion.

Specifically, our recent studies have provided evidence that

the reduction of E-cadherin N-glycosylation
promoted the recruitment of stabilizing components,
vinculin and serine/ threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs)
and enhanced the association of AJs with the actin cytoskeleton.

Here, we examined the details of how

N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions.

Using the hypoglycosylated E-cadherin variant, V13, we show that

V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein.

This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that

increased association of PP2A with V13-containing AJs promoted their tethering to microtubules.

These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through

the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components

Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle

MacKinnon. Fig 1 Ion channels exhibit three basic properties

In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes

depend on their highly evolved and specialized cytoskeletal apparatus.

Defects in components of the cytoskeleton, in the long term,

affect the ability of the cell to compensate at both functional and structural levels.

In addition to the structural remodeling,

the myocardium becomes increasingly susceptible to altered electrical activity leading to arrhythmogenesis.

The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive.

subjects with severe left ventricular chamber dilation such as in DCM can have left bundle branch block (LBBB), while right bundle branch block (RBBB) is more characteristic of right ventricular failure. LBBB and RBBB have both been repeatedly associated with AV block in heart failure.

The impact of volume overload on structural and electro-cardiographic alterations has been noted in cardiomyopathy patients treated with left ventricular assist device (LVAD) therapy, which puts the heart at mechanical rest.

In LVAD-treated subjects,

QRS- and both QT- and QTc duration decreased,

suggesting that QRS- and QT-duration are significantly influenced by mechanical load and

that the shortening of the action potential duration contributes to the improved contractile performance after LVAD support.

An early postoperative period study after cardiac unloading therapy in 17 HF patients showed that in the first two weeks after LVAD implantation,

HF was associated with a relatively high incidence of ventricular arrhythmias associated with QTc interval prolongation.

In addition, a recent retrospective study of 100 adult patients with advanced HF, treated with an axial-flow HeartMate LVAD suggested that

the rate of new-onset monomorphic ventricular tachycardia (MVT) was increased in LVAD treated patients compared to patients given only medical treatment,

The myocardium is exposed to severe and continuous biomechanical stress during each contraction-relaxation cycle. When fiber tension remains uncompensated or simply unbalanced,

it may represent a trigger for arrhythmogenesis caused by cytoskeletal stretching,

which ultimately leads to altered ion channel localization, and subsequent action potential and conduction alterations.

Cytoskeletal proteins not only provide the backbone of the cellular structure, but they also

maintain the shape and flexibility of the different sub-cellular compartments, including the

1.plasma membrane,

2.the double lipid layer, which defines the boundaries of the cell and where

ion channels are mainly localized.

The interaction between the sarcomere, which is the basic for the passive force during diastole and for the restoring force during systole.

Sarcomeric Proteins and Ion Channels

besides fiber stretch associated with mechanical and hemodynamic impairment, cytoskeletal alterations due to primary genetic defects or indirectly to alterations in response to cellular injury can potentially

1.affect ion channel anchoring, and trafficking, as well as

2.functional regulation by second messenger pathways,

3.causing an imbalance in cardiac ionic homeostasis that will trigger arrhythmogenesis.

Intense investigation of

the sarcomeric actin network,

the Z-line structure, and

chaperone molecules docking in the plasma membrane,

has shed new light on the molecular basis of

cytoskeletal interactions in regulating ion channels

Actin disruption using cytochalasin D, an agent that interferes with actin polymerization, increased Na+ channel activity in 90% of excised patches tested within 2 min, which indicated that

the integrity of the filamentous actin (F-actin) network was essential for the maintenance of normal Na+ channel function

These data were the first to support a role for the cytoskeleton in cardiac arrhythmias.

Molecular interactions between the cytoskeleton and ion channels

The figure illustrates the interactions between the ion channels on the sarcolemma, and the sarcomere in cardiac myocytes. Note that the Z-line is connected to the cardiac T-tubules. The diagram illustrates the complex protein-protein interactions that occur between structural components of the cytoskeleton and ion channels. The cytoskeleton is involved in regulating the metabolism of ion channels, modifying their expression, localization, and electrical properties.

sarcomere structure

It is important to be aware of the enormous variety of clinical presentations that derive from distinct variants in the same pool of genetic factors. Knowledge of these variants could facilitate tailoring the therapy of choice for each patient. In particular,

the recent findings of structural and functional links between

the cytoskeleton and ion channels

could expand the therapeutic interventions in

arrhythmia management in structurally abnormal myocardium, where aberrant binding

between cytoskeletal proteins can directly or indirectly alter ion channel function.

Read Full Post »

Summary of Signaling and Signaling Pathways

Posted in Academic Publishing, Alzheimer's Disease, Best evidence, Biochemical pathways, Bone Disease and Musculoskeletal Disease, Ca2+ triggered activation, Calcium Signaling, Calmodulin Kinase and Contraction, Cardiac muscle regeneration, Cardiomyopathy, Cardiovascular Research, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Curation, Cytoskeleton, Developmental biology, Dialectic, Discovery process, Disease Biology, Electrophysiology, Endocrine Diseases, Enzyme Induction, Epigenetics and Cardiovascular Risks, Experimental validation, Explanatory, Fatty acids, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Genomic Expression, Human Immune System in Health and in Disease, Inferential analysis, Kinase, Lipid metabolism, Lipids, Mechanical Assist Devices: LVAD, Metabolism, Metabolomics, Mg++, MS, Mutant Gene Expression, Myocardial Metabolism, Neurohumoral Transmission, Neurological Diseases, Nitric Oxide in Health and Disease, Nutrition, Nutritional Supplements: Atherogenesis, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmaceutical R&D Investment, Pharmacodynamics and Pharmacokinetics, Phosphorylase, Phosphorylation, PKC, Population Health Management, Proteins, Proteomics, RNA Biology, Signaling, Signaling & Cell Circuits, Skeletal muscle regeneration, Small Molecules in Development of Therapeutic Drugs, Systemic Inflammatory Response Related Disorders, Transcriptomics, Translational Research, Translational Science, Ubiquitinylation, tagged Alzheimers Disease, a_b catenin, cell signaling, Cytoskeleton, Extracellular matrix, G-protein coupled receptors (GPCR), integrin, intracellular localization, myasthenia gravis, N-glycosylation, neuroendocrine signal, Neuroendocrine Transcriptome, nuclear receptor, Phosphorylation, protein signals, Receptor tyrosine kinases, second messenger, signaling, signaling cascades, transduction, Tyrosine Kinase, zinc on November 1, 2014| Leave a Comment »

Summary of Signaling and Signaling Pathways

Author and Curator: Larry H Bernstein, MD, FCAP

In the imtroduction to this series of discussions I pointed out JEDS Rosalino’s observation about the construction of a complex molecule of acetyl coenzyme A, and the amount of genetic coding that had to go into it. Furthermore, he observes – Millions of years later, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

acetylCoA

In the tutorial that follows we find support for the view that mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, are achieving a separation from inconsistent views introduced by the classical model of molecular biology and genomics, toward a more functional cellular dynamics that is not dependent on the classic view. The classical view fits a rigid framework that is to genomics and metabolomics as Mendelian genetics if to multidimentional, multifactorial genetics. The inherent difficulty lies in two places:

Interactions between differently weighted determinants
A large part of the genome is concerned with regulatory function, not expression of the code

The goal of the tutorial was to achieve an understanding of how cell signaling occurs in a cell. Completion of the tutorial would provide

a basic understanding signal transduction and
the role of phosphorylation in signal transduction.

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

In addition – detailed knowledge of –

the role of Tyrosine kinases and
G protein-coupled receptors in cell signaling.

serine

threonine

protein kinase

We are constantly receiving and interpreting signals from our environment, which can come

in the form of light, heat, odors, touch or sound.

The cells of our bodies are also

constantly receiving signals from other cells.

These signals are important to

keep cells alive and functioning as well as
to stimulate important events such as
cell division and differentiation.

Signals are most often chemicals that can be found

in the extracellular fluid around cells.

These chemicals can come

from distant locations in the body (endocrine signaling by hormones), from
nearby cells (paracrine signaling) or can even
be secreted by the same cell (autocrine signaling).

Signaling molecules may trigger any number of cellular responses, including

changing the metabolism of the cell receiving the signal or
result in a change in gene expression (transcription) within the nucleus of the cell or both.

controlling the output of ribosomes.

To which I would now add..

result in either an inhibitory or a stimulatory effect

The three stages of cell signaling are:

Cell signaling can be divided into 3 stages:

Reception: A cell detects a signaling molecule from the outside of the cell.

Transduction: When the signaling molecule binds the receptor it changes the receptor protein in some way. This change initiates the process of transduction. Signal transduction is usually a pathway of several steps. Each relay molecule in the signal transduction pathway changes the next molecule in the pathway.

Response: Finally, the signal triggers a specific cellular response.

signal transduction

http://www.hartnell.edu/tutorials/biology/images/signaltransduction_simple.jpg

The initiation is depicted as follows:

Signal Transduction – ligand binds to surface receptor

Membrane receptors function by binding the signal molecule (ligand) and causing the production of a second signal (also known as a second messenger) that then causes a cellular response. These types of receptors transmit information from the extracellular environment to the inside of the cell.

by changing shape or
by joining with another protein
once a specific ligand binds to it.

Examples of membrane receptors include

G Protein-Coupled Receptors and

Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.

Receptor Tyrosine Kinases.

intracellular signaling

http://www.hartnell.edu/tutorials/biology/images/membrane_receptor_tk.jpg

Intracellular receptors are found inside the cell, either in the cytopolasm or in the nucleus of the target cell (the cell receiving the signal).

Note that though change in gene expression is stated, the change in gene expression does not here imply a change in the genetic information – such as – mutation. That does not have to be the case in the normal homeostatic case.

This point is the differentiating case between what JEDS Roselino has referred as

a fast, adaptive reaction, that is the feature of protein molecules, and distinguishes this interaction from
a one-to-one transcription of the genetic code.

The rate of transcription can be controlled, or it can be blocked. This is in large part in response to the metabolites in the immediate interstitium.

This might only be

a change in the rate of a transcription or a suppression of expression through RNA.
Or through a conformational change in an enzyme

Swinging domains in HECT E3 enzymes

Since signaling systems need to be

responsive to small concentrations of chemical signals and act quickly,
cells often use a multi-step pathway that transmits the signal quickly,
while amplifying the signal to numerous molecules at each step.

Signal transduction pathways are shown (simplified):

Signal Transduction

Signal transduction occurs when an

extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell.
In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.
Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.
The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.

In 1970, Martin Rodbell examined the effects of glucagon on a rat’s liver cell membrane receptor. He noted that guanosine triphosphate disassociated glucagon from this receptor and stimulated the G-protein, which strongly influenced the cell’s metabolism. Thus, he deduced that the G-protein is a transducer that accepts glucagon molecules and affects the cell. For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.

Guanosine monophosphate structure

In 2007, a total of 48,377 scientific papers—including 11,211 e-review papers—were published on the subject. The term first appeared in a paper’s title in 1979. Widespread use of the term has been traced to a 1980 review article by Rodbell: Research papers focusing on signal transduction first appeared in large numbers in the late 1980s and early 1990s.

Signal transduction involves the binding of extracellular signaling molecules and ligands to cell-surface receptors that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation of the receptor, known as receptor activation.

This activation is always the initial step (the cause) leading to the cell’s ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.

Integrin

Most steroid hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of steroid-responsive genes.

steroid hormone receptor

Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye, and odorants binding to odorant receptors in the nasal epithelium. Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune system response against invading pathogens mediated by signal transduction processes. This may occur independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.

Signal transduction cascades amplify the signal output

G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including adrenergic receptors and chemokine receptors.

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

signal transduction pathways

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer consisting of Gα, Gβ, and Gγ. Once the GPCR recognizes a ligand, the conformation of the receptor changes to activate the G protein, causing Gα to bind a molecule of GTP and dissociate from the other two G-protein subunits.

The dissociation exposes sites on the subunits that can interact with other molecules. The activated G protein subunits detach from the receptor and initiate signaling from many downstream effector proteins such as phospholipases and ion channels, the latter permitting the release of second messenger molecules.

Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.

insulin receptor and and insulin receptor signaling pathway (IRS)

To perform signal transduction, RTKs need to form dimers in the plasma membrane; the dimer is stabilized by ligands binding to the receptor.

RTKs

The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes.

Subsequent to this, the receptors’ kinase domains are activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that facilitate various cellular processes such as cell differentiation and metabolism.

Signal-Transduction-Pathway

As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK into the cell. In this case, the G proteins are

members of the Ras, Rho, and Raf families, referred to collectively as small G proteins.

They act as molecular switches usually

tethered to membranes by isoprenyl groups linked to their carboxyl ends.

Upon activation, they assign proteins to specific membrane subdomains where they participate in signaling. Activated RTKs in turn activate

small G proteins that activate guanine nucleotide exchange factors such as SOS1.

Once activated, these exchange factors can activate more small G proteins, thus

amplifying the receptor’s initial signal.

The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors that exist in a constitutively activate state; such mutated genes may act as oncogenes.

Integrin

Integrin

Integrin-mediated signal transduction

An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).

Integrins are produced by a wide variety of cells; they play a role in

cell attachment to other cells and the extracellular matrix and
in the transduction of signals from extracellular matrix components such as fibronectin and collagen.

Ligand binding to the extracellular domain of integrins

changes the protein’s conformation,
clustering it at the cell membrane to
initiate signal transduction.

Integrins lack kinase activity; hence, integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules, the main coordinator being integrin-linked kinase.

As shown in the picture, cooperative integrin-RTK signaling determines the

timing of cellular survival,
apoptosis,
proliferation, and
differentiation.

integrin-mediated signal transduction

Integrin signaling

ion channel

A ligand-gated ion channel, upon binding with a ligand, changes conformation

to open a channel in the cell membrane
through which ions relaying signals can pass.

An example of this mechanism is found in the receiving cell of a neural synapse. The influx of ions that occurs in response to the opening of these channels

induces action potentials, such as those that travel along nerves,
by depolarizing the membrane of post-synaptic cells,
resulting in the opening of voltage-gated ion channels.

RyR and Ca+ release from SR

An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca2+;

it acts as a second messenger
initiating signal transduction cascades and
altering the physiology of the responding cell.

This results in amplification of the synapse response between synaptic cells

by remodelling the dendritic spines involved in the synapse.

In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic AMP and IP3,

cAMP

Inositol_1,4,5-trisphosphate.svg

the latter controlling the release of intracellular calcium stores into the cytoplasm.

Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that bind to specific secondary messenger molecules. For example,

calcium ions bind to the EF hand domains of calmodulin,
allowing it to bind and activate calmodulin-dependent kinase.

calcium movement and RyR2 receptor

PIP3 and other phosphoinositides do the same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.

Signals can be generated within organelles, such as chloroplasts and mitochondria, modulating the nuclear
gene expression in a process called retrograde signaling.

Recently, integrative genomics approaches, in which correlation analysis has been applied on transcript and metabolite profiling data of Arabidopsis thaliana, revealed the identification of metabolites which are putatively acting as mediators of nuclear gene expression.

http://fpls.com/unraveling_retrograde_signaling_pathways:_finding_candidate_signaling_molecules_via_metabolomics_and_systems_biology_driven_approaches

Systems Biology Approach Reveals Genome to Phenome Correlation in Type 2 Diabetes (plosone.org)
Gene Expression and Thiopurine Metabolite Profiling in Inflammatory Bowel Disease – Novel Clues to Drug Targets and Disease Mechanisms? (plosone.org)
Activation of the Jasmonic Acid Plant Defence Pathway Alters the Composition of Rhizosphere

Nutrients 2014, 6, 3245-3258; http://dx.doi.org:/10.3390/nu6083245

Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are

α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain,

DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total.

DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on.

On the other hand,

zinc is the most abundant trace metal in the human brain.

There are many scientific studies linking zinc, especially

excess amounts of free zinc, to cellular death.

Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between

omega-3 fatty acids, zinc transporter levels and
free zinc availability at cellular levels.

Many other studies have also suggested a possible

omega-3 and zinc effect on neurodegeneration and cellular death.

Therefore, in this review, we will examine

the effect of omega-3 fatty acids on zinc transporters and
the importance of free zinc for human neuronal cells.

Moreover, we will evaluate the collective understanding of

mechanism(s) for the interaction of these elements in neuronal research and their
significance for the diagnosis and treatment of neurodegeneration.

Epidemiological studies have linked high intake of fish and shellfish as part of the daily diet to

reduction of the incidence and/or severity of Alzheimer’s disease (AD) and senile mental decline in

Omega-3 fatty acids are one of the two main families of a broader group of fatty acids referred to as polyunsaturated fatty acids (PUFAs). The other main family of PUFAs encompasses the omega-6 fatty acids. In general, PUFAs are essential in many biochemical events, especially in early post-natal development processes such as

cellular differentiation,
photoreceptor membrane biogenesis and
active synaptogenesis.

Despite the significance of these

two families, mammals cannot synthesize PUFA de novo, so they must be ingested from dietary sources. Though belonging to the same family, both

omega-3 and omega-6 fatty acids are metabolically and functionally distinct and have
opposing physiological effects. In the human body,
high concentrations of omega-6 fatty acids are known to increase the formation of prostaglandins and
thereby increase inflammatory processes [10].

the reverse process can be seen with increased omega-3 fatty acids in the body.

Many other factors, such as

thromboxane A2 (TXA2),
leukotriene
B4 (LTB4),
IL-1,
IL-6,
tumor necrosis factor (TNF) and
C-reactive protein,

which are implicated in various health conditions, have been shown to be increased with high omega-6 fatty acids but decreased with omega-3 fatty acids in the human body.

Dietary fatty acids have been identified as protective factors in coronary heart disease, and PUFA levels are known to play a critical role in

immune responses,
gene expression and
intercellular communications.

omega-3 fatty acids are known to be vital in

the prevention of fatal ventricular arrhythmias, and
are also known to reduce thrombus formation propensity by decreasing platelet aggregation, blood viscosity and fibrinogen levels

.Since omega-3 fatty acids are prevalent in the nervous system, it seems logical that a deficiency may result in neuronal problems, and this is indeed what has been identified and reported.

The main

In another study conducted with individuals of 65 years of age or older (n = 6158), it was found that

only high fish consumption, but
not dietary omega-3 acid intake,
had a protective effect on cognitive decline

In 2005, based on a meta-analysis of the available epidemiology and preclinical studies, clinical trials were conducted to assess the effects of omega-3 fatty acids on cognitive protection. Four of the trials completed have shown

a protective effect of omega-3 fatty acids only among those with mild cognitive impairment conditions.

A trial of subjects with mild memory complaints demonstrated

an improvement with 900 mg of DHA.

We review key findings on

the effect of the omega-3 fatty acid DHA on zinc transporters and the
importance of free zinc to human neuronal cells.

DHA is the most abundant fatty acid in neural membranes, imparting appropriate

fluidity and other properties,

and is thus considered as the most important fatty acid in neuronal studies. DHA is well conserved throughout the mammalian species despite their dietary differences. It is mainly concentrated

in membrane phospholipids at synapses and
in retinal photoreceptors and
also in the testis and sperm.

In adult rats’ brain, DHA comprises approximately

17% of the total fatty acid weight, and
in the retina it is as high as 33%.

DHA is believed to have played a major role in the evolution of the modern human –

in particular the well-developed brain.

Premature babies fed on DHA-rich formula show improvements in vocabulary and motor performance.

Analysis of human cadaver brains have shown that

people with AD have less DHA in their frontal lobe
and hippocampus compared with unaffected individuals

Furthermore, studies in mice have increased support for the

protective role of omega-3 fatty acids.

Mice administrated with a dietary intake of DHA showed

an increase in DHA levels in the hippocampus.

Errors in memory were decreased in these mice and they demonstrated

reduced peroxide and free radical levels,
suggesting a role in antioxidant defense.

Another study conducted with a Tg2576 mouse model of AD demonstrated that dietary

DHA supplementation had a protective effect against reduction in
drebrin (actin associated protein), elevated oxidation, and to some extent, apoptosis via
decreased caspase activity.

Zinc

Zinc is a trace element, which is indispensable for life, and it is the second most abundant trace element in the body. It is known to be related to

growth,
development,
differentiation,
immune response,
receptor activity,
DNA synthesis,
gene expression,
neuro-transmission,
enzymatic catalysis,
hormonal storage and release,
tissue repair,
memory,
the visual process

and many other cellular functions. Moreover, the indispensability of zinc to the body can be discussed in many other aspects, as

a component of over 300 different enzymes
an integral component of a metallothioneins
a gene regulatory protein.

Approximately 3% of all proteins contain

zinc binding motifs .

The broad biological functionality of zinc is thought to be due to its stable chemical and physical properties. Zinc is considered to have three different functions in enzymes;

catalytic,
coactive and

Indeed, it is the only metal found in all six different subclasses

of enzymes. The essential nature of zinc to the human body can be clearly displayed by studying the wide range of pathological effects of zinc deficiency. Anorexia, embryonic and post-natal growth retardation, alopecia, skin lesions, difficulties in wound healing, increased hemorrhage tendency and severe reproductive abnormalities, emotional instability, irritability and depression are just some of the detrimental effects of zinc deficiency.

Proper development and function of the central nervous system (CNS) is highly dependent on zinc levels. In the mammalian organs, zinc is mainly concentrated in the brain at around 150 μm. However, free zinc in the mammalian brain is calculated to be around 10 to 20 nm and the rest exists in either protein-, enzyme- or nucleotide bound form. The brain and zinc relationship is thought to be mediated

through glutamate receptors, and
it inhibits excitatory and inhibitory receptors.

Vesicular localization of zinc in pre-synaptic terminals is a characteristic feature of brain-localized zinc, and

its release is dependent on neural activity.

Retardation of the growth and development of CNS tissues have been linked to low zinc levels. Peripheral neuropathy, spina bifida, hydrocephalus, anencephalus, epilepsy and Pick’s disease have been linked to zinc deficiency. However, the body cannot tolerate excessive amounts of zinc.

The relationship between zinc and neurodegeneration, specifically AD, has been interpreted in several ways. One study has proposed that β-amyloid has a greater propensity to

form insoluble amyloid in the presence of
high physiological levels of zinc.

Insoluble amyloid is thought to

aggregate to form plaques,

which is a main pathological feature of AD. Further studies have shown that

chelation of zinc ions can deform and disaggregate plaques.

In AD, the most prominent injuries are found in

hippocampal pyramidal neurons, acetylcholine-containing neurons in the basal forebrain, and in
somatostatin-containing neurons in the forebrain.

All of these neurons are known to favor

rapid and direct entry of zinc in high concentration
leaving neurons frequently exposed to high dosages of zinc.

This is thought to promote neuronal cell damage through oxidative stress and mitochondrial dysfunction. Excessive levels of zinc are also capable of

inhibiting Ca2+ and Na+ voltage gated channels
and up-regulating the cellular levels of reactive oxygen species (ROS).

High levels of zinc are found in Alzheimer’s brains indicating a possible zinc related neurodegeneration. A study conducted with mouse neuronal cells has shown that even a 24-h exposure to high levels of zinc (40 μm) is sufficient to degenerate cells.

If the human diet is deficient in zinc, the body

efficiently conserves zinc at the tissue level by compensating other cellular mechanisms

to delay the dietary deficiency effects of zinc. These include reduction of cellular growth rate and zinc excretion levels, and

redistribution of available zinc to more zinc dependent cells or organs.

A novel method of measuring metallothionein (MT) levels was introduced as a biomarker for the

assessment of the zinc status of individuals and populations.

In humans, erythrocyte metallothionein (E-MT) levels may be considered as an indicator of zinc depletion and repletion, as E-MT levels are sensitive to dietary zinc intake. It should be noted here that MT plays an important role in zinc homeostasis by acting

as a target for zinc ion binding and thus
assisting in the trafficking of zinc ions through the cell,
which may be similar to that of zinc transporters

Zinc Transporters

Deficient or excess amounts of zinc in the body can be catastrophic to the integrity of cellular biochemical and biological systems. The gastrointestinal system controls the absorption, excretion and the distribution of zinc, although the hydrophilic and high-charge molecular characteristics of zinc are not favorable for passive diffusion across the cell membranes. Zinc movement is known to occur

via intermembrane proteins and zinc transporter (ZnT) proteins

These transporters are mainly categorized under two metal transporter families; Zip (ZRT, IRT like proteins) and CDF/ZnT (Cation Diffusion Facilitator), also known as SLC (Solute Linked Carrier) gene families: Zip (SLC-39) and ZnT (SLC-30). More than 20 zinc transporters have been identified and characterized over the last two decades (14 Zips and 8 ZnTs).

Members of the SLC39 family have been identified as the putative facilitators of zinc influx into the cytosol, either from the extracellular environment or from intracellular compartments (Figure 1).

The identification of this transporter family was a result of gene sequencing of known Zip1 protein transporters in plants, yeast and human cells. In contrast to the SLC39 family, the SLC30 family facilitates the opposite process, namely zinc efflux from the cytosol to the extracellular environment or into luminal compartments such as secretory granules, endosomes and synaptic vesicles; thus decreasing intracellular zinc availability (Figure 1). ZnT3 is the most important in the brain where

it is responsible for the transport of zinc into the synaptic vesicles of
glutamatergic neurons in the hippocampus and neocortex,

Figure 1: Subcellular localization and direction of transport of the zinc transporter families, ZnT and ZIP. Arrows show the direction of zinc mobilization for the ZnT (green) and ZIP (red) proteins. A net gain in cytosolic zinc is achieved by the transportation of zinc from the extracellular region and organelles such as the endoplasmic reticulum (ER) and Golgi apparatus by the ZIP transporters. Cytosolic zinc is mobilized into early secretory compartments such as the ER and Golgi apparatus by the ZnT transporters. Figures were produced using Servier Medical Art, http://www.servier.com/. http://www.hindawi.com/journals/jnme/2012/173712.fig.001.jpg

Figure 2: Early zinc signaling (EZS) and late zinc signaling (LZS). EZS involves transcription-independent mechanisms where an extracellular stimulus directly induces an increase in zinc levels within several minutes by releasing zinc from intracellular stores (e.g., endoplasmic reticulum). LSZ is induced several hours after an external stimulus and is dependent on transcriptional changes in zinc transporter expression. Components of this figure were produced using Servier Medical Art, http://www.servier.com/ and adapted from Fukada et al. [30].

omega-3 fatty acids in the mammalian body are

α-linolenic acid (ALA),
docosahexenoic acid (DHA) and
eicosapentaenoic acid (EPA).

In general, seafood is rich in omega-3 fatty acids, more specifically DHA and EPA (Table 1). Thus far, there are nine separate epidemiological studies that suggest a possible link between

increased fish consumption and reduced risk of AD
and eight out of ten studies have reported a link between higher blood omega-3 levels

DHA and Zinc Homeostasis

Many studies have identified possible associations between DHA levels, zinc homeostasis, neuroprotection and neurodegeneration. Dietary DHA deficiency resulted in

increased zinc levels in the hippocampus and
elevated expression of the putative zinc transporter, ZnT3, in the rat brain.

Altered zinc metabolism in neuronal cells has been linked to neurodegenerative conditions such as AD. A study conducted with transgenic mice has shown a significant link between ZnT3 transporter levels and cerebral amyloid plaque pathology. When the ZnT3 transporter was silenced in transgenic mice expressing cerebral amyloid plaque pathology,

a significant reduction in plaque load
and the presence of insoluble amyloid were observed.

In addition to the decrease in plaque load, ZnT3 silenced mice also exhibited a significant

reduction in free zinc availability in the hippocampus
and cerebral cortex.

Collectively, the findings from this study are very interesting and indicate a clear connection between

zinc availability and amyloid plaque formation,

thus indicating a possible link to AD.

DHA supplementation has also been reported to limit the following:

amyloid presence,
synaptic marker loss,
hyper-phosphorylation of Tau,
oxidative damage and
cognitive deficits in transgenic mouse model of AD.

In addition, studies by Stoltenberg, Flinn and colleagues report on the modulation of zinc and the effect in transgenic mouse models of AD. Given that all of these are classic pathological features of AD, and considering the limiting nature of DHA in these processes, it can be argued that DHA is a key candidate in preventing or even curing this debilitating disease.

In order to better understand the possible links and pathways of zinc and DHA with neurodegeneration, we designed a study that incorporates all three of these aspects, to study their effects at the cellular level. In this study, we were able to demonstrate a possible link between omega-3 fatty acid (DHA) concentration, zinc availability and zinc transporter expression levels in cultured human neuronal cells.

When treated with DHA over 48 h, ZnT3 levels were markedly reduced in the human neuroblastoma M17 cell line. Moreover, in the same study, we were able to propose a possible

neuroprotective mechanism of DHA,

which we believe is exerted through

a reduction in cellular zinc levels (through altering zinc transporter expression levels)
that in turn inhibits apoptosis.

DHA supplemented M17 cells also showed a marked depletion of zinc uptake (up to 30%), and

free zinc levels in the cytosol were significantly low compared to the control

This reduction in free zinc availability was specific to DHA; cells treated with EPA had no significant change in free zinc levels (unpublished data). Moreover, DHA-repleted cells had

low levels of active caspase-3 and
high Bcl-2 levels compared to the control treatment.

These findings are consistent with previous published data and further strengthen the possible

correlation between zinc, DHA and neurodegeneration.

On the other hand, recent studies using ZnT3 knockout (ZnT3KO) mice have shown the importance of

ZnT3 in memory and AD pathology.

For example, Sindreu and colleagues have used ZnT3KO mice to establish the important role of

ZnT3 in zinc homeostasis that modulates presynaptic MAPK signaling
required for hippocampus-dependent memory

Results from these studies indicate a possible zinc-transporter-expression-level-dependent mechanism for DHA neuroprotection.

Read Full Post »

Complex Models of Signaling: Therapeutic Implications

Posted in Academic Publishing, Alzheimer's Disease, Best evidence, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Bone Disease and Musculoskeletal Disease, Cancer and Current Therapeutics, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Curation, Cytoskeleton, Developmental biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Toxicity, Electrophysiology, Epigenetics and Cardiovascular Risks, Etiology, Experimental validation, Explanatory, Fatty acids, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Human Immune System in Health and in Disease, Inferential analysis, Innovation in Immunology Diagnostics, Innovations in Neurophysiology & Neuropsychology, Kinase, Lipid metabolism, Liver & Digestive Diseases Research, Metabolism, Metabolomics, MS, Myocardial Ischemia, Neurodegenerative Diseases, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Nutrition, Nutritional Supplements: Atherogenesis, lipid metabolism, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmacologic toxicities, Phosphorylation, Population Health Management, Proteins, Proteomics, Regenerative Biology and Medicine, Signaling, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, Translational Effectiveness, Translational Research, Translational Science, Ubiquitinylation, tagged Alzheimers Disease, Amyloid, B-type natriuretic peptide, cell differentiation, cell signaling, DHA, Extracellular matrix, Heart Failure, intracellular localization, multiple schlerosis, neuritogenesis, Omega-3 fatty acid, plasticity, prion protein, zinc on October 31, 2014| Leave a Comment »

Complex Models of Signaling: Therapeutic Implications

Curator: Larry H. Bernstein, MD, FCAP

Updated 6/24/2019

Fishy Business: Effect of Omega-3 Fatty Acids on Zinc Transporters and Free Zinc Availability in Human Neuronal Cells

Damitha De Mel and Cenk Suphioglu *

NeuroAllergy Research Laboratory (NARL), School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Waurn Ponds, Victoria, Australia.

Nutrients 2014, 6, 3245-3258; http://dx.doi.org:/10.3390/nu6083245

Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are

α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain,

DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total.

DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on.

On the other hand,

zinc is the most abundant trace metal in the human brain.

There are many scientific studies linking zinc, especially

excess amounts of free zinc, to cellular death.

Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between

omega-3 fatty acids, zinc transporter levels and
free zinc availability at cellular levels.

Many other studies have also suggested a possible

omega-3 and zinc effect on neurodegeneration and cellular death.

Therefore, in this review, we will examine

the effect of omega-3 fatty acids on zinc transporters and
the importance of free zinc for human neuronal cells.

Moreover, we will evaluate the collective understanding of

mechanism(s) for the interaction of these elements in neuronal research and their
significance for the diagnosis and treatment of neurodegeneration.

Epidemiological studies have linked high intake of fish and shellfish as part of the daily diet to

reduction of the incidence and/or severity of Alzheimer’s disease (AD) and senile mental decline in

cellular differentiation,
photoreceptor membrane biogenesis and
active synaptogenesis.

Despite the significance of these

two families, mammals cannot synthesize PUFA de novo, so they must be ingested from dietary sources. Though belonging to the same family, both

omega-3 and omega-6 fatty acids are metabolically and functionally distinct and have
opposing physiological effects. In the human body,
high concentrations of omega-6 fatty acids are known to increase the formation of prostaglandins and
thereby increase inflammatory processes [10].

the reverse process can be seen with increased omega-3 fatty acids in the body.

Many other factors, such as

thromboxane A2 (TXA2),
leukotriene
B4 (LTB4),
IL-1,
IL-6,
tumor necrosis factor (TNF) and
C-reactive protein,

which are implicated in various health conditions, have been shown to be increased with high omega-6 fatty acids but decreased with omega-3 fatty acids in the human body.

Dietary fatty acids have been identified as protective factors in coronary heart disease, and PUFA levels are known to play a critical role in

immune responses,
gene expression and
intercellular communications.

omega-3 fatty acids are known to be vital in

the prevention of fatal ventricular arrhythmias, and
are also known to reduce thrombus formation propensity by decreasing platelet aggregation, blood viscosity and fibrinogen levels

.Since omega-3 fatty acids are prevalent in the nervous system, it seems logical that a deficiency may result in neuronal problems, and this is indeed what has been identified and reported.

The main omega-3 fatty acids in the mammalian body are

α-linolenic acid (ALA),
docosahexenoic acid (DHA) and
eicosapentaenoic acid (EPA).

In general, seafood is rich in omega-3 fatty acids, more specifically DHA and EPA (Table 1). Thus far, there are nine separate epidemiological studies that suggest a possible link between

increased fish consumption and reduced risk of AD
and eight out of ten studies have reported a link between higher blood omega-3 levels

Table 1. Total percentage of omega-3 fatty acids in common foods and supplements.

Food/Supplement	EPA	DHA	ALA	Total %
Fish
SalmonSardine Anchovy Halibut Herring Mackerel Tuna Fresh Bluefin	XX X X X X X X	XX X X X X X X		>50%>50% >50% >50% >50% >50% >50% >50%
Oils/Supplements
Fish oil capsulesCod liver oils Salmon oil Sardine oil	XX X X	XX X X		>50%>50% >50% >50%
Black currant oilCanola oil Mustard seed oils Soybean oil Walnut oil Wheat germ oil			XX X X X X	10%–50%10%–50% 10%–50% 10%–50% 10%–50% 10%–50%
Seeds and other foods
Flaxseeds/LinseedsSpinach Wheat germ Human milk Peanut butter Soybeans Olive oil Walnuts			XX X X X X X X	>50%>50% 10%–50% 10%–50% <10% <10% <10% <10%

Table adopted from Maclean C.H. et al. [18].

In another study conducted with individuals of 65 years of age or older (n = 6158), it was found that

only high fish consumption, but
not dietary omega-3 acid intake,
had a protective effect on cognitive decline

a protective effect of omega-3 fatty acids only among those with mild cognitive impairment conditions.

A trial of subjects with mild memory complaints demonstrated

an improvement with 900 mg of DHA.

We review key findings on

the effect of the omega-3 fatty acid DHA on zinc transporters and the
importance of free zinc to human neuronal cells.

DHA is the most abundant fatty acid in neural membranes, imparting appropriate

fluidity and other properties,

and is thus considered as the most important fatty acid in neuronal studies. DHA is well conserved throughout the mammalian species despite their dietary differences. It is mainly concentrated

in membrane phospholipids at synapses and
in retinal photoreceptors and
also in the testis and sperm.

In adult rats’ brain, DHA comprises approximately

17% of the total fatty acid weight, and
in the retina it is as high as 33%.

DHA is believed to have played a major role in the evolution of the modern human –

in particular the well-developed brain.

Premature babies fed on DHA-rich formula show improvements in vocabulary and motor performance.

Analysis of human cadaver brains have shown that

people with AD have less DHA in their frontal lobe
and hippocampus compared with unaffected individuals

Furthermore, studies in mice have increased support for the

protective role of omega-3 fatty acids.

Mice administrated with a dietary intake of DHA showed

an increase in DHA levels in the hippocampus.

Errors in memory were decreased in these mice and they demonstrated

reduced peroxide and free radical levels,
suggesting a role in antioxidant defense.

Another study conducted with a Tg2576 mouse model of AD demonstrated that dietary

DHA supplementation had a protective effect against reduction in
drebrin (actin associated protein), elevated oxidation, and to some extent, apoptosis via
decreased caspase activity.

Zinc

Zinc is a trace element, which is indispensable for life, and it is the second most abundant trace element in the body. It is known to be related to

growth,
development,
differentiation,
immune response,
receptor activity,
DNA synthesis,
gene expression,
neuro-transmission,
enzymatic catalysis,
hormonal storage and release,
tissue repair,
memory,
the visual process

and many other cellular functions. Moreover, the indispensability of zinc to the body can be discussed in many other aspects, as

a component of over 300 different enzymes
an integral component of a metallothioneins
a gene regulatory protein.

Approximately 3% of all proteins contain

zinc binding motifs .

The broad biological functionality of zinc is thought to be due to its stable chemical and physical properties. Zinc is considered to have three different functions in enzymes;

catalytic,
coactive and

Indeed, it is the only metal found in all six different subclasses

through glutamate receptors, and
it inhibits excitatory and inhibitory receptors.

Vesicular localization of zinc in pre-synaptic terminals is a characteristic feature of brain-localized zinc, and

its release is dependent on neural activity.

The relationship between zinc and neurodegeneration, specifically AD, has been interpreted in several ways. One study has proposed that β-amyloid has a greater propensity to

form insoluble amyloid in the presence of
high physiological levels of zinc.

Insoluble amyloid is thought to

aggregate to form plaques,

which is a main pathological feature of AD. Further studies have shown that

chelation of zinc ions can deform and disaggregate plaques.

In AD, the most prominent injuries are found in

hippocampal pyramidal neurons, acetylcholine-containing neurons in the basal forebrain, and in
somatostatin-containing neurons in the forebrain.

All of these neurons are known to favor

rapid and direct entry of zinc in high concentration
leaving neurons frequently exposed to high dosages of zinc.

This is thought to promote neuronal cell damage through oxidative stress and mitochondrial dysfunction. Excessive levels of zinc are also capable of

inhibiting Ca2+ and Na+ voltage gated channels
and up-regulating the cellular levels of reactive oxygen species (ROS).

If the human diet is deficient in zinc, the body

efficiently conserves zinc at the tissue level by compensating other cellular mechanisms

to delay the dietary deficiency effects of zinc. These include reduction of cellular growth rate and zinc excretion levels, and

redistribution of available zinc to more zinc dependent cells or organs.

A novel method of measuring metallothionein (MT) levels was introduced as a biomarker for the

assessment of the zinc status of individuals and populations.

as a target for zinc ion binding and thus
assisting in the trafficking of zinc ions through the cell,
which may be similar to that of zinc transporters

Zinc Transporters

via intermembrane proteins and zinc transporter (ZnT) proteins

Members of the SLC39 family have been identified as the putative facilitators of zinc influx into the cytosol, either from the extracellular environment or from intracellular compartments (Figure 1).

it is responsible for the transport of zinc into the synaptic vesicles of
glutamatergic neurons in the hippocampus and neocortex,

Figure 1. Putative cellular localization of some of the different human zinc transporters (i.e., Zip1- Zip4 and ZnT1- ZnT7). Arrows indicate the direction of zinc passage by the appropriate putative zinc transporters in a generalized human cell. Although there are fourteen Zips and eight ZnTs known so far, only the main zinc transporters are illustrated in this figure for clarity and brevity.

zinc transporters

Early zinc signaling (EZS) and late zinc signaling (LZS)

http://www.hindawi.com/journals/jnme/2012/floats/173712/thumbnails/173712.fig.002_th.jpg

DHA and Zinc Homeostasis

Many studies have identified possible associations between DHA levels, zinc homeostasis, neuroprotection and neurodegeneration. Dietary DHA deficiency resulted in

increased zinc levels in the hippocampus and
elevated expression of the putative zinc transporter, ZnT3, in the rat brain.

a significant reduction in plaque load
and the presence of insoluble amyloid were observed.

In addition to the decrease in plaque load, ZnT3 silenced mice also exhibited a significant

reduction in free zinc availability in the hippocampus
and cerebral cortex.

Collectively, the findings from this study are very interesting and indicate a clear connection between

zinc availability and amyloid plaque formation,

thus indicating a possible link to AD.

DHA supplementation has also been reported to limit the following:

amyloid presence,
synaptic marker loss,
hyper-phosphorylation of Tau,
oxidative damage and
cognitive deficits in transgenic mouse model of AD.

When treated with DHA over 48 h, ZnT3 levels were markedly reduced in the human neuroblastoma M17 cell line. Moreover, in the same study, we were able to propose a possible

neuroprotective mechanism of DHA,

which we believe is exerted through

a reduction in cellular zinc levels (through altering zinc transporter expression levels)
that in turn inhibits apoptosis.

DHA supplemented M17 cells also showed a marked depletion of zinc uptake (up to 30%), and

free zinc levels in the cytosol were significantly low compared to the control

This reduction in free zinc availability was specific to DHA; cells treated with EPA had no significant change in free zinc levels (unpublished data). Moreover, DHA-repleted cells had

low levels of active caspase-3 and
high Bcl-2 levels compared to the control treatment.

These findings are consistent with previous published data and further strengthen the possible

correlation between zinc, DHA and neurodegeneration.

On the other hand, recent studies using ZnT3 knockout (ZnT3KO) mice have shown the importance of

ZnT3 in memory and AD pathology.

For example, Sindreu and colleagues have used ZnT3KO mice to establish the important role of

ZnT3 in zinc homeostasis that modulates presynaptic MAPK signaling
required for hippocampus-dependent memory

Results from these studies indicate a possible zinc-transporter-expression-level-dependent mechanism for DHA neuroprotection.

Collectively from these studies, the following possible mechanism can be proposed (Figure 2).

possible benefits of DHA in neuroprotection through reduction of ZnT3 transporter

Figure 2. Proposed neuroprotection mechanism of docosahexaenoic acid (DHA) in reference to synaptic zinc. Schematic diagram showing possible benefits of DHA in neuroprotection through reduction of ZnT3 transporter expression levels in human neuronal cells, which results in a reduction of zinc flux and thus lowering zinc concentrations in neuronal synaptic vesicles, and therefore contributing to a lower incidence of neurodegenerative diseases (ND), such as Alzheimer’s disease (AD).

More recent data from our research group have also shown a link between the expression levels of histone H3 and H4 proteins in human neuronal cells in relation to DHA and zinc. Following DHA treatment, both H3 and H4 levels were up-regulated. In contrast, zinc treatment resulted in a down-regulation of histone levels. Both zinc and DHA have shown opposing effects on histone post-translational modifications, indicating a possible distinctive epigenetic pattern. Upon treatment with zinc, M17 cells displayed an increase in histone deacetylase (HDACs) and a reduction in histone acetylation. Conversely, with DHA treatment, HDAC levels were significantly reduced and the acetylation of histones was up-regulated. These findings also support a possible interaction between DHA and zinc availability.

Conclusions

It is possible to safely claim that there is more than one potential pathway by which DHA and zinc interact at a cellular level, at least in cultured human neuronal cells. Significance and importance of both DHA and zinc in neuronal survival is attested by the presence of these multiple mechanisms.
Most of these reported studies were conducted using human neuroblastoma cells, or similar cell types, due to the lack of live mature human neuronal cells. Thus, the results may differ from results achieved under actual human physiological conditions due to the structural and functional differences between these cells and mature human neurons. Therefore, an alternative approach that can mimic the human neuronal cells more effectively would be advantageous.

Sphingosine-1-phosphate signaling as a therapeutic target

E Giannoudaki, DJ Swan, JA Kirby, S Ali

Applied Immunobiology and Transplantation Research Group, Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK

Cell Health and Cytoskeleton 2012; 4: 63–72

S1P is a 379Da member of the lysophospholipid family. It is the direct metabolite of sphingosine through the action of two sphingosine kinases, SphK1 and SphK2. The main metabolic pathway starts with the hydrolysis of sphingomyelin, a membrane sphingolipid, into ceramide by the enzyme sphingomyelinase and the subsequent production of sphingosine by ceramidase (Figure 1). Ceramide can also be produced de novo in the endoplasmic reticulum (ER) from serine and palmitoyl coenzyme A through multiple intermediates. S1P production is regulated by various S1P-specific and general lipid phosphatases, as well as S1P lyase, which irreversibly degrades S1P into phosphoethanolamine and hexadecanal. The balance between intracellular S1P and its metabolite ceramide can determine cellular fate. Ceramide promotes apoptosis, while S1P suppresses cell death and promotes cell survival. This creates an S1P ceramide “rheostat” inside the cells. S1P lyase expression in tissue is higher than it is in erythrocytes and platelets, the main “suppliers” of S1P in blood. This causes a tissue–blood gradient of S1P, which is important in many S1P-mediated responses, like the lymphocyte egress from lymphoid organs.

S1P signaling overview

S1P is produced inside cells; however, it can also be found extracellularly, in a variety of different tissues. It is abundant in the blood, at concentrations of 0.4–1.5 μM, where it is mainly secreted by erythrocytes and platelets. Blood S1P can be found separately, but mainly it exists in complexes with high-density lipoprotein (HDL) (∼60%). Many of the cardioprotective effects of HDL are hypothesized to involve S1P. Before 1996, S1P was thought to act mainly intracellularly as a second messenger. However, the identification of several GPCRs that bind S1P led to the initiation of many studies on

extracellular S1P signaling through those receptors.

There are five receptors that have been identified currently. These can be coupled with different G-proteins. Assuming that each receptor coupling with a G protein has a slightly different function, one can recognize the complexity of S1P receptor signaling.

S1P as a second messenger

S1P is involved in many cellular processes through its GPCR signaling; studies demonstrate that S1P also acts at an intracellular level. Intracellular S1P plays a role in maintaining the balance of cell survival signal toward apoptotic signals, creating a

cell “rheostat” between S1P and its precursor ceramide.

Important evidence that S1P can act intracellularly as a second messenger came from yeast (Saccharomyces cerevisiae) and plant (Arabidopsis thaliana) cells. Yeast cells do not express any S1P receptors, although they can be affected by S1P during heat-shock responses. Similarly, Arabidopsis has only one GPCR-like protein, termed “GCR1,” which does not bind S1P, although S1P regulates stomata closure during drought.

Sphingosine-1-phosphate

In mammals, the sphingosine kinases have been found to localize in different cell compartments, being responsible for the accumulation of S1P in those compartments to give intracellular signals. In mitochondria, for instance,

S1P was recently found to interact with prohibitin 2,

a conserved protein that maintains mitochondria assembly and function. According to the same study,

SphK2 is the major producer of S1P in mitochondria and the knockout of its gene can cause

disruption of mitochondrial respiration and cytochrome c oxidase function.

SphK2 is also present in the nucleus of many cells and has been implicated to cause cell cycle arrest, and it causes S1P accumulation in the nucleus. It seems that nuclear S1P is affiliated with the histone deacetylases HDAC1 and HDAC2,

inhibiting their activity, thus having an indirect effect in epigenetic regulation of gene expression.

In the ER, SphK2 has been identified to translocate during stress, and promote apoptosis. It seems that S1P has specific targets in the ER that cause apoptosis, probably through calcium mobilization signals.

Sphingosine 1-phosphate (S1P) is a small bioactive lipid molecule that is involved in several processes both intracellularly and extracellularly. It acts intracellularly

to promote the survival and growth of the cell,

through its interaction with molecules in different compartments of the cell.

It can also exist at high concentrations extracellularly, in the blood plasma and lymph. This causes an S1P gradient important for cell migration. S1P signals through five G protein-coupled receptors, S1PR1–S1PR5, whose expression varies in different types of cells and tissue. S1P signaling can be involved in physiological and pathophysiological conditions of the cardiovascular, nervous, and immune systems and diseases such as ischemia/reperfusion injury, autoimmunity, and cancer. In this review, we discuss how it can be used to discover novel therapeutic targets.

The involvement of S1P signaling in disease

In a mouse model of myocardial ischemia-reperfusion injury (IRI), S1P and its carrier, HDL, can help protect myocardial tissue and decrease the infarct size. It seems they reduce cardiomyocyte apoptosis and neutrophil recruitment to the ischemic tissue and may decrease leukocyte adhesion to the endothelium. This effect appears to be S1PR3 mediated, since in S1PR3 knockout mice it is alleviated.

Ischemia activates SphK1, which is then translocated to the plasma membrane. This leads to an increase of intracellular S1P, helping to promote cardiomyocyte survival against apoptosis, induced by ceramide. SphK1 knockout mice cannot be preconditioned against IRI, whereas SphK1 gene induction in the heart protects it from IRI. Interestingly, a recent study shows SphK2 may also play a role, since its knockout reduces the cardioprotective effects of preconditioning. Further, administration of S1P or sphingosine during reperfusion results in better recovery and attenuation of damage to cardiomyocytes. As with preconditioning, SphK1 deficiency also affects post-conditioning of mouse hearts after ischemia reperfusion (IR).

S1P does not only protect the heart from IRI. During intestinal IR, multiple organs can be damaged, including the lungs. S1P treatment of mice during intestinal IR seems to have a protective effect on lung injury, probably due to suppression of iNOS-induced nitric oxide generation. In renal IRI, SphK1 seems to be important, since its deficiency increased the damage in kidney tissue, whereas the lentiviral overexpression of the SphK1 gene protected from injury. Another study suggests that, after IRI, apoptotic renal cells release S1P, which recruits macrophages through S1PR3 activation and might contribute to kidney regeneration and restoration of renal epithelium. However, SphK2 is negatively implicated in hepatic IRI, its inhibition helping protect hepatocytes and restoring mitochondrial function.

Further studies are implicating S1P signaling or sphingosine kinases in several kinds of cancer as well as autoimmune diseases.

Figure 2 FTY720-P causes retention of T cells in the lymph nodes.

Notes: C57BL/6 mice were injected with BALB/c splenocytes in the footpad to create an allogenic response then treated with FTY720-P or vehicle every day on days 2 to 5. On day 6, the popliteal lymph nodes were removed. Popliteal node-derived cells were mixed with BALB/c splenocytes in interferon gamma (IFN-γ) cultured enzyme-linked immunosorbent spot reactions. Bars represent the mean number of IFN-γ spot-forming cells per 1000 popliteal node-derived cells, from six mice treated with vehicle and seven with FTY720-P. **P , 0.01. (not shown)

Fingolimod (INN, trade name Gilenya, Novartis) is an immunomodulating drug, approved for treating multiple sclerosis. It has reduced the rate of relapses in relapsing-remitting multiple sclerosis by over half. Fingolimod is a sphingosine-1-phosphate receptor modulator, which sequesters lymphocytes in lymph nodes, preventing them from contributing to an autoimmune reaction.

Fingolimod3Dan

http://upload.wikimedia.org/wikipedia/commons/thumb/4/48/Fingolimod3Dan.gif/200px-Fingolimod3Dan.gif

The S1P antagonist FTY720 has been approved by the US Food and Drug Administration to be used as a drug against multiple sclerosis (MS). FTY720 is in fact a prodrug, since it is phosphorylated in vivo by SphK2 into FTY720-P, an S1P structural analog, which can activate S1PR1, 3, 4, and 5. FTY720-P binding to S1PR1 causes internalization of the receptor, as does S1P – but instead of recycling it back to the cell surface, it promotes its ubiquitination and degradation at the proteasome. This has a direct effect on lymphocyte trafficking through the lymph nodes, since it relies on S1PR1 signaling and S1P gradient (Figure 2). In MS, it stops migrating lymphocytes into the brain, but it may also have direct effects on the CNS through neuroprotection. FTY720 can pass the blood–brain barrier and it could be phosphorylated by local sphingosine kinases to act through S1PR1 and S1PR3 receptors that are mainly expressed in the CNS. In MS lesions, astrocytes upregulate those two receptors and it has been shown that FTY720-P treatment in vitro inhibits astrocyte production of inflammatory cytokines. A recent study confirms the importance of S1PR3 signaling on activated astrocytes, as well as SphK1, that are upregulated and promote the secretion of the potentially neuroprotective cytokine CXCL-1.

There are several studies implicating the intracellular S1P ceramide rheostat to cancer cell survival or apoptosis and resistance to chemotherapy or irradiation in vitro. Studies with SphK1 inhibition in pancreatic, prostate cancers, and leukemia, show increased ceramide/S1P ratio and induction of apoptosis. However, S1P receptor signaling plays conflicting roles in cancer cell migration and metastasis.

Modulation of S1P signaling: therapeutic potential

S1P signaling can be involved in many pathophysiological conditions. This means that we could look for therapeutic targets in all the molecules taking part in S1P signaling and production, most importantly the S1P receptors and the sphingosine kinases. S1P agonists and antagonists could also be used to modulate S1P signaling during pathological conditions.

S1P can have direct effects on the cardiovascular system. During IRI, intracellular S1P can protect the cardiomyocytes and promote their survival. Pre- or post-conditioning of the heart with S1P could be used as a treatment, but upregulation of sphingosine kinases could also increase intracellular S1P bioavailability. S1P could also have effects on endothelial cells and neutrophil trafficking. Vascular endothelial cells mainly express S1PR1 and S1PR3; only a few types express S1PR2. S1PR1 and S1PR3 activation on these cells has been shown to enhance their chemotactic migration, probably through direct phosphorylation of S1PR1 by Akt, in a phosphatidylinositol 3-kinase and Rac1-dependent signaling pathway. Moreover, it stimulates endothelial cell proliferation through an ERK pathway. S1PR2 activation, however, inhibits endothelial cell migration, morphogenesis, and angiogenesis, most likely through Rho-dependent inhibition of Rac signaling pathway, as Inoki et al showed in mouse cells with the use of S1PR1 and S1PR3 specific antagonists.

Regarding permeability of the vascular endothelium and endothelial barrier integrity, S1P receptors can have different effects. S1PR1 activation enhances endothelial barrier integrity by stimulation of cellular adhesion and upregulation of adhesion molecules. However, S1PR2 and S1PR3 have been shown to have barrier-disrupting effects in vitro, and vascular permeability increasing effects in vivo. All the effects S1P can have on vascular endothelium and smooth muscle cells suggest that activation of S1PR2, not S1PR1 and S1PR3, signaling, perhaps with the use of S1PR2 specific agonists, could be used therapeutically to inhibit angiogenesis and disrupt vasculature, suppressing tumor growth and progression.

An important aspect of S1P signaling that is being already therapeutically targeted, but could be further investigated, is immune cell trafficking. Attempts have already been made to regulate lymphocyte cell migration with the use of the drug FTY720, whose phosphorylated form can inhibit the cells S1PR1-dependent egress from the lymph nodes, causing lymphopenia. FTY720 is used as an immunosuppressant for MS but is also being investigated for other autoimmune conditions and for transplantation. Unfortunately, Phase II and III clinical trials for the prevention of kidney graft rejection have not shown an advantage over standard therapies. Moreover, FTY720 can have some adverse cardiac effects, such as bradycardia. However, there are other S1PR1 antagonists that could be considered instead, including KRP-203, AUY954, and SEW2871. KRP-203 in particular has been shown to prolong rat skin and heart allograft survival and attenuate chronic rejection without causing bradycardia, especially when combined with other immunomodulators.

There are studies that argue S1P pretreatment has a negative effect on neutrophil chemotaxis toward the chemokine CXCL-8 (interleukin-8) or the potent chemoattractant formyl-methionyl-leucyl-phenylalanine. S1P pretreatment might also inhibit trans-endothelial migration of neutrophils, without affecting their adhesion to the endothelium. S1P effects on neutrophil migration toward CXCL-8 might be the result of S1PRs cross-linking with the CXCL-8 receptors in neutrophils, CXCR-1 and CXCR-2. Indeed, there is evidence suggesting S1PR4 and S1PR3 form heterodimers with CXCR-1 in neutrophils. Another indication that S1P plays a role in neutrophil trafficking is a recent paper on S1P lyase deficiency, a deficiency that impairs neutrophil migration from blood to tissue in knockout mice.

S1P lyase and S1PRs in neutrophils may be new therapeutic targets against IRI and inflammatory conditions in general. Consistent with these results, another study has shown that inhibition of S1P lyase can have a protective effect on the heart after IRI and this effect is alleviated when pretreated with an S1PR1 and S1PR3 antagonist. Inhibition was achieved with a US Food and Drug Administration-approved food additive, 2-acetyl-4-tetrahydroxybutylimidazole, providing a possible new drug perspective. Another S1P lyase inhibitor, LX2931, a synthetic analog of 2-acetyl-4-tetrahydroxybutylimidazole, has been shown to cause peripheral lymphopenia when administered in mice, providing a potential treatment for autoimmune diseases and prevention of graft rejection in transplantation. This molecule is currently under Phase II clinical trials in rheumatoid arthritis patients.

S1P signaling research has the potential to discover novel therapeutic targets. S1P signaling is involved in many physiological and pathological processes. However, the complexity of S1P signaling makes it necessary to consider every possible pathway, either through its GPCRs, or intracellularly, with S1P as a second messenger. Where the activation of one S1P receptor may lead to the desired outcome, the simultaneous activation of another S1P receptor may lead to the opposite outcome. Thus, if we are to target a specific signaling pathway, we might need specific agonists for S1P receptors to activate one S1P receptor pathway, while, at the same time, we might need to inhibit another through S1P receptor antagonists.

Evidence of sphingolipid signaling in cancer

Biologically active lipids are important cellular signaling molecules and play a role in cell communication and cancer cell proliferation, and cancer stem cell biology. A recent study in ovarian cancer cell lines shows that exogenous sphingosine 1 phosphate (SIP1) or overexpression of the sphingosine kinase (SPHK1) increases ovarian cancer cell proliferation, invasion and contributes to cancer stem cell like phenotype. The diabetes drug metformin was shown to be an inhibitor of SPHK1 and reduce ovarian cancer tumor growth.

2019 Apr;17(4):870-881. doi: 10.1158/1541-7786.MCR-18-0409. Epub 2019 Jan 17.

SPHK1 Is a Novel Target of Metformin in Ovarian Cancer.

Hart PC^#¹, Chiyoda T^#^1,², Liu X³, Weigert M¹, Curtis M¹, Chiang CY¹, Loth R¹, Lastra R⁴, McGregor SM^4,⁵, Locasale JW³, Lengyel E¹, Romero IL⁶.

Author information

Abstract

The role of phospholipid signaling in ovarian cancer is poorly understood. Sphingosine-1-phosphate (S1P) is a bioactive metabolite of sphingosine that has been associated with tumor progression through enhanced cell proliferation and motility. Similarly, sphingosine kinases (SPHK), which catalyze the formation of S1P and thus regulate the sphingolipid rheostat, have been reported to promote tumor growth in a variety of cancers. The findings reported here show that exogenous S1P or overexpression of SPHK1 increased proliferation, migration, invasion, and stem-like phenotypes in ovarian cancer cell lines. Likewise, overexpression of SPHK1 markedly enhanced tumor growth in a xenograft model of ovarian cancer, which was associated with elevation of key markers of proliferation and stemness. The diabetes drug, metformin, has been shown to have anticancer effects. Here, we found that ovarian cancer patients taking metformin had significantly reduced serum S1P levels, a finding that was recapitulated when ovarian cancer cells were treated with metformin and analyzed by lipidomics. These findings suggested that in cancer the sphingolipid rheostat may be a novel metabolic target of metformin. In support of this, metformin blocked hypoxia-induced SPHK1, which was associated with inhibited nuclear translocation and transcriptional activity of hypoxia-inducible factors (HIF1α and HIF2α). Further, ovarian cancer cells with high SPHK1 were found to be highly sensitive to the cytotoxic effects of metformin, whereas ovarian cancer cells with low SPHK1 were resistant. Together, the findings reported here show that hypoxia-induced SPHK1 expression and downstream S1P signaling promote ovarian cancer progression and that tumors with high expression of SPHK1 or S1P levels might have increased sensitivity to the cytotoxic effects of metformin. IMPLICATIONS: Metformin targets sphingolipid metabolism through inhibiting SPHK1, thereby impeding ovarian cancer cell migration, proliferation, and self-renewal.

Nrf2:INrf2(Keap1) Signaling in Oxidative Stress

James W. Kaspar, Suresh K. Niture, and Anil K. Jaiswal*

Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD

Free Radic Biol Med. 2009 Nov 1; 47(9): 1304–1309. http://dx.doi.org:/10.1016/j.freeradbiomed.2009.07.035

Nrf2:INrf2(Keap1) are cellular sensors of chemical and radiation induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that

controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins.

This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor INrf2. INrf2 functions as an adapter for

Cul3/Rbx1 mediated degradation of Nrf2.
In response to oxidative/electrophilic stress,
Nrf2 is switched on and then off by distinct

early and delayed mechanisms.

Oxidative/electrophilic modification of INrf2cysteine151 and/or PKC phosphorylation of Nrf2serine40 results in the escape or release of Nrf2 from INrf2. Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with unknown proteins, and binds antioxidant response element (ARE) that leads to coordinated activation of gene expression. It takes less than fifteen minutes from the time of exposure

to switch on nuclear import of Nrf2.

This is followed by activation of a delayed mechanism that controls

switching off of Nrf2 activation of gene expression.

GSK3β phosphorylates Fyn at unknown threonine residue(s) leading to

nuclear localization of Fyn.

Fyn phosphorylates Nrf2tyrosine568 resulting in

nuclear export of Nrf2,
binding with INrf2 and
degradation of Nrf2.

The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis and promotes cell survival.

NPRA-mediated suppression of AngII-induced ROS production contributes to the antiproliferative effects of B-type natriuretic peptide in VSMC

Pan Gao, De-Hui Qian, Wei Li, Lan Huang
Mol Cell Biochem (2009) 324:165–172

http://dx.doi.org/10.1007/s11010-008-9995-y

Excessive proliferation of vascular smooth cells (VSMCs) plays a critical role in the pathogenesis of diverse vascular disorders, and inhibition of VSMCs proliferation has been proved to be beneficial to these diseases.

In this study, we investigated the antiproliferative effect of

B-type natriuretic peptide (BNP), a natriuretic peptide with potent antioxidant capacity,

on rat aortic VSMCs, and the possible mechanisms involved. The results indicate that

BNP potently inhibited Angiotensin II (AngII)-induced VSMCs proliferation,

as evaluated by [3H]-thymidine incorporation assay. Consistently, BNP significantly decreased

AngII-induced intracellular reactive oxygen species (ROS)
and NAD(P)H oxidase activity.

8-Br-cGMP, a cGMP analog,

mimicked these effects.

To confirm its mechanism, siRNA of natriuretic peptide receptor-A(NRPA) strategy technology was used

to block cGMP production in VSMCs, and
siNPRA attenuated the inhibitory effects of BNP in VSMCs.

Taken together, these results indicate that

BNP was capable of inhibiting VSMCs proliferation by
NPRA/cGMP pathway,

which might be associated with

the suppression of ROS production.

These results might be related, at least partly, to the anti-oxidant property of BNP.

Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics

A Alleaume-Butaux, C Dakowski, M Pietri, S Mouillet-Richard, Jean-Marie Launay, O Kellermann, B Schneider

1INSERM, UMR-S 747, 2Paris Descartes University, Sorbonne Paris, 3Public Hospital of Paris, Department of Biochemistry, Paris, France; 4Pharma Research Department, Hoffmann La Roche Ltd, Basel, Switzerland

Cell Health and Cytoskeleton 2013; 5: 1–12

Neuritogenesis is a complex morphological phenomena accompanying neuronal differentiation. Neuritogenesis relies on the initial breakage of the rather spherical symmetry of neuroblasts and the formation of buds emerging from the postmitotic neuronal soma. Buds then evolve into neurites, which later convert into an axon or dendrites. At the distal tip of neurites, the growth cone integrates extracellular signals and guides the neurite to its target. The acquisition of neuronal polarity depends on deep modifications of the neuroblast cytoskeleton characterized by the remodeling and activation of focal adhesions (FAs) and localized destabilization of the actin network in the neuronal sphere.Actin instability in unpolarized neurons allows neurite sprouting, ie, the protrusion of microtubules, and subsequent neurite outgrowth. Once the neurite is formed, actin microfilaments recover their stability and exert a sheathed action on neurites, a dynamic process necessary for the maintenance and integrity of neurites.

A combination of extrinsic and intrinsic cues pilots the architectural and functional changes in FAs and the actin network along neuritogenesis. This process includes neurotrophic factors (nerve growth factor, brain derived neurotrophic factor, neurotrophin, ciliary neurotrophic factor, glial derived neurotrophic factor) and their receptors, protein components of the extracellular matrix (ECM) (laminin, vitronectin, fibronectin), plasma membrane integrins and neural cell adhesion molecules (NCAM), and intracellular molecular protagonists such as small G proteins (RhoA, Rac, Cdc42) and their downstream targets.

Neuritogenesis is a dynamic phenomenon associated with neuronal differentiation that allows a rather spherical neuronal stem cell to develop dendrites and axon, a prerequisite for the integration and transmission of signals. The acquisition of neuronal polarity occurs in three steps:

(1) neurite sprouting, which consists of the formation of buds emerging from the postmitotic neuronal soma;

(2) neurite outgrowth, which represents the conversion of buds into neurites, their elongation and evolution into axon or dendrites; and

(3) the stability and plasticity of neuronal polarity.

In neuronal stem cells, remodeling and activation of focal adhesions (FAs) associated with deep modifications of the actin cytoskeleton is a prerequisite for neurite sprouting and subsequent neurite outgrowth. A multiple set of growth factors and interactors located in the extracellular matrix and the plasma membrane orchestrate neuritogenesis

by acting on intracellular signaling effectors,
notably small G proteins such as RhoA, Rac, and Cdc42,
which are involved in actin turnover and the dynamics of FAs.

The cellular prion protein (PrPC), a glycosylphosphatidylinositol

(GPI)-anchored membrane protein

mainly known for its role in a group of fatal

neurodegenerative diseases,

has emerged as a central player in neuritogenesis.

Here, we review the contribution of PrPC to neuronal polarization and detail the current knowledge on the

signaling pathways fine-tuned by PrPC
to promote neurite sprouting, outgrowth, and maintenance.

We emphasize that PrPC-dependent neurite sprouting is a process in which PrPC

governs the dynamics of FAs and the actin cytoskeleton
via β1 integrin signaling.

The presence of PrPC is necessary to render neuronal stem cells

competent to respond to neuronal inducers and
to develop neurites.

In differentiating neurons, PrPC exerts

a facilitator role towards neurite elongation.

This function relies on the interaction of PrPC with a set of diverse partners such as

elements of the extracellular matrix,
plasma membrane receptors,
adhesion molecules, and
soluble factors that control actin cytoskeleton turnover through Rho-GTPase signaling.

Once neurons have reached their terminal stage of differentiation and acquired their polarized morphology, PrPC also

takes part in the maintenance of neurites.

By acting on tissue nonspecific alkaline phosphatase, or

matrix metalloproteinase type 9,

PrPC stabilizes interactions between

neurites and the extracellular matrix.

Keywords: prion, neuronal differentiation

Read Full Post »

Introduction to Lipid Metabolism

Posted in Academic Publishing, Best evidence, Biochemical pathways, Fatty acids, Lipid metabolism, Lipids, Liver & Digestive Diseases Research, Metabolism, Metabolomics, Nephrology, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Phosphorylation, Population Health Management, Proteins, Proteomics, Signaling, Translational Research, Translational Science, tagged carbon length, Cytoskeleton, esseantial fatty acids, fatty acid oxidation, fatty acid synthesis, Fatty acids, Lipid metabolism, lipidomics, Lipids, pathophysiology on October 25, 2014| Leave a Comment »

Introduction to Lipid Metabolism

Author, Curator: Larry H. Bernstein, MD, FCAP

Introduction to Lipid Metabolism

This series of articles is concerned with lipid metabolism. These discussions lay
the groundwork to proceed to discussions that will take on a somewhat different
approach, but they are critical to developing a more complete point of view of life
processes.  I have indicated that there are protein-protein interactions or protein-membrane interactions and associated regulatory features, but the focus of the
discussion or points made were different, and will be returned to.  The role of
lipids in circulating plasma proteins as biomarkers for coronary vascular disease
can be traced to the early work of Frederickson and the classification of lipid disorders.  The very critical role of lipids in membrane structure in health and
disease has had much less attention, despite the enormous importance,
especially in the nervous system.

This portion of the discussions of metabolism will have several topics on lipid
metabolism. The first is concerned with the basic types of lipids -which are defined structurally and have different carbon chain length, and have
two basic types of indispensible fatty acid derivations – along pro-inflammatory
and anti-inflammatory pathways:

Alpha-linolenic acid (ALA) and LA, n-3 polyunsaturated fatty acids LCPUFAs (EPA, DHA, and AA), eicosanoids,
delta-3-desaturase, prostaglandins, and leukotrienes.
the role of the mitochondrial electron transport chain in hydrogen transfers
and oxidative phosphorylation with respect to the oxidation of fatty acids
and fatty acid synthesis.
The membrane structures of the cell, including

the cytoskeleton, essential organelles, and the intercellular matrix, which
is a critical consideration for
cell motility, membrane conductivity, flexibility, and signaling.
The membrane structure involves aggregation of lipids with proteins,
and is associated with hydrophobicity.

The pathophysiology of systemic circulating lipid disorders.
The fifth is the pathophysiology of cell structures under oxidative
stress.
Lipid disposal and storage diseases.

Read Full Post »

Diabetes Mellitus

Posted in Academic Publishing, Alzheimer's Disease, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Curation, Diabetes Mellitus, Disease Biology, Epigenetics and Cardiovascular Risks, Etiology, Gene Regulation, Innovations, Lipid metabolism, Metabolism, Metabolomics, Nephrology, Neurological Diseases, Nitric Oxide in Health and Disease, Nutrition, Nutrition and Phytochemistry, Nutritional Supplements: Atherogenesis, Origins of Cardiovascular Disease, Plant extract, tagged amylin, Amyloid, Beta cell, Diabetes Mellitus, glomerulosclerosis, Insulin resistance, Insulin Sensitivity, islet cells of pancreas, pharmacotherapy, proinsulin on October 24, 2014| Leave a Comment »

Diabetes Mellitus

Author & Curator: Larry H. Bernstein, MD, FCAP

Diabetes mellitus (DM) is a group of metabolic diseases defined by high blood glucose levels, which, depending on the fasting blood glucose, may be pre-diabetes or overt diabetes (110 mg/dl. 124 mg/dl). This blood glucose level reflects a disorder of control of glucose metabolism, which is mediated through the pituitary growth hormone acting on the liver, which produces insulin growth factor 1 (IGF1). Diabetes is due to either the pancreas not producing enough insulin, or the cells of the body not responding properly to the insulin produced. That said, there is much to be understood about the long term systemic effects of this disorder, a multisystem disease. The presence of pre-diabetes glucose levels is sufficient to proactively take measures to reduce the circulating glucose.

Globally, as of 2013, an estimated 382 million people have diabetes worldwide, with type 2 diabetes making up about 90% of the cases. This is equal to 8.3% of the adults population, with equal rates in both women and men. Worldwide in 2012 and 2013 diabetes resulted in 1.5 to 5.1 million deaths per year, making it the 8th leading cause of death. Diabetes overall at least doubles the risk of death. The number of people with diabetes is expected to rise to 592 million by 2035. The economic costs of diabetes globally was estimated in 2013 at $548 billion and in the United States in 2012 $245 billion.

The observation of symptoms of frequent urination, increased thirst, and increased hunger is symptomatic of overt DM, and is seen with diabetic ketoacidosis, with very high hyperglycemia and glucosuria, particularly in Type 1 DM. Untreated, diabetes leads to serious complications. Acute complications include diabetic ketoacidosis. Serious long-term complications include heart disease, stroke, kidney failure, foot ulcers and damage to the eyes.

There are three main types of diabetes mellitus:

Type 1 DM results from the body’s failure to produce enough insulin. This form was previously referred to as “insulin-dependent diabetes mellitus” (IDDM) or “juvenile diabetes”. The cause is unknown.
Type 2 DM begins with insulin resistance, a condition in which cells fail to respond to insulin properly. As the disease progresses a lack of insulin may also develop. This form was previously referred to as “non insulin-dependent diabetes mellitus” (NIDDM) or “adult-onset diabetes”. The primary cause is excessive body weight and not enough exercise.
Gestational diabetes, the third, occurs when pregnant women without a previous history of diabetes develop a high blood glucose level.

Type 1 DM, which presents suddenly in children or young adults, is possibly an as yet unidentified post-translational or epigenetic form, unrelated to Type 2, which is becoming more common in children. It results in the destruction of islet beta cells that then have no capacity to produce insulin. A family history of the disease would be a signal to raise a child with great care to not stress the pancreas. Even though I raised the possibility of an epigenetic factor, it is important to keep in mind that the regulation of glucose is responsive to a number of stresses, even in a healthy person. These are:

Corticosteroids
Glucagon
Growth hormone
Catecholamines
Proinflammatory cytokines
Anxiety disorder
Eating disorder

Gestational diabetes is perhaps Type 2 diabetes in a pregnant woman initiated by the condition of pregnancy. Whether these women were not diabetic, with a glucose level between 100-110 prior to pregnancy, is an open question. However, the pregnant state is accompanied by large effects by hormone levels.

Type 2 diabetes has been increasing worldwide, not only in western nations. However, in non-western countries that have large populations of underserved, there is still a major problem with protein energy malnutrition (PEM). Globally, as of 2013, an estimated 382 million people have diabetes worldwide, with type 2 diabetes making up about 90% of the cases. This is equal to 8.3% of the adults population, with equal rates in both women and men. Worldwide in 2012 and 2013 diabetes resulted in 1.5 to 5.1 million deaths per year, making it the 8th leading cause of death. Diabetes overall at least doubles the risk of death. The number of people with diabetes is expected to rise to 592 million by 2035. The economic costs of diabetes globally was estimated in 2013 at $548 billion and in the United States in 2012 $245 billion.

The major long-term complications relate to damage to blood vessels. Diabetes doubles the risk of cardiovascular disease and about 75% of deaths in diabetics are due to coronary artery disease. Other “macrovascular” diseases are stroke, and peripheral vascular disease. The primary microvascular complications of diabetes include damage to the eyes, kidneys, and nerves. Damage to the eyes, known as diabetic retinopathy, is caused by damage to the blood vessels in the retina of the eye, and can result in gradual vision loss and potentially blindness. Damage to the kidneys, known as diabetic nephropathy, can lead to tissue scarring, urine protein loss, and eventually chronic kidney disease, sometimes requiring dialysis or kidney transplant. Damage to the nerves of the body, known as diabetic neuropathy, is the most common complication of diabetes.

Prevention and treatment involves a healthy diet, physical exercise, not using tobacco and being a normal body weight. Blood pressure control and proper foot care are also important for people with the disease. Type 1 diabetes must be managed with insulin injections. Type 2 diabetes may be treated with medications with or without insulin. Insulin and some oral medications can cause low blood sugar. Weight loss surgery in those with obesity is an effective measure in those with type 2 DM. Gestational diabetes usually resolves after the birth of the baby.

A number of articles in http://pharmaceuticalintelligence,com (this journal) have presented the relationship of DM to heart and vascular disease. The complexity of the disease is not to be underestimated, and there havr been serious controversies with adverse consequences over the use of the class of drugs that includes rosiglitazone and piaglitazone, which has opened serious issues about how clinical trials are conducted, and how the data obtained in studies may be compromised.

Pharmaceutical Insights

Management of Diabetes Mellitus: Could Simultaneous Targeting of Hyperglycemia and Oxidative Stress Be a Better Panacea?

Omotayo O. Erejuwa
Int. J. Mol. Sci. 2012, 13, 2965-2972; http://www.mdpi.com/journal/ijms http://dx.doi.org:/10.3390/ijms13032965

The primary aim of the current management of diabetes mellitus is to achieve and/or maintain a glycated hemoglobin level of ≤6.5%. However, recent evidence indicates that intensive treatment of hyperglycemia is characterized by increased weight gain, severe hypoglycemia and higher mortality. Besides, evidence suggests that it is difficult to achieve and/or maintain optimal glycemic control in many diabetic patients; and that the benefits of intensively-treated hyperglycemia are restricted to microvascular complications only. Evidence also indicates that multiple drugs are required to achieve optimal glycemic target in many diabetic patients. In fact, in many diabetic patients in whom optimal glycemic goal is achieved, glycemic control deteriorates even with optimal drug therapy. It does suggest that with the current hypoglycemic or antidiabetic drugs, it is difficult to achieve and/or maintain tight glycemic control in diabetic patients. In many developing countries, the vast majority of diabetic patients have limited or lack access to quality healthcare providers and good therapeutic monitoring.

While increased weight gain could be due to some component drugs (such as sulphonylureas or insulin) of the intensive therapy regimens, hypoglycemia could be drug-induced or comorbidity-induced. Considering the evidence that associates hypoglycemia with increased mortality, higher incidence of mortality in intensive therapy group could be due to hypoglycemia or too low levels of glycosylated hemoglobin. However, it is difficult to contend that increased mortality was entirely due to hypoglycemia. The possibility of drug-induced or drug-associated toxicities could not be ruled out. For instance, rosiglitazone, which has been prohibited and withdrawn from the market in Europe, was one of the hypoglycemic drugs used to achieve intensive therapy of hyperglycemia in Action to Control Cardiovascular Risk in Diabetes (ACCORD). If these findings are anything to go by, does it not suggest that targeting hyperglycemia as the only therapeutic goal in the management of diabetes mellitus could be detrimental to diabetic patients? In addition, the current hypoglycemic drugs are characterized by limitations and adverse effects. Together with the limitations of intensive glycemic treatment (only beneficial in reducing the risk of microvascular complications, but not macrovascular disease complications), does it not imply that targeting hyperglycemia alone is not only deleterious but also limited and ineffective?

The latest figures predict that the global incidence of diabetes mellitus, which was estimated to be 366 million in 2011, will rise to 522 million by 2030. In view of these frightening statistics on the prevalence of diabetes mellitus and on the lack of adequate healthcare, together with the associated diabetic complications, morbidity and mortality, does it not suggest that there is an urgent need for a better therapeutic management of this disorder? Taken together, with these findings and statistics, it can be contended that it is high time alternative and/or complementary therapies to the currently available hypoglycemic agents (which target primarily hyperglycemia only) were sought.

All these may contribute to the unabated increase in global prevalence of diabetes mellitus and its complications In view of these adverse effects and limitations of intensive treatment of hyperglycemia in preventing diabetic complications, which is linked to oxidative stress,

this commentary proposes a hypothesis that “simultaneous targeting of hyperglycemia and oxidative stress” could be more effective than “intensive treatment of hyperglycemia” in the management of diabetes mellitus.

Oxidative stress is defined as

an “imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage”.

It is implicated in the pathogenesis and complications of diabetes mellitus. The role of oxidative stress is more definite in the pathogenesis of type 2 diabetes mellitus than in type 1 diabetes mellitus. In regard to diabetic complications, there is compelling evidence in support of the role of oxidative stress in both types of diabetes mellitus. Evidence suggests that elevated reactive oxygen species (ROS), which causes factor of increased ROS production, causes tissue damage or diabetic complications have been identified. These include:

hyperglycemia-enhanced polyol pathway;
hyperglycemia-enhanced formation of advanced glycation endproducts (AGEs);
hyperglycemia-activated protein kinase C (PKC) pathway;
hyperglycemia-enhanced hexosamine pathway; and
hyperglycemia-activated Poly-ADP ribose polymerase (PARP) pathway.

These pathways are activated or enhanced by hyperglycemia-driven mitochondrial superoxide overproduction.

Even though oxidative stress plays an important role in its pathogenesis and complications,

unlike other diseases characterized by oxidative stress, diabetes mellitus is unique.

Its cure (restoration of euglycemia, e.g., via pancreas transplants) does not prevent oxidative stress and diabetic complications. This is very important because hyperglycemia exacerbates oxidative stress which is linked to diabetic complications. Theoretically, restoration of euglycemia should prevent oxidative stress and diabetic complications. However, this is not the case. At present, it remains unclear why restoration of euglycemia does not automatically prevent oxidative stress and diabetic complications. The development of diabetes-related complications (both microvascular and macrovascular) may occur in diabetic patients after normoglycemia has been restored. It is a phenomenon whereby previous hyperglycemic milieu is remembered in many target organs such as heart, eyes, kidneys and nerves. This phenomenon is also documented in diabetic animals. Compelling evidence implicates the role of oxidative stress as an important mechanism by which glycemic memory causes tissue damage and diabetic complications. In view of higher incidence of diabetic complications (of which oxidative stress plays an important role) in conventionally-treated diabetic patients, targeting oxidative stress in these patients might be beneficial. In other words, it is possible that the combination of a conventional therapy of hyperglycemia and antioxidant therapy might be more effective and beneficial than intensive therapy of hyperglycemia alone, which is the gold standard at the moment.

Loss of ACE 2 Exaggerates High-Calorie Diet-Induced Insulin Resistance by Reduction of GLUT4 in Mice

M Takeda, K Yamamoto, Y Takemura, H Takeshita, K Hongyo, et al. Diabetes 61:1–11, 2012

ACE type 2 (ACE2) functions as

a negative regulator of the renin angiotensin system
by cleaving angiotensin II (AII) into angiotensin 1–7 (A1–7).

This study assessed the role of

endogenous ACE2 in maintaining insulin sensitivity.

Twelve-week-old male ACE2 knockout (ACE2KO) mice had normal insulin sensitivities when fed a standard diet. AII infusion or a high-fat high-sucrose (HFHS) diet impaired glucose tolerance and insulin sensitivity more severely

in ACE2KO mice than in their wild-type (WT) littermates.

The strain difference in glucose tolerance

was not eliminated by an AII receptor type 1 (AT1) blocker
but was eradicated by A1–7 or an AT1 blocker combined with the A1–7 inhibitor (A779).

The expression of GLUT4 and a transcriptional factor, myocyte enhancer factor (MEF) 2A,

was dramatically reduced in the skeletal muscles of the standard diet–fed ACE2KO mice.

The expression of GLUT4 and MEF2A was increased

by A1–7 in ACE2KO mice and
decreased by A779 in WT mice.

A1–7 enhanced upregulation of MEF2A and GLUT4 during differentiation of myoblast cells. In conclusion,

ACE2 protects against high calorie diet-induced insulin resistance in mice.

This mechanism may involve the transcriptional regulation of GLUT4 via an A1–7-dependent pathway.
Modulation of the action of insulin by angiotensin-(1–7)
FP. Dominici, V Burghi, MC. Munoz, JF. Giani

Clinical Science (2014) 126, 613–630 http://dx.doi.org:/10.1042/CS20130333

The prevalence of Type 2 diabetes mellitus is predicted to increase dramatically over the coming years and the clinical implications and healthcare costs from this disease are overwhelming. In many cases, this pathological condition is linked to a cluster of metabolic disorders, such as

obesity,
systemic hypertension and
dyslipidaemia,

defined as the metabolic syndrome.

Insulin resistance has been proposed as the key mediator of all of these features and contributes to the associated high cardiovascular morbidity and mortality. Although the molecular mechanisms behind insulin resistance are not completely understood, a negative cross-talk between

AngII (angiotensin II) and the insulin signalling pathway

has been the focus of great interest in the last decade. Indeed,

substantial evidence has shown that

anti-hypertensive drugs that block the RAS (renin–angiotensin system) may also act to prevent diabetes.

Despite its long history, new components within the RAS continue to be discovered.

Among them, Ang-(1–7) [angiotensin-(1–7)] has gained special attention as a counter-regulatory hormone

opposing many of the AngII-related deleterious effects.

Specifically, we and others have demonstrated that Ang-(1–7) improves the action of insulin and opposes the negative effect that AngII exerts at this level. In the present review, we provide evidence showing that

insulin and Ang-(1–7) share a common intracellular signalling pathway.

We also address the molecular mechanisms behind the beneficial effects of Ang-(1–7) on

AngII-mediated insulin resistance.

Finally, we discuss potential therapeutic approaches leading to modulation of the

ACE2 (angiotensin-converting enzyme 2)/Ang-(1–7)/Mas receptor axis

as a very attractive strategy in the therapy of the metabolic syndrome and diabetes-associated diseases.

Increased Skeletal Muscle Capillarization After Aerobic Exercise Training and Weight Loss Improves Insulin Sensitivity in Adults With IGT

Prior, JB. Blumenthal, LI. Katzel, AP. Goldberg, AS. Ryan. Diabetes Care 2014;37:1469–1475
http://dx.doi.org:/10.2337/dc13-2358

Transcapillary transport of insulin is one determinant of glucose uptake by skeletal muscle; thus,

a reduction in capillary density (CD) may worsen insulin sensitivity.

Skeletal muscle CD is lower in older adults with impaired glucose tolerance (IGT) compared with those with normal glucose tolerance and

may be modifiable through aerobic exercise training and weight loss (AEX+WL).

Insulin sensitivity (M) and 120-min postprandial glucose (G120) correlated with CD at baseline (r = 0.58 and r = 20.60, respectively, P < 0.05).

AEX+WL increased maximal oxygen consumption (VO2max) 18%(P = 0.02) and reduced weight and fat mass 8% (P < 0.02).

Regression analyses showed that the AEX+WL-induced increase in CD

independently predicted the increase in M (r = 0.74, P < 0.01)
as well as the decrease in G120 (r = 20.55, P < 0.05).

AEX+WL increases skeletal muscle CD in older adults with IGT. This represents one mechanism by which AEX+WL improves insulin sensitivity in older adults with IGT.

Glycaemic durability with dipeptidyl peptidase-4 inhibitors in type 2 diabetes: a systematic review and meta-analysis of long-term randomised controlled trials.

K Esposito, P Chiodini, MI Maiorino, G Bellastella, A Capuano, D Giugliano. BMJ Open 2014;4:e005442.
http://dx.doi.org:/10.1136/bmjopen-2014-005442

A systematic review and meta-analysis of longterm randomised trials of DPP-4 inhibitors (sitagliptin, vildagliptin, saxagliptin, linagliptin and alogliptin). on haemoglobin A1c (HbA1c) was conducted. The difference between final and intermediate HbA1c assessment was the primary outcome. All trials were of 76 weeks duration at least. The difference in HbA1c changes between final and intermediate points averaged 0.22% (95% CI 0.15% to 0.29%), with high heterogeneity (I2=91%, p<0.0001). Estimates
of differences were not affected by the analysis of six extension trials (0.24%, 0.02 to 0.46), or five trials in which a DPP-4 inhibitor was added to metformin (0.24%, 0.16 to 0.32).

The effect of DPP-4 inhibitors on HbA1c in type 2 diabetes significantly declines during the second year of treatment.

Overcoming Diabetes Mellitus & Borderline Diabetes
By Max Stanley Chartrand, Ph.D. (Behavioral Medicine)

The over-arching biomarker that has more to do with the ability to restore normal metabolic processes is in achieving a cellular pH 7.45 (via the Kreb’s Cycle). To say the least, getting one’s cellular pH to 7.45 and A1C score below 6.0 can be a daunting task!

SIRCLE®: Naturally Achieved Targets

 Cellular pH 7.35-7.45

 Oxygen 99-100% @55-65 bpm

 Resting Blood Pressure: 110-135/ 65-80

mmHg (differs male vs female)

 Fasting blood sugar consistently <70-99

mg/dL or 3.5-5.5 mmol/L

 HgA1C score: .04-5.8

 HDL: 40-60 mg/dL; LDL: 100 -140 mg/dL;

triglycerides: <85 mg/dL

 C-Reactive Protein (CRP) Score <.5

 Galectin-3 Assay <17.8 ng/mL

Antidiabetic Activity of Hydroalcoholic Extracts of Nardostachys jatamansi in Alloxan-induced Diabetic Rats

M.A. Aleem, B.S. Asad, T Mohammed, R.A. Khan, M.F. Ahmed, A. Anjum, M. Ibrahim. Brit J Med & Medical Res 4(28): 4665-4673, 2014. http://www.sciencedomain.org/review-history.php?iid=579&id=12&aid=5024

The antidiabetic study was carried out to estimate the anti hyperglycemic potential of Nardostachys Jatamansi rhizome’s hydroalcoholic extracts in alloxan induced diabetic rats over a period of two weeks. The hydroalcoholic extract HAE1 at a dose (500mg/kg) exhibited significantly greater antihyperglycemic activity than extract HAE2 at a dose (500mg/kg) in diabetic rats. The hydroalcoholic extracts showed improvement in different parameters associated with diabetes, like body weight, lipid
profile and biochemical parameters. Extracts also showed improvement in

regeneration of β-cells of pancreas in diabetic rats.

Histopathological studies support the healing of pancreas by hydro alcoholic extracts (HAE1& HAE2) of Nardostachys Jatamansi, as a probable mechanism of their antidiabetic activity.

Antidiabetic and Antihyperlipidemic Effect of Parmelia Perlata. Ach. in Alloxan Induced Diabetic Rats.
Jothi G and Brindha P
Internat J of Pharmacy and Pharmaceut Sciences 2014; 6(suppl 1)

The aqueous extract of the selected plant was administered at dose levels of 200mg and 400mg/kg body weight for 60 days. After the experimental period the blood and tissue samples were collected and subjected to various biochemical and enzymic parameters. There were profound alteration in

fasting blood glucose,
serum insulin,
glycosylated hemoglobin (HbA1C) and
liver glycogen levels in alloxanized rats.

Glucose-6-phosphatase,
glucokinase, and
fructose 1-6 bisphosphatase activity

were also altered in diabetic rats.

Administration of plant extract significantly (P<0.05)

reduced the fasting blood glucose and HbA1C level and increased the level of plasma insulin.

The activities of glucose metabolizing enzymes were also resumed to normal. There was a profound improvement in serum lipid profiles by

reducing serum triglyceride, cholesterol, LDL, VLDL, free fatty acids, phospholipids and increasing the HDL level in a dose dependent manner.

The effects of leaf extract were compared with standard drug glibenclamide (600μg/Kg bw). The results indicate that Parmelia perlata. Ach., Linn. could be a good natural source for developing an antidiabetic drug that can effectively maintained the blood glucose levels and lipid profile to near normal values.

Pathophysiological Insights
Diabetic glomerulosclerosis

Reviewers: Nikhil Sangle, M.D.
Revised: 21 February 2014,
Copyright: (c) 2003-2012, PathologyOutlines.com, Inc.

General

==================================================

Diffuse capillary basement membrane thickening, diffuse and nodular glomerulosclerosis
Causes glomerular disease, arteriolar sclerosis, pyelonephritis, papillary necrosis; similar between type I and II patients
Accounts for 30% of long term dialysis patients in US; causes 20% of deaths in patients with diabetes < age 40
Changes may be related to nephronectin, which functions in the assembly of extracellular matrix (Nephrol Dial Transplant 2012;27:1889)

Clinical features

==================================================

Proteinuria occurs in 50%, usually 12-22 years after onset of diabetes
End stage renal disease occurs in 30% of type I patients
Early increased GFR and microalbuminemia (30-300 mg/day) are predictive of future diabetic nephropathy
Renal disease reduced by tight diabetic control; may recur with renal allografts; ACE inhibitors may reduce progression

Micro description

==================================================

Basement membrane thickening and increased mesangial matrix in ALL patients
Diffuse glomerulosclerosis: increase in mesangial matrix associated with PAS+ basement membrane thickening, eventually obliterates mesangial cells
Nodular glomerulosclerosis: also called intercapillary glomerulosclerosis or Kimmelstiel-Wilson disease; ovoid, spherical, laminated hyaline masses in peripheral of glomerulus, PAS+, eventually obliterates glomerular tuft; specific for diabetes and membranoproliferative glomerulonephritis, light-chain disease and amyloidosis (Hum Pathol 1993;24:77 (pathogenesis of Kimmelstiel-Wilson nodule))
Profound hyalinization of afferent arterioles (insudative lesion-intramural): specific for diabetes in afferent arterioles, but non-specific if in periphery of glomerular loop, Bowman’s capsule or mesangium; insudative material composed of proteins, lipids and mucopolysaccharides
Organizing fibroepithelial crescents: associated with aggressive clinical course
Diffuse thickening of tubular basement membrane, tubular atrophy and interstitial fibrosis
Isolated thickened glomerular basement membrane and proteinuria may be an early predictor of diabetic disease (Mod Pathol 2004;17:1506)

Nodular glomerulosclerosis, Kidney

Glomeruli:

Acellular, homogeneous, eosinophilic, globular nodules in the mesangial orintercapillary region of a glomerular tuft with capillary displaced to the periphery.
Diffuse intercapillary glomerulosclerosis: increasing eosinophilic mesangial matrix materials.
Capsular drop: eosinophilic small nodules on Bowman’s capsule.
Fibrin cap: eosinophilic, waxy, fatty structure within the lumen of one or more capillary loops of glomerular tufts.

nodular glomeruloschlerosis

http://www.kidneypathology.com/Imagenes/Diabetes/Imagen.Hial.jul.w.jpg

Islet amyloid polypeptide, islet amyloid, and diabetes mellitus.

Westermark P1, Andersson A, Westermark GT.
Physiol Rev. 2011 Jul;91(3):795-826.
http://dx.doi.org:/10.1152/physrev.00042.2009.

Islet amyloid polypeptide (IAPP), or amylin, was named for its tendency to

aggregate into insoluble amyloid fibrils, features typical of islets of most individuals with type 2 diabetes.

This pathological characteristic is most probably of

great importance for the development of the β-cell failure in this disease,
but the molecule also has regulatory properties in normal physiology.

In addition, it possibly contributes to the diabetic condition. This review deals with both these facets of IAPP.

Islet amyloid polypeptide (IAPP, or amylin) is one of the major secretory products of β-cells of the pancreatic islets of Langerhans. It is

a regulatory peptide with putative function
both locally in the islets, where it inhibits insulin and glucagon secretion, and at distant targets.

It has binding sites in the brain, possibly contributing also to satiety regulation and inhibits gastric emptying. Effects on several other organs have also been described.

IAPP was discovered through its ability to

aggregate into pancreatic islet amyloid deposits,

which are seen particularly in association with type 2 diabetes in humans and with diabetes in a few other mammalian species, especially monkeys and cats.

Aggregated IAPP has cytotoxic properties and is believed to be

of critical importance for the loss of β-cells in type 2 diabetes

and also in pancreatic islets transplanted into individuals with type 1 diabetes. This review deals both with physiological aspects of IAPP and with the

pathophysiological role of aggregated forms of IAPP,
including mechanisms whereby human IAPP forms toxic aggregates and amyloid fibrils.

Islet amyloid, initially named “islet hyalinization,” was described in 1901 by two researchers independently and for a long time was considered an enigma. It was found to occur in association with diabetes mellitus, particularly in elderly individuals, but its possible pathogenetic importance was often denied. The similarity of the hyaline substance to amyloid was noted at an early date, and some researchers reported staining reactions typical of amyloid. It had been shown in 1959 that

amyloid of several types has a characteristic ultrastructure,
and islet deposits were found to share this appearance.

When biochemical analyses of amyloid fibrils from systemic primary and secondary amyloidoses showed that

these consisted of distinctive proteins,
it was suspected that the islet deposits might also be a polymerized protein.

The chemical composition of islet amyloid did not attract much attention even after the characteristics of other amyloid fibrils had been elucidated. The finding that the amyloid in C cell-derived medullary thyroid carcinoma is of polypeptide hormonal origin was an important indication that amyloid in other endocrine tissues also comes from the local secretory products, and it was believed that

insulin, or proinsulin, or split products thereof constitute the islet amyloid fibrils.

Immunological trials to characterize the amyloid yielded equivocal results. Only when concentrated formic acid was used on amyloid,

extracted from an amyloid-rich insulinoma, was it possible to purify the major fibril protein
and characterize it by NH2-terminal amino acid sequence analysis,

which very unexpectedly revealed a novel peptide,

not resembling any part of proinsulin
but with partial identity to the neuropeptide calcitonin gene-related peptide (CGRP).

Further characterization of the peptide purified from an insulinoma and from islet amyloid of human and feline origin proved it to be a 37-amino acid (aa) residue peptide. The peptide was initially named “insulinoma amyloid peptide” , later diabetes-associated peptide (DAP), and finally islet amyloid polypeptide (IAPP), or “amylin”.

IAPP is a 37-aa residue long peptide, but by the application of molecular biological methods it was quickly shown that IAPP is expressed initially as

part of an 89-aa residue preproprotein containing a 22-aa signal peptide and
two short flanking peptides, the latter cleaved off at double basic aa residues similar to proinsulin.

IAPP is expressed by one single-copy gene on the short arm of chromosome 12,

in contrast to insulin and the other members of the calcitonin family, including
CGRP,
adrenomedullin, and
calcitonin,

all of which are encoded by genes on the evolutionary related chromosome 11.

The preproIAPP gene contains three exons, of which

the last two encode the full prepromolecule.

The signal peptide is cleaved

off in the endoplasmic reticulum (ER), and
conversion of proIAPP to IAPP takes place in the secretory vesicles.

ProIAPP and proinsulin are both processed by the two endoproteases

prohormone convertase 2 (PC2) and
prohormone convertase 1/3 (PC1/3) and
by carboxypeptidase E (CPE) (Figure 1).

amylin

A: the amino acid sequence of human pro-islet amyloid polypeptide (proIAPP) with the cleavage site for PC2 at the NH2 terminus and the cleavage site for PC1/3 at the COOH terminus, indicated by arrows. The KR residues (blue) that remain at the COOH terminus after PC1/3 processing are removed by carboxypeptidase E. This event exposes the glycine residue that is used for COOH-terminal amidation.
Below is a cartoon of IAPP in blue with the intramolecular S-S bond between residues 2–7 and the amidated COOH terminus.

B: the amino acid sequence of human proinsulin with the basic residues at the B-chain/C-peptide junction and the A-chain/C-peptide/junction indicated in blue and the processing sites indicated by arrows. PC1/3 does almost exclusively process proinsulin at the B-chain/C-peptide junction while PC2 preferentially processes proinsulin at the A-chain/C-peptide junction. The basic residues (RR) (position 31, 32) that remain at the COOH terminus of the B-chain is removed by the carboxypeptidase CPE. Below is a cartoon of insulin A-chain and B-chain in red with intermolecular SS bonds between cystein residues 7 in the A and B chains, between cystein residues at position 19 in the B-chain and 20 in the A-chain and the intermolecular SS bond between cystein residues at position 6 and 11 of the A-chain.

http://physrev.physiology.org/content/physrev/91/3/795/F1.large.jpg

IAPP and insulin genes contain similar promoter elements,
and the transcription factor PDX1 regulates the effects of glucose on both genes.
Glucose stimulated β-cells respond with a parallel expression pattern of IAPP and insulin in the rat.

However, this parallel secretion of IAPP and insulin is altered in experimental diabetes models in rodents. Perfused rat pancreas secreted relatively

more IAPP than insulin when exposed to dexamethasone, whereas
high doses of streptozotocin or alloxan reduced insulin secretion more than that of IAPP.

Oleat and palmitate increased the expression of IAPP but not of insulin in MIN6 cells. In mice fed a diet high in fat for 6 mo, plasma IAPP increased 4.5 times more than insulin compared with mice fed standard food containing 4% fat.

In human recipients who had become insulin-independent by intrahepatically transplanted islets, there was disproportionately

more IAPP than normal secreted during hyperglycemia.

These examples show that the strictly parallel expression of IAPP and insulin may be disturbed under certain conditions.

The crystalline structure of insulin in granules is well characterized.

Hexameric insulin, together with zinc, constitutes the core of the mature granules, while
IAPP, together with a large number of additional components, including the C peptide, is found in the halo region.

The highly fibrillogenic human IAPP has to be protected in some way from aggregation, which otherwise would take place spontaneously. The fact that very fibril-prone proteins can be kept in solution at high concentrations is known from studies of arthropod silk. The composition of the β-cell granule is extremely complex, and it has many components in addition to insulin and C peptide, in micromolar concentrations.

It is probable that IAPP is protected from aggregation by interaction with other components. Plausible candidates are

proinsulin, insulin, or their processing intermediates.

Insulin has been found to be

a strong inhibitor of IAPP fibril formation.

This finding has been verified in a number of subsequent studies, which have also shown the potency of the inhibition. The inhibition seems to depend

solely on the B-chain,
which binds specifically to a short segment of IAPP.

An insulin-to-IAPP ratio of between 1:5 and 1:100 had a strong inhibitory effect. The molar ratio between IAPP and insulin in the granule as a whole is ∼1–2:50.

Type 2 Diabetes, APOE Gene, and the Risk for Dementia and Related Pathologies. The Honolulu-Asia Aging Study

Rita Peila, Beatriz L. Rodriguez and Lenore J. Launer
Diabetes Apr 2002; 51(4): 1256-1262
http://dx.doi.org:/10.2337/diabetes.51.4.1256

Type 2 diabetes may be a risk factor for dementia, but the associated pathological mechanisms remains unclear. We evaluated the association of diabetes

alone or combined with the apolipoprotein E (APOE) gene
with incident dementia and neuropathological outcomes

in a population-based cohort of 2,574 Japanese-American men enrolled in the Honolulu-Asia Aging Study, including 216 subjects who underwent autopsy. Type 2 diabetes was ascertained by interview and direct glucose testing. Dementia was assessed in 1991 and 1994 by clinical examination and magnetic resonance imaging and was diagnosed according to international guidelines. Logistic regression was used to assess the RR of developing dementia, and log-linear regression was used to estimate the incident rate ratio (IRR) of neuropathological outcomes.

Diabetes was associated with

total dementia (RR 1.5 [95% CI 1.01–2.2]),
Alzheimer’s disease (AD; 1.8 [1.1–2.9]), and
vascular dementia (VsD; 2.3 [1.1–5.0]).

Individuals with both type 2 diabetes and the APOE ε4 allele

had an RR of 5.5 (CI 2.2–13.7) for AD compared with those with neither risk factor.

Participants with type 2 diabetes and the ε4 allele had

a higher number of hippocampal neuritic plaques (IRR 3.0 [CI 1.2–7.3]) and
neurofibrillary tangles in the cortex (IRR 3.5 [1.6–7.5]) and hippocampus (IRR 2.5 [1.5–3.7]), and
they had a higher risk of cerebral amyloid angiopathy (RR 6.6, 1.5–29.6).

Type 2 diabetes is a risk factor for AD and VsD. The association between diabetes and AD is particularly strong among carriers of the APOE ε4 allele. The neuropathological data are consistent with the clinical results.

Role of insulin signaling impairment, adiponectin and dyslipidemia in peripheral and central neuropathy in mice

Anderson, MR. King, L Delbruck, CG. Jolivalt
Dis. Model. Mech. June 2014; 7(6): 625-633
http://dx.doi.org:/10.1242/dmm.015750

One of the tissues or organs affected by diabetes is the nervous system,

predominantly the peripheral system (peripheral polyneuropathy and/or painful peripheral neuropathy)
but also the central system with impaired learning, memory and mental flexibility.

The aim of this study was to test the hypothesis that the pre-diabetic or diabetic condition caused by a high-fat diet (HFD) can damage both the peripheral and central nervous systems. Groups of C57BL6 and Swiss Webster mice were fed a diet containing 60% fat for 8 months and compared to control and streptozotocin (STZ)-induced diabetic groups that were fed a standard diet containing 10% fat. Aspects of peripheral nerve function (conduction velocity, thermal sensitivity) and central nervous system function (learning ability, memory) were measured at assorted times during the study. Both strains of mice on HFD developed impaired glucose tolerance, indicative of insulin resistance, but

only the C57BL6 mice showed statistically significant hyperglycemia.

STZ-diabetic C57BL6 mice

developed learning deficits in the Barnes maze after 8 weeks of diabetes, whereas
neither C57BL6 nor Swiss Webster mice fed a HFD showed signs of defects at that time point.

By 6 months on HFD, Swiss Webster mice developed

learning and memory deficits in the Barnes maze test,
whereas their peripheral nervous system remained normal.

In contrast, C57BL6 mice fed the HFD developed peripheral nerve dysfunction,

as indicated by nerve conduction slowing and thermal hyperalgesia,
but showed normal learning and memory functions.

Our data indicate that STZ-induced diabetes or a HFD can damage

both peripheral and central nervous systems,
but learning deficits develop more rapidly in insulin-deficient than in insulin-resistant conditions
and only in Swiss Webster mice.

In addition to insulin impairment, dyslipidemia or adiponectinemia might determine the neuropathy phenotype.

Neuroinflammation and neurologic deficits in diabetes linked to brain accumulation of amylin

S Srodulski, S Sharma, AB Bachstetter, JM Brelsfoard, et al.
Molecular Neurodegeneration 2014; 9(30):
http://dx.doi.org:/10.1186/1750-1326-9-30

Background: We recently found that brain tissue from patients with type-2 diabetes (T2D) and cognitive impairment

contains deposits of amylin, an amyloidogenic hormone synthesized and co-secreted with insulin by pancreatic β-cells.

Amylin deposition is promoted by

chronic hypersecretion of amylin (hyperamylinemia), which is common in humans with obesity or pre-diabetic insulin resistance.

Human amylin oligomerizes quickly when oversecreted, which is toxic,

induces inflammation in pancreatic islets and
contributes to the development of T2D.

Here, we tested the hypothesis that accumulation of oligomerized amylin affects brain function.

Methods: In contrast to amylin from humans,

rodent amylin is neither amyloidogenic nor cytotoxic.

We exploited this fact by comparing

rats overexpressing human amylin in the pancreas (HIP rats) with their littermate rats

which express only wild-type (WT) non-amyloidogenic rodent amylin. Cage activity, rotarod and novel object recognition tests were performed on animals nine months of age or older. Amylin deposition in the brain was documented by immunohistochemistry, and western blot. We also measured neuroinflammation by immunohistochemistry, quantitative real-time PCR and cytokine protein levels.

Results: Compared to WT rats, HIP rats show

i) reduced exploratory drive,
ii) impaired recognition memory and
iii) no ability to improve the performance on the rotarod.

The development of neurological deficits is

associated with amylin accumulation in the brain.

The level of oligomerized amylin in supernatant fractions and pellets from brain homogenates

is almost double in HIP rats compared with WT littermates (P < 0.05).

Large amylin deposits (>50 μm diameter) were also occasionally seen in HIP rat brains. Accumulation of oligomerized amylin

alters the brain structure at the molecular level.

Immunohistochemistry analysis with an ED1 antibody indicates possible activated microglia/macrophages which

are clustering in areas positive for amylin infiltration.

Multiple inflammatory markers are expressed in HIP rat brains as opposed to WT rats, confirming that

amylin deposition in the brain induces a neuroinflammatory response.

Conclusions:

Hyperamylinemia promotes accumulation of oligomerized amylin in the brain
leading to neurological deficits through an oligomerized amylin-mediated inflammatory response.

Additional studies are needed to determine

whether brain amylin accumulation may predispose to diabetic brain injury and cognitive decline.

Keywords: Diabetes, Alzheimer’s Disease, Amylin, Pre-diabetes, Insulin Resistance, Inflammation, Behavior

Read Full Post »

Introduction to Metabolomics

Posted in Academic Publishing, Anaerobic Glycolysis, Anemia, Biochemical pathways, Biological Networks, Biomarkers & Medical Diagnostics, Biomedical Measurement Science, Cachexia, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Computational Biology/Systems and Bioinformatics, Curation, Cytoskeleton, Developmental biology, Diabetes Mellitus, Disease Biology, Endocrine Diseases, Enzyme Induction, Enzymes and isoenzymes, Folate and B12, Gene Regulation, Gene Regulation and Evolution, Genetics & Pharmaceutical, Genomic Expression, Glycobiology: Biopharmaceutical Production, Health Economics and Outcomes Research, Hematopoiesis, Hexokinase, Human Immune System in Health and in Disease, Infectious Disease & New Antibiotic Targets, Intellectual Property, Iron deficiency, Lipid metabolism, Liver & Digestive Diseases Research, Lymphoma, Metabolism, Metabolomics, Methylation, Molecular Genetics & Pharmaceutical, Mutant Gene Expression, Myelodysplasia, Myocardial adenine nucleotide metabolism, Myocardial Ischemia, Myocardial Metabolism, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Nutrition, Nutrition and Phytochemistry, Oxidative phosphorylation, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmacodynamics and Pharmacokinetics, Pharmacologic toxicities, Plant extract, Population Health Management, Proteomics, Pyruvate Kinase, Rapid automation of plasma protein pools, RNA Biology, Signaling, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, Transcriptomics, Translational Science, Warburg effect, tagged carbohyudrate metabolism, metabolic maps, Metabolism, Metabolomics, pharmaceutical targets, Proteomics on October 21, 2014| Leave a Comment »

Introduction to Metabolomics

Author: Larry H. Bernstein, MD, FCAP

This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases. It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time bachalaureate degree reader in the biological sciences. Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career.

In the Preface, I failed to disclose that the term Metabolomics applies to plants, animals, bacteria, and both prokaryotes and eukaryotes. The metabolome for each organism is unique, but from an evolutionary perspective has metabolic pathways in common, and expressed in concert with the environment that these living creatures exist. The metabolome of each has adaptive accommodation with suppression and activation of pathways that are functional and necessary in balance, for its existence. Was it William Faulkner who said in his Nobel Prize acceptance that mankind shall not merely exist, but survive? That seems to be the overlying theme for all of life. If life cannot persist, a surviving “remnant” might continue. The history of life may well be etched into the genetic code, some of which is not expressed.

This work is apportioned into chapters in a sequence that is first directed at the major sources for the energy and the structure of life, in the carbohydrates, lipids, and fats, which are sourced from both plants and animals, and depending on their balance, results in an equilibrium, and a disequilibrium we refer to as disease. There is also a need to consider the nonorganic essentials which are derived from the soil, from water, and from the energy of the sun and the air we breathe, or in the case of water-bound metabolomes, dissolved gases.

In addition to the basic essential nutrients and their metabolic utilization, they are under cellular metabolic regulation that is tied to signaling pathways. In addition, the genetic expression of the organism is under regulatory control by the interaction of RNAs that interact with the chromatin genetic framework, with exosomes, and with protein modulators.This is referred to as epigenetics, but there are also drivers of metabolism that are shaped by the interactions between enzymes and substartes, and are related to the tertiary structure of a protein. The framework for diseases in a separate chapter. Pharmaceutical interventions that are designed to modulate specific metabolic targets are addressed as the pathways are unfolded. Neutraceuticals and plant based nutrition are covered in Chapter 8.

Chapter 1: Metabolic Pathways

Chapter 2. Lipid Metabolism

Chapter 3. Cell Signaling

Chapter 4. Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8. Impairments in Pathological States: Endocrine Disorders; Stress Hypermetabolism and Cancer

Read Full Post »

Metabolomics Summary and Perspective

Posted in Acute lymphocytic leukemia, Acute myelocytic leukemia, Amino acids, Anaerobic Glycolysis, Best evidence, Bias measurement tools, Biochemical pathways, Biological Networks, Biomarkers & Medical Diagnostics, Biomedical Measurement Science, Ca2+ triggered activation, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cardiovascular Pharmacogenomics, Cardiovascular Research, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Clinical Diagnostics, Commercialization, Computational Biology/Systems and Bioinformatics, Curation, Cytoskeleton, Developmental biology, Diabetes Mellitus, Diagnostic Immunology, Disease Biology, Drug Toxicity, Endocrine Diseases, Enzyme Induction, Enzymes and isoenzymes, Gene Regulation, Gene Regulation and Evolution, Genetics & Pharmaceutical, Genome Biology, Genomic Expression, Hematology, Human Immune System in Health and in Disease, Image Processing/Computing, Immunodiagnostics, Infectious Disease & New Antibiotic Targets, Inferential analysis, Innovations, Innovations in Neurophysiology & Neuropsychology, Intellectual Property, International Global Work in Pharmaceutical, Investment in Technological Breakthrough, Lipid metabolism, Lipids, Liver & Digestive Diseases Research, Lymphoma, Mass automation of plasma proteins, Metabolism, Metabolomics, Methods, Methylation, Molecular Genetics & Pharmaceutical, Neurohumoral Transmission, Neurological Diseases, Nitric Oxide in Health and Disease, Nutrigenomics, Nutrition, Nutrition and Phytochemistry, Pentose monophosphate shunt, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmaceutical Industry Competitive Intelligence, Pharmacogenomics, Pharmacologic toxicities, Phosphorylation, Population Health Management, Proteins, Proteomics, Quality Assurance, Rapid automation of plasma protein pools, RNA Biology, Signaling, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, Statistical Methods for Research Evaluation, Synaptic vesicle, Systemic Inflammatory Response Related Disorders, Technology Advance Assessment of, Technology Transfer: Biotech and Pharmaceutical, Transcriptomics, Translational Science, Ubiquitinylation, Warburg effect, tagged analytical methods, Autophagy, Cancer - General, Diabetes Mellitus, enzymes, Epigenetics, graphical presentations, metabolic maps, metabolites, Metabolomics, neural science, pathway interactions, Proteins, proteome, signaling, transcription control on October 16, 2014| Leave a Comment »

Metabolomics Summary and Perspective

Author and Curator: Larry H Bernstein, MD, FCAP

This is the final article in a robust series on metabolism, metabolomics, and the “-OMICS-“ biological synthesis that is creating a more holistic and interoperable view of natural sciences, including the biological disciplines, climate science, physics, chemistry, toxicology, pharmacology, and pathophysiology with as yet unforeseen consequences.

There have been impressive advances already in the research into developmental biology, plant sciences, microbiology, mycology, and human diseases, most notably, cancer, metabolic , and infectious, as well as neurodegenerative diseases.

Acknowledgements:

I write this article in honor of my first mentor, Harry Maisel, Professor and Emeritus Chairman of Anatomy, Wayne State University, Detroit, MI and to my stimulating mentors, students, fellows, and associates over many years:

Masahiro Chiga, MD, PhD, Averill A Liebow, MD, Nathan O Kaplan, PhD, Johannes Everse, PhD, Norio Shioura, PhD, Abraham Braude, MD, Percy J Russell, PhD, Debby Peters, Walter D Foster, PhD, Herschel Sidransky, MD, Sherman Bloom, MD, Matthew Grisham, PhD, Christos Tsokos, PhD, IJ Good, PhD, Distinguished Professor, Raool Banagale, MD, Gustavo Reynoso, MD,Gustave Davis, MD, Marguerite M Pinto, MD, Walter Pleban, MD, Marion Feietelson-Winkler, RD, PhD, John Adan,MD, Joseph Babb, MD, Stuart Zarich, MD, Inder Mayall, MD, A Qamar, MD, Yves Ingenbleek, MD, PhD, Emeritus Professor, Bette Seamonds, PhD, Larry Kaplan, PhD, Pauline Y Lau, PhD, Gil David, PhD, Ronald Coifman, PhD, Emeritus Professor, Linda Brugler, RD, MBA, James Rucinski, MD, Gitta Pancer, Ester Engelman, Farhana Hoque, Mohammed Alam, Michael Zions, William Fleischman, MD, Salman Haq, MD, Jerard Kneifati-Hayek, Madeleine Schleffer, John F Heitner, MD, Arun Devakonda,MD, Liziamma George,MD, Suhail Raoof, MD, Charles Oribabor,MD, Anthony Tortolani, MD, Prof and Chairman, JRDS Rosalino, PhD, Aviva Lev Ari, PhD, RN, Rosser Rudolph, MD, PhD, Eugene Rypka, PhD, Jay Magidson, PhD, Izaak Mayzlin, PhD, Maurice Bernstein, PhD, Richard Bing, Eli Kaplan, PhD, Maurice Bernstein, PhD.

This article has EIGHT parts, as follows:

Part 1

Metabolomics Continues Auspicious Climb

Part 2

Biologists Find ‘Missing Link’ in the Production of Protein Factories in Cells

Part 3

Neuroscience

Part 4

Cancer Research

Part 5

Metabolic Syndrome

Part 6

Biomarkers

Part 7

Epigenetics and Drug Metabolism

Part 8

Pictorial

genome cartoon

iron metabolism

personalized reference range within population range

Part 1. MetabolomicsSurge

metagraph _OMICS

Metabolomics Continues Auspicious Climb

Jeffery Herman, Ph.D.
GEN May 1, 2012 (Vol. 32, No. 9)

Aberrant biochemical and metabolite signaling plays an important role in

the development and progression of diseased tissue.

This concept has been studied by the science community for decades. However, with relatively

recent advances in analytical technology and bioinformatics as well as
the development of the Human Metabolome Database (HMDB),

metabolomics has become an invaluable field of research.

At the “International Conference and Exhibition on Metabolomics & Systems Biology” held recently in San Francisco, researchers and industry leaders discussed how

the underlying cellular biochemical/metabolite fingerprint in response to

a specific disease state,
toxin exposure, or
pharmaceutical compound

is useful in clinical diagnosis and biomarker discovery and
in understanding disease development and progression.

Developed by BASF, MetaMap® Tox is

a database that helps identify in vivo systemic effects of a tested compound, including

targeted organs,
mechanism of action, and
adverse events.

Based on 28-day systemic rat toxicity studies, MetaMap Tox is composed of

differential plasma metabolite profiles of rats
after exposure to a large variety of chemical toxins and pharmaceutical compounds.

“Using the reference data,

we have developed more than 110 patterns of metabolite changes, which are
specific and predictive for certain toxicological modes of action,”

said Hennicke Kamp, Ph.D., group leader, department of experimental toxicology and ecology at BASF.

With MetaMap Tox, a potential drug candidate

can be compared to a similar reference compound
using statistical correlation algorithms,
which allow for the creation of a toxicity and mechanism of action profile.

“MetaMap Tox, in the context of early pre-clinical safety enablement in pharmaceutical development,” continued Dr. Kamp,

has been independently validated “
by an industry consortium (Drug Safety Executive Council) of 12 leading biopharmaceutical companies.”

Dr. Kamp added that this technology may prove invaluable

allowing for quick and accurate decisions and
for high-throughput drug candidate screening, in evaluation

on the safety and efficacy of compounds
during early and preclinical toxicological studies,
by comparing a lead compound to a variety of molecular derivatives, and

the rapid identification of the most optimal molecular structure
with the best efficacy and safety profiles might be streamlined.

Dynamic Construct of the –Omics

Targeted Tandem Mass Spectrometry

Biocrates Life Sciences focuses on targeted metabolomics, an important approach for

the accurate quantification of known metabolites within a biological sample.

Originally used for the clinical screening of inherent metabolic disorders from dried blood-spots of newborn children, Biocrates has developed

a tandem mass spectrometry (MS/MS) platform, which allows for

the identification,
quantification, and
mapping of more than 800 metabolites to specific cellular pathways.

It is based on flow injection analysis and high-performance liquid chromatography MS/MS.

common drug targets

The MetaDisIDQ® Kit is a

“multiparamatic” diagnostic assay designed for the “comprehensive assessment of a person’s metabolic state” and
the early determination of pathophysiological events with regards to a specific disease.

MetaDisIDQ is designed to quantify

a diverse range of 181 metabolites involved in major metabolic pathways
from a small amount of human serum (10 µL) using isotopically labeled internal standards,

This kit has been demonstrated to detect changes in metabolites that are commonly associated with the development of

metabolic syndrome, type 2 diabetes, and diabetic nephropathy,

Dr. Dallman reports that data generated with the MetaDisIDQ kit correlates strongly with

routine chemical analyses of common metabolites including glucose and creatinine

Biocrates has also developed the MS/MS-based AbsoluteIDQ® kits, which are

an “easy-to-use” biomarker analysis tool for laboratory research.

The kit functions on MS machines from a variety of vendors, and allows for the quantification of 150-180 metabolites.

The SteroIDQ® kit is a high-throughput standardized MS/MS diagnostic assay,

validated in human serum, for the rapid and accurate clinical determination of 16 known steroids.

Initially focusing on the analysis of steroid ranges for use in hormone replacement therapy, the SteroIDQ Kit is expected to have a wide clinical application.

Hormone-Resistant Breast Cancer

Scientists at Georgetown University have shown that

breast cancer cells can functionally coordinate cell-survival and cell-proliferation mechanisms,
while maintaining a certain degree of cellular metabolism.

To grow, cells need energy, and energy is a product of cellular metabolism. For nearly a century, it was thought that

the uncoupling of glycolysis from the mitochondria,
leading to the inefficient but rapid metabolism of glucose and
the formation of lactic acid (the Warburg effect), was

the major and only metabolism driving force for unchecked proliferation and tumorigenesis of cancer cells.

Other aspects of metabolism were often overlooked.

“.. we understand now that

cellular metabolism is a lot more than just metabolizing glucose,”

said Robert Clarke, Ph.D., professor of oncology and physiology and biophysics at Georgetown University. Dr. Clarke, in collaboration with the Waters Center for Innovation at Georgetown University (led by Albert J. Fornace, Jr., M.D.), obtained

the metabolomic profile of hormone-sensitive and -resistant breast cancer cells through the use of UPLC-MS.

They demonstrated that breast cancer cells, through a rather complex and not yet completely understood process,

can functionally coordinate cell-survival and cell-proliferation mechanisms,
while maintaining a certain degree of cellular metabolism.

This is at least partly accomplished through the upregulation of important pro-survival mechanisms; including

the unfolded protein response;
a regulator of endoplasmic reticulum stress and
initiator of autophagy.

Normally, during a stressful situation, a cell may

enter a state of quiescence and undergo autophagy,
a process by which a cell can recycle organelles
in order to maintain enough energy to survive during a stressful situation or,

if the stress is too great,

undergo apoptosis.

By integrating cell-survival mechanisms and cellular metabolism

advanced ER+ hormone-resistant breast cancer cells
can maintain a low level of autophagy
to adapt and resist hormone/chemotherapy treatment.

This adaptation allows cells

to reallocate important metabolites recovered from organelle degradation and
provide enough energy to also promote proliferation.

With further research, we can gain a better understanding of the underlying causes of hormone-resistant breast cancer, with

the overall goal of developing effective diagnostic, prognostic, and therapeutic tools.

NMR

Over the last two decades, NMR has established itself as a major tool for metabolomics analysis. It is especially adept at testing biological fluids. [Bruker BioSpin]

Historically, nuclear magnetic resonance spectroscopy (NMR) has been used for structural elucidation of pure molecular compounds. However, in the last two decades, NMR has established itself as a major tool for metabolomics analysis. Since

the integral of an NMR signal is directly proportional to
the molar concentration throughout the dynamic range of a sample,

“the simultaneous quantification of compounds is possible

without the need for specific reference standards or calibration curves,” according to Lea Heintz of Bruker BioSpin.

NMR is adept at testing biological fluids because of

high reproducibility,
standardized protocols,
low sample manipulation, and
the production of a large subset of data,

Bruker BioSpin is presently involved in a project for the screening of inborn errors of metabolism in newborn children from Turkey, based on their urine NMR profiles. More than 20 clinics are participating to the project that is coordinated by INFAI, a specialist in the transfer of advanced analytical technology into medical diagnostics. The construction of statistical models are being developed

for the detection of deviations from normality, as well as
automatic quantification methods for indicative metabolites

Bruker BioSpin recently installed high-resolution magic angle spinning NMR (HRMAS-NMR) systems that can rapidly analyze tissue biopsies. The main objective for HRMAS-NMR is to establish a rapid and effective clinical method to assess tumor grade and other important aspects of cancer during surgery.

Combined NMR and Mass Spec

There is increasing interest in combining NMR and MS, two of the main analytical assays in metabolomic research, as a means

to improve data sensitivity and to
fully elucidate the complex metabolome within a given biological sample.
to realize a potential for cancer biomarker discovery in the realms of diagnosis, prognosis, and treatment.

Using combined NMR and MS to measure the levels of nearly 250 separate metabolites in the patient’s blood, Dr. Weljie and other researchers at the University of Calgary were able to rapidly determine the malignancy of a pancreatic lesion (in 10–15% of the cases, it is difficult to discern between benign and malignant), while avoiding unnecessary surgery in patients with benign lesions.

When performing NMR and MS on a single biological fluid, ultimately “we are,” noted Dr. Weljie,

“splitting up information content, processing, and introducing a lot of background noise and error and
then trying to reintegrate the data…
It’s like taking a complex item, with multiple pieces, out of an IKEA box and trying to repackage it perfectly into another box.”

By improving the workflow between the initial splitting of the sample, they improved endpoint data integration, proving that

a streamlined approach to combined NMR/MS can be achieved,
leading to a very strong, robust and precise metabolomics toolset.

Metabolomics Research Picks Up Speed

Field Advances in Quest to Improve Disease Diagnosis and Predict Drug Response

John Morrow Jr., Ph.D.
GEN May 1, 2011 (Vol. 31, No. 9)

As an important discipline within systems biology, metabolomics is being explored by a number of laboratories for

its potential in pharmaceutical development.

Studying metabolites can offer insights into the relationships between genotype and phenotype, as well as between genotype and environment. In addition, there is plenty to work with—there are estimated to be some 2,900 detectable metabolites in the human body, of which

309 have been identified in cerebrospinal fluid,
1,122 in serum,
458 in urine, and
roughly 300 in other compartments.

Guowang Xu, Ph.D., a researcher at the Dalian Institute of Chemical Physics. is investigating the causes of death in China,

and how they have been changing over the years as the country has become a more industrialized nation.
the increase in the incidence of metabolic disorders such as diabetes has grown to affect 9.7% of the Chinese population.

Dr. Xu, collaborating with Rainer Lehman, Ph.D., of the University of Tübingen, Germany, compared urinary metabolites in samples from healthy individuals with samples taken from prediabetic, insulin-resistant subjects. Using mass spectrometry coupled with electrospray ionization in the positive mode, they observed striking dissimilarities in levels of various metabolites in the two groups.

“When we performed a comprehensive two-dimensional gas chromatography, time-of-flight mass spectrometry analysis of our samples, we observed several metabolites, including

2-hydroxybutyric acid in plasma,
as potential diabetes biomarkers,” Dr. Xu explains.

In other, unrelated studies, Dr. Xu and the German researchers used a metabolomics approach to investigate the changes in plasma metabolite profiles immediately after exercise and following a 3-hour and 24-hour period of recovery. They found that

medium-chain acylcarnitines were the most distinctive exercise biomarkers, and
they are released as intermediates of partial beta oxidation in human myotubes and mouse muscle tissue.

Dr. Xu says. “The traditional approach of assessment based on a singular biomarker is being superseded by the introduction of multiple marker profiles.”

Typical of the studies under way by Dr. Kaddurah-Daouk and her colleaguesat Duke University

is a recently published investigation highlighting the role of an SNP variant in
the glycine dehydrogenase gene on individual response to antidepressants.
patients who do not respond to the selective serotonin uptake inhibitors citalopram and escitalopram
carried a particular single nucleotide polymorphism in the GD gene.

“These results allow us to pinpoint a possible

role for glycine in selective serotonin reuptake inhibitor response and
illustrate the use of pharmacometabolomics to inform pharmacogenomics.

These discoveries give us the tools for prognostics and diagnostics so that

we can predict what conditions will respond to treatment.

“This approach to defining health or disease in terms of metabolic states opens a whole new paradigm.

By screening hundreds of thousands of molecules, we can understand

the relationship between human genetic variability and the metabolome.”

Dr. Kaddurah-Daouk talks about statins as a current

model of metabolomics investigations.

It is now known that the statins have widespread effects, altering a range of metabolites. To sort out these changes and develop recommendations for which individuals should be receiving statins will require substantial investments of energy and resources into defining the complex web of biochemical changes that these drugs initiate.
Furthermore, Dr. Kaddurah-Daouk asserts that,

“genetics only encodes part of the phenotypic response.

One needs to take into account the

net environment contribution in order to determine
how both factors guide the changes in our metabolic state that determine the phenotype.”

Interactive Metabolomics

Researchers at the University of Nottingham use diffusion-edited nuclear magnetic resonance spectroscopy to assess the effects of a biological matrix on metabolites. Diffusion-edited NMR experiments provide a way to

separate the different compounds in a mixture
based on the differing translational diffusion coefficients (which reflect the size and shape of the molecule).

The measurements are carried out by observing

the attenuation of the NMR signals during a pulsed field gradient experiment.

Clare Daykin, Ph.D., is a lecturer at the University of Nottingham, U.K. Her field of investigation encompasses “interactive metabolomics,”which she defines as

“the study of the interactions between low molecular weight biochemicals and macromolecules in biological samples ..

without preselection of the components of interest.

“Blood plasma is a heterogeneous mixture of molecules that

undergo a variety of interactions including metal complexation,
chemical exchange processes,
micellar compartmentation,
enzyme-mediated biotransformations, and
small molecule–macromolecular binding.”

Many low molecular weight compounds can exist

freely in solution,
bound to proteins, or
within organized aggregates such as lipoprotein complexes.

Therefore, quantitative comparison of plasma composition from

diseased individuals compared to matched controls provides an incomplete insight to plasma metabolism.

“It is not simply the concentrations of metabolites that must be investigated,

but their interactions with the proteins and lipoproteins within this complex web.

Rather than targeting specific metabolites of interest, Dr. Daykin’s metabolite–protein binding studies aim to study

the interactions of all detectable metabolites within the macromolecular sample.

Such activities can be studied through the use of diffusion-edited nuclear magnetic resonance (NMR) spectroscopy, in which one can assess

the effects of the biological matrix on the metabolites.

“This can lead to a more relevant and exact interpretation

for systems where metabolite–macromolecule interactions occur.”

Diffusion-edited NMR experiments provide a way to separate the different compounds in a mixture based on

the differing translational diffusion coefficients (which reflect the size and shape of the molecule).

The measurements are carried out by observing

the attenuation of the NMR signals during a pulsed field gradient experiment.

Pushing the Limits

It is widely recognized that many drug candidates fail during development due to ancillary toxicity. Uwe Sauer, Ph.D., professor, and Nicola Zamboni, Ph.D., researcher, both at the Eidgenössische Technische Hochschule, Zürich (ETH Zürich), are applying

high-throughput intracellular metabolomics to understand
the basis of these unfortunate events and
head them off early in the course of drug discovery.

“Since metabolism is at the core of drug toxicity, we developed a platform for

measurement of 50–100 targeted metabolites by
a high-throughput system consisting of flow injection
coupled to tandem mass spectrometry.”

Using this approach, Dr. Sauer’s team focused on

the central metabolism of the yeast Saccharomyces cerevisiae, reasoning that
this core network would be most susceptible to potential drug toxicity.

Screening approximately 41 drugs that were administered at seven concentrations over three orders of magnitude, they observed changes in metabolome patterns at much lower drug concentrations without attendant physiological toxicity.

The group carried out statistical modeling of about

60 metabolite profiles for each drug they evaluated.

This data allowed the construction of a “profile effect map” in which

the influence of each drug on metabolite levels can be followed, including off-target effects, which
provide an indirect measure of the possible side effects of the various drugs.

Dr. Sauer says.“We have found that this approach is

at least 100 times as fast as other omics screening platforms,”

“Some drugs, including many anticancer agents,

disrupt metabolism long before affecting growth.”

killing cancer cells

Furthermore, they used the principle of 13C-based flux analysis, in which

metabolites labeled with 13C are used to follow the utilization of metabolic pathways in the cell.

These 13C-determined intracellular responses of metabolic fluxes to drug treatment demonstrate

the functional performance of the network to be rather robust,

conformational changes leading to substrate efflux.

leading Dr. Sauer to the conclusion that

the phenotypic vigor he observes to drug challenges
is achieved by a flexible make up of the metabolome.

Dr. Sauer is confident that it will be possible to expand the scope of these investigations to hundreds of thousands of samples per study. This will allow answers to the questions of

how cells establish a stable functioning network in the face of inevitable concentration fluctuations.

Is Now the Hour?

There is great enthusiasm and agitation within the biotech community for

metabolomics approaches as a means of reversing the dismal record of drug discovery

that has accumulated in the last decade.

While the concept clearly makes sense and is being widely applied today, there are many reasons why drugs fail in development, and metabolomics will not be a panacea for resolving all of these questions. It is too early at this point to recognize a trend or a track record, and it will take some time to see how this approach can aid in drug discovery and shorten the timeline for the introduction of new pharmaceutical agents.

Degree of binding correlated with function

Diagram_of_a_two-photon_excitation_microscope_

Part 2. Biologists Find ‘Missing Link’ in the Production of Protein Factories in Cells

Biologists at UC San Diego have found

the “missing link” in the chemical system that
enables animal cells to produce ribosomes

—the thousands of protein “factories” contained within each cell that

manufacture all of the proteins needed to build tissue and sustain life.

‘Missing Link’

Their discovery, detailed in the June 23 issue of the journal Genes & Development, will not only force

a revision of basic textbooks on molecular biology, but also
provide scientists with a better understanding of
how to limit uncontrolled cell growth, such as cancer,
that might be regulated by controlling the output of ribosomes.

Ribosomes are responsible for the production of the wide variety of proteins that include

enzymes;
structural molecules, such as hair,
skin and bones;
hormones like insulin; and
components of our immune system such as antibodies.

Regarded as life’s most important molecular machine, ribosomes have been intensively studied by scientists (the 2009 Nobel Prize in Chemistry, for example, was awarded for studies of its structure and function). But until now researchers had not uncovered all of the details of how the proteins that are used to construct ribosomes are themselves produced.

In multicellular animals such as humans,

ribosomes are made up of about 80 different proteins
(humans have 79 while some other animals have a slightly different number) as well as
four different kinds of RNA molecules.

In 1969, scientists discovered that

the synthesis of the ribosomal RNAs is carried out by specialized systems using two key enzymes:
RNA polymerase I and RNA polymerase III.

But until now, scientists were unsure if a complementary system was also responsible for

the production of the 80 proteins that make up the ribosome.

That’s essentially what the UC San Diego researchers headed by Jim Kadonaga, a professor of biology, set out to examine. What they found was the missing link—the specialized

system that allows ribosomal proteins themselves to be synthesized by the cell.

Kadonaga says that he and coworkers found that ribosomal proteins are synthesized via

a novel regulatory system with the enzyme RNA polymerase II and
a factor termed TRF2,”

“For the production of most proteins,

RNA polymerase II functions with
a factor termed TBP,
but for the synthesis of ribosomal proteins, it uses TRF2.”

this specialized TRF2-based system for ribosome biogenesis
provides a new avenue for the study of ribosomes and
its control of cell growth, and

“it should lead to a better understanding and potential treatment of diseases such as cancer.”

Coordination of the transcriptome and metabolome

the potential advantages conferred by distal-site protein synthesis

Other authors of the paper were UC San Diego biologists Yuan-Liang Wang, Sascha Duttke and George Kassavetis, and Kai Chen, Jeff Johnston, and Julia Zeitlinger of the Stowers Institute for Medical Research in Kansas City, Missouri. Their research was supported by two grants from the National Institutes of Health (1DP2OD004561-01 and R01 GM041249).

Turning Off a Powerful Cancer Protein

Scientists have discovered how to shut down a master regulatory transcription factor that is

key to the survival of a majority of aggressive lymphomas,
which arise from the B cells of the immune system.

The protein, Bcl6, has long been considered too complex to target with a drug since it is also crucial

to the healthy functioning of many immune cells in the body, not just B cells gone bad.

The researchers at Weill Cornell Medical College report that it is possible

to shut down Bcl6 in diffuse large B-cell lymphoma (DLBCL)
while not affecting its vital function in T cells and macrophages
that are needed to support a healthy immune system.

If Bcl6 is completely inhibited, patients might suffer from systemic inflammation and atherosclerosis. The team conducted this new study to help clarify possible risks, as well as to understand

how Bcl6 controls the various aspects of the immune system.

The findings in this study were inspired from

preclinical testing of two Bcl6-targeting agents that Dr. Melnick and his Weill Cornell colleagues have developed
to treat DLBCLs.

These experimental drugs are

RI-BPI, a peptide mimic, and
the small molecule agent 79-6.

“This means the drugs we have developed against Bcl6 are more likely to be

significantly less toxic and safer for patients with this cancer than we realized,”

says Ari Melnick, M.D., professor of hematology/oncology and a hematologist-oncologist at NewYork-Presbyterian Hospital/Weill Cornell Medical Center.

Dr. Melnick says the discovery that

a master regulatory transcription factor can be targeted
offers implications beyond just treating DLBCL.

Recent studies from Dr. Melnick and others have revealed that

Bcl6 plays a key role in the most aggressive forms of acute leukemia, as well as certain solid tumors.

Bcl6 can control the type of immune cell that develops in the bone marrow—playing many roles

in the development of B cells, T cells, macrophages, and other cells—including a primary and essential role in
enabling B-cells to generate specific antibodies against pathogens.

According to Dr. Melnick, “When cells lose control of Bcl6,

lymphomas develop in the immune system.

Lymphomas are ‘addicted’ to Bcl6, and therefore

Bcl6 inhibitors powerfully and quickly destroy lymphoma cells,” .

The big surprise in the current study is that rather than functioning as a single molecular machine,

Bcl6 functions like a Swiss Army knife,
using different tools to control different cell types.

This multifunction paradigm could represent a general model for the functioning of other master regulatory transcription factors.

“In this analogy, the Swiss Army knife, or transcription factor, keeps most of its tools folded,

opening only the one it needs in any given cell type,”

He makes the following analogy:

“For B cells, it might open and use the knife tool;
for T cells, the cork screw;
for macrophages, the scissors.”

“this means that you only need to prevent the master regulator from using certain tools to treat cancer. You don’t need to eliminate the whole knife,” . “In fact, we show that taking out the whole knife is harmful since

the transcription factor has many other vital functions that other cells in the body need.”

Prior to these study results, it was not known that a master regulator could separate its functions so precisely. Researchers hope this will be a major benefit to the treatment of DLBCL and perhaps other disorders that are influenced by Bcl6 and other master regulatory transcription factors.

The study is published in the journal Nature Immunology, in a paper titled “Lineage-specific functions of Bcl-6 in immunity and inflammation are mediated by distinct biochemical mechanisms”.

Part 3. Neuroscience

Vesicles influence function of nerve cells
Oct, 06 2014 source: http://feeds.sciencedaily.com

Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.

Tiny vesicles containing protective substances

which they transmit to nerve cells apparently
play an important role in the functioning of neurons.

As cell biologists at Johannes Gutenberg University Mainz (JGU) have discovered,

nerve cells can enlist the aid of mini-vesicles of neighboring glial cells
to defend themselves against stress and other potentially detrimental factors.

These vesicles, called exosomes, appear to stimulate the neurons on various levels:

they influence electrical stimulus conduction,
biochemical signal transfer, and
gene regulation.

Exosomes are thus multifunctional signal emitters

that can have a significant effect in the brain.

Exosome

The researchers in Mainz already observed in a previous study that

oligodendrocytes release exosomes on exposure to neuronal stimuli.
these are absorbed by the neurons and improve neuronal stress tolerance.

Oligodendrocytes, a type of glial cell, form an

insulating myelin sheath around the axons of neurons.

The exosomes transport protective proteins such as

heat shock proteins,
glycolytic enzymes, and
enzymes that reduce oxidative stress from one cell type to another,
but also transmit genetic information in the form of ribonucleic acids.

“As we have now discovered in cell cultures, exosomes seem to have a whole range of functions,” explained Dr. Eva-Maria Krmer-Albers. By means of their transmission activity, the small bubbles that are the vesicles

not only promote electrical activity in the nerve cells, but also
influence them on the biochemical and gene regulatory level.

“The extent of activities of the exosomes is impressive,” added Krmer-Albers. The researchers hope that the understanding of these processes will contribute to the development of new strategies for the treatment of neuronal diseases. Their next aim is to uncover how vesicles actually function in the brains of living organisms.

http://labroots.com/user/news/article/id/217438/title/vesicles-influence-function-of-nerve-cells

The above story is based on materials provided by Universitt Mainz.

Universitt Mainz. “Vesicles influence function of nerve cells.” ScienceDaily. ScienceDaily, 6 October 2014. www.sciencedaily.com/releases/2014/10/141006174214.htm

Neuroscientists use snail research to help explain “chemo brain”

10/08/2014
It is estimated that as many as half of patients taking cancer drugs experience a decrease in mental sharpness. While there have been many theories, what causes “chemo brain” has eluded scientists.

In an effort to solve this mystery, neuroscientists at The University of Texas Health Science Center at Houston (UTHealth) conducted an experiment in an animal memory model and their results point to a possible explanation. Findings appeared in The Journal of Neuroscience.

In the study involving a sea snail that shares many of the same memory mechanisms as humans and a drug used to treat a variety of cancers, the scientists identified

memory mechanisms blocked by the drug.

Then, they were able to counteract or

unblock the mechanisms by administering another agent.

“Our research has implications in the care of people given to cognitive deficits following drug treatment for cancer,” said John H. “Jack” Byrne, Ph.D., senior author, holder of the June and Virgil Waggoner Chair and Chairman of the Department of Neurobiology and Anatomy at the UTHealth Medical School. “There is no satisfactory treatment at this time.”

Byrne’s laboratory is known for its use of a large snail called Aplysia californica to further the understanding of the biochemical signaling among nerve cells (neurons). The snails have large neurons that relay information much like those in humans.

When Byrne’s team compared cell cultures taken from normal snails to

those administered a dose of a cancer drug called doxorubicin,

the investigators pinpointed a neuronal pathway

that was no longer passing along information properly.

With the aid of an experimental drug,

the scientists were able to reopen the pathway.

Unfortunately, this drug would not be appropriate for humans, Byrne said. “We want to identify other drugs that can rescue these memory mechanisms,” he added.

According the American Cancer Society, some of the distressing mental changes cancer patients experience may last a short time or go on for years.

Byrne’s UT Health research team includes co-lead authors Rong-Yu Liu, Ph.D., and Yili Zhang, Ph.D., as well as Brittany Coughlin and Leonard J. Cleary, Ph.D. All are affiliated with the W.M. Keck Center for the Neurobiology of Learning and Memory.

Byrne and Cleary also are on the faculty of The University of Texas Graduate School of Biomedical Sciences at Houston. Coughlin is a student at the school, which is jointly operated by UT Health and The University of Texas MD Anderson Cancer Center.

The study titled “Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase” received support from National Institutes of Health grant (NS019895) and the Zilkha Family Discovery Fellowship.

Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase

Source: Univ. of Texas Health Science Center at Houston

http://www.rdmag.com/news/2014/10/neuroscientists-use-snail-research-help-explain-E2_9_Cchemo-brain

Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase

Rong-Yu Liu*, Yili Zhang*, Brittany L. Coughlin, Leonard J. Cleary, and John H. Byrne +Show Affiliations
The Journal of Neuroscience, 1 Oct 2014, 34(40): 13289-13300;
http://dx.doi.org:/10.1523/JNEUROSCI.0538-14.2014

Doxorubicin (DOX) is an anthracycline used widely for cancer chemotherapy. Its primary mode of action appears to be

topoisomerase II inhibition, DNA cleavage, and free radical generation.

However, in non-neuronal cells, DOX also inhibits the expression of

dual-specificity phosphatases (also referred to as MAPK phosphatases) and thereby

inhibits the dephosphorylation of extracellular signal-regulated kinase (ERK) and
p38 mitogen-activated protein kinase (p38 MAPK),
two MAPK isoforms important for long-term memory (LTM) formation.

Activation of these kinases by DOX in neurons, if present,

could have secondary effects on cognitive functions, such as learning and memory.

The present study used cultures of rat cortical neurons and sensory neurons (SNs) of Aplysia

to examine the effects of DOX on levels of phosphorylated ERK (pERK) and
phosphorylated p38 (p-p38) MAPK.

In addition, Aplysia neurons were used to examine the effects of DOX on

long-term enhanced excitability, long-term synaptic facilitation (LTF), and
long-term synaptic depression (LTD).

DOX treatment led to elevated levels of

pERK and p-p38 MAPK in SNs and cortical neurons.

In addition, it increased phosphorylation of

the downstream transcriptional repressor cAMP response element-binding protein 2 in SNs.

DOX treatment blocked serotonin-induced LTF and enhanced LTD induced by the neuropeptide Phe-Met-Arg-Phe-NH2. The block of LTF appeared to be attributable to

overriding inhibitory effects of p-p38 MAPK, because
LTF was rescued in the presence of an inhibitor of p38 MAPK
(SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]) .

These results suggest that acute application of DOX might impair the formation of LTM via the p38 MAPK pathway.
Terms: Aplysia chemotherapy ERK p38 MAPK serotonin synaptic plasticity

Technology that controls brain cells with radio waves earns early BRAIN grant

10/08/2014

bright spots = cells with increased calcium after treatment with radio waves, allows neurons to fire

BRAIN control: The new technology uses radio waves to activate or silence cells remotely. The bright spots above represent cells with increased calcium after treatment with radio waves, a change that would allow neurons to fire.

A proposal to develop a new way to

remotely control brain cells

from Sarah Stanley, a research associate in Rockefeller University’s Laboratory of Molecular Genetics, headed by Jeffrey M. Friedman, is

among the first to receive funding from U.S. President Barack Obama’s BRAIN initiative.

The project will make use of a technique called

radiogenetics that combines the use of radio waves or magnetic fields with
nanoparticles to turn neurons on or off.

The National Institutes of Health is one of four federal agencies involved in the BRAIN (Brain Research through Advancing Innovative Neurotechnologies) initiative. Following in the ambitious footsteps of the Human Genome Project, the BRAIN initiative seeks

to create a dynamic map of the brain in action,

a goal that requires the development of new technologies. The BRAIN initiative working group, which outlined the broad scope of the ambitious project, was co-chaired by Rockefeller’s Cori Bargmann, head of the Laboratory of Neural Circuits and Behavior.

Stanley’s grant, for $1.26 million over three years, is one of 58 projects to get BRAIN grants, the NIH announced. The NIH’s plan for its part of this national project, which has been pitched as “America’s next moonshot,” calls for $4.5 billion in federal funds over 12 years.

The technology Stanley is developing would

enable researchers to manipulate the activity of neurons, as well as other cell types,
in freely moving animals in order to better understand what these cells do.

Other techniques for controlling selected groups of neurons exist, but her new nanoparticle-based technique has a

unique combination of features that may enable new types of experimentation.
it would allow researchers to rapidly activate or silence neurons within a small area of the brain or
dispersed across a larger region, including those in difficult-to-access locations.

Stanley also plans to explore the potential this method has for use treating patients.

“Francis Collins, director of the NIH, has discussed

the need for studying the circuitry of the brain,
which is formed by interconnected neurons.

Our remote-control technology may provide a tool with which researchers can ask new questions about the roles of complex circuits in regulating behavior,” Stanley says.
Rockefeller University’s Laboratory of Molecular Genetics
Source: Rockefeller Univ.

Part 4. Cancer

Two Proteins Found to Block Cancer Metastasis

Why do some cancers spread while others don’t? Scientists have now demonstrated that

metastatic incompetent cancers actually “poison the soil”
by generating a micro-environment that blocks cancer cells
from settling and growing in distant organs.

The “seed and the soil” hypothesis proposed by Stephen Paget in 1889 is now widely accepted to explain how

cancer cells (seeds) are able to generate fertile soil (the micro-environment)
in distant organs that promotes cancer’s spread.

However, this concept had not explained why some tumors do not spread or metastasize.

The researchers, from Weill Cornell Medical College, found that

two key proteins involved in this process work by
dramatically suppressing cancer’s spread.

The study offers hope that a drug based on these

potentially therapeutic proteins, prosaposin and Thrombospondin 1 (Tsp-1),

might help keep human cancer at bay and from metastasizing.

Scientists don’t understand why some tumors wouldn’t “want” to spread. It goes against their “job description,” says the study’s senior investigator, Vivek Mittal, Ph.D., an associate professor of cell and developmental biology in cardiothoracic surgery and director of the Neuberger Berman Foundation Lung Cancer Laboratory at Weill Cornell Medical College. He theorizes that metastasis occurs when

the barriers that the body throws up to protect itself against cancer fail.

But there are some tumors in which some of the barriers may still be intact. “So that suggests

those primary tumors will continue to grow, but that
an innate protective barrier still exists that prevents them from spreading and invading other organs,”

The researchers found that, like typical tumors,

metastasis-incompetent tumors also send out signaling molecules
that establish what is known as the “premetastatic niche” in distant organs.

These niches composed of bone marrow cells and various growth factors have been described previously by others including Dr. Mittal as the fertile “soil” that the disseminated cancer cell “seeds” grow in.

Weill Cornell’s Raúl Catena, Ph.D., a postdoctoral fellow in Dr. Mittal’s laboratory, found an important difference between the tumor types. Metastatic-incompetent tumors

systemically increased expression of Tsp-1, a molecule known to fight cancer growth.
increased Tsp-1 production was found specifically in the bone marrow myeloid cells
that comprise the metastatic niche.

These results were striking, because for the first time Dr. Mittal says

the bone marrow-derived myeloid cells were implicated as
the main producers of Tsp-1,.

In addition, Weill Cornell and Harvard researchers found that

prosaposin secreted predominantly by the metastatic-incompetent tumors
increased expression of Tsp-1 in the premetastatic lungs.

Thus, Dr. Mittal posits that prosaposin works in combination with Tsp-1

to convert pro-metastatic bone marrow myeloid cells in the niche
into cells that are not hospitable to cancer cells that spread from a primary tumor.
“The very same myeloid cells in the niche that we know can promote metastasis
can also be induced under the command of the metastatic incompetent primary tumor to inhibit metastasis,”

The research team found that

the Tsp-1–inducing activity of prosaposin
was contained in only a 5-amino acid peptide region of the protein, and
this peptide alone induced Tsp-1 in the bone marrow cells and
effectively suppressed metastatic spread in the lungs
in mouse models of breast and prostate cancer.

This 5-amino acid peptide with Tsp-1–inducing activity

has the potential to be used as a therapeutic agent against metastatic cancer,

The scientists have begun to test prosaposin in other tumor types or metastatic sites.

Dr. Mittal says that “The clinical implications of the study are:

“Not only is it theoretically possible to design a prosaposin-based drug or drugs
that induce Tsp-1 to block cancer spread, but
you could potentially create noninvasive prognostic tests
to predict whether a cancer will metastasize.”

The study was reported in the April 30 issue of Cancer Discovery, in a paper titled “Bone Marrow-Derived Gr1+ Cells Can Generate a Metastasis-Resistant Microenvironment Via Induced Secretion of Thrombospondin-1”.

Disabling Enzyme Cripples Tumors, Cancer Cells

First Step of Metastasis

Published: Sep 05, 2013 http://www.technologynetworks.com/Metabolomics/news.aspx?id=157138

Knocking out a single enzyme dramatically cripples the ability of aggressive cancer cells to spread and grow tumors.

The paper, published in the journal Proceedings of the National Academy of Sciences, sheds new light on the importance of lipids, a group of molecules that includes fatty acids and cholesterol, in the development of cancer.

Researchers have long known that cancer cells metabolize lipids differently than normal cells. Levels of ether lipids – a class of lipids that are harder to break down – are particularly elevated in highly malignant tumors.

“Cancer cells make and use a lot of fat and lipids, and that makes sense because cancer cells divide and proliferate at an accelerated rate, and to do that,

they need lipids, which make up the membranes of the cell,”

said study principal investigator Daniel Nomura, assistant professor in UC Berkeley’s Department of Nutritional Sciences and Toxicology. “Lipids have a variety of uses for cellular structure, but what we’re showing with our study is that

lipids can send signals that fuel cancer growth.”

In the study, Nomura and his team tested the effects of reducing ether lipids on human skin cancer cells and primary breast tumors. They targeted an enzyme,

alkylglycerone phosphate synthase, or AGPS,
known to be critical to the formation of ether lipids.

The researchers confirmed that

AGPS expression increased when normal cells turned cancerous.
inactivating AGPS substantially reduced the aggressiveness of the cancer cells.

“The cancer cells were less able to move and invade,” said Nomura.

The researchers also compared the impact of

disabling the AGPS enzyme in mice that had been injected with cancer cells.

Nomura. observes -“Among the mice that had the AGPS enzyme inactivated,

the tumors were nonexistent,”

“The mice that did not have this enzyme

disabled rapidly developed tumors.”

The researchers determined that

inhibiting AGPS expression depleted the cancer cells of ether lipids.
AGPS altered levels of other types of lipids important to the ability of the cancer cells to survive and spread, including
- prostaglandins and acyl phospholipids.

“What makes AGPS stand out as a treatment target is that the enzyme seems to simultaneously

regulate multiple aspects of lipid metabolism
important for tumor growth and malignancy.”

Future steps include the

development of AGPS inhibitors for use in cancer therapy,

“This study sheds considerable light on the important role that AGPS plays in ether lipid metabolism in cancer cells, and it suggests that

inhibitors of this enzyme could impair tumor formation,”

said Benjamin Cravatt, Professor and Chair of Chemical Physiology at The Scripps Research Institute, who is not part of the UC.

Agilent Technologies Thought Leader Award Supports Translational Research Program
Published: Mon, March 04, 2013

The award will support Dr DePinho’s research into

metabolic reprogramming in the earliest stages of cancer.

Agilent Technologies Inc. announces that Dr. Ronald A. DePinho, a world-renowned oncologist and researcher, has received an Agilent Thought Leader Award.

DePinho is president of the University of Texas MD Anderson Cancer Center. DePinho and his team hope to discover and characterize

alterations in metabolic flux during tumor initiation and maintenance, and to identify biomarkers for early detection of pancreatic cancer together with
novel therapeutic targets.

Researchers on his team will work with scientists from the university’s newly formed Institute of Applied Cancer Sciences.

The Agilent Thought Leader Award provides funds to support personnel as well as a state-of-the-art Agilent 6550 iFunnel Q-TOF LC/MS system.

“I am extremely pleased to receive this award for metabolomics research, as the survival rates for pancreatic cancer have not significantly improved over the past 20 years,” DePinho said. “This technology will allow us to

rapidly identify new targets that drive the formation, progression and maintenance of pancreatic cancer.

Discoveries from this research will also lead to

the development of effective early detection biomarkers and novel therapeutic interventions.”

“We are proud to support Dr. DePinho’s exciting translational research program, which will make use of

metabolomics and integrated biology workflows and solutions in biomarker discovery,”

said Patrick Kaltenbach, Agilent vice president, general manager of the Liquid Phase Division, and the executive sponsor of this award.

The Agilent Thought Leader Program promotes fundamental scientific advances by support of influential thought leaders in the life sciences and chemical analysis fields.

The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis

Ping Xie, Minghua Zhang, Shan He, Kefeng Lu, Yuhan Chen, Guichun Xing, et al.
Nature Communications 2014; 5(3733). http://dx.doi.org:/10.1038/ncomms4733

Neddylation, the covalent attachment of ubiquitin-like protein Nedd8, of the Cullin-RING E3 ligase family

regulates their ubiquitylation activity.

However, regulation of HECT ligases by neddylation has not been reported to date. Here we show that

the C2-WW-HECT ligase Smurf1 is activated by neddylation.

Smurf1 physically interacts with

Nedd8 and Ubc12,
forms a Nedd8-thioester intermediate, and then
catalyses its own neddylation on multiple lysine residues.

Intriguingly, this autoneddylation needs

an active site at C426 in the HECT N-lobe.

Neddylation of Smurf1 potently enhances

ubiquitin E2 recruitment and
augments the ubiquitin ligase activity of Smurf1.

The regulatory role of neddylation

is conserved in human Smurf1 and yeast Rsp5.

Furthermore, in human colorectal cancers,

the elevated expression of Smurf1, Nedd8, NAE1 and Ubc12
correlates with cancer progression and poor prognosis.

These findings provide evidence that

neddylation is important in HECT ubiquitin ligase activation and
shed new light on the tumour-promoting role of Smurf1.

Swinging domains in HECT E3

Subject terms: Biological sciences Cancer Cell biology

Figure 1: Smurf1 expression is elevated in colorectal cancer tissues.

Smurf1 expression is elevated in colorectal cancer tissues.

(a) Smurf1 expression scores are shown as box plots, with the horizontal lines representing the median; the bottom and top of the boxes representing the 25th and 75th percentiles, respectively; and the vertical bars representing the ra

Figure 2: Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer.

Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer

(a) Representative images from immunohistochemical staining of Smurf1, Ubc12, NAE1 and Nedd8 in the same colorectal cancer tumour. Scale bars, 100 μm. (b–d) The expression scores of Nedd8 (b, n=283 ), NAE1 (c, n=281) and Ubc12 (d, n=19…

Figure 3: Smurf1 interacts with Ubc12.

Smurf1 interacts with Ubc12

(a) GST pull-down assay of Smurf1 with Ubc12. Both input and pull-down samples were subjected to immunoblotting with anti-His and anti-GST antibodies. Smurf1 interacted with Ubc12 and UbcH5c, but not with Ubc9. (b) Mapping the regions…

Figure 4: Nedd8 is attached to Smurf1through C426-catalysed autoneddylation.

Nedd8 is attached to Smurf1through C426-catalysed autoneddylation

(a) Covalent neddylation of Smurf1 in vitro.Purified His-Smurf1-WT or C699A proteins were incubated with Nedd8 and Nedd8-E1/E2. Reactions were performed as described in the Methods section. Samples were analysed by western blotting wi…

Figure 5: Neddylation of Smurf1 activates its ubiquitin ligase activity.

Neddylation of Smurf1 activates its ubiquitin ligase activity.

(a) In vivo Smurf1 ubiquitylation assay. Nedd8 was co-expressed with Smurf1 WT or C699A in HCT116 cells (left panels). Twenty-four hours post transfection, cells were treated with MG132 (20 μM, 8 h). HCT116 cells were transfected with…

http://www.nature.com/ncomms/2014/140513/ncomms4733/carousel/ncomms4733-f1.jpg

http://www.nature.com/ncomms/2014/140513/ncomms4733/carousel/ncomms4733-f2.jpg

http://www.nature.com/ncomms/2014/140513/ncomms4733/carousel/ncomms4733-f3.jpg

http://www.nature.com/ncomms/2014/140513/ncomms4733/carousel/ncomms4733-f4.jpg

http://www.nature.com/ncomms/2014/140513/ncomms4733/carousel/ncomms4733-f5.jpg

http://www.nature.com/ncomms/2014/140513/ncomms4733/carousel/ncomms4733-f6.jpg

http://www.nature.com/ncomms/2014/140513/ncomms4733/carousel/ncomms4733-f7.jpg

http://www.nature.com/ncomms/2014/140513/ncomms4733/carousel/ncomms4733-f8.jpg

The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response

M Simicek, S Lievens, M Laga, D Guzenko, VN. Aushev, et al.
Nature Cell Biology 2013; 15, 1220–1230 http://dx.doi.org:/10.1038/ncb2847

The RAS-like GTPase RALB mediates cellular responses to nutrient availability or viral infection by respectively

engaging two components of the exocyst complex, EXO84 and SEC5.

RALB employs SEC5 to trigger innate immunity signalling, whereas
RALB–EXO84 interaction induces autophagocytosis.

How this differential interaction is achieved molecularly by the RAL GTPase remains unknown.

We found that whereas GTP binding

turns on RALB activity,

ubiquitylation of RALB at Lys 47

tunes its activity towards a particular effector.

Specifically, ubiquitylation at Lys 47

sterically inhibits RALB binding to EXO84, while
facilitating its interaction with SEC5.

Double-stranded RNA promotes

RALB ubiquitylation and
SEC5–TBK1 complex formation.

In contrast, nutrient starvation

induces RALB deubiquitylation
by accumulation and relocalization of the deubiquitylase USP33
to RALB-positive vesicles.

Deubiquitylated RALB

promotes the assembly of the RALB–EXO84–beclin-1 complexes
driving autophagosome formation. Thus,
ubiquitylation within the effector-binding domain
provides the switch for the dual functions of RALB in
- autophagy and innate immune responses.

Part 5. Metabolic Syndrome

Single Enzyme is Necessary for Development of Diabetes

Published: Aug 20, 2014 http://www.technologynetworks.com/Metabolomics/news.aspx?ID=169416

12-LO enzyme promotes the obesity-induced oxidative stress in the pancreatic cells.

An enzyme called 12-LO promotes the obesity-induced oxidative stress in the pancreatic cells that leads

to pre-diabetes, and diabetes.

12-LO’s enzymatic action is the last step in

the production of certain small molecules that harm the cell,

according to a team from Indiana University School of Medicine, Indianapolis.

The findings will enable the development of drugs that can interfere with this enzyme, preventing or even reversing diabetes. The research is published ahead of print in the journal Molecular and Cellular Biology.

In earlier studies, these researchers and their collaborators at Eastern Virginia Medical School showed that

12-LO (which stands for 12-lipoxygenase) is present in these cells
only in people who become overweight.

The harmful small molecules resulting from 12-LO’s enzymatic action are known as HETEs, short for hydroxyeicosatetraenoic acid.

HETEs harm the mitochondria, which then
fail to produce sufficient energy to enable
the pancreatic cells to manufacture the necessary quantities of insulin.

For the study, the investigators genetically engineered mice that

lacked the gene for 12-LO exclusively in their pancreas cells.

Mice were either fed a low-fat or high-fat diet.

Both the control mice and the knockout mice on the high fat diet

developed obesity and insulin resistance.

The investigators also examined the pancreatic beta cells of both knockout and control mice, using both microscopic studies and molecular analysis. Those from the knockout mice were intact and healthy, while

those from the control mice showed oxidative damage,
demonstrating that 12-LO and the resulting HETEs
caused the beta cell failure.

Mirmira notes that fatty diet used in the study was the Western Diet, which comprises mostly saturated-“bad”-fats. Based partly on a recent study of related metabolic pathways, he says that

the unsaturated and mono-unsaturated fats-which comprise most fats in the healthy,
relatively high fat Mediterranean diet-are unlikely to have the same effects.

“Our research is the first to show that 12-LO in the beta cell

is the culprit in the development of pre-diabetes, following high fat diets,” says Mirmira.

“Our work also lends important credence to the notion that

the beta cell is the primary defective cell in virtually all forms of diabetes and pre-diabetes.”

A New Player in Lipid Metabolism Discovered

Published: Aug18, 2014 http://www.technologynetworks.com/Metabolomics/news.aspx?ID=169356

Specially engineered mice gained no weight, and normal counterparts became obese

on the same high-fat, obesity-inducing Western diet.

Specially engineered mice that lacked a particular gene did not gain weight

when fed a typical high-fat, obesity-inducing Western diet.

Yet, these mice ate the same amount as their normal counterparts that became obese.

The mice were engineered with fat cells that lacked a gene called SEL1L,

known to be involved in the clearance of mis-folded proteins
in the cell’s protein making machinery called the endoplasmic reticulum (ER).

When mis-folded proteins are not cleared but accumulate,

they destroy the cell and contribute to such diseases as

mad cow disease,
Type 1 diabetes and
cystic fibrosis.

“The million-dollar question is why don’t these mice gain weight? Is this related to its inability to clear mis-folded proteins in the ER?” said Ling Qi, associate professor of molecular and biochemical nutrition and senior author of the study published online July 24 in Cell Metabolism. Haibo Sha, a research associate in Qi’s lab, is the paper’s lead author.

Interestingly, the experimental mice developed a host of other problems, including

postprandial hypertriglyceridemia,
and fatty livers.

“Although we are yet to find out whether these conditions contribute to the lean phenotype, we found that

there was a lipid partitioning defect in the mice lacking SEL1L in fat cells,
where fat cells cannot store fat [lipids], and consequently
fat goes to the liver.

During the investigation of possible underlying mechanisms, we discovered

a novel function for SEL1L as a regulator of lipid metabolism,” said Qi.

Sha said “We were very excited to find that

SEL1L is required for the intracellular trafficking of
lipoprotein lipase (LPL), acting as a chaperone,” .

and added that “Using several tissue-specific knockout mouse models,

we showed that this is a general phenomenon,”

Without LPL, lipids remain in the circulation;

fat and muscle cells cannot absorb fat molecules for storage and energy combustion,

People with LPL mutations develop

postprandial hypertriglyceridemia similar to
conditions found in fat cell-specific SEL1L-deficient mice, said Qi.

Future work will investigate the

role of SEL1L in human patients carrying LPL mutations and
determine why fat cell-specific SEL1L-deficient mice remain lean under Western diets, said Sha.

Co-authors include researchers from Cedars-Sinai Medical Center in Los Angeles; Wageningen University in the Netherlands; Georgia State University; University of California, Los Angeles; and the Medical College of Soochow University in China.

The study was funded by the U.S. National Institutes of Health, the Netherlands Organization for Health Research and Development National Institutes of Health, the Cedars-Sinai Medical Center, Chinese National Science Foundation, the American Diabetes Association, Cornell’s Center for Vertebrate Genomics and the Howard Hughes Medical Institute.

Part 6. Biomarkers

Biomarkers Take Center Stage

Josh P. Roberts
GEN May 1, 2013 (Vol. 33, No. 9) http://www.genengnews.com/

While work with biomarkers continues to grow, scientists are also grappling with research-related bottlenecks, such as

affinity reagent development,
platform reproducibility, and
sensitivity.

Biomarkers by definition indicate some state or process that generally occurs

at a spatial or temporal distance from the marker itself, and

it would not be an exaggeration to say that biomedicine has become infatuated with them:

where to find them,
when they may appear,
what form they may take, and
how they can be used to diagnose a condition or
predict whether a therapy may be successful.

Biomarkers are on the agenda of many if not most industry gatherings, and in cases such as Oxford Global’s recent “Biomarker Congress” and the GTC “Biomarker Summit”, they hold the naming rights. There, some basic principles were built upon, amended, and sometimes challenged.

In oncology, for example, biomarker discovery is often predicated on the premise that

proteins shed from a tumor will traverse to and persist in, and be detectable in, the circulation.

By quantifying these proteins—singularly or as part of a larger “signature”—the hope is

to garner information about the molecular characteristics of the cancer
that will help with cancer detection and
personalization of the treatment strategy.

Yet this approach has not yet turned into the panacea that was hoped for. Bottlenecks exist in

affinity reagent development,
platform reproducibility, and
sensitivity.

There is also a dearth of understanding of some of the

fundamental principles of biomarker biology that we need to know the answers to,

said Parag Mallick, Ph.D., whose lab at Stanford University is “working on trying to understand where biomarkers come from.”

There are dogmas saying that

circulating biomarkers come solely from secreted proteins.

But Dr. Mallick’s studies indicate that fully

50% of circulating proteins may come from intracellular sources or
proteins that are annotated as such.

“We don’t understand the processes governing

which tumor-derived proteins end up in the blood.”

Other questions include “how does the size of a tumor affect how much of a given protein will be in the blood?”—perhaps

the tumor is necrotic at the center, or
it’s hypervascular or hypovascular.

He points out “The problem is that these are highly nonlinear processes at work, and

there is a large number of factors that might affect the answer to that question,” .

Their research focuses on using

mass spectrometry and
computational analysis

to characterize the biophysical properties of the circulating proteome, and
relate these to measurements made of the tumor itself.

Furthermore, he said – “We’ve observed that the proteins that are likely to

first show up and persist in the circulation, ..
are more stable than proteins that don’t,”
“we can quantify how significant the effect is.”

The goal is ultimately to be able to

build rigorous, formal mathematical models that will allow something measured in the blood
to be tied back to the molecular biology taking place in the tumor.

And conversely, to use those models

to predict from a tumor what will be found in the circulation.

“Ultimately, the models will allow you to connect the dots between

what you measure in the blood and the biology of the tumor.”

Bound for Affinity Arrays

Affinity reagents are the main tools for large-scale protein biomarker discovery. And while this has tended to mean antibodies (or their derivatives), other affinity reagents are demanding a place in the toolbox.

Affimers, a type of affinity reagent being developed by Avacta, consist of

a biologically inert, biophysically stable protein scaffold
containing three variable regions into which
distinct peptides are inserted.

The resulting three-dimensional surface formed by these peptides

interacts and binds to proteins and other molecules in solution,
much like the antigen-binding site of antibodies.

Unlike antibodies, Affimers are relatively small (13 KDa),

non-post-translationally modified proteins
that can readily be expressed in bacterial culture.

They may be made to bind surfaces through unique residues

engineered onto the opposite face of the Affimer,
allowing the binding site to be exposed to the target in solution.

“We don’t seem to see in what we’ve done so far

any real loss of activity or functionality of Affimers when bound to surfaces—

they’re very robust,” said CEO Alastair Smith, Ph.D.

Avacta is taking advantage of this stability and its large libraries of Affimers to develop

very large affinity microarrays for
drug and biomarker discovery.

To date they have printed arrays with around 20–25,000 features, and Dr. Smith is “sure that we can get toward about 50,000 on a slide,” he said. “There’s no real impediment to us doing that other than us expressing the proteins and getting on with it.”

Customers will be provided with these large, complex “naïve” discovery arrays, readable with standard equipment. The plan is for the company to then “support our customers by providing smaller arrays with

the Affimers that are binding targets of interest to them,” Dr. Smith foretold.

And since the intellectual property rights are unencumbered,

Affimers in those arrays can be licensed to the end users
to develop diagnostics that can be validated as time goes on.

Around 20,000-Affimer discovery arrays were recently tested by collaborator Professor Ann Morgan of the University of Leeds with pools of unfractionated serum from patients with symptoms of inflammatory disease. The arrays

“rediscovered” elevated C-reactive protein (CRP, the clinical gold standard marker)
as well as uncovered an additional 22 candidate biomarkers.
other candidates combined with CRP, appear able to distinguish between different diseases such as

rheumatoid arthritis,
psoriatic arthritis,
SLE, or
giant cell arteritis.

Epigenetic Biomarkers

Sometimes biomarkers are used not to find disease but

to distinguish healthy human cell types, with
examples being found in flow cytometry and immunohistochemistry.

These widespread applications, however, are difficult to standardize, being

subject to arbitrary or subjective gating protocols and other imprecise criteria.

Epiontis instead uses an epigenetic approach. “What we need is a unique marker that is

demethylated only in one cell type and
methylated in all the other cell types,”

Each cell of the right cell type will have

two demethylated copies of a certain gene locus,
allowing them to be enumerated by quantitative PCR.

The biggest challenge is finding that unique epigenetic marker. To do so they look through the literature for proteins and genes described as playing a role in the cell type’s biology, and then

look at the methylation patterns to see if one can be used as a marker,

They also “use customized Affymetrix chips to look at the

differential epigenetic status of different cell types on a genomewide scale.”

explained CBO and founder Ulrich Hoffmueller, Ph.D.

The company currently has a panel of 12 assays for 12 immune cell types. Among these is an assay for

regulatory T (Treg) cells that queries the Foxp3 gene—which is uniquely demethylated in Treg
even though it is transiently expressed in activated T cells of other subtypes.

Also assayed are Th17 cells, difficult to detect by flow cytometry because

“the cells have to be stimulated in vitro,” he pointed out.

Developing New Assays for Cancer Biomarkers

Researchers at Myriad RBM and the Cancer Prevention Research Institute of Texas are collaborating to develop

new assays for cancer biomarkers on the Myriad RBM Multi-Analyte Profile (MAP) platform.

The release of OncologyMAP 2.0 expanded Myriad RBM’s biomarker menu to over 250 analytes, which can be measured from a small single sample, according to the company. Using this menu, L. Stephen et al., published a poster, “Analysis of Protein Biomarkers in Prostate and Colorectal Tumor Lysates,” which showed the results of

a survey of proteins relevant to colorectal (CRC) and prostate (PC) tumors
to identify potential proteins of interest for cancer research.

The study looked at CRC and PC tumor lysates and found that 102 of the 115 proteins showed levels above the lower limit of quantification.

Four markers were significantly higher in PC and 10 were greater in CRC.

For most of the analytes, duplicate sections of the tumor were similar, although some analytes did show differences. In four of the CRC analytes, tumor number four showed differences for CEA and tumor number 2 for uPA.

Thirty analytes were shown to be

different in CRC tumor compared to its adjacent tissue.
Ten of the analytes were higher in adjacent tissue compared to CRC.
Eighteen of the markers examined demonstrated —-

significant correlations of CRC tumor concentration to serum levels.

“This suggests.. that the Oncology MAP 2.0 platform “provides a good method for studying changes in tumor levels because many proteins can be assessed with a very small sample.”

Clinical Test Development with MALDI-ToF

While there have been many attempts to translate results from early discovery work on the serum proteome into clinical practice, few of these efforts have progressed past the discovery phase.

Matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry on unfractionated serum/plasma samples offers many practical advantages over alternative techniques, and does not require

a shift from discovery to development and commercialization platforms.

Biodesix claims it has been able to develop the technology into

a reproducible, high-throughput tool to
routinely measure protein abundance from serum/plasma samples.

“.. we improved data-analysis algorithms to

reproducibly obtain quantitative measurements of relative protein abundance from MALDI-ToF mass spectra.

Heinrich Röder, CTO points out that the MALDI-ToF measurements

are combined with clinical outcome data using
modern learning theory techniques
to define specific disease states
based on a patient’s serum protein content,”

The clinical utility of the identification of these disease states can be investigated through a retrospective analysis of differing sample sets. For example, Biodesix clinically validated its first commercialized serum proteomic test, VeriStrat®, in 85 different retrospective sample sets.

Röder adds that “It is becoming increasingly clear that

the patients whose serum is characterized as VeriStrat Poor show
consistently poor outcomes irrespective of

tumor type,
histology, or
molecular tumor characteristics,”

MALDI-ToF mass spectrometry, in its standard implementation,

allows for the observation of around 100 mostly high-abundant serum proteins.

Further, “while this does not limit the usefulness of tests developed from differential expression of these proteins,

the discovery potential would be greatly enhanced
if we could probe deeper into the proteome
while not giving up the advantages of the MALDI-ToF approach,”

Biodesix reports that its new MALDI approach, Deep MALDI™, can perform

simultaneous quantitative measurement of more than 1,000 serum protein features (or peaks) from 10 µL of serum in a high-throughput manner.
it increases the observable signal noise ratio from a few hundred to over 50,000,
resulting in the observation of many lower-abundance serum proteins.

Breast cancer, a disease now considered to be a collection of many complexes of symptoms and signatures—the dominant ones are labeled Luminal A, Luminal B, Her2, and Basal— which suggests different prognose, and

these labels are considered too simplistic for understanding and managing a woman’s cancer.

Studies published in the past year have looked at

somatic mutations,
gene copy number aberrations,
gene expression abnormalities,
protein and miRNA expression, and
DNA methylation,

coming up with a list of significantly mutated genes—hot spots—in different categories of breast cancers. Targeting these will inevitably be the focus of much coming research.

“We’ve been taking these large trials and profiling these on a variety of array or sequence platforms. We think we’ll get

prognostic drivers
predictive markers for taxanes and
monoclonal antibodies and
tamoxifen and aromatase inhibitors,”
explained Brian Leyland-Jones, Ph.D., director of Edith Sanford Breast Cancer Research. “We will end up with 20–40 different diseases, maybe more.”

Edith Sanford Breast Cancer Research is undertaking a pilot study in collaboration with The Scripps Research Institute, using a variety of tests on 25 patients to see how the information they provide complements each other, the overall flow, and the time required to get and compile results.

Laser-captured tumor samples will be subjected to low passage whole-genome, exome, and RNA sequencing (with targeted resequencing done in parallel), and reverse-phase protein and phosphorylation arrays, with circulating nucleic acids and circulating tumor cells being queried as well. “After that we hope to do a 100- or 150-patient trial when we have some idea of the best techniques,” he said.

Dr. Leyland-Jones predicted that ultimately most tumors will be found

to have multiple drivers,
with most patients receiving a combination of two, three, or perhaps four different targeted therapies.

Reduce to Practice

According to Randox, the evidence Investigator is a sophisticated semi-automated biochip system designed for research, clinical, forensic, and veterinary applications.

Once biomarkers that may have an impact on therapy are discovered, it is not always routine to get them into clinical practice. Leaving regulatory and financial, intellectual property and cultural issues aside, developing a diagnostic based on a biomarker often requires expertise or patience that its discoverer may not possess.

Andrew Gribben is a clinical assay and development scientist at Randox Laboratories, based in Northern Ireland, U.K. The company utilizes academic and industrial collaborators together with in-house discovery platforms to identify biomarkers that are

augmented or diminished in a particular pathology
relative to appropriate control populations.

Biomarkers can be developed to be run individually or

combined into panels of immunoassays on its multiplex biochip array technology.

Specificity can also be gained—or lost—by the affinity of reagents in an assay. The diagnostic potential of Heart-type fatty acid binding protein (H-FABP) abundantly expressed in human myocardial cells was recognized by Jan Glatz of Maastricht University, The Netherlands, back in 1988. Levels rise quickly within 30 minutes after a myocardial infarction, peaking at 6–8 hours and return to normal within 24–30 hours. Yet at the time it was not known that H-FABP was a member of a multiprotein family, with which the polyclonal antibodies being used in development of an assay were cross-reacting, Gribben related.

Randox developed monoclonal antibodies specific to H-FABP, funded trials investigating its use alone, and multiplexed with cardiac biomarker assays, and, more than 30 years after the biomarker was identified, in 2011, released a validated assay for H-FABP as a biomarker for early detection of acute myocardial infarction.

Ultrasensitive Immunoassays for Biomarker Development

Research has shown that detection and monitoring of biomarker concentrations can provide

insights into disease risk and progression.

Cytokines have become attractive biomarkers and candidates

for targeted therapies for a number of autoimmune diseases, including rheumatoid arthritis (RA), Crohn’s disease, and psoriasis, among others.

However, due to the low-abundance of circulating cytokines, such as IL-17A, obtaining robust measurements in clinical samples has been difficult.

Singulex reports that its digital single-molecule counting technology provides

increased precision and detection sensitivity over traditional ELISA techniques,
helping to shed light on biomarker verification and validation programs.

The company’s Erenna® immunoassay system, which includes optimized immunoassays, offers LLoQ to femtogram levels per mL resolution—even in healthy populations, at an improvement of 1-3 fold over standard ELISAs or any conventional technology and with a dynamic range of up to 4-logs, according to a Singulex official, who adds that

this sensitivity improvement helps minimize undetectable samples that
could otherwise delay or derail clinical studies.

The official also explains that the Singulex solution includes an array of products and services that are being applied to a number of programs and have enabled the development of clinically relevant biomarkers, allowing translation from discovery to the clinic.

In a poster entitled “Advanced Single Molecule Detection: Accelerating Biomarker Development Utilizing Cytokines through Ultrasensitive Immunoassays,” a case study was presented of work performed by Jeff Greenberg of NYU to show how the use of the Erenna system can provide insights toward

improving the clinical utility of biomarkers and
accelerating the development of novel therapies for treating inflammatory diseases.

A panel of inflammatory biomarkers was examined in DMARD (disease modifying antirheumatic drugs)-naïve RA (rheumatoid arthritis) vs. knee OA (osteoarthritis) patient cohorts. Markers that exhibited significant differences in plasma concentrations between the two cohorts included

CRP, IL-6R alpha, IL-6, IL-1 RA, VEGF, TNF-RII, and IL-17A, IL-17F, and IL-17A/F.

Among the three tested isoforms of IL-17,

the magnitude of elevation for IL-17F in RA patients was the highest.

“Singulex provides high-resolution monitoring of baseline IL-17A concentrations that are present at low levels,” concluded the researchers. “The technology also enabled quantification of other IL-17 isoforms in RA patients, which have not been well characterized before.”

The Singulex Erenna System has also been applied to cardiovascular disease research, for which its

cardiac troponin I (cTnI) digital assay can be used to measure circulating
levels of cTnI undetectable by other commercial assays.

Recently presented data from Brigham and Women’s Hospital and the TIMI-22 study showed that

using the Singulex test to serially monitor cTnI helps
stratify risk in post-acute coronary syndrome patients and
can identify patients with elevated cTnI
who have the most to gain from intensive vs. moderate-dose statin therapy,

according to the scientists involved in the research.

The study poster, “Prognostic Performance of Serial High Sensitivity Cardiac Troponin Determination in Stable Ischemic Heart Disease: Analysis From PROVE IT-TIMI 22,” was presented at the 2013 American College of Cardiology (ACC) Annual Scientific Session & Expo by R. O’Malley et al.

Biomarkers Changing Clinical Medicine

Better Diagnosis, Prognosis, and Drug Targeting Are among Potential Benefits

John Morrow Jr., Ph.D.

Researchers at EMD Chemicals are developing biomarker immunoassays

to monitor drug-induced toxicity including kidney damage.

The pace of biomarker development is accelerating as investigators report new studies on cancer, diabetes, Alzheimer disease, and other conditions in which the evaluation and isolation of workable markers is prominently featured.

Wei Zheng, Ph.D., leader of the R&D immunoassay group at EMD Chemicals, is overseeing a program to develop biomarker immunoassays to

monitor drug-induced toxicity, including kidney damage.

“One of the principle reasons for drugs failing during development is because of organ toxicity,” says Dr. Zheng.
“proteins liberated into the serum and urine can serve as biomarkers of adverse response to drugs, as well as disease states.”

Through collaborative programs with Rules-Based Medicine (RBM), the EMD group has released panels for the profiling of human renal impairment and renal toxicity. These urinary biomarker based products fit the FDA and EMEA guidelines for assessment of drug-induced kidney damage in rats.

The group recently performed a screen for potential protein biomarkers in relation to

kidney toxicity/damage on a set of urine and plasma samples
from patients with documented renal damage.

Additionally, Dr. Zheng is directing efforts to move forward with the multiplexed analysis of

organ and cellular toxicity.

Diseases thought to involve compromised oxidative phosphorylation include

diabetes, Parkinson and Alzheimer diseases, cancer, and the aging process itself.

Good biomarkers allow Dr. Zheng to follow the mantra, “fail early, fail fast.” With robust, multiplexible biomarkers, EMD can detect bad drugs early and kill them before they move into costly large animal studies and clinical trials. “Recognizing the severe liability that toxicity presents, we can modify the structure of the candidate molecule and then rapidly reassess its performance.”

Scientists at Oncogene Science a division of Siemens Healthcare Diagnostics, are also focused on biomarkers. “We are working on a number of antibody-based tests for various cancers, including a test for the Ca-9 CAIX protein, also referred to as carbonic anhydrase,” Walter Carney, Ph.D., head of the division, states.

CAIX is a transmembrane protein that is

overexpressed in a number of cancers, and, like Herceptin and the Her-2 gene,
can serve as an effective and specific marker for both diagnostic and therapeutic purposes.
It is liberated into the circulation in proportion to the tumor burden.

Dr. Carney and his colleagues are evaluating patients after tumor removal for the presence of the Ca-9 CAIX protein. If

the levels of the protein in serum increase over time,
this suggests that not all the tumor cells were removed and the tumor has metastasized.

Dr. Carney and his team have developed both an immuno-histochemistry and an ELISA test that could be used as companion diagnostics in clinical trials of CAIX-targeted drugs.

The ELISA for the Ca-9 CAIX protein will be used in conjunction with Wilex’ Rencarex®, which is currently in a

Phase III trial as an adjuvant therapy for non-metastatic clear cell renal cancer.

Additionally, Oncogene Science has in its portfolio an FDA-approved test for the Her-2 marker. Originally approved for Her-2/Neu-positive breast cancer, its indications have been expanded over time, and was approved

for the treatment of gastric cancer last year.

It is normally present on breast cancer epithelia but

overexpressed in some breast cancer tumors.

“Our products are designed to be used in conjunction with targeted therapies,” says Dr. Carney. “We are working with companies that are developing technology around proteins that are

overexpressed in cancerous tissues and can be both diagnostic and therapeutic targets.”

The long-term goal of these studies is to develop individualized therapies, tailored for the patient. Since the therapies are expensive, accurate diagnostics are critical to avoid wasting resources on patients who clearly will not respond (or could be harmed) by the particular drug.

“At this time the rate of response to antibody-based therapies may be very poor, as

they are often employed late in the course of the disease, and patients are in such a debilitated state
that they lack the capacity to react positively to the treatment,” Dr. Carney explains.

Nanoscale Real-Time Proteomics

Stanford University School of Medicine researchers, working with Cell BioSciences, have developed a

nanofluidic proteomic immunoassay that measures protein charge,
similar to immunoblots, mass spectrometry, or flow cytometry.
unlike these platforms, this approach can measure the amount of individual isoforms,
specifically, phosphorylated molecules.

“We have developed a nanoscale device for protein measurement, which I believe could be useful for clinical analysis,” says Dean W. Felsher, M.D., Ph.D., associate professor at Stanford University School of Medicine.

Critical oncogenic transformations involving

the activation of the signal-related kinases ERK-1 and ERK-2 can now be followed with ease.

“The fact that we measure nanoquantities with accuracy means that

we can interrogate proteomic profiles in clinical patients,

by drawing tiny needle aspirates from tumors over the course of time,” he explains.

“This allows us to observe the evolution of tumor cells and

their response to therapy
from a baseline of the normal tissue as a standard of comparison.”

According to Dr. Felsher, 20 cells is a large enough sample to obtain a detailed description. The technology is easy to automate, which allows

the inclusion of hundreds of assays.

Contrasting this technology platform with proteomic analysis using microarrays, Dr. Felsher notes that the latter is not yet workable for revealing reliable markers.

Dr. Felsher and his group published a description of this technology in Nature Medicine. “We demonstrated that we could take a set of human lymphomas and distinguish them from both normal tissue and other tumor types. We can

quantify changes in total protein, protein activation, and relative abundance of specific phospho-isoforms
from leukemia and lymphoma patients receiving targeted therapy.

Even with very small numbers of cells, we are able to show that the results are consistent, and

our sample is a random profile of the tumor.”

Splice Variant Peptides

“Aberrations in alternative splicing may generate

much of the variation we see in cancer cells,”

says Gilbert Omenn, Ph.D., director of the center for computational medicine and bioinformatics at the University of Michigan School of Medicine. Dr. Omenn and his colleague, Rajasree Menon, are

using this variability as a key to new biomarker identification.

It is becoming evident that splice variants play a significant role in the properties of cancer cells, including

initiation, progression, cell motility, invasiveness, and metastasis.

Alternative splicing occurs through multiple mechanisms

when the exons or coding regions of the DNA transcribe mRNA,
generating initiation sites and connecting exons in protein products.

Their translation into protein can result in numerous protein isoforms, and

these isoforms may reflect a diseased or cancerous state.

Regulatory elements within the DNA are responsible for selecting different alternatives; thus

the splice variants are tempting targets for exploitation as biomarkers.

Analyses of the splice-site mutation

Despite the many questions raised by these observations, splice variation in tumor material has not been widely studied. Cancer cells are known for their tremendous variability, which allows them to

grow rapidly, metastasize, and develop resistance to anticancer drugs.

Dr. Omenn and his collaborators used

mass spec data to interrogate a custom-built database of all potential mRNA sequences
to find alternative splice variants.

When they compared normal and malignant mammary gland tissue from a mouse model of Her2/Neu human breast cancers, they identified a vast number (608) of splice variant proteins, of which

peptides from 216 were found only in the tumor sample.

“These novel and known alternative splice isoforms

are detectable both in tumor specimens and in plasma and
represent potential biomarker candidates,” Dr. Omenn adds.

Dr. Omenn’s observations and those of his colleague Lewis Cantley, Ph.D., have also

shed light on the origins of the classic Warburg effect,
the shift to anaerobic glycolysis in tumor cells.

The novel splice variant M2, of muscle pyruvate kinase,

is observed in embryonic and tumor tissue.

It is associated with this shift, the result of

the expression of a peptide splice variant sequence.

It is remarkable how many different areas of the life sciences are tied into the phenomenon of splice variation. The changes in the genetic material can be much greater than point mutations, which have been traditionally considered to be the prime source of genetic variability.

“We now have powerful methods available to uncover a whole new category of variation,” Dr. Omenn says. “High-throughput RNA sequencing and proteomics will be complementary in discovery studies of splice variants.”

Splice variation may play an important role in rapid evolutionary changes, of the sort discussed by Susumu Ohno and Stephen J. Gould decades ago. They, and other evolutionary biologists, argued that

gene duplication, combined with rapid variability, could fuel major evolutionary jumps.

At the time, the molecular mechanisms of variation were poorly understood, but today

the tools are available to rigorously evaluate the role of
splice variation and other contributors to evolutionary change.

“Biomarkers derived from studies of splice variants, could, in the future, be exploited

both for diagnosis and prognosis and
for drug targeting of biological networks,
in situations such as the Her-2/Neu breast cancers,” Dr. Omenn says.

Aminopeptidase Activities

“By correlating the proteolytic patterns with disease groups and controls, we have shown that

exopeptidase activities contribute to the generation of not only cancer-specific
but also cancer type specific serum peptides.

according to Paul Tempst, Ph.D., professor and director of the Protein Center at the Memorial Sloan-Kettering Cancer Center.

So there is a direct link between peptide marker profiles of disease and differential protease activity.” For this reason Dr. Tempst argues that “the patterns we describe may have value as surrogate markers for detection and classification of cancer.”

To investigate this avenue, Dr. Tempst and his colleagues have followed

the relationship between exopeptidase activities and metastatic disease.

“We monitored controlled, de novo peptide breakdown in large numbers of biological samples using mass spectrometry, with relative quantitation of the metabolites,” Dr. Tempst explains. This entailed the use of magnetic, reverse-phase beads for analyte capture and a MALDI-TOF MS read-out.

“In biomarker discovery programs, functional proteomics is usually not pursued,” says Dr. Tempst. “For putative biomarkers, one may observe no difference in quantitative levels of proteins, while at the same time, there may be substantial differences in enzymatic activity.”

In a preliminary prostate cancer study, the team found a significant difference

in activity levels of exopeptidases in serum from patients with metastatic prostate cancer
as compared to primary tumor-bearing individuals and normal healthy controls.

However, there were no differences in amounts of the target protein, and this potential biomarker would have been missed if quantitative levels of protein had been the only criterion of selection.

It is frequently stated that “practical fusion energy is 30 years in the future and always will be.” The same might be said of functional, practical biomarkers that can pass muster with the FDA. But splice variation represents a new handle on this vexing problem. It appears that we are seeing the emergence of a new approach that may finally yield definitive diagnostic tests, detectable in serum and urine samples.

Part 7. Epigenetics and Drug Metabolism

DNA Methylation Rules: Studying Epigenetics with New Tools

The tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.

Patricia Fitzpatrick Dimond, Ph.D.

http://www.genengnews.com/media/images/AnalysisAndInsight/Feb7_2013_24454248_GreenPurpleDNA_EpigeneticsToolsII3576166141.jpg

New tools may help move the field of epigenetic analysis forward and potentially unveil novel biomarkers for cellular development, differentiation, and disease.

DNA sequencing has had the power of technology behind it as novel platforms to produce more sequencing faster and at lower cost have been introduced. But the tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.

Among these mechanisms, DNA methylation, or the enzymatically mediated addition of a methyl group to cytosine or adenine dinucleotides,

serves as an inherited epigenetic modification that
stably modifies gene expression in dividing cells.

The unique methylomes are largely maintained in differentiated cell types, making them critical to understanding the differentiation potential of the cell.

In the DNA methylation process, cytosine residues in the genome are enzymatically modified to 5-methylcytosine,

which participates in transcriptional repression of genes during development and disease progression.

5-methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. DNA methylation affects gene transcription by physically

interfering with the binding of proteins involved in gene transcription.

Methylated DNA may be bound by methyl-CpG-binding domain proteins (MBDs) that can

then recruit additional proteins. Some of these include histone deacetylases and other chromatin remodeling proteins that modify histones, thereby
forming compact, inactive chromatin, or heterochromatin.

While DNA methylation doesn’t change the genetic code,

it influences chromosomal stability and gene expression.

Epigenetics and Cancer Biomarkers

multistage chemical carcinogenesis

And because of the increasing recognition that DNA methylation changes are involved in human cancers, scientists have suggested that these epigenetic markers may provide biological markers for cancer cells, and eventually point toward new diagnostic and therapeutic targets. Cancer cell genomes display genome-wide abnormalities in DNA methylation patterns,

some of which are oncogenic and contribute to genome instability.

In particular, de novo methylation of tumor suppressor gene promoters

occurs frequently in cancers, thereby silencing them and promoting transformation.

Cytosine hydroxymethylation (5-hydroxymethylcytosine, or 5hmC), the aforementioned DNA modification resulting from the enzymatic conversion of 5mC into 5-hydroxymethylcytosine by the TET family of oxygenases, has been identified

as another key epigenetic modification marking genes important for
pluripotency in embryonic stem cells (ES), as well as in cancer cells.

The base 5-hydroxymethylcytosine was recently identified as an oxidation product of 5-methylcytosine in mammalian DNA. In 2011, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2′-deoxycytidine (5hmdC) and 5-methyl-2′-deoxycytidine (5mdC) in genomic DNA, scientists at the Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California investigated

whether levels of 5hmC can distinguish normal tissue from tumor tissue.

They showed that in squamous cell lung cancers, levels of 5hmdC showed

up to five-fold reduction compared with normal lung tissue.

In brain tumors,5hmdC showed an even more drastic reduction

with levels up to more than 30-fold lower than in normal brain,
but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1, the enzyme that converts 5hmC to 5hmdC.

Immunohistochemical analysis indicated that 5hmC is “remarkably depleted” in many types of human cancer.

there was an inverse relationship between 5hmC levels and cell proliferation with lack of 5hmC in proliferating cells.

Their data suggest that 5hmdC is strongly depleted in human malignant tumors,

a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue.

In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis.

Enzymatic Mapping

But according to New England Biolabs’ Sriharsa Pradhan, Ph.D., methods for distinguishing 5mC from 5hmC and analyzing and quantitating the cell’s entire “methylome” and “hydroxymethylome” remain less than optimal.

The protocol for bisulphite conversion to detect methylation remains the “gold standard” for DNA methylation analysis. This method is generally followed by PCR analysis for single nucleotide resolution to determine methylation across the DNA molecule. According to Dr. Pradhan, “.. bisulphite conversion does not distinguish 5mC and 5hmC,”

Recently we found an enzyme, a unique DNA modification-dependent restriction endonuclease, AbaSI, which can

decode the hydryoxmethylome of the mammalian genome.

You easily can find out where the hydroxymethyl regions are.”

AbaSI, recognizes 5-glucosylatedmethylcytosine (5gmC) with high specificity when compared to 5mC and 5hmC, and

cleaves at narrow range of distances away from the recognized modified cytosine.

By mapping the cleaved ends, the exact 5hmC location can, the investigators reported, be determined.

Dr. Pradhan and his colleagues at NEB; the Department of Biochemistry, Emory University School of Medicine, Atlanta; and the New England Biolabs Shanghai R&D Center described use of this technique in a paper published in Cell Reports this month, in which they described high-resolution enzymatic mapping of genomic hydroxymethylcytosine in mouse ES cells.

In the current report, the authors used the enzyme technology for the genome-wide high-resolution hydroxymethylome, describing simple library construction even with a low amount of input DNA (50 ng) and the ability to readily detect 5hmC sites with low occupancy.

As a result of their studies, they propose that

factors affecting the local 5mC accessibility to TET enzymes play important roles in the 5hmC deposition

including include chromatin compaction, nucleosome positioning, or TF binding.
the regularly oscillating 5hmC profile around the CTCF-binding sites, suggests 5hmC ‘‘writers’’ may be sensitive to the nucleosomal environment.
some transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.

“We were able to do complete mapping in mouse embryonic cells and are pleased about what this enzyme can do and how it works,” Dr. Pradhan said.

And the availability of novel tools that make analysis of the methylome and hypomethylome more accessible will move the field of epigenetic analysis forward and potentially novel biomarkers for cellular development, differentiation, and disease.

Patricia Fitzpatrick Dimond, Ph.D. (pdimond@genengnews.com), is technical editor at Genetic Engineering & Biotechnology News.

Epigenetic Regulation of ADME-Related Genes: Focus on Drug Metabolism and Transport

Published: Sep 23, 2013

Epigenetic regulation of gene expression refers to heritable factors that are functionally relevant genomic modifications but that do not involve changes in DNA sequence.

Examples of such modifications include

DNA methylation, histone modifications, noncoding RNAs, and chromatin architecture.

Epigenetic modifications are crucial for

packaging and interpreting the genome, and they have fundamental functions in regulating gene expression and activity under the influence of physiologic and environmental factors.

In this issue of Drug Metabolism and Disposition, a series of articles is presented to demonstrate the role of epigenetic factors in regulating

the expression of genes involved in drug absorption, distribution, metabolism, and excretion in organ development, tissue-specific gene expression, sexual dimorphism, and in the adaptive response to xenobiotic exposure, both therapeutic and toxic.

The articles also demonstrate that, in addition to genetic polymorphisms, epigenetics may also contribute to wide inter-individual variations in drug metabolism and transport. Identification of functionally relevant epigenetic biomarkers in human specimens has the potential to improve prediction of drug responses based on patient’s epigenetic profiles.

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=157804

This study is published online in Drug Metabolism and Disposition

Part 8. Pictorial Maps

Prediction of intracellular metabolic states from extracellular metabolomic data

MK Aurich, G Paglia, Ottar Rolfsson, S Hrafnsdottir, M Magnusdottir, MM Stefaniak, BØ Palsson, RMT Fleming &

Ines Thiele

Metabolomics Aug 14, 2014;

http://dx.doi.org:/10.1007/s11306-014-0721-3

http://link.springer.com/article/10.1007/s11306-014-0721-3/fulltext.html#Sec1

http://link.springer.com/static-content/images/404/art%253A10.1007%252Fs11306-014-0721-3/MediaObjects/11306_2014_721_Fig1_HTML.gif

Metabolic models can provide a mechanistic framework

to analyze information-rich omics data sets, and are
increasingly being used to investigate metabolic alternations in human diseases.

An expression of the altered metabolic pathway utilization is the selection of metabolites consumed and released by cells. However, methods for the

inference of intracellular metabolic states from extracellular measurements in the context of metabolic models remain underdeveloped compared to methods for other omics data.

Herein, we describe a workflow for such an integrative analysis

emphasizing on extracellular metabolomics data.

We demonstrate,

using the lymphoblastic leukemia cell lines Molt-4 and CCRF-CEM,

how our methods can reveal differences in cell metabolism. Our models explain metabolite uptake and secretion by predicting

a more glycolytic phenotype for the CCRF-CEM model and
a more oxidative phenotype for the Molt-4 model,
which was supported by our experimental data.

Gene expression analysis revealed altered expression of gene products at

key regulatory steps in those central metabolic pathways, and

literature query emphasized the role of these genes in cancer metabolism.

Moreover, in silico gene knock-outs identified unique

control points for each cell line model, e.g., phosphoglycerate dehydrogenase for the Molt-4 model.

Thus, our workflow is well suited to the characterization of cellular metabolic traits based on

-extracellular metabolomic data, and it allows the integration of multiple omics data sets
into a cohesive picture based on a defined model context.

Keywords Constraint-based modeling _ Metabolomics _ Multi-omics _ Metabolic network _ Transcriptomics

1 Introduction

Modern high-throughput techniques have increased the pace of biological data generation. Also referred to as the ‘‘omics avalanche’’, this wealth of data provides great opportunities for metabolic discovery. Omics data sets

contain a snapshot of almost the entire repertoire of mRNA, protein, or metabolites at a given time point or

under a particular set of experimental conditions. Because of the high complexity of the data sets,

computational modeling is essential for their integrative analysis.

Currently, such data analysis is a bottleneck in the research process and methods are needed to facilitate the use of these data sets, e.g., through meta-analysis of data available in public databases [e.g., the human protein atlas (Uhlen et al. 2010) or the gene expression omnibus (Barrett et al. 2011)], and to increase the accessibility of valuable information for the biomedical research community.

Constraint-based modeling and analysis (COBRA) is

a computational approach that has been successfully used to
investigate and engineer microbial metabolism through the prediction of steady-states (Durot et al.2009).

The basis of COBRA is network reconstruction: networks are assembled in a bottom-up fashion based on

genomic data and extensive
organism-specific information from the literature.

Metabolic reconstructions capture information on the

known biochemical transformations taking place in a target organism
to generate a biochemical, genetic and genomic knowledge base (Reed et al. 2006).

Once assembled, a

metabolic reconstruction can be converted into a mathematical model (Thiele and Palsson 2010), and
model properties can be interrogated using a great variety of methods (Schellenberger et al. 2011).

The ability of COBRA models

to represent genotype–phenotype and environment–phenotype relationships arises
through the imposition of constraints, which
limit the system to a subset of possible network states (Lewis et al. 2012).

Currently, COBRA models exist for more than 100 organisms, including humans (Duarte et al. 2007; Thiele et al. 2013).

Since the first human metabolic reconstruction was described [Recon 1 (Duarte et al. 2007)],

biomedical applications of COBRA have increased (Bordbar and Palsson 2012).

One way to contextualize networks is to

define their system boundaries according to the metabolic states of the system, e.g., disease or dietary regimes.

The consequences of the applied constraints can

then be assessed for the entire network (Sahoo and Thiele 2013).

Additionally, omics data sets have frequently been used

to generate cell-type or condition-specific metabolic models.

Models exist for specific cell types, such as

enterocytes (Sahoo and Thiele2013),
macrophages (Bordbar et al. 2010),
adipocytes (Mardinoglu et al. 2013),
even multi-cell assemblies that represent the interactions of brain cells (Lewis et al. 2010).

All of these cell type specific models, except the enterocyte reconstruction

were generated based on omics data sets.

Cell-type-specific models have been used to study

diverse human disease conditions.

For example, an adipocyte model was generated using

transcriptomic, proteomic, and metabolomics data.

This model was subsequently used to investigate metabolic alternations in adipocytes

that would allow for the stratification of obese patients (Mardinoglu et al. 2013).

The biomedical applications of COBRA have been

cancer metabolism (Jerby and Ruppin, 2012).
predicting drug targets (Folger et al. 2011; Jerby et al. 2012).

A cancer model was generated using

multiple gene expression data sets and subsequently used
to predict synthetic lethal gene pairs as potential drug targets
selective for the cancer model, but non-toxic to the global model (Recon 1),

a consequence of the reduced redundancy in the cancer specific model (Folger et al. 2011).

In a follow up study, lethal synergy between FH and enzymes of the heme metabolic pathway

were experimentally validated and resolved the mechanism by which FH deficient cells,
e.g., in renal-cell cancer cells survive a non-functional TCA cycle (Frezza et al. 2011).

Contextualized models, which contain only the subset of reactions active in a particular tissue (or cell-) type,

can be generated in different ways (Becker and Palsson, 2008; Jerby et al. 2010).

However, the existing algorithms mainly consider

gene expression and proteomic data
to define the reaction sets that comprise the contextualized metabolic models.

These subset of reactions are usually defined

based on the expression or absence of expression of the genes or proteins (present and absent calls),
or inferred from expression values or differential gene expression.

Comprehensive reviews of the methods are available (Blazier and Papin, 2012; Hyduke et al. 2013). Only the compilation of a large set of omics data sets

can result in a tissue (or cell-type) specific metabolic model, whereas

the representation of one particular experimental condition is achieved

through the integration of omics data set generated from one experiment only (condition-specific cell line model).

Recently, metabolomic data sets have become more comprehensive and

using these data sets allow direct determination of the metabolic network components (the metabolites).

Additionally, metabolomics has proven to be stable, relatively inexpensive, and highly reproducible (Antonucci et al. 2012). These factors make metabolomic data sets particularly valuable for

interrogation of metabolic phenotypes.

Thus, the integration of these data sets is now an active field of research (Li et al. 2013; Mo et al. 2009; Paglia et al. 2012b; Schmidt et al. 2013).

Generally, metabolomic data can be incorporated into metabolic networks as

qualitative, quantitative, and thermodynamic constraints (Fleming et al. 2009; Mo et al. 2009).

Mo et al. used metabolites detected in the

spent medium of yeast cells to determine intracellular flux states through a sampling analysis (Mo et al. 2009),
which allowed unbiased interrogation of the possible network states (Schellenberger and Palsson 2009) and
prediction of internal pathway use.

Modes of transcriptional regulation during the YMC

Such analyses have also been used to reveal the effects of

enzymopathies on red blood cells (Price et al. 2004),
to study effects of diet on diabetes (Thiele et al. 2005) and
to define macrophage metabolic states (Bordbar et al. 2010).

This type of analysis is available as a function in the COBRA toolbox (Schellenberger et al. 2011).

In this study, we established a workflow

for the generation and analysis of condition-specific metabolic cell line models
that can facilitate the interpretation of metabolomic data.

Our modeling yields meaningful predictions regarding

metabolic differences between two lymphoblastic leukemia cell lines (Fig. 1A).

Fig. 1

metabol leukem cell lines11306_2014_721_Fig1_HTML

A Combined experimental and computational pipeline to study human metabolism.

Experimental work and omics data analysis steps precede computational modeling.
Model predictions are validated based on targeted experimental data.
Metabolomic and transcriptomic data are used for model refinement and submodel extraction.
Functional analysis methods are used to characterize the metabolism of the cell-line models and compare it to additional experimental data.
The validated models are subsequently used for the prediction of drug targets.

B Uptake and secretion pattern of model metabolites. All metabolite uptakes and secretions that were mapped during model generation are shown.

Metabolite uptakes are depicted on the left, and
secreted metabolites are shown on the right.

A number of metabolite exchanges mapped to the model were unique to one cell line.
Differences between cell lines were used to set quantitative constraints for the sampling analysis.

C Statistics about the cell line-specific network generation.

D Quantitative constraints.

For the sampling analysis, an additional set of constraints was imposed on the cell line specific models,

emphasizing the differences in metabolite uptake and secretion between cell lines.

Higher uptake of a metabolite was allowed

in the model of the cell line that consumed more of the metabolite in vitro, whereas
the supply was restricted for the model with lower in vitro uptake.

This was done by establishing the same ratio between the models bounds as detected in vitro.

X denotes the factor (slope ratio) that distinguishes the bounds, and

which was individual for each metabolite.

(a) The uptake of a metabolite could be x times higher in CCRF-CEM cells,

(b) the metabolite uptake could be x times higher in Molt-4,

(d) metabolite secretion could be x times higher in Molt-4 cells.LOD limit of detection.

The consequence of the adjustment was, in case of uptake, that one model was constrained to a lower metabolite uptake (A, B), and the difference depended on the ratio detected in vitro. In case of secretion, one model

had to secrete more of the metabolite, and again
the difference depended on the experimental difference detected between the cell lines

2 Results

We set up a pipeline that could be used to infer intracellular metabolic states

from semi-quantitative data regarding metabolites exchanged between cells and their environment.

Our pipeline combined the following four steps:

data acquisition,
data analysis,
metabolic modeling and
experimental validation of the model predictions (Fig. 1A).

We demonstrated the pipeline and the predictive potential to predict metabolic alternations in diseases such as cancer based on

^two lymphoblastic leukemia cell lines.

The resulting Molt-4 and CCRF-CEM condition-specific cell line models could explain

^ metabolite uptake and secretion
^ by predicting the distinct utilization of central metabolic pathways by the two cell lines.
^ the CCRF-CEM model resembled more a glycolytic, commonly referred to as ‘Warburg’ phenotype,
^ our model predicted a more respiratory phenotype for the Molt-4 model.

We found these predictions to be in agreement with measured gene expression differences

at key regulatory steps in the central metabolic pathways, and they were also
consistent with additional experimental data regarding the energy and redox states of the cells.

After a brief discussion of the data generation and analysis steps, the results derived from model generation and analysis will be described in detail.

2.1 Pipeline for generation of condition-specific metabolic cell line models

integration of exometabolomic (EM) data

2.1.1 Generation of experimental data

We monitored the growth and viability of lymphoblastic leukemia cell lines in serum-free medium (File S2, Fig. S1). Multiple omics data sets were derived from these cells.Extracellular metabolomics (exo-metabolomic) data,

integration of exometabolomic (EM) data

^ comprising measurements of the metabolites in the spent medium of the cell cultures (Paglia et al. 2012a),
^ were collected along with transcriptomic data, and these data sets were used to construct the models.

2.1.4 Condition-specific models for CCRF-CEM and Molt-4 cells

To determine whether we had obtained two distinct models, we evaluated the reactions, metabolites, and genes of the two models. Both the Molt-4 and CCRF-CEM models contained approximately half of the reactions and metabolites present in the global model (Fig. 1C). They were very similar to each other in terms of their reactions, metabolites, and genes (File S1, Table S5A–C).

(1) The Molt-4 model contained seven reactions that were not present in the CCRF-CEM model (Co-A biosynthesis pathway and exchange reactions).
(2) The CCRF-CEM contained 31 unique reactions (arginine and proline metabolism, vitamin B6 metabolism, fatty acid activation, transport, and exchange reactions).
(3) There were 2 and 15 unique metabolites in the Molt-4 and CCRF-CEM models, respectively (File S1, Table S5B).
(4) Approximately three quarters of the global model genes remained in the condition-specific cell line models (Fig. 1C).
(5) The Molt-4 model contained 15 unique genes, and the CCRF-CEM model had 4 unique genes (File S1, Table S5C).
(6) Both models lacked NADH dehydrogenase (complex I of the electron transport chain—ETC), which was determined by the absence of expression of a mandatory subunit (NDUFB3, Entrez gene ID 4709).

Rather, the ETC was fueled by FADH2 originating from succinate dehydrogenase and from fatty acid oxidation, which through flavoprotein electron transfer

FADH2

could contribute to the same ubiquinone pool as complex I and complex II (succinate dehydrogenase).

Despite their different in vitro growth rates (which differed by 11 %, see File S2, Fig. S1) and
^^^ differences in exo-metabolomic data (Fig. 1B) and transcriptomic data,
^^^ the internal networks were largely conserved in the two condition-specific cell line models.

2.1.5 Condition-specific cell line models predict distinct metabolic strategies

Despite the overall similarity of the metabolic models, differences in their cellular uptake and secretion patterns suggested distinct metabolic states in the two cell lines (Fig. 1B and see “Materials and methods” section for more detail). To interrogate the metabolic differences, we sampled the solution space of each model using an Artificial Centering Hit-and-Run (ACHR) sampler (Thiele et al. 2005). For this analysis, additional constraints were applied, emphasizing the quantitative differences in commonly uptaken and secreted metabolites. The maximum possible uptake and maximum possible secretion flux rates were reduced
^^^ according to the measured relative differences between the cell lines (Fig. 1D, see “Materials and methods” section).

We plotted the number of sample points containing a particular flux rate for each reaction. The resulting binned histograms can be understood as representing the probability that a particular reaction can have a certain flux value.

A comparison of the sample points obtained for the Molt-4 and CCRF-CEM models revealed

a considerable shift in the distributions, suggesting a higher utilization of glycolysis by the CCRF-CEM model
(File S2, Fig. S2).

This result was further supported by differences in medians calculated from sampling points (File S1, Table S6).
The shift persisted throughout all reactions of the pathway and was induced by the higher glucose uptake (34 %) from the extracellular medium in CCRF-CEM cells.

The sampling median for glucose uptake was 34 % higher in the CCRF-CEM model than in Molt-4 model (File S2, Fig. S2).

The usage of the TCA cycle was also distinct in the two condition-specific cell-line models (Fig. 2). Interestingly,
the models used succinate dehydrogenase differently (Figs. 2, 3).

TCA_reactions

The Molt-4 model utilized an associated reaction to generate FADH2, whereas

in the CCRF-CEM model, the histogram was shifted in the opposite direction,
toward the generation of succinate.

Additionally, there was a higher efflux of citrate toward amino acid and lipid metabolism in the CCRF-CEM model (Fig. 2). There was higher flux through anaplerotic and cataplerotic reactions in the CCRF-CEM model than in the Molt-4 model (Fig. 2); these reactions include

(1) the efflux of citrate through ATP-citrate lyase,
(2) uptake of glutamine,
(3) generation of glutamate from glutamine,
(4) transamination of pyruvate and glutamate to alanine and to 2-oxoglutarate,
(5) secretion of nitrogen, and
(6) secretion of alanine.

energetics-of-cellular-respiration

The Molt-4 model showed higher utilization of oxidative phosphorylation (Fig. 3), again supported by
elevated median flux through ATP synthase (36 %) and other enzymes, which contributed to higher oxidative metabolism. The sampling analysis therefore revealed different usage of central metabolic pathways by the condition-specific models.

Fig. 2

Differences in the use of the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).

The table provides the median values of the sampling results. Negative values in histograms and in the table describe reversible reactions with flux in the reverse direction. There are multiple reversible reactions for the transformation of isocitrate and α-ketoglutarate, malate and fumarate, and succinyl-CoA and succinate. These reactions are unbounded, and therefore histograms are not shown. The details of participating cofactors have been removed.

Figure 3.

Molt-4 has higher median flux through ETC reactions II–IV 11306_2014_721_Fig3_HTML

Atp ATP, cit citrate, adp ADP, pi phosphate, oaa oxaloacetate, accoa acetyl-CoA, coa coenzyme-A, icit isocitrate, αkg α-ketoglutarate, succ-coa succinyl-CoA, succ succinate, fumfumarate, mal malate, oxa oxaloacetate,
pyr pyruvate, lac lactate, ala alanine, gln glutamine, ETC electron transport chain

Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes

metabolic pathways 1476-4598-10-70-1

Metabolic Systems Research Team fig2

Metabolic control analysis of respiration in human cancer tissue. fphys-04-00151-g001

Metabolome Informatics Research fig1

Modelling of Central Metabolism network3

N. gaditana metabolic pathway map ncomms1688-f4

protein changes in biological mechanisms

Read Full Post »

Mechanisms of Drug Resistance

Posted in Biochemical pathways, Ca2+ triggered activation, Calcium Signaling, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Clinical Diagnostics, Cytoskeleton, Disease Biology, Drug Toxicity, Enzyme Induction, Gene Regulation, Gene Regulation and Evolution, Genetics & Pharmaceutical, Genomic Testing: Methodology for Diagnosis, Genomics Pharmacy, Infectious Disease & New Antibiotic Targets, Innovations, Intellectual Property, Ionic Transporters Na+, Lipid metabolism, Lipids, Metabolism, Metabolomics, Methods, Molecular Genetics & Pharmaceutical, Mutant Gene Expression, Nitric Oxide in Health and Disease, Nutrigenomics, Nutrition and Phytochemistry, Patents, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmaceutical Drug Discovery, Phosphorylation, Plant extract, Proteins, Proteomics, Signaling, Systemic Inflammatory Response Related Disorders, Transcriptomics, Translational Research, Translational Science, tagged Antibiotic resistance, biofilm, cancer stem cell (CSC), cartwheel method, conformational changes, drug repurposing, E. Coli, efflux pump, enterobacter aerogenes, enterococuus, food-associated diarrhea, gram negative organisms, gram positive organisms, Klebsiella, M. tubercuslosis, methiocillin-resistant S. aureas (MRSA), multidrug resistance (MDR), neutraceuticals, phenothiazines, plant extracts, S. aureus, Salmonella, substrate binding on October 9, 2014| Leave a Comment »

Mechanisms of Drug Resistance

Curator: Larry H. Bernstein, MD, FCAP

Leaders in Pharmaceutical Intelligence, CSO

Mechanisms of Drug Resistance

This discussion is a continuing discussion of matters of metabolomics and the
essential role of genomic or epigenetic mechanisms to guide the development of
proteomic driven effectors of resistance to drug therapy.
We start with the elucidation of efflux pumps in bacteria, and we conclude with
consideration of cancer cells.

Part 1. Antimicrobial Resistance

Antimicrobial resistance is the ability of microbes, such as bacteria, viruses,
parasites, or
fungi, to grow in the presence of a chemical (drug) that would normally kill it
or limit its growth.

difference between non-resistant bacteria and drug resistant bacteria

http://www.niaid.nih.gov/SiteCollectionImages/topics/antimicrobialresistance/1whatIs
DrugResistance.gif

Non-resistant bacteria multiply, and upon drug treatment, the bacteria die. Drug
resistant bacteria multiply as well, but upon drug treatment, the bacteria continue
to spread.

Many infectious diseases are increasingly difficult to treat because of antimicrobial-resistant organisms, including HIV infection, staphylococcal infection, tuberculosis,
influenza, gonorrhea, candida infection, and malaria.

Between 5 and 10 percent of all hospital patients develop an infection. About 90,000
of these patients die each year as a result of their infection, up from 13,300 patient
deaths in 1992.

According to the Centers for Disease Control and Prevention (April 2011), antibiotic
resistance in the United States costs an estimated $20 billion a year in excess health
care costs. In addition, a cost of $35 million in other societal costs and more than 8
million additional days that people spend in the hospital. This is because people
infected with antimicrobial-resistant organisms are more likely to have longer hospital stays and may require more complicated treatment.

Diagnostic tests designed to determine which microbe is causing infection and to
which antimicrobials the microbe might be resistant take a few days or weeks to give
results because of a requirement for the microbe to grow for it to be identified.

Part 2. Antibiotic Tolerance
Reported By Jef Akst | June 25, 2014

Optimization of lag time underlies antibiotic tolerance in evolved bacterial
populations
O. Fridma et al. Nature, 2014
http://dx.doi.org://10.1038/nature13469

Populations of Escherichia coli grown in the lab develop tolerance when exposed to
repeated treatments with the antibiotic ampicillin. The bacteria evolved to stay in a
dormant “lag” phase for just longer than three-, five-, or eight-hour-long treatment
courses. Antibiotic tolerance, which allows bacteria to survive even high levels of
antibiotics by remaining dormant. Tolerance may lead to an inaccurate assumption
that an unsuccessful antibiotic treatment failed as a result of resistance, in which
the microbe has evolved to grow in the presence of the drug. Resistance is very well
known; but the issue of tolerance is much less known,” according to Tom Coenye of
the Laboratory of Pharmaceutical Microbiology (LPM) at Gent University in Belgium,
who was not involved in the research. This is a new phenomenon, extended lag,
where mutants have a longer lag time, and that extended lag allows them to survive
an attack by antibiotics.

To gain a better understanding of how bacterial populations might evolve to tolerate
antibiotic exposure, Nathalie Q. Balaban, a microbiologist and physicist at The Hebrew
University of Jerusalem in Israel and her colleagues exposed cultures of E. coli to high
concentrations of ampicillin for three, five, or eight hours, then washed the drug away
and suspended the bacteria in fresh media to be grown overnight. The next day, the
team repeated these treatments. In 10 cycles we could see that tolerance had evolved,
” Balaban said. Indeed, while the ampicillin treatments killed more than 99.9 percent of
the E. coli, by day 10, bacterial survival had increased 100-fold.

Moreover, the bacteria were also tolerant to norfloxacin, an antibiotic with a different mechanism of action than ampicillin but also ineffective during the dormant stage,
further supporting the idea that the E. coli populations had evolved to tolerate certain
durations of antibiotic exposure. “This is characteristic of tolerance,” said Balaban.
“The bacteria that have evolved tolerance under ampicillin are also more tolerant to
this completely different class of antibiotics.” Resistance, on the other hand, is usually
class-specific, she noted.

The researchers identified three genes that seemed to play a functional role in antibiotic
tolerance. While the exact mechanism of how mutations in these genes may have
lengthened the bacteria’s lag time is not yet known, two of the genes are part of pathways
that were previously implicated in bacterial persistence, including an antitoxin in a
common toxin-antitoxin module that may help regulate that bacteria’s growth.

Part 3. Multidrug Resistance Perspective

Mechanisms of antibiotic resistance in salmonella: efflux pumps, genetics,
quorum sensing and biofilm formation.

Perspectives in Drug Discovery and Design 02/2011; 8:114-123.
Martins M, McCusker, Amaral, Fanning S

Multidrug resistance (MDR) to antibiotics presents a serious therapeutic problem
in the treatment of bacterial infections. The importance of this mechanism of resistance
in clinical settings is reflected in the increasing number of reports of multidrug resistant
isolates. In Salmonella enterica, the most common etiological agent of food borne
salmonellosis worldwide, MDR is becoming a major concern.

In Salmonella the main mechanisms of antibiotic resistance are mutations in target
genes (such as DNA gyrase and topoisomerase IV) and the over-expression of efflux pumps. However, other mechanisms such as

changes in the cell envelope;
down regulation of membrane porins;
increased lipopolysaccharide (LPS) component of the outer cell membrane;
quorum sensing and
biofilm formation

can also contribute to the resistance seen in this microorganism. To overcome
this problem new therapeutic approaches are urgently needed.

In the case of efflux-mediated multidrug resistant isolates, one of the treatment
options could be

the use of efflux pump inhibitors (EPIs)
in combination with the antibiotics to which the bacteria is resistant.

By blocking the efflux pumps

resistance is partly or wholly reversed,
allowing antibiotics showing no activity against the MDR strains
to be used to treat these infections.

Compounds that show potential as an EPI are therefore of interest, as well as new
strategies to target the efflux systems. Quorum sensing (QS) and biofilm formation
are systems also known to be involved in antibiotic resistance. Consequently,
compounds that

can disrupt or inhibit these bacterial “communication systems” will be of use in
the treatment of these infections.

Part 5. Effux pumps and S. Aureus

Multidrug Efflux Pumps in Staphylococcus aureus: an Update

SS Costa, M Viveiros, L Amaral and I Couto
1Grupo de Micobactérias, Unidade de Microbiologia Médica, Instituto de Higiene e
Medicina Tropical, Universidade Nova de Lisboa (IHMT, UNL), 2Centro de Recursos
Microbiológicos (CREM), UNL, Portugal,3COST ACTION BM0701 (ATENS), Brussels,
Belgium
The Open Microbiology Journal 2013;(Suppl 1-M5): 59-71

The emergence of infections caused by multi- or pan-resistant bacteria in the hospital
or in the community settings is an increasing health concern. Albeit there is no single
resistance mechanism behind multi-resistance, multidrug efflux pumps,

proteins that cells use to detoxify from noxious compounds,

seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria.
During the last decades, experimental data has established their contribution to low
level resistance to antimicrobials in bacteria and their

potential role in the appearance of MDR phenotypes, by the extrusion of multiple,
unrelated compounds.

Recent studies suggest that

efflux pumps may be used by the cell as a first-line defense mechanism,

avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration
occurs, that allows survival in the presence of that agent.

In this paper we review the current knowledge on

MDR efflux pumps and their
intricate regulatory network in Staphylococcus aureus,

a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that

aureus MDR efflux pumps,
either chromosomal or plasmid-encoded, have
on resistance towards different antimicrobial agents and
on the selection of drug – resistant strains.

We will also discuss the many questions that still remain on the role of each specific
efflux pump and the need to establish appropriate methodological approaches to
address all these questions.

Table 1. Multidrug Efflux Pumps Described for Staphylococcus aureus

Efflux Pump	Familya	Regulator(s)b	Substrate Specificity	References
Chromosomally-encoded Efflux Systems
NorA	MFS	MgrA, NorG(?)	Hydrophilic fluoroquinolones (ciprofloxacin, norfloxacin) QACs (tetraphenylphosphonium, benzalkonium chloride) Dyes (e.g. ethidium bromide, rhodamine)	[16,18,19]
NorB	MFS	MgrA, NorG	Fluoroquinolones (e.g. hydrophilic: ciprofloxacin, norfloxacin and hydrophobic: moxifloxacin, sparfloxacin) Tetracycline QACs (e.g. tetraphenylphosphonium, cetrimide) Dyes (e.g. ethidium bromide)	[31]
NorC	MFS	MgrA(?), NorG	Fluoroquinolones (e.g. hydrophilic: ciprofloxacin and hydrophobic: moxifloxacin) Dyes (e.g. rhodamine)	[35,36]
MepA	MATE	MepR	Fluoroquinolones (e.g. hydrophilic: ciprofloxacin, norfloxacin and hydrophobic: moxifloxacin, sparfloxacin) Glycylcyclines (e.g. tigecycline) QACs (e.g. tetraphenylphosphonium, cetrimide, benzalkonium chloride) Dyes (e.g. ethidium bromide)	[37,38]
MdeA	MFS	n.i.	Hydrophilic fluoroquinolones (e.g. ciprofloxacin, norfloxacin) Virginiamycin, novobiocin, mupirocin, fusidic acid QACs (e.g. tetraphenylphosphonium, benzalkonium chloride, dequalinium) Dyes (e.g. ethidium bromide)	[39,40]

SepA	n.d.	n.i.	QACs (e.g. benzalkonium chloride) Biguanidines (e.g. chlorhexidine) Dyes (e.g. acriflavine)	[41]
SdrM	MFS	n.i.	Hydrophilic fluoroquinolones (e.g. norfloxacin) Dyes (e.g. ethidium bromide, acriflavine)	[42]
LmrS	MFS	n.i.	Oxazolidinone (linezolid) Phenicols (e.g. choramphenicol, florfenicol) Trimethoprim, erythromycin, kanamycin, fusidic acid QACs (e.g. tetrapheny- lphosphonium) Detergents (e.g. sodium docecyl sulphate) Dyes (e.g. ethidium bromide)	[43]

Plasmid-encoded Efflux Systems

QacA	MFS	QacR	QACs (e.g. tetraphenylphosphonium, benzalkonium chloride, dequalinium) Biguanidines (e.g. chlorhexidine) Diamidines (e.g. pentamidine) Dyes (e.g. ethidium bromide, rhodamine, acriflavine)	[45,49]
QacB	MFS	QacR	QACs (e.g. tetraphenylphosphonium, benzalkonium chloride)Dyes (e.g. ethidium bromide, rhodamine, acriflavine)	[53]
Smr	SMR	n.i.	QACs (e.g. benzalkonium chloride, cetrimide) Dyes (e.g. ethidium bromide)	[58,61]
QacG	SMR	n.i.	QACs (e.g. benzalkonium chloride, cetyltrymethylammonium) Dyes (e.g. ethidium bromide)	[67]
QacH	SMR	n.i.	QACs (e.g. benzalkonium chloride, cetyltrymethylammonium) Dyes (e.g. ethidium bromide)	[68]
QacJ	SMR	n.i.	QACs (e.g. benzalkonium chloride, cetyltrymethylammonium) Dyes (e.g. ethidium bromide)	[69]

a n.d.: The family of transporters to which SepA belongs is not elucidated to date.
b n.i.: The transporter has no regulator identified to date.
QACs: quaternary ammonium compounds

Identification of the plasmid-encoded qacA efflux pump gene
in meticillin-resistant Staphylococcus aureus (MRSA)
strain HPV107, a representative of the MRSA Iberian clone

S.S. Costaa,b, E. Ntokouc, A. Martinsa,d, M. Viveirosa,e, S. Pournarasc,
I. Coutoa,b, L. Amarala,d,e,∗
a Unidade de Micobactérias, Instituto de Higiene e Medicina Tropical,
Universidade Nova de Lisboa (IHMT, UNL), b Centro de Recursos Microbiológicos,
Universidade Nova de Lisboa (CREM, UNL), d Unidade de Parasitologia e
Microbiologia Médica (UPMM), Instituto de Higiene e Medicina Tropical, Universidade
Nova de Lisboa (IHMT, UNL), Lisbon, Portugal; e COST ACTION BM0701 (ATENS)
c Department of Microbiology, Medical School, University of Thessaly, Larissa, Greece;
Int J Antimicrobial Agents 2010; 36: 557–561
http://www.elsevier.com/locate/ijantimicag

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial
bacterium for which prevention and control measures consist mainly of

the application of biocides with antiseptic and disinfectant activity.

In this study, we demonstrated the presence of

the plasmid-located efflux pump gene qacA in MRSA strain HPV107,

a clinical isolate representative of the MRSA Iberian clone. The existence
of efflux activity in strain HPV107 due to the QacA pump was found and

this QacA efflux activity was linked with a phenotype of
reduced susceptibility towards several biocide compounds.

No association could be made with antibiotic resistance. This work
emphasises the potential of QacA pump activity in

the maintenance and dissemination of important MRSA strains in
the hospital setting and, increasingly, in the community.

Efflux-mediated response of Staphylococcus aureus exposed to
ethidium bromide

I Couto1,2, S S Costa1, M Viveiros1, M Martins1,3 and L Amaral1,3*
1Unidade de Micobacterias, Instituto de Higiene e Medicina Tropical,
Universidade Nova de Lisboa (UNL), 2Centro de Recursos Microbiolo´gicos (CREM), Faculdade de Cieˆncias e Tecnologia, UNL,3UPMM,
Instituto de Higiene e Medicina Tropical, UNL, Portugal
J Antimicrob Chemother (2008) 62, 504–513
http://dx.doi.org:/10.1093/jac/dkn217

By adapting an antibiotic-susceptible Staphylococcus aureus strain to
increasing concentrations of ethidium bromide, a known substrate
of efflux pumps (EPs), and

by phenotypically and genotypically analysing the resulting progeny,
we characterized the molecular mechanisms of S. aureus
adaptation to ethidium bromide.

ATCC 25923 was grown in increasing concentrations of ethidium bromide.
The MICs of representatives of eight classes of antibiotics, eight biocides
and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny
ATCC 25923EtBr were determined

with or without six efflux pump inhibitors (EPIs).

Efflux activity in the presence/absence of EPIs was evaluated by realtime
fluorometry. The presence and expression of eight EP genes were assayed
by PCR and quantitative RT–PCR (qRT–PCR), respectively. Mutations in
grlA, gyrA and norA promoter regions were screened by DNA sequencing.

Compared with its parental strain, ATCC 25923EtBr was

32-fold more resistant to ethidium bromide and
also more resistant to biocides and hydrophilic fluoroquinolones.
Resistance to these could be reduced by the EPIs chlorpromazine,
thioridazine and reserpine.

Increased efflux of ethidium bromide by ATCC 25923EtBr could be
inhibited by the same EPIs. qRT–PCR showed that

norA was 35-fold over-expressed in ATCC 25923EtBr,

whereas the remaining EP genes showed no significant increase in their

expression. Sequencing of the norA promoter region revealed

a 70 bp deletion in ATCC 25923EtBr.

Exposure of S. aureus to quaternary compounds such as ethidium bromide
results in decreased susceptibility of the organism to a wide variety of
compounds, including quinolones and biocides

through an efflux-mediated response, which
for strain ATCC 25923 is mainly NorA-mediated.

This altered expression may result from alterations in the norA
promoter region.

Ethnic consumption of plant leaf extracts and appraisal of
their nutraceutical efficacy against multidrug resistant
staphylococcus aureus

Kaushik S1, 2*, Tomar Rs1, Shrivastav V1, Shrivastav A2 And Jain Sk3
Amity Institute of Biotechnology, Amity University Madhya Pradesh,
Gwalior (M.P.); 2: College of Life Sciences, Cancer Hospital and
Research Institute, Gwalior (M.P.); 3: Department of Microbiology,
Vikram University, Ujjain (M.P.), INDIA
IJBPAS, Feb, 2014, 3(2): 204-209

Nutraceuticals are natural bioactive chemical compounds that have
health promoting, disease preventing or medicinal properties.
Emergence of Multi Drug Resistant Staphylococci is increasing at
alarming rates and diseases caused by these strains leave patients
against multiple resistant Staphylococcus aureus.

The test bacteria were isolated and characterized by standard and
NCCLS recommended microbiological techniques. A total of eighteen
plant extracts were analysed for their antimicrobial activity. The
selection of medicinal plants was based on their traditional uses in
India. However most of these plants were not previously screened.
Antibacterial activity of these components was performed by standard
Kirby Bauer Disk Diffusion method approved by NCCLS and the
inhibitory effect was analysed by calculating Zone of inhibition.

Among the eighteen plant extracts analysed we found highest
activity in the effect of chemotherapy and as promising bio control agents

Guava,
Mango,
Jamun and
Pomengrate plant extracts,

while most of the other plants were either showing very moderate/
least activity against test bacteria. Our recent experiment indicated
that phytochemicals extracted with methanol can be utilized as
nutraceutical to lower the side.

Part 6. Efflux pumps and gram-negative organisms

Efflux Pumps that Bestow Multi-Drug Resistance of Pathogenic Gram-
negative Bacteria

Amaral L1,2*, Spengler G2, Martins A2,3 and Molnar J2
1Travel Medicine of the Centre for Malaria and Other Tropical Diseases (CMDT),
Institute of Hygiene and Tropical Medicine, Lisbon, Portugal 2Department of
Medical Microbiology and Immunobiology, Faculty of Medicine, University of
Szeged, Szeged, Hungary 3Unit of Parasitology and Medical Microbiology
(UPMM), Institute of Hygiene and Tropical Medicine, Lisbon, Portugal
Amaral et al., Biochem Pharmacol 2013; 2:3
http://dx.doi.org/10.4172/2167-0501.1000119

The efflux pump

Efflux pumps are integral plasma membrane protein systems that recognize and bind
noxious compounds present in the cytoplasm (toxic products produced by metabolism;
compounds that have penetrated the cell), or periplasm of the bacterial cell and extrude
it into the environment in which the bacterium resides [1].

The efflux pump machinery gives the cell additional protection to the one provided by

the constituents of its cell wall (example: lipopolysaccharides), and
provides an initial protection to noxious agents present in its
natural environment that have penetrated into the cell (example: bile
salts in the colon) [1].

The efflux pump machinery is divided into five superfamily classes;

the major facilitator (MF),
the ATP-binding cassette (ABC),
the resistance-nodulation-division (RND),
the small multi-drug resistance (SMR) and
the multi-drug and toxic compound extrusion (MATE).

With respect to Gram-negative bacteria, although they all play
important roles in the protection of the bacterium from noxious
agents present in the environment, the

main efflux pump of the Gram negative bacterium is a
member of the RND superfamily, and
because multi-drug resistance of clinical isolates have
been associated with the over-expression of this pump,

it has received a great deal of attention [2].

The first in vitro response of bacteria to a given noxious agent,
such as an antibiotic, is to over-express its main efflux pump [2].
If the bacterium is serially exposed in vitro to increasing
concentrations of that compound, it responds by increasing
the effective number of its main efflux pump, as well as others
that provide redundant protection [2].

However, if that “adapted” bacterium is now maintained at a
constant level of a noxious agent, the level of efflux pump
activity increases up to a maximum, followed by a gradual
return of efflux pump activity to its basal level. Concomitant
to this process, an accumulation of mutations of essential
proteins located in the plasma membrane (example penicillin
binding proteins), mutations 30 S component of the ribosome
and gyrase take place [3]. These events suggest that when
the organism is faced with an environment that contains a
constant toxic level of a compound, and the cost for
maintaining an energy consuming system, such as that
needed for the energy dependent efflux pump, is too
great a price to pay.

Therefore, in order to survive in this unchanging environment,
other mechanisms are activated. For example, activation of a
mutator master gene is thought to be an important step at this
level, which results in the mutation of genes that code for
essential proteins, reversing the over-expression of efflux-
pumps, but still conferring the bacterial resistant to the
environmental pressure via other mechanism(s), yet
to be understood [4,5].

During therapy, the level of resistance increases many fold
higher than that of the initial infecting strain. Hence, clinical
isolates from treated patients often show much higher levels
of antibiotic resistance than that of their wild type counterpart
(sometimes it can even present a 1000 fold increase) [6].
At this stage, resistance is usually related to the presence
of mutations, which reduces the survival of the resistant
bacteria,

once it is transferred to a noxious agent-free environment

that contains the competing wild type counterpart [3,4].

Depending upon when during therapy a clinical strain is isolated,
its resistance to two or more antibiotic classes (multi-drug
resistance (MDR)), may be due entirely to over-expressed
efflux pumps; to a mixture of over-expressed efflux pumps
and increasing accumulation of mutations; and only to mutations [3,4].

The degree of resistance can readily be determined with
methods that employ compounds known for their modulation
of efflux pump activity, such as

phenothiazines [7] or phenyl-arginine-betanaphthylamide
(PAβN),
the latter which competes with the antibiotic as
substrate of the efflux pump [8].

If in presence of such compounds,

the MDR bacterium is rendered fully susceptible
to the antibiotic(s) to which it was initially resistant,
resistance is most likely due to its overexpressed
efflux pump systems.
Contributions made by accumulated mutations
render the organism less and less affected by the EPI.

This type of information is of great value to clinicians faced
with long-term therapy of a bacterial infection that
progresses to an MDR phenotype. It should be understood
that although the Gram-negative bacterium has essentially
one main efflux pump, such as

the AcrAB (Escherichia coli) or
the MexAB (Pseudomonas aeruginosa),

the deletion of the main efflux pump results in the over-
expression of one or more other RND efflux pumps,
such as is the case for deletion of the AcrAB, followed by

the over-expression of the AcrEF pump [2].

Redundancy of as many as nine RND efflux pumps [2],
provides additional protection to the organism.

The pumps belonging to the RND family form

a tripartite complex together with
the periplasmic proteins belonging to the
membrane fusion-protein (MFP) family and
the outer membrane channels.

RND transporters consist of

a transporter protein that recognises and
binds the noxious agent
in the cytoplasm or periplasm and
transports it to the contiguous channel (TolC),
ending at the surface of the outer membrane.

The transporter is attached to the plasma membrane
by two or three fusion proteins, which are believed to assist the

extrusion of the substrate by peristaltic actions [9].

Although the actual structure of RND efflux pumps
in the cell envelop is not completely understood,

the structure of the transporter, TolC and fusion
proteins are well established for major Gram-negative
bacteria [10].

The PMF energy dependent efflux pump most likely needs the
passage of hydronium ions through its internal cavity,

for the release of the substrate that is
in turn ejected into the TolC channel via the
peristaltic action of the fusion proteins [11].

A low pH,

the concentration of hydronium ions at the surface of the cell
results in a pH difference of 2 or 3 pH units compared
to that of the milieu,

the surface concentration of hydronium ions

provides the force for the mobility of hydronium ions
through porins leading to the acidification of the periplasm,
providing the low pH needed by the transporter
for the release of the substrate.

At high pH, these hydronium ions come from

hydrolysis of ATP by ATP synthase, and
are passed into the transporter, thereby
reducing its internal pH, so that
the release of the substrates can take place [11,12].

EPIs, such as the phenothiazines chlorpromazine or thioridazine,

exert their inhibition at pH above 6, and
are thought to affect hydrolysis of ATP
denying the efflux pump transporter hydronium ions needed

for release of the bound substrate [11,12].

The search for EPIs that are clinically useful continues, although

with respect to thioridazine, this old neuroleptic has been shown

to inhibit efflux pumps of pathogenic mycobacteria [13], and
has been successfully used to treat extensively drug resistant
tuberculosis infections [14].

The regulation of the main efflux pump of Escherichia coli may
take place via distinct pathways. The induced synthesis of the
transporter component of the AcrAB efflux pump, when the
organism is exposed in vitro to a noxious agent,

involves the activation of the stress gene soxS,
followed by the activation of the local regulator marA,
then by the activation of the transporter gene acrB [8].

In the case of Salmonella spp. two component resistance
mechanisms, such as the PmrA/PmrB system, directly
activate the master efflux pump regulator ram A gene [15].
The activation of the PmrA/PmrB system takes place
readily when Salmonella spp. is phagocytosed due to
the acidic nature of the phagolysosome [15], as follows:

PmrB is a sensor that self-phosphorylates, and
then transfers the phosphate to PmrA.
PmrA activates a nine gene operon, which
codes for Lipid A introduced into the nascent
lipopolysaccharide layer of the outer membrane.
The increased presence of Lipid A renders the
phagocytosed bacterium practically immune to
everything, including the hydrolases of the
phagolysososome [15].

Although some EPIs are in clinical trials, none have yet to
reach the marketplace, mainly due to their common
toxicity against healthy mammalian cells, affecting
intrinsic mammalian efflux pumps, as for example
those of the blood brain barrier. Lastly, it should be
noted that compounds that inhibit the efflux pump
of bacteria also have the capacity to promote the
removal of plasmids that carry antibiotic resistant
genes [16,17].

Nikaido H, Pages JM (2012) Broad-specificity efflux
pumps and their role in multidrug resistance of Gram-
negative bacteria. FEMSMicrobiol Rev 36: 340-363.
Viveiros M, Jesus A, Brito M, Leandro C, Martins M,
et al. (2005) Inducement and reversal of tetracycline
resistance in Escherichia coli K-12 and expression of
proton gradient-dependent multidrug efflux pump
genes. Antimicrob Agents Chemother 49: 3578-3582.
Martins A, Couto I, Aagaard L, Martins M, Viveiros M
(2007) Prolonged exposure of methicillin-resistant
Staphylococcus aureus (MRSA) COL strain to
increasing concentrations of oxacillin results in a
multidrug-resistant phenotype. Int J Antimicrob
Agent 29: 302-305.
Martins A, Spengler G, Molnar J, Amaral L (2012)
Sequential responses of bacteria to noxious agents
(antibiotics) leading to accumulation of mutations
and permanent resistance. Biochem Pharmacol J
Open Access 1: 7.

Inhibitors of efflux pumps of Gram-negative
bacteria inhibit Quorum Sensing

Leonard Amaral, Joseph Molnar
1 Grupo de Micobacterias, Unidade de Microbacterilogia,
Centro de Malaria e Doenças Tropicais (CMDT), Instituto de
Higiene e Medicina Tropical, Universidade Nova de Lisboa,
Lisbon, Portugal; 2 Cost Action BM0701 (ATENS) of the
European Commission/European Science Foundation;
3 Department of Medical Microbiology and Immunobiology,
University of Szeged, Szeged, Hungary
Open Journal of Pharmacology, 2012, 2-2

Quorum Sensing (QS) systems of bacteria consist of

a producer of the QS signal and the responder.

The generation of a QS signal provides the means by which
a population can behave in a concerted manner such as

swarming, swimming and secretion of biofilm, etc.

Because concerted bahaviour bestows protection to the bacterial
species, and hence factors involved in the severity of an infection
such as virulence are products of QS systems, compounds that
inhibit the QS system have significant clinical relevance. Recent
evidence suggests that

the secretion of QS signals takes place via
the efflux pump system of the producer of the signal.

Interestingly, compounds such as phenothiazines and
trifluoromethyl ketones (TFs)

that inhibit proton motive force (PMF) activities such
as swarming and swimming also
inhibit the PMF dependent efflux pump systems of
bacteria and their QS systems.

This review discusses the relationship between the efflux
pump, the QS system and the compounds that affect both.
Lastly, suggestions are made regarding classes of compounds
that have been shown

to inhibit PMF dependent efflux pumps and the need
to evaluate them for QS inhibitory properties.

Keywords: Quorum Sensing, QS signal, acylated hydroxyl
lactone (AHL), efflux pumps, Proton Motive Force (PMF),
inhibitors of efflux pumps, inhibitors of QS systems,
phenothiazines, Trifluormethyl Ketones (TFs), plants
sources for QS inhibitors

Efflux pumps of bacteria provide protection from noxious
agents that are present in the environment in which they
exist. Noxious agents may be naturally occurring compounds
present in environments outside and within the human.

Because over-expressed efflux pumps render antibiotic
therapy problematic, an intense search for agents that
inhibit specific efflux pumps of specific bacteria has
been conducted during the past decade [9].

Communication between bacteria of the same strain
or species and between species contributes to their
survival [11-13]. Communication involves the secretion
of signals that invoke a specific response from the responder
[11-13]. This communication process is termed Quorum
sensing (QS). When it takes place between strains of the
same species,

communication is directed towards the reduction
of population growth and
reducing the possibility of exceeding the nutritional
support of the environment

Other signals may involve a population response that involves

the secretion of bioactive molecules that inhibit the
replication of a competing population species [14-16]
or even kill [biocidins) [17-21] or
promote a swarming effect that recruits members
of the same species to migrate to a specific location [22-24]
similar to swarming by insects subsequent to signals
indicating site of food [example bees).
biofilm, encase the bacteria at distances from each other
[25-29] and within the matrix of this biofilm are
channels used for further communication [30].

Biofilms are produced in the wild, at sites such as surfaces
of rocks which maintain the bacterial population in situ [31]
and are also produced at sites of the human colonized by
infecting bacteria [32, 33].

Agents that inhibit the QS response of the infecting bacterium
are obviously important and hence, the search for such agents
that inhibit the QS system and biofilm formation has been in
effect for the past two decades [11-13].

There is a relationship between efflux pumps (EP), QS and
biofilm (BF) secretion which has come to the forefront only
recently [13]. Control of this relationship is critical for
successful therapy of MDR bacterial infections which have
become rather commonplace. It is the intent of this review
to identify agents which may serve to interfere with the
complex system of EP-QS-BF interaction.

Proton motive force (PMF) dependent transporters obtain
their energy for function from the proton motive force. The
proton motive force is the result of cellular metabolism which
yields protons that are not used for coupling with molecular
oxygen and which are exported to the surface of the cell [43-45]
where they are distributed and bound to components of
the protective lipopolysaccharide layer that covers the cell
and constitutes a part of the outer cell wall of Gram-negative
[46] and the cell wall of Gram positive bacteria [47].

The larger the concentration of protons (hydronium ions)
on the surface of the cell with respect to their lower
concentration on the medial side of the cytoplasmic
membrane creates an electrochemical gradient that
is termed the proton motive force (PMF) [48].

Because hydronium ions cannot penetrate the cell wall
or the membrane, they may re-enter the cell only
through channels such as porins in general [49, 50].
The movement of these hydromium ions from the
surface of the cell to the periplasm or cytoplasm is
predicated upon systems that use the PMF as source
of energy-namely the resistance nodulation division
(RND) family of transporters.

E. coli has a multiplicity of efflux pumps that may
exceed 30 in number [51]. However, the main
efflux pump of this organism is the AcrAB-TolC
efflux pump [52, 53] which when deleted, its
function is replaced by the AcrEF-TolC efflux
pump [51]. Both efflux pumps are members
of the resistance nodulation division family of
transporters [51] and consist of three proteins:

The transporter AcrB coded by the gene acrB and
is intimately attached to the plasma membrane;
Two fusion proteins AcrA coded by the gene acrA
that flank the AcrB transporter and are thought
to assist the movement of a substrate through
the AcrB transporter [35]; and,
TolC which is also part of other tri-unit efflux pumps
of the organism [35], is contiguous with the AcrB
transporter and provides a conduit for the extrusion
of the substrate [38].

Although the means for the recognition of the substrate to
be extruded appears to involve a pocket within the transporter,
it appears to be

defined by a phenyalanine residue [54].

Nevertheless, studies employing fluorochromes recognised by
the AcrB transporter indicate that the binding and release of
the substrate are pH dependent [55].

At low pH the dissociation of the substrate is high and
at high pH it is very slow.

In a physiological environment of ca. pH 7, if the dissociation
of the substrate is slow or not at all, then the effectiveness of
the pump to extrude a noxious agent would be nullified.
However, since the pump functions at this pH, conditions that
result in the dissociation of the substrate needed for continuous
pump action must involve a

decrease of the pH of the internal cavity of the pump
to which the substrate is bound.

It has been postulated that the lowering of the pH takes place
by the generation of hydronium ions from metabolism [6] which

pass from the cytoplasmic side of the plasma membrane
through the transporter.

At lower pH, there is no need for the generation of metabolically
derived hydronium ions since these ions can be

diverted by the PMF from the surface of the cell
to the periplasm via porins.

Whether hydronium ions are to be generated from the
hydrolysis of ATP at high pH or used for the synthesis
of ATP at low pH is a special

function of ATP synthase [56-58].

Model of the AcrAB-TolC efflux pump of a Gram-
negative bacterium

AcrAB-TolC efflux pump of a Gram-negative bacterium

Hypothesis. At near neutral pH, Hydronium ions from hydrolysis of ATP
by ATP synthase pass through the AcrB

transporter, reduce the pH to a point that causes the release of the
substrate. When the hydronium ions reach the surface of the cell they
are distributed over that surface and bind to lipopolysaccharides
and basic amino acids. When there is a need for hydronium ions for
activity of the efflux pump and the pH is lower than neutral, and
the hydrolysis of ATP is not favoured, hydronium ions from the
surface of cell via the PMF mobilize through the Aqua porins
and reach the transporter where they are pushed through
the transporter by the peristaltic action caused by the fusion
proteins. Substrates bound to the transporter dissociate
when the pH is reduced by the flow of hydronium ions and
are carried out by the flow of water.

Inhibitors of bacterial efflux pumps
Inhibitors of the QS of bacteria

Because phenothiazines inhibit many energy dependent systems
of bacteria such as motility [89, 90, 95], and these phenothiazines
also inhibit efflux pumps of bacteria [6, 7, 9, 41, 51, 73, 74, 76-83],
there seems to be a correlation between an active efflux pump
system and a functional QS system. That this assumption is correct,
recent evidence has been provided showing that the efflux pumps of
the AHL responding environmental Chromobacterium violaceum
(CV026) bacterium and that of E. coli are inhibited by the phenothiazine
thioridazine (TZ) [12]. Because TZ is known to inhibit genes that
regulate and code for efflux pumps of bacteria [41, 119, 120], it is
possible that the inhibition of the responding CV0126 bacterium to
AHLs [12] involves the inhibition of genes that code and regulate
the efflux pump of the responder which is assumed to recognise the
AHL signal as an noxious agent and hence would extrude it to the
environment [12]. The inhibition of an efflux pump should manifest
itself as an inhibitor of the QS component responsible for biofilm
formation.

Since the discovery of berberine a powerful inhibitor of bacterial
efflux pumps [159], plants have become sources of inhibitors of
efflux pumps [160-164]. Given that efflux pumps and the QS of
bacteria have an intimate relationship as described in this review,
attention has been focused on plants for potential sources of inhibitors
of efflux pumps and QS systems. Essential oils from Columbian
plants have yielded a large number of compounds that inhibit the
QS system of responding bacteria such as

limonene-carvone , the
citral (geranial-neral) (isolated from Lippia alba),
α-pinene (from Ocotea sp.),
β-pinene (from Swinglea glutinosa),
cineol (from Elettaria cardamomun),
α-zingiberene (from Zingiber officinale) and
pulegone (from Minthostachys mollis) [165].

Several other essential oils, in particular were shown to present
promising inhibitory properties for the short chain AHL quorum
sensing (QS) system in Escherichia coli containing the biosensor

plasmid pJBA132, in particular Lippia alba.

Citral was the only essential oil that presented some activity for
the long chain AHL QS system in Pseudomonas putida containing

the plasmid pRK-C12 [165].

The essence of this review is to correlate the relationship of the
efflux pump system to the QS system of bacteria via the use of
compounds that inhibit both systems. Simply put, inhibitors of
the efflux pump system also, when studied, inhibit the QS system
as well. Because the PMF dependent efflux pump system of Gram-
negatives that is overexpressed is responsible for the multi-drug
phenotype of the bacterium, compounds that affect the PMF of
the bacterium are candidates that will inhibit the activity of the
pump. Consequently, this inhibition will inhibit the secretion of
biofilm, and because biofilm is a deterrent to the action of antibiotics,
compounds that affect the efflux pump system are promising
candidates for clinical evaluation.

Limiting and controlling carbapenem-resistant
Klebsiella pneumonia

L Saidel-Odes, A Borer.
1Infection Control and Hospital Epidemiology Unit, 2Infectious
Diseases Institute, Soroka University Medical Center and the
Faculty of Health Sciences, Ben-Gurion University of the Negev,
Beer-Sheva, Israel
Infection and Drug Resistance 2014:7 9–14

Carbapenem-resistant Klebsiella pneumoniae (CRKP)

is resistant to almost all antimicrobial agents,
is associated with substantial morbidity and mortality, and
poses a serious threat to public health.

The ongoing worldwide spread of this pathogen emphasizes the
need for immediate intervention. This article reviews the global
spread and risk factors for CRKP colonization/infection, and
provides an overview of the strategy to combat CRKP dissemination

Outbreaks of CRKP that have occurred around the world have
been associated with the plasmid-encoded carbapenemase
K. pneumoniae carbapenemase (KPC),

a carbapenem-hydrolyzing β-lactamase.19

CRKP isolates are resistant to almost all available antimicrobials
and are susceptible

only to polymyxins and tigecycline;
a minority to the few remaining aminoglycosides,
though resistance to these agents is increasingly reported.20,21

Several investigators have evaluated predictors for CRKP colonization.
The following summarizes various studies.

In a multivariate analysis, prior use of macrolides and
any antibiotic exposure $14 days remained the only
independent factors associated with CRKP bacteremia
Nosocomial isolation of CRKP was strongly favored by the
selection pressure of carbapenem. In this study, prior
treatment with fluoroquinolones was associated with
decreased risk for the emergence of CRKP.
Previous use of carbapenem and cephalosporin
Nursing home residency before hospital admission, bedridden
status, and previous antibiotic therapy
exposure to fluoroquinolones
the recipient of antibiotics
intensive care unit (ICU) stay, and
Poor functional status,
Independent predictors of subsequent carbapenem-
resistant Enterobacteriaceae (CRE) infection were

admission to the ICU,
having a central venous catheter,
receipt of antibiotics, and
diabetes mellitus

Schwaber et al and the Israeli CRE Working Group enforced the
Israel Ministry of Health guidelines mandating physical separation
of hospitalized carriers of CRE and dedicated staffing and appointed
a professional task force charged with containment.19 The monthly
incidence of nosocomial CRE was reduced from 55.5 to 11.7 cases
per 100,000 patient days within 15 months.

Part 7. Tuberculosis

The Mechanism by which the Phenothiazine Thioridazine
Contributes to Cure Problematic Drug-Resistant Forms
of Pulmonary Tuberculosis: Recent Patents for “New Use”

L Amaral1*, A Martins2,3, G Spengler2, A Hunyadi4 and J Molnar2
Recent Patents on Anti-Infective Drug Discovery 2013; 8(3):000-000

At this moment, over half million patients suffer from multi-drug
resistant tuberculosis (MDR-TB) according to the data from the WHO.
A large majority is terminally ill with essentially incurable pulmonary
tuberculosis. This herein mini-review provides the experimental and
observational evidence that a specific phenothiazine,

thioridazine,

will contribute to cure any form of drug-resistant tuberculosis. This
antipsychotic agent is no longer under patent protection for its
initial use. The reader is informed on the recent developments

in patenting this compound for “new use” with a special
emphasis on the aspects of drug-resistance.

Given that economic motivation can stimulate the use of this drug
as an antitubercular agent, future prospects are also discussed.

Thioridazine is not the only phenothiazine that has been recommended
for therapy of pulmonary tuberculosis. In general, many phenothiazines
have been implicated for antitubercular activity [62, 80-86]. Among
these are

trifluoperazine [87-94],
methdilazine [95, 96],
promazine [97, 98],
promethazine [97, 98],
fluphenazin [99],
propiomazine [100], and
the methylene blue related toluidine blue [101].

There are phenothiazine compounds derived from the parental
methylene blue for therapy of pathologies unrelated to tuberculosis
that also possess

antitubercular [44, 48] and/or antimalarial properties [44].

Moreover, derivatives made from any of the phenothiazines that
have in vitro activity against Mycobacterium tuberculosis are also
active [61, 67, 102, 103], suggesting ample opportunities for
patenting of new analogs developed from known, active phenothiazines
with even less side effects than those of TZ, as recently suggested by
Musuka and co-authors [104]. It is important to mention, that the
commercially available phenothiazines such as for example

trifluoperazine, methdilazine, promazine, promethazine,
fluphenazin and propiomazine

are beyond patent protection as initially intended. Nevertheless,
these compounds have been patented as adjuvants for the treatment
of MDR cancer (patent expired in 2011 [105]; and, right afterwards,
a new patent has been filed with a priority date of 28th March, 2012,
claiming combination therapy of cancer with a chemotherapeutic
agent and a dopamine receptor antagonist against Cancer stem cells (CSC).

Taking into account that intrinsic MDR is considered as one of the key
properties of CSCs [107], the subject to be covered is indeed related.
According to the MDR, XDR and TDR Mycobacterium tuberculosis,
subjects of this herein paper, the initial step for actually reaching those
in need has been made: a patent has been published in December, 2007,
for the use of TZ and its derivatives for reversing anti-microbial drug
resistance [108]. We must note, however, that, despite the six years
passed since, we were unable to find any related clinical trials, which
would certainly be of outmost importance and urgency in order to
proceed towards an effective therapy of highly resistant mycobacterial
infections.

Mechanism Of Action Of Tz: Why It Cures Multi-Drug,
Extensively Drug Resistant And Probably Totally Drug
Resistant Tuberculosis

Over-expressed efflux pumps of Mycobacterium tuberculosis render
the organism multi-drug resistant [13]. Special attention has been
given to those coded by the

mmpL7, p55, efpA, mmr, Rv1258c and Rv2459 genes [109].

The activity of these efflux pumps can be suppressed by

concentrations of TZ that have no effect on the viability of
Mycobacterium tuberculosis
rendering the organism susceptible to the antibiotic to
which it was initially resistant
as a consequence of the over-expression of its
efflux pumps [109].

TZ has also been shown to inhibit the activity of the main

efflux pumps of bacteria belonging to other species.

TZ has strong inhibitory activity against the genes that code for
essential proteins of M. tuberculosis [122-124]. Consequently, we
may conclude that the in vitro activity of TZ involves

the inhibition of the efflux pumps of M. tuberculosis and that
the in vitro exposure of this organism to TZ renders the organism
susceptible to antibiotics to which it was initially resistant
as a consequence of over-expressed efflux pumps [21].

Phenothiazines such as CPZ, TZ, trifluoperazine, etc., also inhibit

the binding of calcium to calcium binding proteins such as

calmodulin in eukaryotes [125], and

interfere with other proteins involved in
the regulation of cellular activity [126].

They inhibit the transport of calcium and potassium systems

in eukaryotic cells [127-129] as well as in
mycobacteria [89, 130] and
E. coli [113].

In fact, in the latter case, calcium was shown essential to

the continuous activity of the thioridiazine sensitive
efflux system [113].

The killing activity of the human macrophage as well as that
of the neutrophil

is dependent upon the retention of calcium and potassium
within the phagolysosome of the cell [131].

Considering this, several alternative choices are available for
patenting under “new use”, which would allow a “fresh start”
for the compound to be developed. However, the needed
experimental proof that these phenothiazine agents have
activity at the pulmonary macrophage of the alveolar unit
(the site where the causative organism of pulmonary tuberculosis
resides) is still absent.

Targeting the Human Macrophage with Combinations
of Drugs and Inhibitors of Ca2+ and K+ Transport to
Enhance the Killing of Intracellular Multi-Drug Resistant
M. tuberculosis (MDR-TB) – a Novel, Patentable Approach
to Limit the Emergence of XDR-TB

Marta Martins
UCD Centre for Food Safety, School of Agriculture, Food Science and
Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
& Unit of Mycobacteriology and UPMM; Instituto de Higiene e Medicina
Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal
Recent Patents on Anti-Infective Drug Discovery, 2011, 6, 000-000

The emergence of resistance in Tuberculosis has become a serious
problem for the control of this disease. For that reason, new therapeutic
strategies that can be implemented in the clinical setting are urgently
needed. The design of new compounds active against mycobacteria
must take into account that Tuberculosis is mainly an intracellular
infection of the alveolar macrophage and therefore must maintain
activity within the host cells.

An alternative therapeutic approach will be described in this review,
focusing on the activation of the phagocytic cell and the subsequent
killing of the internalized bacteria. This approach explores the combined
use of antibiotics and phenothiazines, or Ca2+ and K+ flux inhibitors,
in the infected macrophage.

Targeting the infected macrophage and not the internalized bacteria
could overcome the problem of bacterial multi-drug resistance. This
will potentially eliminate the appearance of new multi-drug resistant
tuberculosis (MDR-TB) cases and subsequently prevent the emergence
of extensively-drug resistant tuberculosis (XDR-TB).

Patents resulting from this novel and innovative approach could be
extremely valuable if they can be implemented in the clinical setting.
Other patents will also be discussed such as the treatment of TB
using immunomodulator compounds (for example: betaglycans).

Role of Phenothiazines and Structurally Similar
Compounds of Plant Origin in the Fight against
Infections by Drug Resistant Bacteria

SG. Dastidar 1, JE. Kristiansen 2, J Molnar 3 and L Amaral
Antibiotics 2013; 2: 58-71;
http://dx.doi.org:/10.3390/antibiotics2010058

Phenothiazines have their primary effects on the plasma membranes
of prokaryotes and eukaryotes. Among the components of the
prokaryotic plasma membrane affected are

efflux pumps,
their energy sources
and energy providing enzymes, such as ATPase,
and genes that regulate and code for the permeability
aspect of a bacterium.

The response of multidrug and extensively drug resistant
tuberculosis to phenothiazines shows an alternative therapy for the
treatment of these dreaded diseases, which are claiming more and
more lives every year throughout the world.

Many phenothiazines have shown

synergistic activity with several antibiotics thereby
lowering the doses of antibiotics administered to patients
suffering from specific bacterial infections.

Trimeprazine is synergistic with trimethoprim. Flupenthixol (Fp)
has been found to be synergistic with penicillin and chlorpromazine
(CPZ); in addition, some antibiotics are also synergistic. Along with
the antibacterial action described in this review,

many phenothiazines possess plasmid curing activities, which
render the bacterial carrier of the plasmid sensitive to antibiotics.

Thus, simultaneous applications of a phenothiazine like TZ would not
only act as an additional antibacterial agent but also would help

to eliminate drug resistant plasmid from
the infectious bacterial cells.

Part 8. Cancer Cytotherapy

Synthesis and Structure-Activity Relationships of Novel
Dioxolanes as MDR Modulators in Cancer

A Martins 1,2,†,*, J Csábi 3,†, A Balázs 4, DKitka 1, L Amaral 5,
J Molnár 1, A Simon 6, G Tóth 6 and A Hunyadi 3,
Molecules 2013, 18, 15255-15275;
http://dx.doi.org:/10.3390/molecules181215255

Ecdysteroids, molting hormones of insects, can exert several mild,
non-hormonal bioactivities in mammals, including humans. In a
previous study, we have found a significant effect of certain derivatives

on the ABCB1 transporter mediated multi-drug resistance of a
transfected murine leukemia cell line.

In this paper, we present a structure-activity relationship study
focused on

the apolar dioxolane derivatives of 20-hydroxyecdysone.

Semi-synthesis and bioactivity of a total of 32 ecdysteroids, including
20 new compounds, is presented, supplemented with their

complete 1H- and 13C-NMR signal assignment

As published before [9], the 20,22-diol moiety of 20E is more reactive
than the 2,3-diol, probably due to the free rotation of the 20,22-bond
of 20E that allows the 20,22-dioxolane ring to form with less strain.

This allowed us to selectively obtain the 20,22-mono-dioxolane
derivatives 2–14, or, depending on the amount of reagent and the
reaction time, the 2,3;20,22-bis-homo-dioxolanes 17 and 21–25.

By utilizing the 20,22-monodioxolane ecdysteroids, another aldehyde
or ketone could be coupled to position 2,3, resulting in several bis-hetero-
dioxolane derivatives 26–33. For this, however, gradually decreasing
reactivity with the increase of the size of the reagent was a limiting factor:

larger aldehydes or ketones (mainly those containing a
substituted aromatic ring) could not be coupled at the 2,3-position.
The 2,3-monodioxolane derivatives also appeared to be present as
minor side-products of the reactions, and as a consequence of their
low amount, only one such compound (compound 15) was isolated and studied.

To selectively obtain this kind of a compound (16) in a more reasonable
yield, another, three-step approach was successfully applied:

after protecting the 20,22-diol with phenylboronic acid, the
2,3-acetonide could be prepared, and
removal of the 20,22 protecting group afforded the desired
2,3-monoacetonide in a one-pot procedure.

In the case of the reactions with aldehydes or asymmetric ketones,
the new C-28 and C-29 central atoms of the dioxolane rings are
stereogenic centers and thus two possible diastereomers can be
formed at both diols. Their configuration was elucidated by two-
dimensional ROESY or selective one-dimensional ROESY experiments,
e.g., in the doubly substituted

dioxolane derivative 22 (R1 = R4 = n-Bu, R2 = R3 = H)
the unambiguous differentiation of the 1H and 13C signals of
the two n-butyl groups was achieved in the following way
(see Figure 2).

Assignment of the H-C(28) atoms (δ = 4.93/105.9 ppm) was supported by

the H-2/C-28 and H-3/C-28 HMBC correlations, and
that of H-C(29) (δ = 4.91/105.6 ppm) by the H-22/C-29
cross peak, respectively.

The selective ROESY experiment irradiating at 4.93 ppm showed

contacts with the Hα-2 and Hα-3 atoms proving the
α position of the R2 = H atom.

The ROESY response obtained irradiating H = R3 signal (δ = 4.91)
on H-22 (δ = 3.64 ppm) revealed their

cis arrangement and the R configuration around C-29.

The unambiguous assignments of the signals

of the two n-butyl groups R1 and R4 were achieved by
selective TOCSY experiments (irradiation at
δ = 4.93 and 4.91, respectively).

Figure 2

Stereostructure of 22. Red-ROESY proximitries. Blue- 1H. Black-1 001

Stereostructure of 22. Red arrows indicate the detected ROESY
steric proximities, the blue numbers give the characteristic 1H,
and the black numbers the 13C chemical shifts.

Related Material

Identification of Efflux Pump-mediated Multidrug-resistant
Bacteria by the Ethidium Bromide-agar Cartwheel Method
M Martins, M Viveiros, I Couto, SS. Costa, T Pacheco, S Fanning,
Jean-Marie Pagès, and L Amaral
in vivo 2011; 25: 171-178

Index for efflux activity of the MDR strains. The capacity to efflux EtBr
of each bacterial strain was ranked relative to the reference strain
according to the following formula:

Index for efflux activity of the MDR strains

A Simple Method for Assessment of MDR Bacteria for
Over-Expressed Efflux Pumps
M Martinsa,b*, MP. McCuskera,b, M Viveirosa,c, I Coutoc,d,
S Fanninga,b, Jean-Marie Pagès b,e, L Amaral,b,
The Open Microbiol J 2013; 7: 1-5 1874-2858/13 Bentham

Flowchart followed to test bacterial strains using the EtBr-agar
Cartwheel method.

Flowchart followed to test bacterial strains using the EtBr-agar Cartwheel method.

EtBr-agar cartwheel method applied to different bacterial species

The effect of selected EPIs on the resistance of the induced and
MDR Gram-positive bacteria.

					TET
Enterococcus	EFC ATCC29212 HSEFM-D	1.5 >2.5	w/o EPI	+ TZ	+ CPZ	+ RES

			4 16	4 4	4 4	4 8
				(4×)	(4×)	(2×)
MCEtBr NOR (mg/l)				MIC NOR (mg/l)
	HSEFM-E	>2.5	0.125	0.125	0.125	0.125

EPI: Efflux pump inhibitor; w/o: without; TZ: thioridazine; CPZ:
chlorpromazine; PAN: phenyl arginine β-naphthylamide. Values
in bold-type correspond to a decrease of 4-fold or higher on
the MIC values in comparison to those in the absence of inhibitor.
Values in parenthesis indicate the MIC decrease relative to that
of the original culture. The concentration of each EPI used is
defined in the Materials and Methods section.

Macrocyclic diterpenes resensitizing multidrug
resistant phenotypes
MA. Reis a, A Paterna a, RJ. Ferreira a, H Lage b,
Maria-José U. Ferreira a,⇑
a Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade
de Farmácia, Universidade de Lisboa, Lisboa, Portugal
b Charité Campus Mitte, Institute of Pathology, Berlin, Germany
Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx

Herein, collateral sensitivity effect was exploited as a strategy to
select effective compounds to overcome multidrug resistance in
cancer. Thus, eleven macrocyclic diterpenes, namely jolkinol D (1),
isolated from Euphorbia piscatoria, and its derivatives (2–11) were
evaluated for their activity on three different Human cancer entities:

gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29)

each with a variant selected for resistance to mitoxantrone

EPG85-257RN;
EPP85-181RN;
HT-29RN and

one to daunorubicin (EPG85-257RD; EPP85-181RD; HT-29RD).

Jolkinol D (1) and most of its derivatives (2–11) exhibited significant
collateral sensitivity effect towards the cell lines

EPG85-257RN (associated with P-glycoprotein overexpression) and
HT-29RD (altered topoisomerase II expression).

The benzoyl derivative, jolkinoate L (8) demonstrated ability to

target different cellular contexts with
concomitant high antiproliferative activity.

These compounds were previously assessed as
P-glycoprotein modulators,

at non-cytotoxic doses, on MDR1-mouse lymphoma cells.

A regression analysis between

the antiproliferative activity presented herein and
the previously assessed P-glycoprotein modulatory effect

showed a strong relation between the compounds that presented

both high P-glycoprotein modulation and cytotoxicity.

Molecular Docking Characterizes Substrate-Binding Sites
and Efflux Modulation Mechanisms within P‑Glycoprotein.

Ferreira,† Maria-José U. Ferreira,† and DJVA dos Santos*,†,‡
†Research Institute for Medicines and Pharmaceutical Sciences
(iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
‡REQUIMTE, Department of Chemistry & Biochemistry, Faculty of
Sciences, University of Porto, Porto, Portugal
J. Chem. Inf. Model. XXXX, XXX, XXX−XXX
http://dx.doi.org:/10.1021/ci400195v

P-Glycoprotein (Pgp) is one of the best characterized ABC
transporters, often involved

in the multidrug-resistance phenotype
overexpressed by several cancer cell lines.

Experimental studies contributed to important knowledge concerning
substrate polyspecificity, efflux mechanism, and drug binding sites.
This information is, however, scattered through different perspectives,
not existing a unifying model for the knowledge available for this transporter.
Using a previously refined structure of murine Pgp,

three putative drug-binding sites were hereby characterized
by means of molecular docking.

The modulator site (M-site) is characterized by

cross interactions between both Pgp halves

herein defined for the first time, having an important role in

impairing conformational changes leading to substrate efflux.

Two other binding sites, located next to the inner leaflet of the lipid bilayer,

were identified as the substrate binding H and R sites
by matching docking and experimental results.

A new classification model

with the ability to discriminate substrates from modulators

is also proposed, integrating a vast number of theoretical and experimental data.

conformational changes leading to substrate efflux

http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jcisd8/
2013/jcisd8.2013.53.issue-7/ci400195v/production/pdfimages_v02/normal.img-000.jpg

Read Full Post »

Metformin, Thyroid-Pituitary Axis, Diabetes Mellitus, and Metabolism

Posted in Amino acids, Anaerobic Glycolysis, Best evidence, Biochemical pathways, Ca2+ triggered activation, Calcium Signaling, Calmodulin Kinase and Contraction, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Clinical Diagnostics, Curation, Curation methodology, Cytoskeleton, Dehydrogenase, Developmental biology, Discovery process, Disease Biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Endocrine Diseases, Endothelial cells, Enzyme Induction, Enzymes and isoenzymes, Evidence-based decision-making, Experimental validation, Explanatory, Fatty acids, Gene Regulation, Genomic Expression, Hexokinase, Historical relevance, Inferential analysis, Kinase, Lipid metabolism, Liver & Digestive Diseases Research, Metabolism, Metabolomics, Myocardial Ischemia, Myocardial Metabolism, Nitric Oxide in Health and Disease, Nutrition, Nutrition and Phytochemistry, Nutritional Supplements: Atherogenesis, Oxidative phosphorylation, Pharmaceutical Analytics, Pharmaceutical Discovery, Phosphatase, Phosphorylase, Phosphorylation, Plant extract, Population Health Management, Population Health Management, Nutrition and Phytochemistry, Proteins, Proteomics, Signaling, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, Statistical Methods for Research Evaluation, Systemic Inflammatory Response Related Disorders, Theoretic convergence, Transcriptomics, Translational Effectiveness, Translational Research, Translational Science, Warburg effect, tagged AMP, AMP activation, AMPK, AMPKK, ATP, cell metabolic regulation, conformational change, Diabetes Mellitus, electron transport chain, euthyroid, Glycolysis, hypothyroid, metformin, mitochondria, natural dessicated thyroid (NDT), Phosphorylation, protein conformation, signaling pathways, synthroid, T3 on September 28, 2014| Leave a Comment »

Metformin, Thyroid-Pituitary Axis, Diabetes Mellitus, and Metabolism

Larry H, Bernstein, MD, FCAP, Author and Curator
and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/9/27/2014/Metformin,_thyroid-pituitary_ axis,_diabetes_mellitus,_and_metabolism

The following article is a review of the central relationship between the action of
metformin as a diabetic medication and its relationship to AMPK, the important and
essential regulator of glucose and lipid metabolism under normal activity, stress, with
its effects on skeletal muscle, the liver, the action of T3 and more.

We start with a case study and a publication in the J Can Med Assoc. Then we shall look
into key literature on these metabolic relationships.

Part I. Metformin , Diabetes Mellitus, and Thyroid Function

Hypothyroidism, Insulin resistance and Metformin
May 30, 2012 By Janie Bowthorpe
The following was written by a UK hypothyroid patient’s mother –
Sarah Wilson.

My daughter’s epilepsy is triggered by unstable blood sugars. And since taking
Metformin to control her blood sugar, she has significantly reduced the number of
seizures. I have been doing research and read numerous academic medical journals,
which got me thinking about natural thyroid hormone and Hypothyroidism. My hunch
was that when patients develop hypothyroid symptoms, they are actually becoming
insulin resistant (IR). There are many symptoms in common between women with
polycystic ovaries and hypothyroidism–the hair loss, the weight gain, etc.
(http://insulinhub.hubpages.com/hub/PCOS-and-Hypothyroidism).

A hypothyroid person’s body behaves as if it’s going into starvation mode and so, to
preserve resources and prolong life, the metabolism changes. If hypothyroid is prolonged
or pronounced, then perhaps, chemical preservation mode becomes permanent even
with the reintroduction of thyroid hormones. To get back to normal, they need
a “jump-start” reinitiate a higher rate of metabolism. The kick start is initiated through
AMPK, which is known as the “master metabolic regulating enzyme.”
(http://en.wikipedia.org/wiki/AMP-activated protein kinase).

Guess what? This is exactly what happens to Diabetes patients when Metformin is
introduced. http://en.wikipedia.org/wiki/Metformin
Suggested articles: http://www.springerlink.com/content/r81606gl3r603167/ and
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2265.2011.04029.x/pdf

Note the following comments/partial statements:
“Hypothyroidism is characterized by decreased insulin responsiveness”;
“the pivotal regulatory role of T3 in major metabolic pathways”.

The community knows that T3/NTH (natural thyroid hormone [Armour]) makes
hypothyroid patients feel better – but the medical establishment is averse to T3/NTH
(treating subclinical hypoT (T3/T4 euthyroid) with natural dessicated thyroid (NDT).
The medical establishment might find an alternative view about impaired metabolism
more if shown real proof that the old NDT **was/is** having the right result –i.e., the
T3 is jump-starting the metabolism by re-activating AMPK.

If NDT also can be used for hypothyroidism without the surmised “dangers” of NTH,
then they should consider it. [The reality in the choice is actually recombinant TH
(Synthroid)]. Metformin is cheap, stable and has very few serious side effects. I use the
car engine metaphor, and refer to glucose as our petrol, AMPK as the spark plug and
both T3 and Metformin as the ignition switches. Sometimes if you have flat batteries in
the car, it doesn’t matter how much you turn the ignition switch or pump the petrol
pedal, all it does is flatten the battery and flood the engine.

Dr. Skinner in the UK has been treating “pre-hypothyroidism” the way that some
doctors treat “pre-diabetes”. Those hypothyroid patients who get treated early
might not have had their AMPK pathways altered and the T4-T3 conversion still works.
There seems to be no reason why thyroid hormone replacement therapy shouldn’t
logically be given to ward off a greater problem down the line.

It’s my belief that there is clear and abundant academic evidence that the AMPK/
Metformin research should branch out to also look at thyroid disease.

Point – direct T3 is kicking the closed -down metabolic process back into life,
just like Metformin does for insulin resistance.
http://www.hotthyroidology.com/editorial_79.html
There is serotonin resistance! http://www.ncbi.nlm.nih.gov/pubmed/17250776

Metformin Linked to Risk of Low Levels of Thyroid Hormone

CMAJ (Canadian Medical Association Journal) 09/22/2014

Metformin, the drug commonly for treating type 2 diabetes,

is linked to an increased risk of low thyroid-stimulating hormone
(TSH) levels
in patients with underactive thyroids (hypothyroidism),

according to a study in CMAJ (Canadian Medical Association Journal).

Metformin is used to lower blood glucose levels

by reducing glucose production in the liver.

previous studies have raised concerns that

metformin may lower thyroid-stimulating hormone levels.

Study characteristics:

Retrospective long-term
74 300 patient who received metformin and sulfonylurea
25-year study period.
5689 had treated hypothyroidism
59 937 had normal thyroid function.

Metformin and low levels of thyroid-stimulating hormone in
patients with type 2 diabetes mellitus

Jean-Pascal Fournier, Hui Yin, Oriana Hoi Yun Yu, Laurent Azoulay +
Centre for Clinical Epidemiology (Fournier, Yin, Yu, Azoulay), Lady Davis Institute,
Jewish General Hospital; Department of Epidemiology, Biostatistics and Occupational
Health (Fournier), McGill University; Division of Endocrinology (Yu), Jewish General
Hospital; Department of Oncology (Azoulay), McGill University, Montréal, Que., Cananda

CMAJ Sep 22, 2014, http://dx.doi.org:/10.1503/cmaj.140688

Background:

metformin may lower thyroid-stimulating hormone (TSH) levels.

Objective:

determine whether the use of metformin monotherapy, when compared with
sulfonylurea monotherapy,
is associated with an increased risk of low TSH levels(< 0.4 mIU/L)
in patients with type 2 diabetes mellitus.

Methods:

Used the Clinical Practice Research Datalink,
identified patients who began receiving metformin or sulfonylurea monotherapy
between Jan. 1, 1988, and Dec. 31, 2012.
2 subcohorts of patients with treated hypothyroidism or euthyroidism,

followed them until Mar. 31, 2013.

Used Cox proportional hazards models to evaluate the association of low TSH
levels with metformin monotherapy, compared with sulfonylurea monotherapy,
in each subcohort.

Results:

5689 patients with treated hypothyroidism and 59 937 euthyroid patients were
included in the subcohorts.

For patients with treated hypothyroidism:

495 events of low TSH levels were observed (incidence rate 0.1197/person-years).
322 events of low TSH levels were observed (incidence rate 0.0045/person-years)
in the euthyroid group.

metformin monotherapy was associated with a 55% increased risk of low TSH
levels in patients with treated hypothyroidism (incidence rate 0.0795/person-years
vs.0.1252/ person-years, adjusted hazard ratio [HR] 1.55, 95% confidence
interval [CI] 1.09– 1.20), compared with sulfonylurea monotherapy,
the highest risk in the 90–180 days after initiation (adjusted HR 2.30, 95% CI
1.00–5.29).
No association was observed in euthyroid patients (adjusted HR 0.97, 95% CI 0.69–1.36).

Interpretation: The clinical consequences of this needs further investigation.

Crude and adjusted hazard ratios for suppressed thyroid-stimulating hormone levels (< 0.1 mIU/L) associated with the use metformin monotherapy, compared with sulfonylurea monotherapy, in patients with treated hypothyroidism or euthyroidism and type 2 diabetes
Variable	No. events suppressed TSH levels	Person-years of exposure	Incidence rate, per 1000 person-years (95% CI)	Crude HR	Adjusted HR*(95% CI)
Patients with treated hypothyroidism, n = 5689
Sulfonylure, n = 762	18	503	35.8 (21.2–56.6)	1.00	1.00 (reference)
Metformin, n = 4927	130	3 633	35.8 (29.9–42.5)	1.05	0.99 (0.57–1.72)
Euthyroid patients, n = 59 937
Sulfonylurea, n = 7980	12	8 576	1.4 (0.7–2.4)	1.00	1.00 (reference)
Metformin, n = 51 957	75	63 047	1.2 (0.9–1.5)	0.85	1.03 (0.52–2.03)

Part II. Metabolic Underpinning
(Source: Wikipedia, AMPK and thyroid)

5′ AMP-activated protein kinase or AMPK or 5′ adenosine monophosphate-activated protein kinase
is an enzyme that plays a role in cellular energy homeostasis.
It consists of three proteins (subunits) that

together make a functional enzyme, conserved from yeast to humans.
It is expressed in a number of tissues, including the liver, brain, and skeletal
muscle.
The net effect of AMPK activation is stimulation of
1. hepatic fatty acid oxidation and ketogenesis,
2. inhibition of cholesterol synthesis,
3. lipogenesis, and triglyceride synthesis,
4. inhibition of adipocyte lipolysis and lipogenesis,
5. stimulation of skeletal muscle fatty acid oxidation and muscle
  glucose uptake, and
6. modulation of insulin secretion by pancreatic beta-cells.

The heterotrimeric protein AMPK is formed by α, β, and γ subunits. Each of these three
subunits takes on a specific role in both the stability and activity of AMPK.

the γ subunit includes four particular Cystathionine beta synthase (CBS) domains
giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio.
The four CBS domains create two binding sites for AMP commonly referred to as
Bateman domains. Binding of one AMP to a Bateman domain cooperatively
increases the binding affinity of the second AMP to the other Bateman domain.
As AMP binds both Bateman domains the γ subunit undergoes a conformational
change which exposes the catalytic domain found on the α subunit.
It is in this catalytic domain where AMPK becomes activated when
phosphorylation takes place at threonine-172by an upstream AMPK kinase
(AMPKK). The α, β, and γ subunits can also be found in different isoforms.

AMPK acts as a metabolic master switch regulating several intracellular systems

the cellular uptake of glucose,
the β-oxidation of fatty acids and
the biogenesis of glucose transporter 4 (GLUT4) and
mitochondria

The energy-sensing capability of AMPK can be attributed to

its ability to detect and react to fluctuations in the AMP:ATP ratio that take
place during rest and exercise (muscle stimulation).

During muscle stimulation,

AMP increases while ATP decreases, which changes AMPK into a good substrate
for activation.
AMPK activity increases while the muscle cell experiences metabolic stress
brought about by an extreme cellular demand for ATP.
Upon activation, AMPK increases cellular energy levels by
- inhibiting anabolic energy consuming pathways (fatty acid synthesis,
  protein synthesis, etc.) and
- stimulating energy producing, catabolic pathways (fatty acid oxidation,
  glucose transport, etc.).

A recent JBC paper on mice at Johns Hopkins has shown that when the activity of brain
AMPK was pharmacologically inhibited,

the mice ate less and lost weight.

When AMPK activity was pharmacologically raised (AICAR see below)

the mice ate more and gained weight.

Research in Britain has shown that the appetite-stimulating hormone ghrelin also
affects AMPK levels.

The antidiabetic drug metformin (Glucophage) acts by stimulating AMPK, leading to

reduced glucose production in the liver and
reduced insulin resistance in the muscle.

(Metformin usually causes weight loss and reduced appetite, not weight gain and
increased appetite, ..opposite of expected from the Johns Hopkins mouse study results.)

Triggering the activation of AMPK can be carried out provided two conditions are met.

First, the γ subunit of AMPK

must undergo a conformational change so as to
expose the active site(Thr-172) on the α subunit.

The conformational change of the γ subunit of AMPK can be accomplished

under increased concentrations of AMP.

Increased concentrations of AMP will

give rise to the conformational change on the γ subunit of AMPK
as two AMP bind the two Bateman domains located on that subunit.
It is this conformational change brought about by increased concentrations
of AMP that exposes the active site (Thr-172) on the α subunit.

This critical role of AMP is further substantiated in experiments that demonstrate

AMPK activation via an AMP analogue 5-amino-4-imidazolecarboxamide
ribotide (ZMP) which is derived fromthe familiar
5-amino-4-imidazolecarboxamide riboside (AICAR)

AMPK is a good substrate for activation via an upstream kinase complex, AMPKK
AMPKK is a complex of three proteins,

STE-related adaptor (STRAD),
mouse protein 25 (MO25), and
LKB1 (a serine/threonine kinase).

The second condition that must be met is

the phosphorylation/activation of AMPK on its activating loop at
Thr-172of the α subunit
brought about by an upstream kinase (AMPKK).

The complex formed between LKB1 (STK 11), mouse protein 25 (MO25), and the
pseudokinase STE-related adaptor protein (STRAD) has been identified as

the major upstream kinase responsible for phosphorylation of AMPK
on its activating loop at Thr-172

Although AMPK must be phosphorylated by the LKB1/MO25/STRAD complex,

it can also be regulated by allosteric modulators which
directly increase general AMPK activity and
modify AMPK to make it a better substrate for AMPKK
and a worse substrate for phosphatases.

It has recently been found that 3-phosphoglycerate (glycolysis intermediate)

acts to further pronounce AMPK activation via AMPKK

Muscle contraction is the main method carried out by the body that can provide
the conditions mentioned above needed for AMPK activation

As muscles contract, ATP is hydrolyzed, forming ADP.
ADP then helps to replenish cellular ATP by donating a phosphate group to
another ADP,
- forming an ATP and an AMP.
As more AMP is produced during muscle contraction,
- the AMP:ATP ratio dramatically increases,
leading to the allosteric activation of AMPK

For over a decade it has been known that calmodulin-dependent protein kinase
kinase-beta (CaMKKbeta) can phosphorylate and thereby activate AMPK,

but it was not the main AMPKK in liver.

CaMKK inhibitors had no effect on 5-aminoimidazole-4-carboxamide-1-beta-4-
ribofuranoside (AICAR) phosphorylation and activation of AMPK.

AICAR is taken into the celland converted to ZMP,
an AMP analogthat has been shown to activate AMPK.

Recent LKB1 knockout studies have shown that without LKB1,

electrical and AICAR stimulation of muscleresults in very little
phosphorylation of AMPK and of ACC, providing evidence that
LKB1-STRAD-MO25 is the major AMPKK in muscle.

Two particular adipokines, adiponectin and leptin, have even been demonstrated
to regulate AMPK. A main functions of leptin in skeletal muscle is

the upregulation of fatty acid oxidation.

Leptin works by way of the AMPK signaling pathway, and adiponectin also
stimulates the oxidation of fatty acids via the AMPK pathway, and

Adiponectin also stimulates the uptake of glucose in skeletal muscle.

An increase in enzymes which specialize in glucose uptake in cells such as GLUT4
and hexokinase II are thought to be mediated in part by AMPK when it is activated.
Increases in AMPK activity are brought about by increases in the AMP:ATP ratio
during single bouts of exercise and long-term training.

One of the key pathways in AMPK’s regulation of fatty acid oxidation is the

phosphorylation and inactivation of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase (ACC) converts acetyl-CoA (ACA) to malonyl-CoA
(MCA), an inhibitor of carnitine palmitoyltransferase 1 (CPT-1).
CPT-1 transports fatty acids into the mitochondria for oxidation.
Inactivation of ACC results in increased fatty acid transport and oxidation.
the AMPK induced ACC inactivation and reduced conversion to MCA
may occur as a result of malonyl-CoA decarboxylase (MCD)
MCD as an antagonist to ACC, decarboxylatesmalonyl-CoA to acetyl-CoA
(reversal of ACC conversion of ACA to MCA)
This resultsin decreased malonyl-CoA and increased CPT-1 and fatty acid oxidation.

AMPK also plays an important role in lipid metabolism in the liver. It has long been
known that hepatic ACC has been regulated in the liver.

It phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)
acetyl-CoA(ACA) is converted to mevalonic acid (MVA) by ACC
with inhibition of CPT-1
HMGR converts 3-hydroxy-3-methylglutaryl-CoA, which is made from MVA
which then travels down several more metabolic steps to become cholesterol.

Insulin facilitates the uptake of glucose into cells via increased expression and
translocation of glucose transporter GLUT-4. In addition, glucose is phosphorylated
by hexokinase wheni iot enters the cell. The phosphorylated form keeps glucose from
leaving the cell,

The decreasedthe concentration of glucose molecules creates a gradient for more
glucose to be transported into the cell.

AMPK and thyroid hormone regulate some similar processes. Knowing these similarities,
Winder and Hardie et al. designed an experiment to see if AMPK was influenced by thyroid
hormone. They found that all of the subunits of AMPK were increased in skeletal muscle,
especially in the soleus and red quadriceps, with thyroid hormone treatment. There was
also an increase in phospho-ACC, a marker of AMPK activity.

Winder WW, Hardie DG (July 1999). “AMP-activated protein kinase,
a metabolic master switch: possible roles in type 2 diabetes”. J. Physiol. 277
(1 Pt 1): E1–10. PMID 10409121.
Winder WW, Hardie DG (February 1996). “Inactivation of acetyl-CoA
carboxylase and activation of AMP-activated protein kinase in muscle
during exercise”. J. Physiol. 270 (2 Pt 1): E299–304. PMID 8779952.
Hutber CA, Hardie DG, Winder WW (February 1997). “Electrical stimulation
inactivates muscle acetyl-CoA carboxylase and increases AMP-activated
protein kinase”. Am. J. Physiol. 272 (2 Pt 1): E262–6. PMID 9124333
Durante PE, Mustard KJ, Park SH, Winder WW, Hardie DG (July 2002).
“Effects of endurance training on activity and expression of AMP-activated
protein kinase isoforms in rat muscles”. Am. J. Physiol. Endocrinol.
Metab. 283 (1): E178–86. doi:10.1152/ajpendo.00404.2001. PMID 12067859
Corton JM, Gillespie JG, Hardie DG (April 1994). “Role of the AMP-activated
protein kinase in the cellular stress response”. Curr. Biol. 4 (4):
315–24. doi:10.1016/S0960-9822(00)00070-1. PMID 7922340
Winder WW (September 2001). “Energy-sensing and signaling by
AMP-activated protein kinase in skeletal muscle”. J. Appl. Physiol. 91 (3):
1017–28. PMID 11509493
Suter M, Riek U, Tuerk R, Schlattner U, Wallimann T, Neumann D (October
2006). “Dissecting the role of 5′-AMP for allosteric stimulation, activation,
and deactivation of AMP-activated protein kinase”. J. Biol. Chem.
281 (43): 32207–6. doi:10.1074/jbc.M606357200. PMID 16943194

Part III. Pituitary-thyroid axis and diabetes mellitus
The Interface Between Thyroid and Diabetes Mellitus

Leonidas H. Duntas, Jacques Orgiazzi, Georg Brabant Clin Endocrinol. 2011;75(1):1-9.
Interaction of Metformin and Thyroid Function

Metformin acts primarily by

suppressing hepatic gluconeogenesis via activation of AMPK
It has the opposite effects on hypothalamic AMPK,
- inhibiting activity of the enzyme.
the metformin effects on hypothalamic AMPK activity will
- counteractT3 effects at the hypothalamic level.
AMPK therefore represents a direct target for dual regulation
- in the hypothalamic partitioning of energy homeostasis.
metformin crossesthe blood–brain barrier and
- levels in the pituitary gland are substantially increased.
It convincinglysuppresses TSH

A recent study recruiting 66 patients with benign thyroid nodules furthermore
demonstrated that metformin significantly decreases nodule size in patients with
insulin resistance.[76] The effect of metformin, which was produced over a
6-month treatment period, parallelled a fall in TSH concentrations and achieved a
shrinkage amounting to 30% of the initial nodule size when metformin was
administered alone and up to 55% when it was added to ongoing LT4 treatment.

These studies reveal a

suppressive effect of metformin on TSH secretion patterns in
hypothyroid patients, an effect that is apparently
independent of T4 treatment and does not alter the TH profile.
A rebound of TSH secretion occurs at about 3 months following metformin
withdrawal.

It appears that recommendations for more frequent testing, on an annual to
biannual basis, seems justified in higher risk groups like patients over 50 or 55,
particularly with suggestive symptoms, raised antibody titres or dylipidaemia.
We thus would support the suggestion of an initial TSH and TPO antibody testing
which, as discussed, will help to predict the development of hypothyroidism in
patients with diabetes.

Hypothalamic AMPK and fatty acid metabolism mediate thyroid
regulation of energy balance
M López, L Varela, MJ Vázquez, S Rodríguez-Cuenca, CR González, …, & Vidal-Puig
Nature Medicine 29 Aug 2010; 16: 1001–1008 http://dx.doi.org:/10.1038/nm.2207

Thyroid hormones have widespread cellular effects; however it is unclear whether
their effects on the central nervous system (CNS) contribute to global energy balance.
Here we demonstrate that either

whole-body hyperthyroidism or central administration of triiodothyronine
(T3) decreases
- the activity of hypothalamic AMP-activated protein kinase (AMPK),
- increases sympathetic nervous system (SNS) activity and
- upregulates thermogenic markers in brown adipose tissue (BAT).

Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus
(VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses
the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid
hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism.

This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this
enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and
increases expression of thermogenic markers in BAT. These effects are reversed by
pharmacological blockade of the SNS. Thus, thyroid hormone–induced modulation
of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of
whole-body energy homeostasis.

Metabolic Basis for Thyroid Hormone Liver Preconditioning:
Upregulation of AMP-Activated Protein Kinase Signaling
LA Videla,1 V Fernández, P Cornejo, and R Vargas
1Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences,
Faculty of Medicine, University of Chile, 2Faculty of Medicine, Diego Portales University,
Santiago, Chile
Academic Editors: H. M. Abu-Soud and D. Benke
The Scientific World Journal 2012; 2012, ID 475675, 10 pp
http://dx.doi.org/10.1100/2012/475675

The liver is a major organ responsible for most functions of cellular metabolism and

a mediator between dietary and endogenous sources of energy for extrahepatic tissues.
In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
constitutes an intrahepatic energy sensor
regulating physiological energy dynamics by limiting anabolism and stimulating
catabolism, thus increasing ATP availability.
This is achieved by mechanisms involving direct allosteric activation and
reversible phosphorylation of AMPK, in response to signals such as
- energy status,
- serum insulin/glucagon ratio,
- nutritional stresses,
- pharmacological and natural compounds, and
- oxidative stress status.

Reactive oxygen species (ROS) lead to cellular AMPK activation and

downstream signaling under several experimental conditions.

Thyroid hormone (L-3,3′,5-triiodothyronine, T3) administration, a condition
that enhances liver ROS generation,

triggers the redox upregulation of cytoprotective proteins
- affording preconditioning against ischemia-reperfusion (IR) liver injury.

Data discussed in this work suggest that T3-induced liver activation of AMPK

may be of importance in the promotion of metabolic processes
favouring energy supply for the induction and operation of preconditioning
mechanisms.

These include

antioxidant,
antiapoptotic, and
anti-inflammatory mechanisms,
repair or resynthesis of altered biomolecules,
induction of the homeostatic acute-phase response, and
stimulation of liver cell proliferation,

which are required to cope with the damaging processes set in by IR.

The liver functions as a mediator between dietary and endogenous sources
of energy and extrahepatic organs that continuously require energy, mainly
the brain and erythrocytes, under cycling conditions between fed and fasted states.

In the fed state, where insulin action predominates, digestion-derived glucose is
converted to pyruvate via glycolysis, which is oxidized to produce energy, whereas
fatty acid oxidation is suppressed. Excess glucose can be either stored as hepatic
glycogen or channelled into de novo lipogenesis.

In the fasted state, considerable liver fuel metabolism changes occur due to decreased
serum insulin/glucagon ratio, with higher glucose production as a consequence of
stimulated glycogenolysis and gluconeogenesis (from alanine, lactate, and glycerol).

Major enhancement in fatty acid oxidation also occurs to provide energy for liver
processes and ketogenesis to supply metabolic fuels for extrahepatic tissues. For these
reasons, the liver is considered as the metabolic processing organ of the body, and
alterations in liver functioning affect whole-body metabolism and energy homeostasis.

In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
is the downstream component of a protein kinase cascade acting as an

intracellular energy sensor regulating physiological energy dynamics by
limiting anabolic pathways, to prevent excessive adenosine triphosphate (ATP)
utilization, and
by stimulating catabolic processes, to increase ATP production.

Thus, the understanding of the mechanisms by which liver AMPK coordinates hepatic
energy metabolism represents a crucial point of convergence of regulatory signals
monitoring systemic and cellular energy status

Liver AMPK: Structure and Regulation

AMPK, a serine/threonine kinase, is a heterotrimeric complex comprising

a catalytic subunit α and
two regulatory subunits β and γ .

The α subunit has a threonine residue (Thr172) within the activation loop of the kinase
domain, with the C-terminal region being required for association with β and γ subunits.
The β subunit associates with α and γ by means of its C-terminal region , whereas

the γ subunit has four cystathionine β-synthase (CBS) motifs, which
bind AMP or ATP in a competitive manner.

75675.fig.001 (not shown)

Figure 1: Regulation of AMP-activated protein kinase (AMPK) by
(A) direct allosteric activation and
(B) reversible phosphorylation and downstream responses maintaining
intracellular energy balance.

Regulation of liver AMPK activity involves both direct allosteric activation and
reversible phosphorylation. AMPK is allosterically activated by AMP through

binding to the regulatory subunit-γ, which induces a conformational change in
the kinase domain of subunit α that protects AMPK from dephosphorylation
of Thr172, probably by protein phosphatase-2C.

Activation of AMPK requires phosphorylation of Thr172 in its α subunit, which can be
attained by either

(i) tumor suppressor LKB1 kinase following enhancement in the AMP/ATP ratio, a
kinase that plays a crucial role in AMPK-dependent control of liver glucose and
lipid metabolism;

(ii) Ca2+-calmodulin-dependent protein kinase kinase-β (CaMKKβ) that
phosphorylates AMPK in an AMP-independent, Ca2+-dependent manner;

(iii) transforming growth-factor-β-activated kinase-1 (TAK1), an important
kinase in hepatic Toll-like receptor 4 signaling in response to lipopolysaccharide.

Among these kinases, the relevance of CaMKKβ and TAK1 in liver AMPK activation
remains to be established in metabolic stress conditions. Both allosteric and
phosphorylation mechanisms are able to elicit

over 1000-fold increase in AMPK activity, thus allowing
the liver to respond to small changes in energy status in a highly sensitive fashion.

In addition to rapid AMPK regulation through allosterism and reversible phosphorylation

long-term effects of AMPK activation induce changes in hepatic gene expression.

This was demonstrated for

(i) the transcription factor carbohydrate-response element-binding protein (ChREBP),

whose Ser568 phosphorylation by activated AMPK
blocks its DNA binding capacity and glucose-induced gene transcription
under hyperlipidemic conditions;(ii) liver sterol regulatory element-binding
protein-1c (SREBP-1c), whose mRNA and protein expression and those of
its target gene for fatty acid synthase (FAS)
are reduced by metformin-induced AMPK activation,
decreasing lipogenesis and increasing fatty acid oxidation due to
malonyl-CoA depletion;

(iii) transcriptional coactivator transducer of regulated CREB activity-2 (TORC2),
a crucial component of the hepatic gluconeogenic program, was reported
to be phosphorylated by activated AMPK.

This modification leads to subsequent cytoplasmatic sequestration of TORC2 and
inhibition of gluconeogenic gene expression, a mechanism underlying

the plasma glucose-lowering effects of adiponectin and metformin
through AMPK activation by upstream LKB1.

Activation of AMPK in the liver is a key regulatory mechanism controlling glucose
and lipid metabolism,

inhibiting anabolic processes, and
enhancing catabolic pathways in response to different signals, including
1. energy status,
2. serum insulin/glucagon ratio,
3. nutritional stresses,
4. pharmacological and natural compounds, and
5. oxidative stress status

Reactive Oxygen Species (ROS) and AMPK Activation

The high energy demands required to cope with all the metabolic functions
of the liver are met by

fatty acid oxidation under conditions of both normal blood glucose levels and
hypoglycemia, whereas
glucose oxidation is favoured in hyperglycemic states, with consequent
generation of ROS.

Under normal conditions, ROS occur at relatively low levels due to their fast processing
by antioxidant mechanisms, whereas at acute or prolonged high ROS levels, severe
oxidation of biomolecules and dysregulation of signal transduction and gene expression
is achieved, with consequent cell death through necrotic and/or apoptotic-signaling
pathways.

Thyroid Hormone (L-3,3′,5-Triiodothyronine, T3), Metabolic Regulation,
and ROS Production

T3 is important for the normal function of most mammalian tissues, with major actions
on O2 consumption and metabolic rate, thus

determining enhancement in fuel consumption for oxidation processes
and ATP repletion.

T3 acts predominantly through nuclear receptors (TR) α and β, forming

functional complexes with retinoic X receptor that
bind to thyroid hormone response elements (TRE) to activate gene expression.

T3 calorigenesis is primarily due to the

induction of enzymes related to mitochondrial electron transport and ATP
synthesis, catabolism, and
some anabolic processes via upregulation of genomic mechanisms.

The net result of T3 action is the enhancement in the rate of O2 consumption of target
tissues such as liver, which may be effected by secondary processes induced by T3

(i) energy expenditure due to higher active cation transport,

(ii) energy loss due to futile cycles coupled to increase in catabolic and anabolic pathways, and

(iii) O2 equivalents used in hepatic ROS generation both in hepatocytes and Kupffer cells

In addition, T3-induced higher rates of mitochondrial oxidative phosphorylation are
likely to induce higher levels of ATP, which are partially balanced by intrinsic uncoupling
afforded by induction of uncoupling proteins by T3. In agreement with this view, the
cytosolic ATP/ADP ratio is decreased in hyperthyroid tissues, due to simultaneous
stimulation of ATP synthesis and consumption.

Regulation of fatty acid oxidation is mainly attained by carnitine palmitoyltransferase Iα (CPT-Iα),

catalyzing the transport of fatty acids from cytosol to mitochondria for β-oxidation,
and acyl-CoA oxidase (ACO),
catalyzing the first rate-limiting reaction of peroxisomal β-oxidation, enzymes that are
induced by both T3 and peroxisome proliferator-activated receptor α (PPAR-α).

Furthermore, PPAR-α-mediated upregulation of CPT-Iα mRNA is enhanced by PPAR-γ
coactivator 1α (PGC-1α), which in turn

augments T3 induction of CPT-Iα expression.

Interestingly, PGC-1α is induced by

T3,
AMPK activation, and
ROS,

thus establishing potential links between

T3 action, ROS generation, and AMPK activation

with the onset of mitochondrial biogenesis and fatty acid β-oxidation.

Liver ROS generation leads to activation of the transcription factors

nuclear factor-κB (NF-κB),
activating protein 1 (AP-1), and
signal transducer and activator of transcription 3 (STAT3)

at the Kupffer cell level, with upregulation of cytokine expression (TNF-α, IL-1, IL-6),
which upon interaction with specific receptors in hepatocytes trigger the expression of

cytoprotective proteins (Figure 3(A)).

These responses and the promotion of hepatocyte and Kupffer-cell proliferation
represent hormetic effects reestablishing

redox homeostasis,
promoting cell survival, and
protecting the liver against ischemia-reperfusion injury.

T3 liver preconditioning also involves the activation of the

Nrf2-Keap1 defense pathway

upregulating antioxidant proteins,
phase-2 detoxifying enzymes, and
multidrug resistance proteins, members of the ATP binding cassette (ABC)
superfamily of transporters (Figure 3(B))

In agreement with T3-induced liver preconditioning, T3 or L-thyroxin afford
preconditioning against IR injury in the heart, in association with

activation of protein kinase C and
attenuation of p38 and
c-Jun-N-terminal kinase activation ,

and in the kidney, in association with

heme oxygenase-1 upregulation.

475675.fig.002

http://www.hindawi.com/journals/tswj/2012/floats/475675/thumbnails/475675.fig.002_th.jpg

Figure 2: Calorigenic response of thyroid hormone (T3) and its relationship with O2
consumption, reactive oxygen species (ROS) generation, and antioxidant depletion in the liver.
Abbreviations: CYP2E1, cytochrome P450 isoform 2E1; GSH, reduced glutathione; QO2, rate
of O2 consumption; SOD, superoxide dismutase.

475675.fig.003

genomic signaling in T3 calorigenesis and ROS production 475675.fig.003

http://www.hindawi.com/journals/tswj/2012/floats/475675/thumbnails/475675.fig.003_th.jpg

Figure 3: Genomic signaling mechanisms in T3 calorigenesis and liver reactive oxygen
species (ROS) production leading to
(A) upregulation of cytokine expression in Kupffer cells and hepatocyte activation of genes
conferring cytoprotection,
(B) Nrf2 activation controling expression of antioxidant and detoxication proteins, and
(C) activation of the AMPK cascade regulating metabolic functions.Abbreviations: AP-1, activating protein 1; ARE, antioxidant responsive element; CaMKKβ,
Ca2+-calmodulin-dependent kinase kinase-β; CBP, CREB binding protein; CRC, chromatin
remodelling complex; EH, epoxide hydrolase; HO-1, hemoxygenase-1; GC-Ligase,
glutamate cysteine ligase; GPx, glutathione peroxidase; G-S-T, glutathione-S-transferase;
HAT, histone acetyltransferase; HMT, histone arginine methyltransferase; IL1,
interleukin 1; iNOS, inducible nitric oxide synthase; LKB1, tumor suppressor LKB1 kinase;
MnSOD, manganese superoxide dismutase; MRPs, multidrug resistance proteins; NF-κB,
nuclear factor-κB; NQO1, NADPH-quinone oxidoreductase-1; NRF-1, nuclear respiratory
factor-1; Nrf2, nuclear receptor-E2-related factor 2; PCAF, p300/CBP-associated
factor; RXR, retinoic acid receptor; PGC-1, peroxisome proliferator-activated receptor-γ
coactivator-1; QO2, rate of O2 consumption; STAT3, signal transducer and activator
of transcription 3; TAK1, transforming-growth-factor-β-activated kinase-1; TNF-α, tumor
necrosis factor-α; TR, T 3 receptor; TRAP, T3-receptor-associated protein; TRE, T3 responsive element; UCP, uncoupling proteins; (—), reported mechanisms;
(- - - -), proposed mechanisms.

T3 is a key metabolic regulator coordinating short-term and long-term energy needs,
with major actions on liver metabolism. These include promotion of

(i) gluconeogenesis and hepatic glucose production, and

(ii) fatty acid oxidation coupled to enhanced adipose tissue lipolysis, with

higher fatty acid flux to the liver and
consequent ROS production (Figure 2) and
redox upregulation of cytoprotective proteins

affording liver preconditioning (Figure 3).

Thyroid Hormone and AMPK Activation: Skeletal Muscle and Heart

In skeletal muscle, T3 increases the levels of numerous proteins involved in

glucose uptake (GLUT4),
glycolysis (enolase, pyruvate kinase, triose phosphate isomerase),
fatty acid oxidation (carnitine palmitoyl transferase-1, mitochondrial thioesterase I),
and uncoupling protein-3,

effects that are achieved through enhanced transcription of TRE-containing genes

Skeletal muscle AMPK activation is characterized by

(i) being a rapid and transient response,

(ii) upstream activation by Ca2+-induced mobilization and CaMKKβ activation,

(iii) upstream upregulation of LKB1 expression, which requires association with STRAD
and MO25 for optimal phosphorylation/activation of AMPK, and

(iv) stimulation of mitochondrial fatty acid β-oxidation.

T3-induced muscle AMPK activation was found to trigger two major downstream

signaling pathways, namely,

(i) peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA
expression and phosphorylation, a transcriptional regulator for genes related to

mitochondrial biogenesis,
fatty acid oxidation, and
gluconeogenesis and

(ii) cyclic AMP response element binding protein (CREB) phosphorylation, which

in turn induces PGC-1α expression in liver tissue, thus
reinforcing mechanism (i).

These data indicate that AMPK phosphorylation of PGC-1α initiates many of the
important gene regulatory functions of AMPK in skeletal muscle.

In heart, hyperthyroidism increased glycolysis and sarcolemmal GLUT4 levels by the
combined effects of AMPK activation and insulin stimulation, with concomitant increase
in fatty acid oxidation proportional to enhanced cardiac mass and contractile function.

Thyroid Hormone, AMPK Activation, and Liver Preconditioning

Recent studies by our group revealed that administration of a single dose of 0.1 mg T3/kg
to rats activates liver AMPK (Figure 4; unpublished work).

enhancement in phosphorylated AMPK/nonphosphorylated AMPK ratios in T3-
treated rats over control values thatis significant in the time period of 1 to 48
hours after hormone treatment
Administration of a substantially higher dose (0.4 mg T3/kg) resulted in
decreased liver AMPK activation at 4 h to return to control values at 6 h
after treatment

Activation of liver AMPK by T3 may be of relevance in terms of

promotion of fatty acid oxidation for ATP supply,
supporting hepatoprotection against IR injury (Figure 3(C)).

This proposal is based on the high energy demands underlying effective liver
preconditioning for full operation of hepatic

antioxidant, antiapoptotic, and anti-inflammatory mechanisms,
oxidized biomolecules repair or resynthesis,
induction of the homeostatic acute-phase response, and
promotion of hepatocyte and Kupffer cell proliferation,

mechanisms that are needed to cope with the damaging processes set in by IR.
T3 liver preconditioning , in addition to that afforded by

n-3 long-chain polyunsaturated fatty acids given alone or
combined with T3 at lower dosages, or
by iron supplementation,

constitutes protective strategies against hepatic IR injury.

Studies on the molecular mechanisms underlying T3-induced liver AMPK
activation (Figure 4) are currently under assessment in our laboratory.

References

Fernández and L. A. Videla, “Kupffer cell-dependent signaling in thyroid hormone
calorigenesis: possible applications for liver preconditioning,” Current Signal
Transduction Therapy 2009; 4(2): 144–151.

Viollet, B. Guigas, J. Leclerc et al., “AMP-activated protein kinase in the regulation
of hepatic energy metabolism: from physiology to therapeutic perspectives,” Acta
Physiologica 2009; 196(1): 81–98.

Carling, “The AMP-activated protein kinase cascade – A unifying system
for energy control,” Trends in Biochemical Sciences, 2004;. 29(1): 18–24.

E. Kemp, D. Stapleton, D. J. Campbell et al., “AMP-activated protein kinase,
super metabolic regulator,” Biochemical Society Transactions 2003; 31(1):
162–168.

G. Hardie, “AMP-activated protein kinase-an energy sensor that
regulates all ;aspects of cell function,” Genes and Development,
2011; 25(18): 1895–1908.

Woods, P. C. F. Cheung, F. C. Smith et al., “Characterization of AMP-activated
protein kinase βandγ subunits Assembly of the heterotrimeric complex in vitro,”
Journal of Biological Chemistry 1996;271(17): 10282–10290.

Xiao, R. Heath, P. Saiu et al., “Structural basis for AMP binding to mammalian AMP-
activated protein kinase,” Nature 2007; 449(7161): 496–500.

more…

Impact of Metformin and compound C on NIS expression and iodine uptake in vitro and in vivo: a role for CRE in AMPK modulation of thyroid function.
Abdulrahman RM1, Boon MR, Sips HC, Guigas B, Rensen PC, Smit JW, Hovens GC.
Author information
Thyroid. 2014 Jan;24(1):78-87. Epub 2013 Sep 25. PMID: 23819433
http://dx.doi.org:/10.1089/thy.2013.0041.

Although adenosine monophosphate activated protein kinase (AMPK) plays a crucial role
in energy metabolism, a direct effect of AMPK modulation on thyroid function has only
recently been reported, and much of its function in the thyroid is currently unknown.

The aim of this study was

to investigate the mechanism of AMPK modulation in iodide uptake.
to investigate the potential of the AMPK inhibitor compound C as an enhancer of
iodide uptake by thyrocytes.

Metformin reduced NIS promoter activity (0.6-fold of control), whereas compound C
stimulated its activity (3.4-fold) after 4 days. This largely coincides with

CRE activation (0.6- and 3.0-fold).

These experiments show that AMPK exerts its effects on iodide uptake, at least partly,
through the CRE element in the NIS promoter. Furthermore, we have used AMPK-alpha1
knockout mice to determine the long-term effects of AMPK inhibition without chemical compounds.
These mice have a less active thyroid, as shown by reduced colloid volume and reduced
responsiveness to thyrotropin.

NIS expression and iodine uptake in thyrocytes

can be modulated by metformin and compound C.

These compounds exert their effect by

modulation of AMPK, which, in turn, regulates
the activation of the CRE element in the NIS promoter.

Overall, this suggests that AMPK modulating compounds may be useful for the
enhancement of iodide uptake by thyrocytes, which could be useful for the
treatment of thyroid cancer patients with radioactive iodine.

AMPK: Master Metabolic Regulator

AMPK-activating drugs metformin or phenformin might provide protection against cancer 1741-7007-11-36-5

AMPK and AMPK-related kinase (ARK) family 1741-7007-11-36-4

AMP-activated protein kinase (AMPK) was first discovered as an activity that

inhibited preparations of acetyl-CoA carboxylase (ACC) and 3-hydroxy-
3-methylglutaryl-CoA reductase (HMG-CoA reductase, HMGR) and was
induced by AMP.

AMPK induces a cascade of events within cells in response to the ever changing energy
charge of the cell. The role of AMPK in regulating cellular energy charge places this
enzyme at a central control point in maintaining energy homeostasis.

More recent evidence has shown that AMPK activity can also be regulated by physiological stimuli, independent of the energy charge of the cell, including hormones and nutrients.

Once activated, AMPK-mediated phosphorylation events

switch cells from active ATP consumption (e.g. fatty acid and cholesterol
biosynthesis) to active ATP production (e.g. fatty acid and glucose oxidation).

These events are rapidly initiated and are referred to as

short-term regulatory processes.

The activation of AMPK also exerts

long-term effects at the level of both gene expression and protein synthesis.

Other important activities attributable to AMPK are

regulation of insulin synthesis and
secretion in pancreatic islet β-cells and
modulation of hypothalamic functions involved in the regulation of satiety.

How these latter two functions impact obesity and diabetes will be discussed below.

Regulation of AMPK

In the presence of AMP the activity of AMPK is increased approximately 5-fold.
However, more importantly is the role of AMP in regulating the level of phosphorylation
of AMPK. An increased AMP to ATP ratio leads to a conformational change in the γ-subunit
leading to increased phosphorylation and decreased dephosphorylation of AMPK.

The phosphorylation of AMPK results in activation by at least 100-fold. AMPK is
phosphorylated by at least three different upstream AMPK kinases (AMPKKs).
Phosphorylation of AMPK occurs in the α subunit at threonine 172 (T172) which

lies in the activation loop.

One kinase activator of AMPK is

Ca2+-calmodulin-dependent kinase kinase β (CaMKKβ)
which phosphorylates and activates AMPK in response to increased calcium.

The distribution of CaMKKβ expression is primarily in the brain with detectable levels
also found in the testes, thymus, and T cells. As described for the Ca2+-mediated
regulation of glycogen metabolism,

increased release of intracellular stores of Ca2+ create a subsequent demand for
ATP.

Activation of AMPK in response to Ca fluxes

provides a mechanism for cells to anticipate the increased demand for ATP.

Evidence has also demonstrated that the serine-threonine kinase, LKB1 (also called
serine-threonine kinase 11, STK11) which is encoded by the Peutz-Jeghers syndrome
tumor suppressor gene, is required for activation of AMPK in response to stress.

The active LKB1 kinase is actually a complex of three proteins:

LKB1,
Ste20-related adaptor (STRAD) and
mouse protein 25 (MO25).

Thus, the enzyme complex is often referred to as LKB1-STRAD-MO25. Phosphorylation
of AMPK by LKB1 also occurs on T172. Unlike the limited distribution of CaMKKβ,

LKB1 is widely expressed, thus making it the primary AMPK-regulating kinase.

Loss of LKB1 activity in adult mouse liver leads to

near complete loss of AMPK activity and
is associated with hyperglycemia.

The hyperglycemia is, in part, due to an increase in the transcription of gluconeogenic
genes. Of particular significance is the increased expression of

the peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator 1α
(PGC-1α), which drives gluconeogenesis.
Reduction in PGC-1α activity results in normalized blood glucose levels in
LKB1-deficient mice.

The third AMPK phosphorylating kinase is transforming growth factor-β-activated
kinase 1 (TAK1). However, the normal physiological conditions under which TAK1
phosphorylates AMPK are currently unclear.

The effects of AMP are two-fold:

a direct allosteric activation and making AMPK a poorer substrate for
dephosphorylation.

Because AMP affects both
the rate of AMPK phoshorylation in the positive direction and
dephosphorylation in the negative direction,

the cascade is ultrasensitive. This means that

a very small rise in AMP levels can induce a dramatic increase in the activity of
AMPK.

The activity of adenylate kinase, catalyzing the reaction shown below, ensures that

AMPK is highly sensitive to small changes in the intracellular [ATP]/[ADP] ratio.

2 ADP ——> ATP + AMP

Negative allosteric regulation of AMPK also occurs and this effect is exerted by
phosphocreatine. As indicated above, the β subunits of AMPK have a glycogen-binding domain, GBD. In muscle, a high glycogen content

represses AMPK activity and
this is likely the result of interaction between the GBD and glycogen,
the GBD of AMPK allows association of the enzyme with the regulation of glycogen metabolism
by placing AMPK in close proximity to one of its substrates glycogen synthase.

AMPK has also been shown to be activated by receptors that are coupled to

phospholipase C-β (PLC-β) and by
hormones secreted by adipose tissue (termed adipokines) such as leptinand adiponectin (discussed below).

Targets of AMPK

The signaling cascades initiated by the activation of AMPK exert effects on

glucose and lipid metabolism,
gene expression and
protein synthesis.

These effects are most important for regulating metabolic events in the liver, skeletal
muscle, heart, adipose tissue, and pancreas.

Demonstration of the central role of AMPK in the regulation of metabolism in response
to events such as nutrient- or exercise-induced stress. Several of the known physiologic
targets for AMPK are included as well as several pathways whose flux is affected by
AMPK activation. Arrows indicate positive effects of AMPK, whereas, T-lines indicate
the resultant inhibitory effects of AMPK action.

The uptake, by skeletal muscle, accounts for >70% of the glucose removal from the
serum in humans. Therefore, it should be obvious that this event is extremely important
for overall glucose homeostasis, keeping in mind, of course, that glucose uptake by
cardiac muscle and adipocytes cannot be excluded from consideration. An important fact
related to skeletal muscle glucose uptake is that this process is markedly impaired in
individuals with type 2 diabetes.

The uptake of glucose increases dramatically in response to stress (such as ischemia) and
exercise and is stimulated by insulin-induced recruitment of glucose transporters
to the plasma membrane, primarily GLUT4. Insulin-independent recruitment of glucose
transporters also occurs in skeletal muscle in response to contraction (exercise).

The activation of AMPK plays an important, albeit not an exclusive, role in the induction of
GLUT4 recruitment to the plasma membrane. The ability of AMPK to stimulate
GLUT4 translocation to the plasma membrane in skeletal muscle is by a different mechanism
than that stimulated by insulin and insulin and AMPK effects are additive.

Under ischemic/hypoxic conditions in the heart the activation of AMPK leads to the
phosphorylation and activation of the kinase activity of phosphofructokinase-2, PFK-2
(6-phosphofructo-2-kinase). The product of the action of PFK-2 (fructose-2,6-bisphosphate,
F2,6BP) is one of the most potent regulators of the rate of flux through
glycolysis and gluconeogenesis.

In liver the PKA-mediated phosphorylation of PFK-2 results in conversion of the
enzyme from a kinase that generates F2,6BP to a phosphatase that removes the
2-phosphate thus reducing the levels of the potent allosteric activator of the glycolytic
enzyme 6-phosphfructo-1-kinase, PFK-1 and the potent allosteric inhibitor
of the gluconeogenic enzyme fructose-1,6-bisphosphatase (F1,-6BPase).

It is important to note that like many enzymes, there are multiple isoforms of PFK-2
(at least 4) and neither the liver or the skeletal muscle isoforms contain the AMPK
phosphorylation sites found in the cardiac and inducible (iPFK2) isoforms of PFK-2.

Inducible PFK-2 is expressed in the monocyte/macrophage lineage in response to pro-
inflammatory stimuli. The ability to activate the kinase activity by phosphorylation of
PFK-2 in cardiac tissue and macrophages in response to ischemic conditions allows these
cells to continue to have a source of ATP via anaerobic glycolysis. This phenomenon is
recognized as the Pasteur effect: an increased rate of glycolysis in response to hypoxia.

Of pathological significance is the fact that the inducible form of PFK-2 is commonly
expressed in many tumor cells and this may allow AMPK to play an important role in
protecting tumor cells from hypoxic stress. Indeed, techniques for depleting AMPK in
tumor cells have shown that these cells become sensitized to nutritional stress upon loss
of AMPK activity.

Whereas, stress and exercise are powerful inducers of AMPK activity in skeletal muscle,
additional regulators of its activity have been identified.

Insulin-sensitizing drugs of the thiazolidinedione family (activators of PPAR-γ, see
below) as well as the hypoglycemia drug metformin exert a portion of their effects
through regulation of the activity of AMPK.

As indicated above, the activity of the AMPK activating kinase, LKB1, is critical for
regulating gluconeogenic flux and consequent glucose homeostasis. The action of
metformin in reducing blood glucose levels

requires the activity of LKB1 in the liver for this function.

Also, several adipokines (hormones secreted by adipocytes) either stimulate or inhibit
AMPK activation:

leptin and adiponectin have been shown to stimulate AMPK activation, whereas,
resistininhibits AMPK activation.

Cardiac effects exerted by activation of AMPK also include

phosphorylation of endothelial nitric oxide synthase, eNOSin cardiac endothelium.

AMPK-mediated phosphorylation of eNOS leads to increased activity and consequent
NO production and provides a link between metabolic stresses and cardiac function.

In platelets, insulin action leads to an increase in eNOS activity that is

due to its phosphorylation by AMPK.

Activation of NO production in platelets leads to

a decrease in thrombin-induced aggregation, thereby,
limiting the pro-coagulant effects of platelet activation.

The response of platelets to insulin function clearly indicates why disruption in insulin
action is a major contributing factor in the development of the metabolic syndrome

Activation of AMPK leads to a reduction in the level of SREBP

a transcription factor &regulator of the expression of numerous
lipogenic enzymes

Another transcription factor reduced in response to AMPK activation is

hepatocyte nuclear factor 4α, HNF4α
- a member of the steroid/thyroid hormone superfamily.
- HNF4α is known to regulate the expression of several liver and
  pancreatic β-cell genes such as GLUT2, L-PK and preproinsulin.
Of clinical significance is that mutations in HNF4α are responsible for
- maturity-onset diabetes of the young, MODY-1.

Recent evidence indicates that the gene for the carbohydrate-response-element-
binding protein (ChREBP) is a target for AMPK-mediated transcriptional regulation
in the liver. ChREBP is rapidly being recognized as a master regulator of lipid
metabolism in liver, in particular in response to glucose uptake.

The target of the thiazolidinedione (TZD) class of drugs used to treat type 2 diabetes is
the peroxisome proliferator-activated receptor γ, PPARγ which

itself may be a target for the action of AMPK.

The transcription co-activator, p300, is phosphorylated by AMPK

which inhibits interaction of p300 with not only PPARγ but also
the retinoic acid receptor, retinoid X receptor, and
thyroid hormone receptor.

PPARγ is primarily expressed in adipose tissue and thus it was difficult to reconcile how
a drug that was apparently acting only in adipose tissue could lead to improved insulin
sensitivity of other tissues. The answer to this question came when it was discovered that the TZDs stimulated the expression and release of the adipocyte hormone (adipokine),
adiponectin. Adiponectin stimulates glucose uptake and fatty acid oxidation in skeletal
muscle. In addition, adiponectin stimulates fatty acid oxidation in liver while inhibiting
expression of gluconeogenic enzymes in this tissue.

These responses to adiponectin are exerted via activation of AMPK. Another
transcription factor target of AMPK is the forkhead protein, FKHR (now referred to as
FoxO1). FoxO1 is involved in the activation of glucose-6-phosphatase expression and,
therefore, loss of FoxO1 activity in response to AMPK activation will lead to reduced
hepatic output of glucose.

This concludes a very complicated perspective that ties together the thyroid hormone
activity, the hypophysis, diabetes mellitus, and AMPK tegulation of metabolism in the
liver, skeletal muscle, adipose tissue, and heart. I also note at this time that there
nongenetic points to be made here:

The tissue specificity of isoenzymes
The modulatory role of AMP:ATP ratio in phosphorylation/dephosphorylation
effects on metabolism tied to AMPK
The tie in of stress or ROS with fast reactions to protect harm to tissues
The relationship of cytokine activation and release to the above metabolic events
The relationship of effective and commonly used diabetes medications to AMPK
mediated processes
The preceding presentation is notable for the importance of proteomic and
metabolomic invetigations in elucidation common chronic and nongenetic diseases

Read Full Post »

Somatic, germ-cell, and whole sequence DNA in cell lineage and disease profiling

Posted in Amino acids, Best evidence, CANCER BIOLOGY & Innovations in Cancer Therapy, Computational Biology/Systems and Bioinformatics, Curation, Developmental biology, Disease Biology, Evolutionary cognition, Experimental validation, Explanatory, Gene Regulation, Gene Regulation and Evolution, Genetics & Pharmaceutical, Genomic Expression, Historical relevance, Innovations, Metabolism, Molecular Genetics & Pharmaceutical, mtDNA, Nitric Oxide in Health and Disease, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Drug Discovery, Pharmacologic toxicities, Phosphorylation, Proteins, Proteomics, Signaling, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, Transcriptomics, Translational Science, Ubiquitinylation, tagged ablation of toxicity, breast cancer, breast cancer genes, Cancer - General, gene maps, Mitochondrial DNA, mtDNA mutations, novel therapeutic targets, phylogeography, population migration, somatic DNA, transcription, transfer RNA on September 14, 2014| Leave a Comment »

Somatic, germ-cell, and whole sequence DNA in cell lineage and disease profiling

Curator: Larry H Bernstein, MD, FCAP

In humans, mitochondrial DNA spans about 16,500 DNA building blocks (base pairs), representing a small fraction of the total DNA in cells. Mitochondrial DNA contains 37 genes, essential for normal mitochondrial function and thirteen of them provide instructions for making enzymes involved in inner membrane function. The remaining 24 genes are transcribed into transfer RNA (tRNA) and ribosomal RNA (rRNA), which are needed to transfer amino acids into proteins.

Somatic mutations occur in the DNA of certain cells during a person’s lifetime and typically are not passed to future generations. They differ from germ-line mutations that have a lineal descent from the maternal parent, and they occur later in life. Mutations in the sperm DNA are not carried on to future generations, as the sperm mitochondria are destroyed after the egg is fertilized.

There is limited evidence linking somatic mutations in mitochondrial DNA with certain cancers, including breast, colon, stomach, liver, and kidney tumors. These mutations might also be associated with cancer of blood-forming tissue (leukemia) and cancer of immune system cells (lymphoma). There are many heritable diseases that are related to germ-line mutations, and germ-line mutations have a role in many common diseases. Mitochondrial DNA is particularly vulnerable to the effects of reactive oxygen species (ROS), and with a limited ability of the mitochondrion to repair itself, ROS easily damage mitochondrial DNA. The repair mechanism is tied to ubiquitinylation system. A list of disorders associated with mitochondrial genes is provided from Wikipedia.

Inherited changes in mitochondrial DNA may be associated with pathologies in growth and development, and multiorgan system disorders, as mutations disrupt the mitochondria’s ability to generate the cell’s energy. The effects of these conditions are most pronounced in organs and tissues with high energy requirements (such as the heart, brain, and muscles). Although the health consequences of inherited mitochondrial DNA mutations vary widely, some frequently observed features include muscle weakness and wasting, problems with movement, diabetes, kidney failure, heart disease, loss of intellectual functions (dementia), hearing loss, and abnormalities involving the eyes and vision.

A buildup of somatic mutations in mitochondrial DNA has been considered to have a role in or associated with increased risk of certain age-related disorders such as heart disease, Alzheimer disease, and Parkinson disease, and the severity of many mitochondrial disorders is thought to be associated with the percentage of mitochondria affected by a particular genetic change. Consequently, the progressive accumulation of these mutations over a person’s lifetime may play a role in aging.

Mitochondrial DNA is typically diagrammed as a circular structure with genes and regulatory regions labeled.

Mitochondrial DNA

http://ghr.nlm.nih.gov/html/images/chromosomeIdeograms/mitochondria/wholeMitochondria.jpg

Additional Resources:

Additional NIH Resources – National Institutes of Health

NHGRI Talking Glossary: Mitochondrial DNA

Gene Reviews – Clinical summary (6 links)
Research Resources – Tools for researchers (5 links)
OMIM – Genetic disorder catalog (2 links)
Genetics Education
Human Genome Project
Resources for Genetics Researchers

mtDNA : The Eve Gene – by Stephen Oppenheimer

Mutations are a cumulative dossier of our own maternal prehistory. The main task of DNA is to copy itself to each new generation. We can use these mutations to reconstruct a genetic tree of mtDNA, because each new mtDNA mutation in a prospective mother’s ovum will be transferred in perpetuity to all her descendants down the female line. Each new female line is thus defined by the old mutations as well as the new ones.

By looking at the DNA code in a sample of people alive today, and piecing together the changes in the code that have arisen down the generations, biologists can trace the line of descent back in time to a distant shared ancestor. Because we inherit mtDNA only from our mother, this line of descent is a picture of the female genealogy of the human species.

formation of gene trees

The diagram above shows the drawing of gene trees using single mutations

http://www.bradshawfoundation.com/journey/images/gene-diagram3.gif

Not only can we retrace the tree, but by taking into account here the sampled people came from, we can see where certain mutations occurred – for example, whether in Europe, or Asia, or Africa. What’s more, because the changes happen at a statistically consistent (though random) rate, we can approximate the time when they happened. This has made it possible, during the late 1990s and in the new century, for us to do something that anthropologists of the past could only have dreamt of: we can now trace the migrations of modern humans around our planet.

It turns out that the oldest changes in our mtDNA took place in Africa 150,000 – 190,000 years ago. Then new mutations start to appear in Asia, about 60,000 – 80,000 years ago. This tells us that modern humans evolved in Africa, and that some of us migrated out of Africa into Asia after 80,000 years ago. A method established in 1996, which dates each branch of the gene tree by averaging the number of new mutations in daughter types of that branch, has stood the test of time.

A final point on the methods of genetic tracking of migrations: it is important to distinguish this new approach to tracing the history of molecules on a DNA tree, known as phylogeography (literally ‘tree-geography’), from the mathematical study of the history of whole human populations, which has been used for decades and is known as classical population genetics.

The two disciplines are based on the same Mendelian biological principles, but have quite different aims and assumptions, and the difference is the source of much misunderstanding and controversy. The simplest way of explaining it is that phylogeography studies the prehistory of individual DNA molecules, while population genetics studies the prehistory of populations. Put another way, each human population contains multiple versions of any particular DNA molecule, each with its own history and different origin.

gene-diagram

The diagram above shows the tracing of gene spread geographically.
Green disks represent migrant new growth on the tree
http://www.bradshawfoundation.com/journey/images/gene-diagram4.gif

http://www.bradshawfoundation.com/journey/eve.html

David Moskowitz, MD, PhD
Founder and President, GenoMed

Germline genes make the best drug targets

They operate earliest in the disease pathway
Unlike tissue-expressed genes, which operate years after the disease began
But which everybody else is using as drug targets

Variation in germline DNA is where all disease starts

Cancer patients overexpress oncogenes and underexpress tumor suppressors

beginning in their germline DNA

Mutations in tumor DNA are “private”
Each tumor is a “snowflake”

Tumor-expressed genes can be compensatory, not causative

“Passengers, not drivers”
We have the drivers

Tumorigenesis SNPs

Using a SNPnet™ covering only 1/3 of the genome, we found about

2,500 genes associated with each of 6 different cancers in whites

Nobody else has found any yet
This will change in 2-3 years

We estimate 10,000 genes per cancer

What cellular program takes up 1/3-1/2 of the genome?

What program takes up >1/3 of the genome?

Differentiation…

Does sporadic cancer arise when a tissue stem cell fails to differentiate?

In the embryo, the surrounding tissue expresses “fields”

Lent C. Johnson published a “field” based hypothesis of bone tumors that coincides with differentiation at the

METAPHYSIS
HYPOPHYSIS

and the type CELL – chondroblast, osteoblast, giant cell (osteoclast), fibroblast

Orthopedic surgeons use magnetic fields for healing

of powerful transcription factors.
Not so in adult life: a proliferating tissue stem cell is literally on its own.

Germlines hold the key to effective “differentiation therapy”

Ideal for patients with stage 3-4 cancer
Examples of differentiation therapy:

1,25-vitamin D and
retinoic acid

Non-toxic but more effective treatment for late stage disease,

GenoMed’s 2,500 cancer-causing genes:

½ are oncogenes,
½ are tumor suppressors

Design inhibitors to oncogenes

Screen 1st for toxicity;
genomic epidemiology guarantees clinical efficacy

Jewish Heritage Written in DNA

By Kate Yandell | Sept 9, 2014

Fully sequenced genomes of more than 100 Ashkenazi people clarify the group’s history and provide a reference for researchers and physicians trying to pinpoint disease-associated genes.

A whole-genome sequence study from 128 healthy Jewish people is aimed at identifying disease-associated variants in the jewish population of Ashkenazi ancestry, according to a study published Sept 9 in Nature Communications. The library of sequences confirms earlier conclusions about Ashkenazi history hinted at by more limited DNA sequencing studies. The sequences point to an approximate 350-person bottleneck in the Ashkenazi population as recently as 700 years ago (1400 A.D.), and suggest that the population has a mixture of European and Middle Eastern ancestry.

The study “provides a very nice reference panel for the very unique population of Ashkenazi Jews,” said Alon Keinan, who studies human population genomics at Cornell University in New York. Keinan
is acknowledged in the study but was not involved in the research.

“One might have thought that, after many years of genetic studies relating to Ashkenazi Jews . . . there would be little room for additional insights,” Karl Skorecki of the Rambam Healthcare Campus
in Israel who also was not involved in the study wrote in an e-mail to The Scientist. The study, he added, provides “a powerful further validation and further resolution of the demographic history of
the Ashkenazi Jews in relation to non-Jewish Europeans that is reassuringly consistent with inferences drawn from two decades of studies using uniparental regions . . . and from array-based data.”

Itsik Pe’er, coauthor of the new study and an associate professor of computer science at Columbia University in New York City, recalled that several years ago, he and his colleagues kept running into the same problem as they tried to understand the genetics of disease in Ashkenazi populations. They were comparing their Ashkenazi samples to the only control genomes that were available, which were of largely non-Jewish European origin. The Ashkenazi genomes had variation that was absent in these general European genomes, making it hard to distinguish rare variants in Ashkenazi people.

“Technology is there to tell us everything in that [Ashkenazi] patient’s genome, but the genome was not there to distinguish the variants that are there and to tell us whether they are normal or whether we should get worried,” said Pe’er. Pe’er’s group teamed up with researchers from additional universities and hospitals in the U.S., Belgium, and Israel to sequence a collection of healthy Ashkenazi people’s genomes. The panel of reference sequences performs better than a group of European genomes at filtering out harmless variants from Ashkenazi Jewish genomes, thereby making it easier to identify potentially harmful ones. According to Pe’er, researchers will also be able to use the panel to infer
more complete sequences from partially sequenced genomes by looking for familiar sequences from the reference genomes.

The team also used its data to better understand the history of the Ashkenazi Jewish people through analyzing both level of similarity within Ashkenazi genomes and between Ashkenazi and non-Jewish
European genomes. By analyzing the length of identical DNA sequences that Ashkenazi individuals share, the researchers were able to estimate that 25 to 32 generations ago, the Ashkenazi Jewish population shrunk to just several hundred people, before expanding rapidly to eventually include the millions of Ashkenazi Jews alive today. Further, the researchers concluded that modern Ashkenazi Jews likely have an approximately even mixture of European and Middle Eastern ancestry. This suggests that after the Jewish people migrated from the Middle East to Europe, they recruited people from local European populations.

These results are compatible with those of prior work on mitochondrial DNA (mtDNA), which is passed on maternally. This prior work suggested that Ashkenazi men from the Middle East intermarried with local European women. The Ashkenazi population “hasn’t been likely as isolated as at least some researchers considered,” said Keinan.

Finally, the newly sequenced genomes shed light on the deeper history of Europe, showing that the European and Middle Eastern portions of Ashkenazi ancestry diverged just around 20,000 years ago.

“This is, I think, the first evidence from whole human genomes that the most important wave of settlement from the Near East was most likely shortly after the Last Glacial Maximum . . . and, notably, before the Neolithic transition—which is what researchers working on mitochondrial DNA have been arguing for some years,” Martin Richards, an archeogeneticist at the University of Huddersfield in the U.K., told The Scientist in an e-mail.

Skorecki noted that the new study “demonstrates the utility of sequencing whole genomes in a diverse population… with sufficient numbers of samples, parent population information, and
computational analytic power, we can expect important and surprising utilities for personal genomic and insights in terms of human demographic history from whole genomes.”

Carmi et al., “Sequencing an Ashkenazi reference panel supports population-targeted personal genomics and illuminates Jewish and European origins,” Nature
Communications,
http://dx.doi.org:/10.1038/ncomms5835, 2014.

Added Layers of Proteome Complexity

By Anna Azvolinsky | July 17, 2014

Scientists discover a broad spectrum of alternatively spliced human protein variants within a well-studied family of genes.

There may be more to the human proteome than previously thought. Some genes are known to have several different alternatively spliced protein variants, but the Scripps Research Institute’s Paul Schimmel and his colleagues have now uncovered almost 250 protein splice variants of an essential, evolutionarily conserved family of human genes. The results were published today (July 17) in Science.

Focusing on the 20-gene family of aminoacyl tRNA synthetases (AARSs), the team captured AARS transcripts from human tissues—some fetal, some adult—and showed that many of these messenger RNAs (mRNAs) were translated into proteins. Previous studies have identified
several splice variants of these enzymes that have novel functions, but uncovering so many more variants was unexpected, Schimmel said. Most of these new protein products lack the catalytic domain but retain other AARS non-catalytic functional domains. “The main point is that a vast new area of biology, previously missed, has been uncovered,”
said Schimmel.

“This is an incredible study that fundamentally changes how we look at the protein-synthesis machinery,” Michael Ibba, a protein translation researcher at Ohio State University who was not involved in the work, told The Scientist in an e-mail. “The unexpected and potentially vast
expanded functional networks that emerge from this study have the potential to influence virtually any aspect of cell growth.”

The team—including researchers at the Hong Kong University of Science and Technology, Stanford University, and aTyr Pharma, a San Diego-based biotech company that Schimmel co-founded—comprehensively captured and sequenced the AARS mRNAs from six human tissue types using high-throughput deep sequencing. While many of the transcripts were expressed in each of the tissues, there was also some tissue specificity.

Next, the team showed that a proportion of these transcripts, including those missing the catalytic domain, indeed resulted in stable protein products: 48 of these splice variants associated with polysomes. In vitro translation assays and the expression of more than 100 of these variants in cells confirmed that many of these variants could be made into
stable protein products.

The AARS enzymes—of which there’s one for each of the 20 amino acids—bring together an amino acid with its appropriate transfer RNA (tRNA) molecule. This reaction allows a ribosome to add the amino acid to a growing peptide chain during protein translation. AARS
enzymes can be found in all living organisms and are thought to be among the first proteins to have originated on Earth.

To understand whether these non-catalytic proteins had unique biological activities, the researchers expressed and purified recombinant AARS fragments, testing them in cell-based assays for proliferation, cell differentiation, and transcriptional regulation, among other
phenotypes. “We screened through dozens of biological assays and found that these variants operate in many signaling pathways,” said Schimmel.

“This is an interesting finding and fits into the existing paradigm that, in many cases, a single gene is processed in various ways [in the cell] to have alternative functions,” said Steven Brenner, a computational genomics researcher at the University of California, Berkeley.

The team is now investigating the potentially unique roles of these protein splice variants in greater detail—in both human tissue as well as in model organisms. For example, it is not yet clear whether any of these variants directly bind tRNAs.

“I do think [these proteins] will play some biological roles,” said Tao Pan, who studies the functional roles of tRNAs at the University of Chicago. “I am very optimistic that interesting biological functions will come out of future studies on these variants.”

Brenner agreed. “There could be very different biological roles [for some of these proteins]. Biology is very creative that way, [it’s] able to generate highly diverse new functions using combinations of existing protein domains.” However, the low abundance of these variants
is likely to constrain their potential cellular functions, he noted.

Because AARSs are among the oldest proteins, these ancient enzymes were likely subject to plenty of change over time, said Karin Musier-Forsyth, who studies protein translational
at the Ohio State University. According to Musier-Forsyth, synthetases are already known to have non-translational functions and differential localizations. “Like the addition of post-translational modifications, splicing variation has evolved as another way to repurpose protein function,” she said.

One of the protein variants was able to stimulate skeletal muscle fiber formation ex vivo and upregulate genes involved in muscle cell differentiation and metabolism in primary human skeletal myoblasts. “This was really striking,” said Musier-Forsyth. “This suggests
that, perhaps, peptides derived from these splice variants could be used as protein-based therapeutics for a variety of diseases.”

W.S. Lo et al., “Human tRNA synthetase catalytic nulls with diverse functions,” Science, http://dx.doi.org:/10.1126/science.1252943, 2014.

It’s Not Only in DNA’s Hands

By Ilene Schneider LabRoots Aug 22, 2014

Blood stem cells have the potential to turn into any type of blood cell, whether it is the oxygen-carrying red blood cells or the immune system’s many types of white blood cells that help fight infection. How exactly is the fate of these stem cells regulated? Preliminary findings from research conducted by scientists from the Weizmann Institute of Science and the Hebrew University are starting to reshape the conventional understanding of the way blood stem cell fate decisions are controlled, thanks to a new technique for epigenetic analysis developed at these institutions. Understanding epigenetic mechanisms (environmental influences other than genetics) of cell fate could lead to the deciphering of the molecular mechanisms of many diseases,
including immunological disorders, anemia, leukemia, and many more. The study of epigenetics also lends strong support to findings that environmental factors and lifestyle play a more prominent
role in shaping our destiny than previously realized.

The process of differentiation – in which a stem cell becomes a specialized mature cell – is controlled by a cascade of events in which specific genes are turned “on” and “off” in a highly regulated and accurate order. The instructions for this process are contained within the DNA itself in short regulatory sequences.

These regulatory regions are normally in a “closed” state, masked by special proteins called histones to ensure against unwarranted activation. Therefore, to access and “activate”
the instructions,
this DNA mask needs to be “opened” by epigenetic modifications of the histones so it can be read by the necessary machinery.

In a paper published in Science, Dr. Ido Amit and David Lara-Astiaso of the Weizmann Institute’s Department of Immunology, along with Prof. Nir Friedman and Assaf Weiner of the Hebrew University of Jerusalem, charted – for the first time – histone dynamics during blood development. Thanks to the new technique for epigenetic profiling they developed, in which just a handful of cells – as few as 500 – can be sampled and analyzed accurately, they have identified the exact
DNA sequences, as well as the various regulatory proteins, that are involved in regulating the process of blood stem cell fate.

This research has also yielded unexpected results: As many as

50% of these regulatory sequences are established and opened during intermediate stages of cell development.

The meaning of the research is that epigenetics can be active at stages in which it had been thought that cell destiny was already set. “This changes our whole understanding of the process of blood stem cell fate decisions,” says Lara-Astiaso, “suggesting that the process is more
dynamic and flexible than previously thought.”

Although this research was conducted on mouse blood stem cells, the scientists believe that the mechanism may hold true for other types of cells. “This research creates a lot of excitement in the field, as it sets the groundwork to study these regulatory elements in humans,” says Weiner.

Largest Cancer Genetic Analysis Reveals New Way of Classifying Cancer

http://www.biosciencetechnology.com/news/2014/08/largest-cancer-genetic-analysis-reveals-new-way-classifying-cancer

Thu, 08/07/2014 – 2:24pm

Researchers with The Cancer Genome Atlas (TCGA) Research Network have completed the largest, most diverse tumor genetic analysis ever conducted, revealing a new approach to classifying cancers. The work, led by researchers at the UNC Lineberger Comprehensive
Cancer Center at the University of North Carolina at Chapel Hill and other TCGA sites, not only

revamps traditional ideas of how cancers are diagnosed and treated, but could also have
a profound impact on the future landscape of drug development.

“We found that one in 10 cancers analyzed in this study would be classified differently using this new approach,” said Chuck Perou, PhD, professor of genetics and pathology, UNC Lineberger member and senior author of the paper, which appears online Aug. 7 in Cell.
“That means that

10 percent of the patients might be better off getting a different therapy—that’s huge.”

Since 2006, much of the research has identified cancer as not a single disease, but many types and subtypes and has defined these disease types based on the tissue—breast, lung, colon, etc.—in which it originated. In this scenario, treatments were tailored to which
tissue was affected, but questions have always existed because some treatments work, and fail for others, even when a single tissue type is tested.

In their work, TCGA researchers analyzed more than 3,500 tumors across 12 different tissue types to see how they compared to one another — the largest data set of tumor genomics ever assembled, explained Katherine Hoadley, PhD, research assistant professor
in genetics and lead author. They found that

cancers are more likely to be genetically similar based on the type of cell in which the cancer originated, compared to the type of tissue in which it originated.

This is fundamental premise of pathology! (Larry Bernstein) It goes back to Rudolph Virchow.

“In some cases, the cells in the tissue from which the tumor originates are the same,” said Hoadley. “But in other cases, the tissue in which the cancer originates is made up of multiple types of cells that can each give rise to tumors. Understanding the cell in which the cancer originates appears to be very important in determining the subtype of a tumor
and, in turn, how that tumor behaves and how it should be treated.”

Perou and Hoadley explain that the new approach may also shift how cancer drugs are developed, focusing more on the development of drugs targeting larger groups of cancers with genomic similarities, as opposed to a single tumor type as they are currently developed.

One striking example of the genetic differences within a single tissue type is breast cancer.
The breast, a highly complex organ with multiple types of cells, gives rise to multiple types of breast cancer; luminal A, luminal B, HER2-enriched and basal-like, which was previously known. In this analysis, the basal-like breast cancers looked more like ovarian cancer
and cancers of a squamous-cell type origin, a type of cell that composes the lower-layer of a tissue, rather than other cancers that arise in the breast.

“This latest research further solidifies that basal-like breast cancer is an entirely unique disease and is completely distinct from other types of breast cancer,” said Perou. In addition, bladder cancers were also quite diverse and might represent at least three different disease types that also showed differences in patient survival.

As part of the Alliance for Clinical Trials in Oncology, a national network of researchers conducting clinical trials, UNC researchers are already testing the effectiveness of carboplatin—a common treatment for ovarian cancer—on top of standard of care chemotherapy for triple-negative breast cancer (TNBC) patients, of which 80 percent are the basal-like subtype. The results of this study (called CALGB40603)
were just published on Aug. 6 in the Journal of Clinical Oncology and showed a benefit of carboplatin in TNBC patients. This new clinical trial result suggests that there may be great value in comparing clinical results across tumor types for which this study highlights as having common genomic similarities.

As participants in TCGA, UNC Lineberger scientists have been involved in multiple individual tissue type studies including most recently an analysis of a comprehensive genomic profile of lung adenocarcinoma. Perou’s seminal work in 2000 led to the first discovery of breast
cancer as not one, but in fact, four distinct subtypes of disease. These most recent findings should continue to lay the groundwork for what could be the next generation of cancer diagnostics.

Source: University of North Carolina at Chapel Hill School of Medicine

New Gene Tied to Breast Cancer Risk

Wed, 08/06/2014

Marilynn Marchione – AP Chief Medical Writer – Associated Press

It’s long been known that faulty BRCA genes greatly raise the risk for breast cancer. Now, scientists say a more recently identified, less common gene can do the same.

Mutations in the gene can make breast cancer up to nine times more likely to develop, an international team of researchers reports in this week’s New England Journal of Medicine.

About 5 to 10 percent of breast cancers are thought to be due to bad BRCA1 or BRCA2 genes. Beyond those, many other genes are thought to play a role but how much each one raises risk has not been known, said Dr. Jeffrey Weitzel, a genetics expert at City of Hope Cancer Center
in Duarte, Calif.

The new study on the gene- called PALB2 – shows “this one is serious,” and probably is the most dangerous in terms of breast cancer after the BRCA genes, said Weitzel, one of leaders of the study.

It involved 362 members of 154 families with PALB2 mutations – the largest study of its kind. The faulty gene seems to give a woman a 14 percent chance of breast cancer by age 50 and 35 percent by age 70 and an even greater risk if she has two or more close relatives with the disease.

That’s nearly as high as the risk from a faulty BRCA2 gene, Dr. Michele Evans of the National Institute on Aging and Dr. Dan Longo of the medical journal staff write in a commentary in the journal.

The PALB2 gene works with BRCA2 as a tumor suppressor, so when it is mutated, cancer can flourish.

How common the mutations are isn’t well known, but it’s “probably more than we thought because people just weren’t testing for it,” Weitzel said. He found three cases among his own breast cancer
patients in the last month alone.

Among breast cancer patients, BRCA mutations are carried by 5 percent of whites and 12 percent of Eastern European (Ashkenazi) Jews. PALB2 mutations have been seen in up to 4 percent of families with a history of breast cancer.

Men with a faulty PALB2 gene also have a risk for breast cancer that is eight times greater than men in the general population.

Testing for PALB2 often is included in more comprehensive genetic testing, and the new study should give people with the mutation better information on their risk, Weitzel said. Doctors say that people with faulty cancer genes should be offered genetic counseling and may want to consider more frequent screening and prevention options, which can range from hormone-blocking pills to breast removal.

The actress Angelina Jolie had her healthy breasts removed last year after learning she had a defective BRCA1 gene.

The study was funded by many government and cancer groups around the world and was led by Dr. Marc Tischkowitz of the University of Cambridge in England. The authors include Mary-Clare King, the University of Washington scientist who discovered the first breast
cancer predisposition gene, BRCA1.

Study: http://www.nejm.org/doi/full/10.1056/NEJMoa1400382

Gene info: http://ghr.nlm.nih.gov/gene/PALB2

Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide

Eric S. Fischer, Kerstin Böhm, John R. Lydeard, Haidi Yang, …, J. Wade Harper, Jeremy L. Jenkins & Nicolas H. Thomä

Nature (07 Aug 2014); 512, 49–53 http://dx.doi.org:/10.1038/nature13527

Published online 16 July 2014

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide,

these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for
multiple myeloma and 5q-deletion-associated dysplasia.

IMiDs target the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4CRBN) and
promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4CRBN.

Here we present crystal structures of the DDB1–CRBN complex bound to thalidomide,
lenalidomide and pomalidomide. The structure establishes that

CRBN is a substrate receptor within CRL4CRBN and enantioselectively binds IMiDs.

Using an unbiased screen, we identified the

homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN.

Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degradation.

This dual activity implies that

small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

Curator’s Viewpoint:

The short pieces may not appear to be so closely connected, except for the last subject on the pharmaceutical targeting of an E3 ubiquitin ligase ubiquitination of proteins, but even in that case, we have to keep in mind that protein formation by amino acid transcription, remodeling, and recapture of amino acids are in equilibrium through ubiquitylation. So I put it there. The DNA in populations ties some mutations to disease that is tied specifically to populations, not only the sephardic population, but in Asia as well.

The next article for consideration is methodological considerations. The BRCA2 in the sephardic population is one of a number of mutations we can identify, extending to Tay Sachs disease, for instance. How this might have occurred in the history of the jewish people is not so obvious, except perhaps in the segregation of the jewish population for centuries. The mutation would be confined within the population with limited marriage outside of the jewish community. It has been known for some time that there is a Cohen gene that traces back to the priests (Kohanim) of the Holy Temple, the descendents of Aaron (Aharon), the brother of Moses. The priests would stand at the Ark and bless the congregation in the most holy convocation of Yom Kippur, according to tradition. Marriages were arranged between the bride and the groom. Of course, arranged marriages were also the case in other ethnic communities, and between the privileged.

That was dramatically the case during the reign of Queen Victoria of England, with Royal arrangements across Europe.
That would be a factor in the transmission of hemophilia, and in mental disorders in the Royal families. Haemophilia figured prominently in the history of European royalty in the 19th and 20th centuries. Britain’s Queen Victoria, through two of her five daughters (Princess Alice and Princess Beatrice), passed the mutation to various royal houses across the continent, including the royal families of Spain, Germany and Russia. Victoria’s son Prince Leopold, Duke of Albany suffered from the disease. The Prince Leopold, Duke of Albany KG KT GCSI GCMG GCStJ (Leopold George Duncan Albert; 7 April 1853 – 28 March 1884) was the eighth child and fourth son of Queen Victoria and Prince Albert of Saxe-Coburg and Gotha. Leopold was later created Duke of Albany, Earl of Clarence, and Baron Arklow. He had haemophilia, which led to his death at the age of 30. The sex-linked X chromosome disorder manifests almost entirely in males, although the gene for the disorder is located on the X chromosome and may be inherited from either mother or father. Expression of the disorder is much more common in males than in females. This is because, although the trait is recessive, males only inherit one X chromosome, from their mothers. Of course, this is classical Mendelian genetics. Victoria appears to have been a spontaneous or de novo mutation and is usually considered the source of the disease in modern cases of haemophilia among royalty. The mutation would probably be assumed today to have occurred at the conception of Princess Alice, as she was the only known carrier among Victoria and Albert’s first seven children. Leopold was a sufferer of haemophilia and her daughters Alice and Beatrice were confirmed carriers of the gene.

Cousin marriage is marriage between people with a common grandparent or other more distant ancestor. In various cultures and legal jurisdictions, Marriages between first and second cousins account for over 10% of marriages worldwide, and they are common in the Middle East, where in some nations they account for over half of all marriages. Proportions of first-cousin marriage in the United States, Europe and other Western countries like Brazil have declined since the 19th century, though even during that period they were not more than 3.63 percent of all unions in Europe. Cousin marriage is allowed throughout the Middle East for all recorded history, and is used mostly in Syria. It has often been chosen to keep cultural values intact through many generations and preserve familial wealth. In Iraq the right of the cousin has also traditionally been followed and a girl breaking the rule without the consent of the ibn ‘amm could have ended up murdered by him. The Syrian city of Aleppo during the 19th century featured a rate of cousin marriage among the elite of 24% according to one estimate, a figure that masked widespread variation: some leading families had none or only one cousin marriage, while others had rates approaching 70%. Cousin marriage rates were highest among women, merchant families, and older well-established families. The percentage of Iranian cousin marriages increased from 34 to 44% between the 1940s and 1970s. Cousin marriage among native Middle Eastern Jews is generally far higher than among the European Ashkenazim, who assimilated European marital practices after the diaspora.

The essential elements of the marriage contract were now an offer by the man, an acceptance by the woman, and the performance of such conditions as the payment of dowry. According to anthropologist Ladislav Holý, cousin marriage is not an independent phenomenon but rather one expression of a wider Middle Eastern preference for agnatic solidarity, or solidarity with one’s father’s lineage.

A 2009 study found that many Arab countries display some of the highest rates of consanguineous marriages in the world, and that first cousin marriages which may reach 25-30% of all marriages. Research among Arabs and worldwide has indicated that consanguinity could have an effect on some reproductive health parameters such as postnatal mortality and rates of congenital malformations.

In the terraced streets of Bradford, Yorkshire, a child’s death is anything but rare. At the boy’s inquest, coroner Mark Hinchliffe said Hamza Rehman had died because his Pakistan-born parents (shopkeeper Abdul and housewife Rozina) are first cousins. Muslims have practiced marriages between first cousins in non-prohibited countries since the time of the Quran.

Four years before, Hamza’s older sister, three-month-old Khadeja, had died of the same brain disorder which causes fits, sickness and chest infections. The couple had another baby born with equally devastating neurological problems.

A heartbroken Mr Rehman told the inquest that he and his wife were unsure whether to have any more children. The coroner expressed deep sympathy before saying that Hamza’s death should serve as a warning to others.

I have diverged somewhat onto the genetic risks of consanguinous marriages, which George Darwin, son of Charles Darwin, argues were had a small effect in then English society. But most importantly, we see the larger factor here of social and familial inheritance, and also the concept of cultural identity.

Insofar as the somatic and mitochondrial mutations are concerned, I call attention to the finding in the GWAS study above discussed that the results were supportive of the conclusions from mtDNA. This gives some reason to consider whether sufficient information is obtained from the mtDNA, without the more robust GWAS. One cannot fully consider this without some knowledge of the methodology of specimen preparation.

It is not difficult to prepare mitochondria from cells and obtain a very good preparation before further analysis, whether of the membrane structures, the enzymatic activity, or of the DNA and RNA polynucleotides. The separation is easily achieved with differential centrifugation. On the other hand, the finding of the basal layer of epithelium having a different signature than the superficial layer, established by the genomic studies, but known histologically for non-neoplastic tissue, is a matter for cell separation methods that are not easy. It is from the lower layer of cells that we derive carcinoma in-situ. These cells were identified in breast, are expected to be found in uterus, and were like the cells in ovarian-cancer, which suggested the use of a common treatment regimen as adjunct in triple negative breast cancer and ovarian cancer. The importance of a suuficiently prepared cellular specimen as opposed to tissue specimen can’t be taken for granted.

Read Full Post »

« Newer Posts - Older Posts »

Leaders in Pharmaceutical Business Intelligence Group, LLC, Doing Business As LPBI Group, Newton, MA

Archive for the ‘Nitric Oxide in Health and Disease’ Category

Functional Correlates of Signaling Pathways

Functional Correlates of Signaling Pathways

Like this:

Complex Models of Signaling: Therapeutic Implications

Complex Models of Signaling: Therapeutic Implications

Evidence of sphingolipid signaling in cancer

SPHK1 Is a Novel Target of Metformin in Ovarian Cancer.

Author information

Abstract

Like this:

Introduction to Lipid Metabolism

Introduction to Lipid Metabolism

Introduction to Lipid Metabolism

Like this:

Diabetes Mellitus

Diabetes Mellitus

Like this:

Somatic, germ-cell, and whole sequence DNA in cell lineage and disease profiling

Somatic, germ-cell, and whole sequence DNA in cell lineage and disease profiling

Like this:

Follow Blog via Email

Recent Posts

Archives

Categories

Meta

Archive for the ‘Nitric Oxide in Health and Disease’ Category

Functional Correlates of Signaling Pathways

Share this:

Like this:

Summary of Signaling and Signaling Pathways

Share this:

Like this:

Complex Models of Signaling: Therapeutic Implications

Evidence of sphingolipid signaling in cancer

SPHK1 Is a Novel Target of Metformin in Ovarian Cancer.

Abstract

Share this:

Like this:

Introduction to Lipid Metabolism

Introduction to Lipid Metabolism

Share this:

Like this:

Diabetes Mellitus

Share this:

Like this:

Introduction to Metabolomics

Share this:

Like this:

Metabolomics Summary and Perspective

This article has EIGHT parts, as follows:

Share this:

Like this:

Mechanisms of Drug Resistance

Share this:

Like this:

Metformin, Thyroid-Pituitary Axis, Diabetes Mellitus, and Metabolism

Share this:

Like this:

Somatic, germ-cell, and whole sequence DNA in cell lineage and disease profiling

Share this:

Like this:

Follow Blog via Email

Recent Posts

Archives

Categories

Meta