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Integrins, Cadherins, Signaling and the Cytoskeleton

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Integrins, Cadherins, Signaling and the Cytoskeleton

Curator: Larry H. Bernstein, MD, FCAP

We have reviewed the cytoskeleton, cytoskeleton pores and ionic translocation under lipids. We shall now look at this again, with specific attention to proteins, transporters and signaling.

Integrins and extracellular matrix in mechanotransduction

Lindsay Ramage
Queen’s Medical Research Institute, University of Edinburgh,

Edinburgh, UK
Cell Health and Cytoskeleton 2012; 4: 1–9

https://s3.amazonaws.com/academia.edu.documents/37116869/CHC-21829-integrins-and-extracellular-matrix-in-mechanotransduction_122311.pdf?response-content-disposition=inline%3B%20filename%3DCell_Health_and_Cytoskeleton_Integrins_a.pdf&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWOWYYGZ2Y53UL3A%2F20191231%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20191231T021009Z&X-Amz-Expires=3600&X-Amz-SignedHeaders=host&X-Amz-Signature=b376084e0e1c31c399ee7fe96eb81b1b65d3346d647192e9ebeff96f577e117d

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.

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

embryonic development,
inflammatory responses,
wound healing,
and adult tissue homeostasis.

This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.

Integrins are a family of αβ heterodimeric receptors which act as

cell adhesion molecules
connecting the ECM to the actin cytoskeleton.

The actin cytoskeleton is involved in the regulation of

cell motility,
cell polarity,
cell growth, and
cell survival.

The integrin family consists of around 25 members which are composed of differing

combinations of α and β subunits.

The combination of αβ subunits determines

binding specificity and
signaling properties.

In mammals around 19 α and eight β subunits have been characterized.

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.

The ECM is a complex mixture of matrix molecules, including -glycoproteins, collagens, laminins, glycosaminoglycans, proteoglycans,
and nonmatrix proteins, – including growth factors.
These can be categorized as insoluble molecules within the ECM, soluble molecules, and/or matrix-associated biochemicals, such as systemic hormones or growth factors and cytokines that act locally.

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.6 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.

The integrins are composed of well-defined domains used for protein–protein interactions. The α-I domains of α integrin subunits comprise the ligand binding sites. X-ray crystallography has identified an α-I domain within the β subunit and a β propeller domain within the α subunit which complex to form the ligand-binding head of the integrin.

The use of activating and conformation-specific antibodies also suggests that the β chain is extended in the active integrin. It has since been identified that the hybrid domain in the β chain is critical for integrin activation, and a swing-out movement of this leg activates integrins.

http://www.ks.uiuc.edu/Publications/Stories/tcbg_ytt/pdfs/dbp6.pdf

DBP6: Integrin

Integrin

Integrin.large

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.

integrin coupled to F-actin via linker

http://dx.dio.org:/integrin-coupled-to-f-actin-via-linker-nrm3896-f4.jpg

Integrin extracellular binding activity is regulated from inside the cell and binding to the ECM induces signals that are transmitted into the cell.15 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

cell polarity,
directed cell migration, and
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

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

The binding of the adaptor protein talin to the β subunit cytoplasmic tail is known to have a key role in integrin activation. This is thought to occur through the disruption of

inhibitory interactions between α and β subunit cytoplasmic tails.

Talin also binds

to actin and to cytoskeletal and signaling proteins.

This allows talin to directly link activated integrins

to signaling events and the cytoskeleton.

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

cell type,
matrix composition, and
integrins activated.

One way in which integrin expression is important in genetic programming is in the fate and differentiation of stem cells.
Osteoblast differentiation occurs through ECM interactions

with specific integrins

to initiate intracellular signaling pathways leading to osteoblast-specific gene expression
disruption of interactions between integrins and collagen;
fibronectin blocks osteoblast differentiation and

Disruption of α2 integrin prevents osteoblast differentiation, and activation of the transcription factor

osteoblast-specific factor 2/core-binding factor α1.

It was found that the ECM-integrin interaction induces osteoblast-specific factor 2/core-binding factor α1 to

increase its activity as a transcriptional enhancer
rather than increasing protein levels.

It was also found that modification of α2 integrin alters

induction of the osteocalcin promoter;
inhibition of α2 prevents activation of the osteocalcin promoter,
overexpression enhanced osteocalcin promoter activity.

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.

A variety of growth factors and cytokines have been shown to be important in the regulation of integrin expression and function in chondrocytes. Mechanotransduction in chondrocytes occurs through several different receptors and ion channels including integrins. During osteoarthritis the expression of integrins by chondrocytes is altered, resulting in different cellular transduction pathways which contribute to tissue pathology.

In normal adult cartilage, chondrocytes express α1β1, α10β1 (collagen receptors), α5β1, and αvβ5 (fibronectin) receptors. 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 (Figure 3). Early events are similar; α5β1 integrin and stretch activated ion channels are activated and result in rapid tyrosine phosphorylation events. The actin cytoskeleton is required for the integrin-dependent Mechanotransduction leading to changes in membrane potential in normal but not osteoarthritic chondrocytes.

Cell–matrix interactions are essential for maintaining the integrity of tissues. An intact matrix is essential for cell survival and proliferation and to allow efficient mechanotransduction and tissue homeostasis. Cell–matrix interactions have been extensively studied in many tissues and this knowledge is being used to develop strategies to treat pathology. This is particularly important in tissues subject to abnormal mechanical loading, such as musculoskeletal tissues. Integrin-ECM interactions are being used to enhance tissue repair mechanisms in these tissues through differentiation of progenitor cells for in vitro and in vivo use. Knowledge of how signaling cascades are differentially regulated in response to physiological and pathological external stimuli (including ECM availability and mechanical loading/stimulation) will enable future strategies to be developed to prevent and treat the progression of pathology associated with integrin-ECM interactions.

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

Matthews, DR. Overby, R Mannix and DE. Ingber
1Vascular Biology Program, Departments of Pathology and Surgery, Children’s Hospital, and 2Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, MA J Cell Sci 2006; 119: 508-518. http://dx.doi.org:/10.1242/jcs.02760

To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead isplacement with sub-micrometer resolution using optical microscopy. 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

J Atherton, I Farabella, I-Mei Yu, SS Rosenfeld, A Houdusse, M Topf, CA Moores

1Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, London, United Kingdom; 2Structural Motility, Institut Curie, Centre National de la Recherche Scientifique, Paris, France; 3Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States
eLife 2014;3:e03680. http://dx.doi.org:/10.7554/eLife.03680

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.

Atherton et al. use a technique called cryo-electron microscopy to study—in more detail than previously seen—the structure of the motor domains of two types of kinesin called kinesin-1 and kinesin-3. Images were taken at different stages of the cycle used by the motor domains to extract the energy from ATP molecules. 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 are relatively small but 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. This mechanism involves tight coupling between track binding and fuel usage and makes kinesins highly efficient motors.

A number of kinesins drive long distance transport of cellular cargo with dimerisation allowing them to take multiple 8 nm ATP-driven steps toward MT plus ends. Their processivity depends on communication between the two motor domains, which is achieved via the neck linker that connects each motor domain to the dimer-forming coiled-coil

Kinesins are a superfamily of microtubule-based

ATP-powered motors, important for multiple, essential cellular functions.

How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy.

All our reconstructions have, as their asymmetric unit, a triangle-shaped motor domain bound to an αβ-tubulin dimer within the MT lattice (Figure 1). The structural comparisons below are made with respect to the MT surface, which, at the resolution of our structures (∼7 Å, Table 1), is the same (CCC > 0.98 for all). As is well established across the superfamily, 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 (Figure 1C, Kikkawa et al., 2001).

However, multiple conformational changes are seen throughout the rest of each domain in response to bound nucleotide (Figure 1D). Below, we describe the conformational changes in functionally important regions of each motor domain 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. In addition, 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 (Figure 3—figure supplement 2); this is required to accommodate rotation of helix-α6 and consequent neck linker docking (Figure 3B–E).

Peeling of the central β-sheet has previously been proposed to arise from tilting of the entire motor domain relative to static MT contacts, pivoting around helix-α4 (the so-called ‘seesaw’ model; Sindelar, 2011). Specifically, this model predicts that the major difference in the motor before and after Mg-ATP binding would be the orientation of the motor domain with respect to helix-α4.

Kinesin mechanochemistry and the extent of mechanistic conservation within the motor superfamily are open questions, critical to explain how MT binding, and ATP binding and hydrolysis drive motor activity. Our structural characterisation of two transport motors now allows us to propose a model that describes the roles of mechanochemical elements that together drive conserved MT-based motor function.

Model of conserved MT-bound kinesin mechanochemistry. Loop11/N-terminus of helix-α4 is flexible in ADP-bound kinesin in solution, the neck linker is also flexible while loop9 chelates ADP. MT binding is sensed by loop11/helix-α4 N-terminus, biasing them towards more ordered conformations.

We propose that this favours crosstalk between loop11 and loop9, stimulating ADP release. In the NN conformation, both loop11 and loop9 are well ordered and primed to favour ATP binding, while helix-α6—which is required for mechanical amplification–is closely associated with the MT on the other side of the motor domain. 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.

Family-specific differences across the kinesin–microtubule interface account for the

distinctive properties of each motor.

Our data thus provide evidence for a

conserved ATP-driven

mechanism for kinesins and
reveal the critical mechanistic contribution of the microtubule interface.

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

I Timmerman, PL Hordijk, JD van Buul

Cell Health and Cytoskeleton 2010; 2: 23–31
Endothelial cell–cell junctions are strictly regulated in order to

control the barrier function of endothelium.

Vascular endothelial (VE)-cadherin is one of the proteins that is crucial in this process. It has been reported that

phosphorylation events control the function of VE-cadherin.

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 postcapillary 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.

Data using purified proteins show that

monomeric α-catenin binds strongly to cadherin-bound β-catenin;
in contrast to the dimer which has a higher affinity for actin filaments,
indicating that α-catenin might function as a molecular switch regulating cadherin-mediated cell–cell adhesion and actin assembly.

Thus, interactions between the cadherin complex and the actin cytoskeleton are more complex than previously thought. Recently, Takeichi and colleagues reported that

the actin binding protein EPLIN (epithelial protein lost in neoplasm)
can associate with α-catenin and thereby
link the E-cadherin–catenin complex to the actin cytoskeleton.

Although this study was performed in epithelial cells,

an EPLIN-like molecule might serve as
a bridge between the cadherin–catenin complex and
the actin cytoskeleton in endothelial cells.

Next to β-catenin and plakoglobin, p120-catenin also binds directly to the intracellular tail of VE-cadherin.

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. Mutant forms of VE-cadherin which

lack either the β-catenin, plakoglobin or p120 binding regions reduce the strength of cell–cell adhesion.

Moreover, our own results showed that

interfering with the interaction between α-catenin and β-catenin,
using a cell-permeable peptide which encodes the binding site in α-catenin for β-catenin,
resulted in an increased permeability of the endothelial monolayer.

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.

One of the first reports that supported this idea showed that the level of phosphorylation of VE-cadherin was

high in loosely confluent endothelial cells, but
low in tightly confluent monolayers,

when intercellular junctions are stabilized.

In addition, several conditions that induce tyrosine phosphorylation

of adherens junction components, like

v-Src transformation
and inhibition of phosphatase activity by pervanadate,

have been shown to shift cell–cell adhesion from a strong to a weak state. More physiologically relevant;

permeability-increasing agents such as

histamine,
tumor necrosis factor-α (TNF-α),
thrombin,
platelet-activating factor (PAF) and
vascular endothelial growth factor (VEGF)

increase tyrosine phosphorylation of various components of the cadherin–catenin complex.

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

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

Notes: A) Permeability-inducing agents such as thrombin, histamine and VEGF, induce tyrosine phosphorylation (pY) of VE-cadherin and the associated catenins. Although the specific consequences of catenin tyrosine phosphorylation in endothelial cells are still unknown, VE-cadherin tyrosine phosphorylation results in opening of the cell–cell junctions (indicated by arrows) and enhanced vascular permeability. How tyrosine phosphorylation affects VE-cadherin adhesiveness is not yet well understood; disrupted binding of catenins, which link the cadherin to the actin cytoskeleton, may be involved. VEGF induces phosphorylation of VE-cadherin at specific residues, Y658 and Y731, which have been reported to regulate p120-catenin and β-catenin binding, respectively. Moreover, VEGF stimulation results in serine phosphorylation (pSer) of VE-cadherin, specifically at residue S665, which leads to its endocytosis. B) Adhesion of leukocytes to endothelial cells via ICAM-1 increases endothelial permeability by inducing phosphorylation of VE-cadherin on tyrosine residues. Essential mediators, such as the kinases Pyk2 and Src, and signaling routes involving reactive oxygen species (ROS) and Rho, have been shown to act downstream of ICAM-1. Different tyrosine residues within the cytoplasmic domain of VE-cadherin are involved in the extravasation of neutrophils and lymphocytes, including Y658 and Y731. (β: β-catenin, α: α-catenin, γ: γ-catenin/plakoglobin).

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

BT Jamal, MN Nita-Lazar, Z Gao, B Amin, J Walker, MA Kukuruzinska
Cell Health and Cytoskeleton 2009; 1: 67–80

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.

On the other hand, V13/γ-catenin complexes associated more with vinculin, suggesting that they

mediated the interaction of AJs with the actin cytoskeleton.
N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin.

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

Matteo Vatta, and Georgine Faulkner,

1 Departments of Pediatrics (Cardiology), Baylor College of Medicine, Houston, TX 2 Department of Reproductive and Developmental Sciences, University of Trieste, Trieste, Italy
3 Muscular Molecular Biology Unit, International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy

Future Cardiol. 2006 July 1; 2(4): 467–476. http://dx.doi.org:/10.2217/14796678.2.4.467

The heart is a force-generating organ that responds to

self-generated electrical stimuli from specialized cardiomyocytes.

This function is modulated

by sympathetic and parasympathetic activity.

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. Here I will review

the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels

and, I will discuss the future impact of new data on molecular cardiology research and clinical practice.

Myocardial dysfunction in the end-stage failing heart is very often associated with increasing

susceptibility to ventricular tachycardia (VT) and ventricular fibrillation (VF),

both of which are common causes of sudden cardiac death (SCD).

Among the various forms of HF,

myocardial remodeling due to ischemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM)

is characterized by alterations in baseline ECG,

which includes the

prolongation of the QT interval,
as well as QT dispersion,
ST-segment elevation, and
T-wave abnormalities,

especially during exercise. In particular, 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.

Despite the increasing use of LVAD supporting either continuous or pulsatile blood flow in patients with severe HF, the benefit of this treatment in dealing with the risk of arrhythmias is still controversial.

Large epidemiological studies, such as the REMATCH study, demonstrated that the

employment of LVAD significantly improved survival rate and the quality of life, in comparison to optimal medical management.

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,

while no effect was observed on the development of polymorphic ventricular tachycardia (PVT)/ventricular fibrillation (VF).

The sarcomere

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

plasma membrane,
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. Titin connects

the Z-line to the M-line of the sarcomeric structure
(Figure 1).

In addition to the strategic

localization and mechanical spring function,
titin is a length-dependent sensor during
stretch and promotes actin-myosin interaction

Titin is stabilized by the cross-linking protein

telethonin (T-Cap), which localizes at the Z-line and is also part of titin sensor machinery (Figure 1).

The complex protein interactions in the sarcomere entwine telethonin to other

Z-line components through the family of the telethonin-binding proteins of the Z-disc, FATZ, also known as calsarcin and myozenin.

FATZ binds to

calcineurin,
γ-filamin as well as the
spectrin-like repeats (R3–R4) of α-actinin-2,

the major component of the Z-line and a pivotal

F-actin cross-linker (Figure 1).contractile unit of striated muscles, and
the sarcolemma,

the plasma membrane surrendering the muscle fibers in skeletal muscle and the muscle cell of the cardiomyocyte,

determines the mechanical plasticity of the cell, enabling it to complete and re-initiate each contraction-relaxation cycle.

At the level of the sarcomere,

actin (thin) and myosin (thick) filaments generate the contractile force,

while other components such as titin, the largest protein known to date, are responsible for

the passive force during diastole and for the restoring force during systole, and (titin).
the Z-line to the M-line of the sarcomeric structure
(Figure 1).

In addition to the strategic

localization and mechanical spring function,
it acts as a length-dependent sensor during stretch and
promotes actin-myosin interaction.

Stabilized by the cross-linking protein telethonin (T-Cap),

titin localizes at the Z-line and is
part of titin sensor machinery

Another cross-linker of α-actinin-2 in the complex Z-line scaffold is

the Z-band alternatively spliced PDZ motif protein (ZASP),
which has an important role in maintaining Z-disc stability

in skeletal and cardiac muscle (Figure 1).

ZASP contains a PDZ motif at its N-terminus,

which interacts with C-terminus of α-actinin-2,
and a conserved sequence called the ZASP like motif (ZM)
found in the alternatively spliced exons 4 and 6.

It has also been reported

to bind to the FATZ (calsarcin) family of Z-disc proteins (Figure 1).

The complex protein interactions in the sarcomere entwine telethonin to other Z-line components through the family of the telethonin-binding proteins of the

Z-disc,
FATZ, also known as calsarcin and
myozenin

FATZ binds to calcineurin,

γ-filamin as well as the
spectrin-like repeats (R3–R4) of α-actinin-2, the major component of the Z-line and a pivotal F-actin cross-linker (Figure 1).

sarcomere structure

Figure 1. Sarcomere structure

The diagram illustrates the sarcomeric structure. The Z-line determines the boundaries of the contractile unit, while Titin connects the Z-line to the M-line and acts as a functional spring during contraction/relaxation cycles.

Sarcomeric Proteins and Ion Channels

In addition to systolic dysfunction characteristic of dilated cardiomyopathy (DCM) and diastolic dysfunction featuring hypertrophic cardiomyopathy (HCM), the clinical phenotype of patients with severe cardiomyopathy is very often associated with a high incidence of cardiac arrhythmias. Therefore, 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

affect ion channel anchoring, and trafficking, as well as
functional regulation by second messenger pathways,
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.

In 1991, Cantiello et al., demonstrated that

although the epithelial sodium channel and F-actin are in close proximity,
they do not co-localize.

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.

Later, the group of Dr. Jonathan Makielski demonstrated that

actin disruption induced a dramatic reduction in Na+ peak current and
slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation.

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

F-actin is intertwined in a multi-protein complex that includes

the composite Z-line structure.

Further, there is a direct binding between

the major protein of the Z-line, α-actinin-2 and
the voltage-gated K+ channel 1.5 (Kv1.5), (Figure 2).

The latter is expressed in human cardiomyocytes and localizes to

the intercalated disk of the cardiomyocyte
in association with connexin and N-cadherin.

Maruoka et al. treated HEK293 cells stably expressing Kv1.5 with cytochalasin D, which led to

a massive increase in ionic and gating IK+ currents.

This was prevented by pre-incubation with phalloidin, an F-actin stabilizing agent. In addition, the Z-line protein telethonin binds to the cytoplasmic domain of minK, the beta subunit of the potassium channel KCNQ1 (Figure 2).

Molecular interactions between the cytoskeleton and ion channels

Figure 2. 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. The cardiac sodium channel Nav1.5 associates with the DGC, while potassium channels such as Kv1.5, associate with the Z-line.

Ion Channel Subunits and Trafficking

Correct localization is essential for ion channel function and this is dependent upon the ability of auxiliary proteins to

shuttle ion channels from the cytoplasm to their final destination such as
the plasma membrane or other sub-cellular compartments.

In this regard, Kvβ-subunits are

cytoplasmic components known to assemble with the α-subunits of voltage-dependent K+ (Kv) channels
at their N-terminus to form stable Kvα/β hetero-oligomeric channels.

When Kvβ is co-expressed with Kv1.4 or Kv1.5, it enhances Kv1.x channel trafficking to the cell membrane without changing the overall protein channel content. The regulatory Kvβ subunits, which are also expressed in cardiomyocytes, directly decrease K+ current by

accelerating Kv1.x channel inactivation.

Therefore, altered expression or mutations in Kvβ subunits could cause abnormal ion channel transport to the cell surface, thereby increasing the risk of cardiac arrhythmias.

Ion Channel Protein Motifs and Trafficking

Cell membrane trafficking in the Kv1.x family may occur in a Kvβ subunit-independent manner through specific motifs in their C-terminus. Mutagenesis of the final asparagine (N) in the Kv1.2 motif restores the leucine (L) of the Kv1.4 motif

re-establishing high expression levels at the plasma membrane in a Kvβ-independent manner

Cytoskeletal Proteins and Ion Channel Trafficking

Until recently, primary arrhythmias such as LQTS have been almost exclusively regarded as ion channelopathies. Other mutations have been identified with regard to channelopathies. However, the conviction that primary mutations in ion channels were solely responsible for

the electrical defects associated with arrhythmias

has been shaken by the identification of mutations in the

ANK2 gene encoding the cytoskeletal protein ankyrin-B

that is associated with LQTS in animal models and humans.

Ankyrin-B acts as a chaperone protein, which shuttles the cardiac sodium channel from the cytoplasm to the membrane. Immunohistochemical analysis has localized ankyrin-B to the Zlines/T-tubules on the plasma membrane in the myocardium. Mutations in ankyrin-B associated with LQTS

alter sodium channel trafficking due to loss of ankyrin-B localization at the Z-line/transverse (T)-tubules.

Reduced levels of ankyrin-B at cardiac Z-lines/T-tubules were associated with the deficiency of ankyrin-B-associated proteins such as Na/K-ATPase, Na/Ca exchanger (NCX) and inositol-1, 4, 5-trisphosphate receptors (InsP3R).

Dystrophin component of the Dystrophin Glycoprotien Complex (DGC)

Synchronized contraction is essential for cardiomyocytes, which are connected to each other via the extracellular matrix (ECM) through the DGC. The N-terminus domain of dystrophin

binds F-actin, and connects it to the sarcomere, while
the cysteine-rich (CR) C-terminus domain ensures its connection to the sarcolemma (Figure 2).

The central portion of dystrophin, the rod domain, is composed of

rigid spectrin-like repeats and four hinge portions (H1–H4) that determine the flexibility of the protein.

Dystrophin possesses another F-actin binding domain in the Rod domain region, between the basic repeats 11- 17 (DysN-R17).

Dystrophin, originally identified as the gene responsible for Duchenne and Becker muscular dystrophies (DMD/BMD), and later for the X-linked form of dilated cardiomyopathy (XLCM), exerts a major function in physical force transmission in striated muscle. In addition to its structural significance, dystrophin and other DGC proteins such as syntrophins are required for the

correct localization,
clustering and
regulation of ion channel function.

Syntrophins have been implicated in ion channel regulation. Syntrophins contain two pleckstrin homology (PH) domains, a PDZ domain, and a syntrophin-unique (SU) C-terminal region. The interaction between syntrophins and dystrophin occurs at the PH domain distal to the syntrophin N-terminus and through the highly conserved SU domain. Conversely, the PH domain proximal to the N-terminal portion of the protein and the PDZ domain interact with other membrane components such as

phosphatidyl inositol-4, 5-bisphosphate,
neuronal NOS (nNOS),
aquaporin-4,
stress-activated protein kinase-3, and
5,

thereby linking all these molecules to the dystrophin complex (Figure 2).

Among the five known isoforms of syntrophin, the 59 KDa α1-syntrophin isoform is the most highly represented in human heart, whereas in skeletal muscle it is only present on the

sarcolemma of fast type II fibers.

In addition, the skeletal muscle γ2-syntrophin was found at high levels only at the

postsynaptic membrane of the neuromuscular junctions.

In addition to syntrophin, other scaffolding proteins such as caveolin-3 (CAV3), which is present in the caveolae, flask-shaped plasma membrane microdomains, are involved

in signal transduction and vesicle trafficking in myocytes,
modulating cardiac remodeling during heart failure.

CAV3 and α1-syntrophin, localizes at the T-tubule and are part of the DGC. In addition, α1-syntrophin binds Nav1.5, while

caveolin-3 binds the Na+/Ca2+ exchanger, Nav1.5 and the L-type Ca2+ channel as well as nNOS and the DGC (Figure 2).

Although ankyrin-B is the only protein found mutated in patients with primary arrhythmias, other proteins such as caveolin-3 and the syntrophins if mutated may alter ion channel function.

Conclusions

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.

Executive Summary

Arrhythmogenesis and myocardial structure

Rhythm alterations can develop as a secondary consequence of myocardial structural abnormalities or as a result of a primary defect in the cardiac electric machinery.
Until recently, no molecular mechanism has been able to fully explain the occurrence of arrhythmogenesis in heart failure, however genetic defects that are found almost exclusively in ion channel genes account for the majority of primary arrhythmias such as long QT syndromes and Brugada syndrome. The contractile apparatus is linked to ion channels
The sarcomere, which represents the contractile unit of the myocardium not only generates the mechanical force necessary to exert the pump function, but also provides localization and anchorage to ion channels.
Alpha-actinin-2, and telethonin, two members of the Z-line scaffolding protein complex in the striated muscle associate with the potassium voltage-gated channel alpha subunit Kv1.5 and the beta subunit KCNE1 respectively.
Mutations in KCNE1 have previously been associated with the development of arrhythmias in LQTS subjects.
Mutations in both alpha-actinin-2, and telethonin were identified in individuals with cardiomyopathy. The primary defect is structural leading to ventricular dysfunction, but the secondary consequence is arrhythmia.

Ion channel trafficking and sub-cellular compartments

Ion channel trafficking from the endoplasmic reticulum (ER) to the Golgi complex is an important check-point for regulating the functional channel molecules on the plasma membrane. Several molecules acting as chaperones bind to and shuttle the channel proteins to their final localization on the cell surface
Ion channel subunits such as Kvβ enhance Kv1.x ion channel presentation on the sarcolemma. The α subunits of the Kv1.x potassium channels can be shuttled in a Kvβ-independent manner through specific sequence motif at Kv1.x protein level.
In addition, cytoskeletal proteins such as ankyrin-G bind Nav1.5 and are involved in the sodium channel trafficking. Another member of the ankyrin family, ankyrin-B was found mutated in patients with LQTS but the pathological mechanism of ankyrin-B mutations is still obscure, although the sodium current intensity is dramatically reduced.

The sarcolemma and ion channels

The sarcolemma contains a wide range of ion channels, which are responsible for the electrical propagating force in the myocardium.
The DGC is a protein complex, which forms a scaffold for cytoskeletal components and ion channels.
Dystrophin is the major component of the DGC and mutations in dystrophin and DGC cause muscular dystrophies and X-linked cardiomyopathies (XLCM) in humans. Cardiomyopathies are associated with arrhythmias
Caveolin-3 and syntrophins associate with Nav1.5, and are part of the DGC. Syntrophins can directly modulate Nav1.5 channel function.

Conclusions

The role of the cytoskeleton in ion channel function has been hypothesized in the past, but only recently the mechanism underlying the development of arrhythmias in structurally impaired myocardium has become clearer.
The recently acknowledged role of the cytoskeleton in ion channel function suggests that genes encoding cytoskeletal proteins should be regarded as potential candidates for variants involved in the susceptibility to arrhythmias, as well as the primary target of genetic mutations in patients with arrhythmogenic syndromes such as LQTS and Brugada syndrome.
Studies of genotype-phenotype correlation and and patient risk stratification for mutations in cytoskeletal proteins will help to tailor the therapy and management of patients with arrhythmias.

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Introduction to Signaling

Posted in Academic Publishing, Cardiovascular Research, Curation, Curation methodology, Cytoskeleton, Developmental biology, Discovery process, Disease Biology, Endocrine Diseases, Enzyme Induction, Enzymes and isoenzymes, Evolutionary cognition, Experimental validation, Explanatory, Gene Regulation and Evolution, Historical relevance, Innovations, Lipid metabolism, Metabolism, Metabolomics, Na-K transport, Na-K-ATPase, Neurohumoral Transmission, Nutrition, Pharmaceutical Analytics, Phosphorylation, Proteins, Proteomics, RNA Biology, Cancer and Therapeutics, S-nitrosylation, Signaling, Signaling & Cell Circuits, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, Transcriptomics, Ubiquitinylation, tagged adaptive compensatory glycolysis, androgen receptor, Chemokine receptor, Endothelin Receptors, Estrogen receptor, glucose transporters, signaling, steroid hormone receptors, transporters on October 30, 2014| Leave a Comment »

Introduction to Signaling

Curator: Larry H. Bernstein, MD, FCAP

We have laid down a basic structure and foundation for the remaining presentations. It was essential to begin with the genome, which changed the course of teaching of biology and medicine in the 20^th century, and introduced a central dogma of translation by transcription. Nevertheless, there were significant inconsistencies and unanswered questions entering the twenty first century, accompanied by vast improvements in technical advances to clarify these issues. We have covered carbohydrate, protein, and lipid metabolism, which function in concert with the development of cellular structure, organ system development, and physiology. To be sure, the progress in the study of the microscopic and particulate can’t be divorced from the observation of the whole. We were left in the not so distant past with the impression of the Sufi story of the elephant and the three blind men, who one at a time held the tail, the trunk, and the ear, each proclaiming that it was the elephant.

I introduce here a story from the Brazilian biochemist, Jose

Eduardo des Salles Rosalino, on a formativr experience he had with the Nobelist, Luis Leloir.

Just at the beginning, when phosphorylation of proteins is presented, I assume you must mention that some proteins are activated by phosphorylation. This is fundamental in order to present self –organization reflex upon fast regulatory mechanisms. Even from an historical point of view. The first observation arrived from a sample due to be studied on the following day of glycogen synthetase. It was unintended left overnight out of the refrigerator. The result was it has changed from active form of the previous day to a non-active form. The story could have being finished here, if the researcher did not decide to spent this day increasing substrate levels (it could be a simple case of denaturation of proteins that changes its conformation despite the same order of amino acids). He kept on trying and found restoration of maximal activity. This assay was repeated with glycogen phosphorylase and the result was the opposite – it increases its activity. This led to the discovery

of cAMP activated protein kinase and
the assembly of a very complex system in the glycogen granule
that is not a simple carbohydrate polymer.

Instead, it has several proteins assembled and

preserves the capacity to receive from a single event (rise in cAMP)
two opposing signals with maximal efficiency,
stops glycogen synthesis,
as long as levels of glucose 6 phosphate are low
and increases glycogen phosphorylation as long as AMP levels are high).

I did everything I was able to do by the end of 1970 in order to repeat the assays with PK I, PKII and PKIII of M. Rouxii and using the Sutherland route to cAMP failed in this case. I then asked Leloir to suggest to my chief (SP) the idea of AA, AB, BB subunits as was observed in lactic dehydrogenase (tetramer) indicating this as his idea. The reason was my “chief”(SP) more than once, had said to me: “Leave these great ideas for the Houssay, Leloir etc…We must do our career with small things.” However, as she also had a faulty ability for recollection she also used to arrive some time later, with the very same idea but in that case, as her idea.
Leloir, said to me: I will not offer your interpretation to her as mine. I think it is not phosphorylation, however I think it is glycosylation that explains the changes in the isoenzymes with the same molecular weight preserved. This dialogue explains why during the reading and discussing “What is life” with him he asked me if as a biochemist in exile, talking to another biochemist, I expressed myself fully. I had considered that Schrödinger would not have confronted Darlington & Haldane because he was in U.K. in exile. This might explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes, in a way that suggested that the pattern could be of covalent changes over a protein. Our FEBS and Eur J. Biochemistry papers on pyruvate kinase of M. Rouxii is wrongly quoted in this way on his review about pyruvate kinase of that year (1971).

Another aspect I think you must call attention to the following. Show in detail with different colors what carbons belongs to CoA, a huge molecule in comparison with the single two carbons of acetate that will produce the enormous jump in energy yield

in comparison with anaerobic glycolysis.

The idea is

how much must have been spent in DNA sequences to build that molecule in order to use only two atoms of carbon.

Very limited aspects of biology could be explained in this way. In case we follow an alternative way of thinking, it becomes clearer that proteins were made more stable by interaction with other molecules (great and small). Afterwards, it’s rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy).

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.

The discussions that follow are concerned with protein interactions and signaling.

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Oxidation and Synthesis of Fatty Acids

Posted in Biochemical pathways, Cell Biology, Enzymes and isoenzymes, Fatty acids, Lipid metabolism, Metabolism, Metabolomics, Myocardial Metabolism, Oxidative phosphorylation, Phosphorylation, Signaling, tagged fatty acid oxidation, Lynen cycle, mitochondria, Oxidative phosphorylation on October 25, 2014| Leave a Comment »

Oxidation and Synthesis of Fatty Acids

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

Lipid Metabolism

http://www.elmhurst.edu/~chm/vchembook/622overview.html

Overview of Lipid Catabolism:

The major aspects of lipid metabolism are involved with

Fatty Acid Oxidation to produce energy or
the synthesis of lipids which is called Lipogenesis.

The metabolism of lipids and carbohydrates are related by the conversion of lipids from carbohydrates. This can be seen in the diagram. Notice the link through actyl-CoA, the seminal discovery of Fritz Lipmann. The metabolism of both is upset by diabetes mellitus, which results in the release of ketones (2/3 betahydroxybutyric acid) into the circulation.

metabolism of fats

http://www.elmhurst.edu/~chm/vchembook/images/590metabolism.gif

The first step in lipid metabolism is the hydrolysis of the lipid in the cytoplasm to produce glycerol and fatty acids.

Since glycerol is a three carbon alcohol, it is metabolized quite readily into an intermediate in glycolysis, dihydroxyacetone phosphate. The last reaction is readily reversible if glycerol is needed for the synthesis of a lipid.

The hydroxyacetone, obtained from glycerol is metabolized into one of two possible compounds. Dihydroxyacetone may be converted into pyruvic acid, a 3-C intermediate at the last step of glycolysis to make energy.

In addition, the dihydroxyacetone may also be used in gluconeogenesis (usually dependent on conversion of gluconeogenic amino acids) to make glucose-6-phosphate for glucose to the blood or glycogen depending upon what is required at that time.

Fatty acids are oxidized to acetyl CoA in the mitochondria using the fatty acid spiral. The acetyl CoA is then ultimately converted into ATP, CO₂, and H₂O using the citric acid cycle and the electron transport chain.

There are two major types of fatty acids – ω-3 and ω-6. There are also saturated and unsaturated with respect to the existence of double bonds, and monounsaturated and polyunsatured. Polyunsaturated fatty acids (PUFAs) are important in long term health, and it will be seen that high cardiovascular risk is most associated with a low ratio of ω-3/ω-6, the denominator being from animal fat. Ω-3 fatty acids are readily available from fish, seaweed, and flax seed. More can be said of this later.

Fatty acids are synthesized from carbohydrates and occasionally from proteins. Actually, the carbohydrates and proteins have first been catabolized into acetyl CoA. Depending upon the energy requirements, the acetyl CoA enters the citric acid cycle or is used to synthesize fatty acids in a process known as LIPOGENESIS.

The relationships between lipid and carbohydrate metabolism are
summarized in Figure 2.

fattyacidspiral

http://www.elmhurst.edu/~chm/vchembook/images/620fattyacidspiral.gif

Energy Production Fatty Acid Oxidation:

“Visible” ATP:

In the fatty acid spiral, there is only one reaction which directly uses ATP and that is in the initiating step. So this is a loss of ATP and must be subtracted later.

A large amount of energy is released and restored as ATP during the oxidation of fatty acids. The ATP is formed from both the fatty acid spiral and the citric acid cycle.

Connections to Electron Transport and ATP:

One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD⁺ (step 3) with the electron transport chain. Total ATP per turn of the fatty acid spiral is:

Electron Transport Diagram – (e.t.c.)

Step 1 – FAD into e.t.c. = 2 ATP
Step 3 – NAD⁺ into e.t.c. = 3 ATP
Total ATP per turn of spiral = 5 ATP

In order to calculate total ATP from the fatty acid spiral, you must calculate the number of turns that the spiral makes. Remember that the number of turns is found by subtracting one from the number of acetyl CoA produced. See the graphic on the left bottom.

Example with Palmitic Acid = 16 carbons = 8 acetyl groups

Number of turns of fatty acid spiral = 8-1 = 7 turns

ATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP.

This would be a good time to remember that single ATP that was needed to get the fatty acid spiral started. Therefore subtract it now.

NET ATP from Fatty Acid Spiral = 35 – 1 = 34 ATP

Review ATP Summary for Citric Acid Cycle:The acetyl CoA produced from the fatty acid spiral enters the citric acid cycle. When calculating ATP production, you have to show how many acetyl CoA are produced from a given fatty acid as this controls how many “turns” the citric acid cycle makes.Starting with acetyl CoA, how many ATP are made using the citric acid cycle? E.T.C = electron transport chain

Step	ATP produced
7	1
Step 4 (NAD⁺ to E.T.C.)	3
Step 6 (NAD⁺ to E.T.C.)	3
Step10 (NAD⁺ to E.T.C.)	3
Step 8 (FAD to E.T.C.)	2
NET	12 ATP

ATP Summary for Palmitic Acid – Complete Metabolism:The phrase “complete metabolism” means do reactions until you end up with carbon dioxide and water. This also means to use fatty acid spiral, citric acid cycle, and electron transport as needed.Starting with palmitic acid (16 carbons) how many ATP are made using fatty acid spiral? This is a review of the above panel E.T.C = electron transport chain

Step	ATP (used -) (produced +)
Step 1 (FAD to E.T.C.)	+2
Step 4 (NAD⁺ to E.T.C.)	+3
Total ATP	+5
7 turns	7 x 5 = 35
initial step	-1
NET	34 ATP

The fatty acid spiral ends with the production of 8 acetyl CoA from the 16 carbon palmitic acid.

Starting with one acetyl CoA, how many ATP are made using the citric acid cycle? Above panel gave the answer of 12 ATP per acetyl CoA.

E.T.C = electron transport chain

Step	ATP produced
One acetyl CoA per turn C.A.C.	+12 ATP
8 Acetyl CoA = 8 turns C.A.C.	8 x 12 = 96 ATP
Fatty Acid Spiral	34 ATP
GRAND TOTAL	130 ATP

Fyodor Lynen

Feodor Lynen was born in Munich on 6 April 1911, the son of Wilhelm Lynen, Professor of Mechanical Engineering at the Munich Technische Hochschule. He received his Doctorate in Chemistry from Munich University under Heinrich Wieland, who had won the Nobel Prize for Chemistry in 1927, in March 1937 with the work: «On the Toxic Substances in Amanita». in 1954 he became head of the Max-Planck-Institut für Zellchemie, newly created for him as a result of the initiative of Otto Warburg and Otto Hahn, then President of the Max-Planck-Gesellschaft zur Förderung der Wissenschaften.

Lynen’s work was devoted to the elucidation of the chemical details of metabolic processes in living cells, and of the mechanisms of metabolic regulation. The problems tackled by him, in conjunction with German and other workers, include the Pasteur effect, acetic acid degradation in yeast, the chemical structure of «activated acetic acid» of «activated isoprene», of «activated carboxylic acid», and of cytohaemin, degradation of fatty acids and formation of acetoacetic acid, degradation of tararic acid, biosynthesis of cysteine, of terpenes, of rubber, and of fatty acids.

In 1954 Lynen received the Neuberg Medal of the American Society of European Chemists and Pharmacists, in 1955 the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker, in 1961 the Carus Medal of the Deutsche Akademie der Naturforscher «Leopoldina», and in 1963 the Otto Warburg Medal of the Gesellschaft für Physiologische Chemie. He was also a member of the U>S> National Academy of Sciences, and shared the Nobel Prize in Physiology and Medicine with Konrad Bloch in 1964, and was made President of the Gesellschaft Deutscher Chemiker (GDCh) in 1972.

This biography was written at the time of the award and first published in the book series Les Prix Nobel. It was later edited and republished in Nobel Lectures, and shortened by myself.

The Pathway from “Activated Acetic Acid” to the Terpenes and Fatty Acids

My first contact with dynamic biochemistry in 1937 occurred at an exceedingly propitious time. The remarkable investigations on the enzyme chain of respiration, on the oxygen-transferring haemin enzyme of respiration, the cytochromes, the yellow enzymes, and the pyridine proteins had thrown the first rays of light on the chemical processes underlying the mystery of biological catalysis, which had been recognised by your famous countryman Jöns Jakob Berzelius. Vitamin B2 , which is essential to the nourishment of man and of animals, had been recognised by Hugo Theorell in the form of the phosphate ester as the active group of an important class of enzymes, and the fermentation processes that are necessary for Pasteur’s “life without oxygen”

had been elucidated as the result of a sequence of reactions centered around “hydrogen shift” and “phosphate shift” with adenosine triphosphate as the phosphate-transferring coenzyme. However, 1,3-diphosphoglyceric acid, the key substance to an understanding of the chemical relation between oxidation and phosphorylation, still lay in the depths of the unknown. Never-

theless, Otto Warburg was on its trail in the course of his investigations on the fermentation enzymes, and he was able to present it to the world in 1939.

This was the period in which I carried out my first independent investigation, which was concerned with the metabolism of yeast cells after freezing in liquid air, and which brought me directly into contact with the mechanism of alcoholic fermentation. This work taught me a great deal, and yielded two important pieces of information.

The first was that in experiments with living cells, special attention must be given to the permeability properties of the cell membranes, and
the second was that the adenosine polyphosphate system plays a vital part in the cell,
- not only in energy transfer, but
- also in the regulation of the metabolic processes.

This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day.

My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acids.

The explanation of these observations was provided-by the Thunberg-Wieland process, according to which two molecules of acetic acid are dehydrogenated to succinic acid, which is converted back into acetic acid via oxaloacetic acid, pyruvic acid, and acetaldehyde, or combines at the oxaloacetic acid stage with a further molecule of acetic acid to form citric acid (Fig. 1). However, an experimental check on this view by a Wieland’s student Robert Sonderhoffs brought a surprise. The citric acid formed when trideuteroacetic acid was supplied to yeast cells contained the expected quantity of deuterium, but the succinic acid contained only half of the four deuterium atoms required by Wieland’s scheme.

This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day. My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acid

The answer provided by Martius was that citric acid is in equilibrium with isocitric acid and is oxidised to cr-ketoglutaric acid, the conversion of which into succinic acid had already been discovered by Carl Neuberg (Fig. 1).

It was possible to assume with fair certainty from these results that the succinic acid produced by yeast from acetate is formed via citric acid. Sonderhoff’s experiments with deuterated acetic acid led to another important discovery.

In the analysis of the yeast cells themselves, it was found that while the carbohydrate fraction contained only insignificant quantities of deuterium, large quantities of heavy hydrogen were present in the fatty acids formed and in the sterol fraction. This showed that

fatty acids and sterols were formed directly from acetic acid, and not indirectly via the carbohydrates.

As a result of Sonderhoff’s early death, these important findings were not pursued further in the Munich laboratory.

This situation was elucidated only by Konrad Bloch’s isotope experiments, on which he reports.

My interest first turned entirely to the conversion of acetic acid into citric acid, which had been made the focus of the aerobic degradation of carbohydrates by the formulation of the citric acid cycle by Hans Adolf Krebs. Unlike Krebs, who regarded pyruvic acid as the condensation partner of acetic acid,

we were firmly convinced, on the basis of the experiments on yeast, that pyruvic acid is first oxidised to acetic acid, and only then does the condensation take place.

Further progress resulted from Wieland’s observation that yeast cells that had been “impoverished” in endogenous fuels by shaking under oxygen were able to oxidise added acetic acid only after a certain “induction period” (Fig. 2). This “induction period” could be shortened by addition of small quantities of a readily oxidisable substrate such as ethyl alcohol, though propyl and butyl alcohol were also effective. I explained this by assuming that acetic acid is converted, at the expense of the oxidation of the alcohol, into an “activated acetic acid”, and can only then condense with oxalacetic acid.

In retrospect, we find that I had come independently on the same group of problems as Fritz Lipmann, who had discovered that inorganic phosphate is indispensable to the oxidation of pyruvic acid by lactobacilli, and had detected acetylphosphate as an oxidation product. Since this anhydride of acetic acid and phosphoric acid could be assumed to be the “activated acetic acid”.

I learned of the advances that had been made in the meantime in the investigation of the problem of “activated acetic acid”. Fritz Lipmann has described the development at length in his Nobel Lecture’s, and I need not repeat it. The main advance was the recognition that the formation of “activated acetic acid” from acetate involved not only ATP as an energy source, but also the newly discovered coenzyme A, which contains the vitamin pantothenic acid, and that “activated acetic acid” was probably an acetylated coenzyme A.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/1964/lynen-bio.html

http://onlinelibrary.wiley.com/store/10.1002/anie.201106003/asset/image_m/mcontent.gif?v=1&s=1e6dc789dfa585fe48947e92cc5dfdcabd8e2677

Fyodor Lynen

Lynen’s most important research at the University of Munich focused on intermediary metabolism, cholesterol synthesis, and fatty acid biosynthesis. Metabolism involves all the chemical processes by which an organism converts matter and energy into forms that it can use. Metabolism supplies the matter—the molecular building blocks an organism needs for the growth of new tissues. These building blocks must either come from the breakdown of molecules of food, such as glucose (sugar) and fat, or be built up from simpler molecules within the organism.

Cholesterol is one of the fatty substances found in animal tissues. The human body produces cholesterol, but this substance also enters the body in food. Meats, egg yolks, and milk products, such as butter and cheese, contain cholesterol. Such organs as the brain and liver contain much cholesterol. Cholesterol is a type of lipid, one of the classes of chemical compounds essential to human health. It makes up an important part of the membranes of each cell in the body. The body also uses cholesterol to produce vitamin D and certain hormones.

All fats are composed of an alcohol called glycerol and substances called fatty acids. A fatty acid consists of a long chain of carbon atoms, to which hydrogen atoms are attached. There are three types of fatty acids: saturated, monounsaturated, and polyunsaturated.

Living cells manufacture complicated chemical compounds from simpler substances through a process called biosynthesis. For example, simple molecules called amino acids are put together to make proteins. The biosynthesis of both fatty acids and cholesterol begins with a chemically active form of acetate, a two-carbon molecule. Lynen discovered that the active form of acetate is a coenzyme, a heat-stabilized, water-soluble portion of an enzyme, called acetyl coenzyme A. Lynen and his colleagues demonstrated that the formation of cholesterol begins with the condensation of two molecules of acetyl coenzyme A to form acetoacetyl coenzyme A, a four-carbon molecule.

http://science.howstuffworks.com/dictionary/famous-scientists/biologists/feodor-lynen-info.htm

Fyodor Lynen

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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.

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Summary, Metabolic Pathways

Posted in Academic Publishing, Amino acids, Biochemical pathways, Curation, Dehydrogenase, Enzymes and isoenzymes, Kinase, Leloir pathway, Metabolism, Myocardial Ischemia, Myocardial Metabolism, Nutrition, Pentose monophosphate shunt, Phosphorylase, Phosphorylation, Pyridine nucleotide transhydrogenase, Pyridine nucleotides, tagged Acetyl-CoA, ATP, Carbohydrate metabolism, galactose, glycogenolysis, Glycolysis, lactic acid, LDH, mitochondrial respiration, Oxidative phosphorylation, pentose phosphate shunt, pyruvate on October 23, 2014| Leave a Comment »

Summary, Metabolic Pathways

Author: Larry H. Bernstein, MD, FCAP

This portion of a series of chapters on metabolism, proteomics and metabolomics dealt mainly with carbohydrate metabolism. Amino acids and lipids are presented more fully in the chapters that follow. There are features on the

functioning of enzymes and proteins,
on sequential changes in a chain reaction, and
on conformational changes that we shall also cover.

These are critical to developing a more complete understanding of life processes.

I needed to lay out the scope of metabolic reactions and pathways, and their complementary changes. These may not appear to be adaptive, if the circumstances and the duration is not clear. The metabolic pathways map in total
is in interaction with environmental conditions – light, heat, external nutrients and minerals, and toxins – all of which give direction and strength to these reactions. A developing goal is to discover how views introduced by molecular biology and genomics don’t clarify functional cellular dynamics that are not related to the classical view. The work is vast.

Carbohydrate metabolism denotes the various biochemical processes responsible for the formation, breakdown and interconversion of carbohydrates in living organisms. The most important carbohydrate is glucose, a simple sugar (monosaccharide) that is metabolized by nearly all known organisms. Glucose and other carbohydrates are part of a wide variety of metabolic pathways across species: plants synthesize carbohydrates from carbon dioxide and water by photosynthesis storing the absorbed energy internally, often in the form of starch or lipids. Plant components are consumed by animals and fungi, and used as fuel for cellular respiration. Oxidation of one gram of carbohydrate yields approximately 4 kcal of energy and from lipids about 9 kcal. Energy obtained from metabolism (e.g. oxidation of glucose) is usually stored temporarily within cells in the form of ATP. Organisms capable of aerobic respiration metabolize glucose and oxygen to release energy with carbon dioxide and water as byproducts.

Carbohydrates are used for short-term fuel, and even though they are simpler to metabolize than fats, they don’t produce as equivalent energy yield measured by ATP. In animals, the concentration of glucose in the blood is linked to the pancreatic endocrine hormone, insulin. . In most organisms, excess carbohydrates are regularly catabolized to form acetyl-CoA, which is a feed stock for the fatty acid synthesis pathway; fatty acids, triglycerides, and other lipids are commonly used for long-term energy storage. The hydrophobic character of lipids makes them a much more compact form of energy storage than hydrophilic carbohydrates.

Glucose is metabolized obtaining ATP and pyruvate by way of first splitting a six-carbon into two three carbon chains, which are converted to lactic acid from pyruvate in the lactic dehydrogenase reaction. The reverse conversion is by a separate unidirectional reaction back to pyruvate after moving through pyruvate dehydrogenase complex.

Pyruvate dehydrogenase complex (PDC) is a complex of three enzymes that convert pyruvate into acetyl-CoA by a process called pyruvate decarboxylation. Acetyl-CoA may then be used in the citric acid cycle to carry out cellular respiration, and this complex links the glycolysis metabolic pathway to the citric acid cycle. This multi-enzyme complex is related structurally and functionally to the oxoglutarate dehydrogenase and branched-chain oxo-acid dehydrogenase multi-enzyme complexes. In eukaryotic cells the reaction occurs inside the mitochondria, after transport of the substrate, pyruvate, from the cytosol. The transport of pyruvate into the mitochondria is via a transport protein and is active, consuming energy. On entry to the mitochondria pyruvate decarboxylation occurs, producing acetyl CoA. This irreversible reaction traps the acetyl CoA within the mitochondria. Pyruvate dehydrogenase deficiency from mutations in any of the enzymes or cofactors results in lactic acidosis.

PDH-rxns The acetyl group is transferred to coenzyme A

http://guweb2.gonzaga.edu/faculty/cronk/biochem/images/PDH-rxns.gif

Typically, a breakdown of one molecule of glucose by aerobic respiration (i.e. involving both glycolysis and Kreb’s cycle) is about 33-35 ATP. This is categorized as:

Glycogenolysis – the breakdown of glycogen into glucose, which provides a glucose supply for glucose-dependent tissues.

Glycogenolysis in liver provides circulating glucose short term.

Glycogenolysis in muscle is obligatory for muscle contraction.

Pyruvate from glycolysis enters the Krebs cycle, also known as the citric acid cycle, in aerobic organisms.

Anaerobic breakdown by glycolysis – yielding 8-10 ATP

Aerobic respiration by Kreb’s cycle – yielding 25 ATP

The pentose phosphate pathway (shunt) converts hexoses into pentoses and regenerates NADPH. NADPH is an essential antioxidant in cells which prevents oxidative damage and acts as precursor for production of many biomolecules.

Glycogenesis – the conversion of excess glucose into glycogen as a cellular storage mechanism; achieving low osmotic pressure.

Gluconeogenesis – de novo synthesis of glucose molecules from simple organic compounds. An example in humans is the conversion of a few amino acids in cellular protein to glucose.

Metabolic use of glucose is highly important as an energy source for muscle cells and in the brain, and red blood cells.

The hormone insulin is the primary glucose regulatory signal in animals. It mainly promotes glucose uptake by the cells, and it causes the liver to store excess glucose as glycogen. Its absence

turns off glucose uptake,
reverses electrolyte adjustments,
begins glycogen breakdown and glucose release into the circulation by some cells,
begins lipid release from lipid storage cells, etc.

The level of circulatory glucose (known informally as “blood sugar”) is the most important signal to the insulin-producing cells.

insulin is made by beta cells in the pancreas,
fat is stored n adipose tissue cells, and
glycogen is both stored and released as needed by liver cells.
no glucose is released to the blood from internal glycogen stores from muscle cells.

The hormone glucagon, on the other hand, opposes that of insulin, forcing the conversion of glycogen in liver cells to glucose, and then release into the blood. Growth hormone, cortisol, and certain catecholamines (such as epinepherine) have glucoregulatory actions similar to glucagon. These hormones are referred to as stress hormones because they are released under the influence of catabolic proinflammatory (stress) cytokines – interleukin-1 (IL1) and tumor necrosis factor α (TNFα).

Net Yield of GlycolysisThe preparatory phase consumes 2 ATP

The pay-off phase produces 4 ATP.

The gross yield of glycolysis is therefore

4 ATP – 2 ATP = 2 ATP

The pay-off phase also produces 2 molecules of NADH + H+ which can be further converted to a total of 5 molecules of ATP* by the electron transport chain (ETC) during oxidative phosphorylation.

Thus the net yield during glycolysis is 7 molecules of ATP*
This is calculated assuming one NADH molecule gives 2.5 molecules of ATP during oxidative phosphorylation.

Cellular respiration involves 3 stages for the breakdown of glucose – glycolysis, Kreb’s cycle and the electron transport system. Kreb’s cycle produces about 60-70% of ATP for release of energy in the body. It directly or indirectly connects with all the other individual pathways in the body.

The Kreb’s Cycle occurs in two stages:

Conversion of Pyruvate to Acetyl CoA
Acetyl CoA Enters the Kreb’s Cycle

Each pyruvate in the presence of pyruvate dehydrogenase (PDH) complex in the mitochondria gets converted to acetyl CoA which in turn enters the Kreb’s cycle. This reaction is called as oxidative decarboxylation as the carboxyl group is removed from the pyruvate molecule in the form of CO2 thus yielding 2-carbon acetyl group which along with the coenzyme A forms acetyl CoA.

The PDH requires the sequential action of five co-factors or co-enzymes for the combined action of dehydrogenation and decarboxylation to take place. These five are TPP (thiamine phosphate), FAD (flavin adenine dinucleotide), NAD (nicotinamide adenine dinucleotide), coenzyme A (denoted as CoA-SH at times to depict role of -SH group) and lipoamide.

Acetyl CoA condenses with oxaloacetate (4C) to form a citrate (6C) by transferring its acetyl group in the presence of enzyme citrate synthase. The CoA liberated in this reaction is ready to participate in the oxidative decarboxylation of another molecule of pyruvate by PDH complex.

Isocitrate undergoes oxidative decarboxylation by the enzyme isocitrate dehydrogenase to form oxalosuccinate (intermediate- not shown) which in turn forms α-ketoglutarate (also known as oxoglutarate) which is a five carbon compound. CO2 and NADH are released in this step. α-ketoglutarate (5C) undergoes oxidative decarboxylation once again to form succinyl CoA (4C) catalysed by the enzyme α-ketoglutarate dehydrogenase complex.

Succinyl CoA is then converted to succinate by succinate thiokinase or succinyl coA synthetase in a reversible manner. This reaction involves an intermediate step in which the enzyme gets phosphorylated and then the phosphoryl group which has a high group transfer potential is transferred to GDP to form GTP.

Succinate then gets oxidised reversibly to fumarate by succinate dehydrogenase. The enzyme contains iron-sulfur clusters and covalently bound FAD which when undergoes electron exchange in the mitochondria causes the production of FADH2.

Fumarate is then by the enzyme fumarase converted to malate by hydration(addition of H2O) in a reversible manner.

Malate is then reversibly converted to oxaloacetate by malate dehydrogenase which is NAD linked and thus produces NADH.

The oxaloacetate produced is now ready to be utilized in the next cycle by the citrate synthase reaction and thus the equilibrium of the cycle shifts to the right.

The NADH formed in the cytosol can yield variable amounts of ATP depending on the shuttle system utilized to transport them into the mitochondrial matrix. This NADH, formed in the cytosol, is impermeable to the mitochondrial inner-membrane where oxidative phosphorylation takes place. Thus to carry this NADH to the mitochondrial matrix there are special shuttle systems in the body. The most active shuttle is the malate-aspartate shuttle via which 2.5 molecules of ATP are generated for 1 NADH molecule. This shuttle is mainly used by the heart, liver and kidneys. The brain and skeletal muscles use the other shuttle known as glycerol 3-phosphate shuttle which synthesizes 1.5 molecules of ATP for 1 NADH.

Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose Phosphate Pathway. This enzyme is regulated by availability of the substrate NADP+. As NADPH is utilized in reductive synthetic pathways, the increasing concentration of NADP+ stimulates the Pentose Phosphate Pathway, to replenish NADPH. The importance of this pathway can easily be underestimated. The main source for energy in respiration was considered to be tied to the high energy phosphate bond in phosphorylation and utilizes NADPH, converting it to NADP+. The pentose phosphate shunt is essential for the generation of nucleic acids, in regeneration of red cells and lens – requiring NADPH.

NAD+ serves as electron acceptor in catabolic pathways in which metabolites are oxidized. The resultant NADH is reoxidized by the respiratory chain, producing ATP.

The pyridine nucleotide transhydrogenase reaction concerns the energy-dependent reduction of TPN by DPNH. In 1959, Klingenberg and Slenczka made the important observation that incubation of isolated liver mitochondria with DPN-specific substrates or succinate in the absence of phosphate acceptor resulted in a rapid and almost complete reduction of the intramitochondrial TPN. These and related findings led Klingenberg and co-workers (1-3) to postulate the occurrence of a ATP-controlled transhydrogenase reaction catalyzing the reduction of TPN by DPNH. (The role of transhydrogenase in the energy-linked reduction of TPN. Fritz Hommes, Ronald W. Estabrook, The Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden. Biochemical and Biophysical Research Communications 11, (1), 2 Apr 1963, Pp 1–6. http://dx.doi.org:/10.1016/0006-291X(63)90017-2/).

Further studies observed the coupling of TPN-specific dehydrogenases with the transhydrogenase and observing the reduction of large amounts of diphosphopyridine nucleotide (DPN) in the presence of catalytic amounts of triphosphopyridine nucleotide (TPN). The studies showed the direct interaction between TPNHz and DPN, in the presence of transhydrogenase to yield products having the properties of TPN and DPNHZ. The reaction involves a transfer of electrons (or hydrogen) rather than a phosphate. (Pyridine Nucleotide Transhydrogenase II. Direct Evidence for and Mechanism of the Transhydrogenase Reaction* by Nathan 0. Kaplan, Sidney P. Colowick, And Elizabeth F. Neufeld. (From The Mccollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland) J. Biol. Chem. 1952, 195:107-119.) http://www.JBC.org/Content/195/1/107.Citation
Notation: TPN, NADP; DPN, NAD+; reduced pyridine nucleotides: TPNH (NADPH₂), DPNH (NADH).

Note: In this discussion there is a detailed presentation of the activity of lactic acid conversion in the mitochondria by way of PDH. In a later section there is mention of the bidirectional reaction of lactate dehydrogenase. However, the forward reaction is dominant (pyruvate to lactate) and is described. This is not related to the kinetics of the LD reaction with respect to the defining characteristic – Km.

Biochemical Education Jan 1977; 5(1):15. Kinetics of Lactate Dehydrogenase: A Textbook Problem.
K.L. MANCHESTER. Department of Biochemistry, University of Witwatersrand, Johannesburg South Africa.

One presupposes that determined Km values are meaningful under intracellular conditions. In relation to teaching it is a simple experiment for students to determine for themselves the Km towards pyruvate of LDH in a post-mitochondrial supernatant of rat heart and thigh muscle. The difference in Km may be a factor of 3 or 4-fold.It is pertinent then to ask what is the range of suhstrate concentrations over which a difference in Km may be expected to lead to significant differences in activity and how these concentrations compare with pyruvate concentrations in the cell. The evidence of Vesell and co-workers that inhibition by pyruvate is more readily seen at low than at high enzyme concentration is important in emphasizing that under intracellular conditions enzyme concentrations may be relatively large in relation to the substrate available. This will be particularly so in relation to [NADH] which in the cytoplasm is likely to be in the ~M range.

A final point concerns the kinetic parameters for LDH quoted by Bergmeyer for lactate estimations a pH of 9 is recommended and the Km towards lactate at that pH is likely to be appreciably different from the quoted values at pH 7 — Though still at pH 9 showing a substantially lower value for lactate with the heart preparation. http://onlinelibrary.wiley.com/doi/10.1016/0307-4412%2877%2990013-9/pdf

Several investigators have established that epidermis converts most of the glucose it uses to lactic acid even in the presence of oxygen. This is in contrast to most tissues where lactic acid production is used for energy production only when oxygen is not available. This large amount of lactic acid being continually produced within the epidermal cell must be excreted by the cell and then carried away by the blood stream to other tissues where the lactate can be utilized. The LDH reaction with pyruvate and NADH is reversible although at physiological pH the equilibrium position for the reaction lies very far to the right, i.e., in favor of lactate production. The speed of this reaction depends not only on the amount of enzyme present but also on the concentrations of the substances involved on both sides of the equation. The net direction in which the reaction will proceed depends solely on the relative concentrations of the substances on each side of the equation.
In vivo there is net conversion of pyruvate (formed from glucose) to lactate. Measurements of the speed of lactate production by sheets of epidermis floating on a medium containing glucose indicate a rate of lactate production of approximately 0.7 rn/sm/
mm/mg of fresh epidermis.Slice incubation experiments are presumably much closer to the actual in vivo conditions than
the homogenate experiments. The discrepancy between the
two indicates that in vivo conditions are far from optimal for the conversion of pyruvate to lactate. Only 1/100th of the maximal activity of the enzyme present is being achieved. The concentrations of the various substances involved are not
optimal in vivo since pyruvate and NADH concentrations are
lower than lactate and NAD concentrations and this might explain the in vivo inhibition of LDH activity. (Lactate Production And Lactate Dehydrogenase In The Human Epidermis*. KM. Halprin, A Ohkawara. J Invest Dermat 1966; 47(3): 222-6.)
http://www.nature.com/jid/journal/v47/n3/pdf/jid1966133a.pdf

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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…

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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

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Mechanisms of Drug Resistance

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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

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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-
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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

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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.

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Compilation of References in Leaders in Pharmaceutical Intelligence about proteomics, metabolomics, signaling pathways, and cell regulation

Posted in Academic Publishing, Alzheimer's Disease, Amino acids, Anaerobic Glycolysis, Atherogenic Processes & Pathology, Best evidence, Biochemical pathways, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Ca2+ triggered activation, Ca2+ triggered activation, Cachexia, Calcium Signaling, Calmodulin, Calmodulin Kinase and Contraction, CANCER BIOLOGY & Innovations in Cancer Therapy, Cardiomyopathy, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Coagulation Therapy and Internal Bleeding, Computational Biology/Systems and Bioinformatics, Curation, Cytoskeleton, Dehydrogenase, Diabetes Mellitus, Discovery process, Epigenetics and Cardiovascular Risks, Evolutionary cognition, Experimental validation, Explanatory, Fatty acids, Gene Regulation, Genome Biology, Genomic Expression, Hematology, Hexokinase, Historical relevance, Human Immune System in Health and in Disease, Inferential analysis, Inosine nucleotides, Kinase, Leloir pathway, Lipid metabolism, Lipids, Metabolism, Metabolomics, Myocardial Ischemia, Myocardial Metabolism, Na-K transport, Nephrology, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Nutrition, Nutritional Supplements: Atherogenesis, Oxidative phosphorylation, Pentose monophosphate shunt, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Drug Discovery, Phosphorylase, Phosphorylation, PKC, Proteins, Proteomics, Pyridine nucleotide transhydrogenase, Pyridine nucleotides, Regenerative Biology and Medicine, RNA Biology, RNA Biology, Cancer and Therapeutics, S-nitrosylation, Sensors & Analytics, Signaling, Signaling & Cell Circuits, Synaptic vesicle, Systemic Inflammatory Response Related Disorders, Technology Advance Assessment of, Theoretic convergence, Transcriptomics, Translational Research, Translational Science, Ubiquitinylation, Warburg effect, tagged 6-phosphogluconate, adaptive compensatory glycolysis, Adaptive immune system, adenine nucleotides, Adenosine, anabolism, anaerobic and aerobic gycolysis, anion, Aptamer affinity chromatography, Aptamer-based multiplexed technology, ATP, Bibliography, Calcium, cancers, carbohydrates, catabolism, cation, Coagulation, Cytoskeleton, drug targets, Endothelial Cell Coagulation Activation, ETC, Gene Regulation, genetics, genomics, hysrogen transfer, inner membrane, inosine nucleotide, ion chromatography, Leaders in Pharmaceutical Intelligence, Lipids, magnesium, metabolic regulation, Metabolism, Metabolomics, mitochondia, mitochondrial dynamics, mitochondrial function, Monounsaturated fat, NADH, NADPH, nitric oxide, NOS, omega 3 FA, omega 6 FA, omega-6/omega-3, platelet endothelial interaction, platelet signaling, PLI references, Polyunsaturated fatty acid, post-translation modifications, protein separation methods, Pyridine nucleotides, saturated fatty acids, separation methods, signaling cascades, signaling pathways, SIRS, Sodium, systemic inflammatory disorders, systemic inflammatory response, Transcriptomics on August 31, 2014| Leave a Comment »

Compilation of References in Leaders in Pharmaceutical Intelligence about
proteomics, metabolomics, signaling pathways, and cell regulation

Curator: Larry H. Bernstein, MD, FCAP

Proteomics

The Human Proteome Map Completed
Reporter and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/28/the-human-proteome-map-completed/

Proteomics – The Pathway to Understanding and Decision-making in Medicine
Author and Curator, Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/06/24/proteomics-the-pathway-to-understanding-and-decision-making-in-medicine/

Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
Author and Curator, Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/10/22/advances-in-separations-technology-for-the-omics-and-clarification-of-therapeutic-targets/

Expanding the Genetic Alphabet and Linking the Genome to the Metabolome
Author and Curator, Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-metabolome/

Synthesizing Synthetic Biology: PLOS Collections
Reporter: Aviva Lev-Ari
http://pharmaceuticalintelligence.com/2012/08/17/synthesizing-synthetic-biology-plos-collections/

Metabolomics

Extracellular evaluation of intracellular flux in yeast cells
Larry H. Bernstein, MD, FCAP, Reviewer and Curator
http://pharmaceuticalintelligence.com/2014/08/25/extracellular-evaluation-of-intracellular-flux-in-yeast-cells/
Metabolomic analysis of two leukemia cell lines. I.
Larry H. Bernstein, MD, FCAP, Reviewer and Curator
http://pha rmaceuticalintelligence.com/2014/08/23/metabolomic-analysis-of-two-leukemia-cell-lines-_i/
Metabolomic analysis of two leukemia cell lines. II.
Larry H. Bernstein, MD, FCAP, Reviewer and Curator
http://pharmaceuticalintelligence.com/2014/08/24/metabolomic-analysis-of-two-leukemia-cell-lines-ii/
Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics
Reviewer and Curator, Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/22/metabolomics-metabonomics-and-functional-nutrition-the-next-step-in-nutritional-metabolism-and-biotherapeutics/
Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation
Larry H. Bernstein, MD, FCAP, Reviewer and curator
http://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-metabolism-provides-homeomeostatic-regulation/

Metabolic Pathways

Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief
Reviewer and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/21/pentose-shunt-electron-transfer-galactose-more-lipids-in-brief/
Mitochondria: More than just the “powerhouse of the cell”
Reviewer and Curator: Ritu Saxena
http://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/
Mitochondrial fission and fusion: potential therapeutic targets?
Reviewer and Curator: Ritu saxena
http://pharmaceuticalintelligence.com/2012/10/31/mitochondrial-fission-and-fusion-potential-therapeutic-target/
Mitochondrial mutation analysis might be “1-step” away
Reviewer and Curator: Ritu Saxena
http://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/
Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com
Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-and-metabolic-pathways-in-leaders-in-pharmaceutical-intelligence/
Metabolic drivers in aggressive brain tumors
Prabodh Kandal, PhD
http://pharmaceuticalintelligence.com/2012/11/11/metabolic-drivers-in-aggressive-brain-tumors/
Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes
Author and Curator: Aviva Lev-Ari, PhD, RD
http://pharmaceuticalintelligence.com/2012/10/22/metabolite-identification-combining-genetic-and-metabolic-information-genetic-association-links-unknown-metabolites-to-functionally-related-genes/
Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation
Author and curator:Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/
Therapeutic Targets for Diabetes and Related Metabolic Disorders
Reporter, Aviva Lev-Ari, PhD, RD
http://pharmaceuticalintelligence.com/2012/08/20/therapeutic-targets-for-diabetes-and-related-metabolic-disorders/
Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation
Larry H. Bernstein, MD, FCAP, Reviewer and curator
http://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-metabolism-provides-homeomeostatic-regulation/
The multi-step transfer of phosphate bond and hydrogen exchange energy
Curator:Larry H. Bernstein, MD, FCAP,
http://pharmaceuticalintelligence.com/2014/08/19/the-multi-step-transfer-of-phosphate-bond-and-hydrogen-exchange-energy/
Studies of Respiration Lead to Acetyl CoA
Author and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/
Lipid Metabolism
Author and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/15/lipid-metabolism/
Carbohydrate Metabolism
Author and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/13/carbohydrate-metabolism/
Prologue to Cancer – e-book Volume One – Where are we in this journey?
Author and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/04/13/prologue-to-cancer-ebook-4-where-are-we-in-this-journey/
Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?
Author and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/04/04/introduction-the-evolution-of-cancer-therapy-and-cancer-research-how-we-got-here/
Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K
Author and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/11/01/inhibition-of-the-cardiomyocyte-specific-kinase-tnni3k/
The Binding of Oligonucleotides in DNA and 3-D Lattice Structures
Author and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/05/15/the-binding-of-oligonucleotides-in-dna-and-3-d-lattice-structures/
Mitochondrial Metabolism and Cardiac Function
Author and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/
How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia
Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-leads_to_hyperhomocysteinemia/
AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo
Author and Curator: SJ. Williams
http://pharmaceuticalintelligence.com/2013/03/12/ampk-is-a-negative-regulator-of-the-warburg-effect-and-suppresses-tumor-growth-in-vivo/
A Second Look at the Transthyretin Nutrition Inflammatory Conundrum
Author and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-conundrum/
Overview of Posttranslational Modification (PTM)
Writer and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/07/29/overview-of-posttranslational-modification-ptm/
Malnutrition in India, high newborn death rate and stunting of children age under five years
Writer and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/07/15/malnutrition-in-india-high-newborn-death-rate-and-stunting-of-children-age-under-five-years/
Update on mitochondrial function, respiration, and associated disorders
Writer and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-disorders/
Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease
Larry H. Bernstein, MD, FCAP, Curator
http://pharmaceuticalintelligence.com/2014/07/06/omega-3-fatty-acids-depleting-the-source-and-protein-insufficiency-in-renal-disease/
Late Onset of Alzheimer’s Disease and One-carbon Metabolism
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
http://pharmaceuticalintelligence.com/2013/05/06/alzheimers-disease-and-one-carbon-metabolism/
Problems of vegetarianism
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
http://pharmaceuticalintelligence.com/2013/04/22/problems-of-vegetarianism/

Signaling Pathways

Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine
Larry H. Bernstein, MD, FCAP, writer, and Aviva Lev- Ari, PhD, RN http://pharmaceuticalintelligence.com/2014/04/27/larryhbernintroduction_to_cardiovascular_diseases-translational_medicine-part_2/
Epilogue: Envisioning New Insights in Cancer Translational Biology
Series C: e-Books on Cancer & Oncology
Author & Curator: Larry H. Bernstein, MD, FCAP, Series C Content Consultant
http://pharmaceuticalintelligence.com/2014/03/29/epilogue-envisioning-new-insights/
Ca2+-Stimulated Exocytosis: The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter Writer and Curator: Larry H Bernstein, MD, FCAP and Curator and Content Editor: Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocy
Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
Author and Curator: Larry H Bernstein, MD, FCAP and Article Curator: Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/
Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Author and Curator: Larry H Bernstein, MD, FCAP Author: Stephen Williams, PhD, and Curator: Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/
Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP, Author and Curator
http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/
Advanced Topics in Sepsis and the Cardiovascular System at its End Stage
Author and Curator: Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-Sepsis-and-the-Cardiovascular-System-at-its-End-Stage/
The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology
Demet Sag, PhD, Author and Curator
http://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase
Demet Sag, PhD, Author and Curator
http://pharmaceuticalintelligence.com/2013/08/04/ido-for-commitment-of-a-life-time-the-origins-and-mechanisms-of-ido-indolamine-2-3-dioxygenase/
Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad
Author and Curator: Demet Sag, PhD, CRA, GCP
http://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-of-immune-responses-for-good-and-bad/
Signaling Pathway that Makes Young Neurons Connect was discovered @ Scripps Research Institute
Reporter: Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/06/26/signaling-pathway-that-makes-young-neurons-connect-was-discovered-scripps-research-institute/
Naked Mole Rats Cancer-Free
Writer and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/06/20/naked-mole-rats-cancer-free/
Amyloidosis with Cardiomyopathy
Writer and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/03/31/amyloidosis-with-cardiomyopathy/
Liver endoplasmic reticulum stress and hepatosteatosis
Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2013/03/10/liver-endoplasmic-reticulum-stress-and-hepatosteatosis/
The Molecular Biology of Renal Disorders: Nitric Oxide – Part III
Curator and Author: Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2012/11/26/the-molecular-biology-of-renal-disorders/
Nitric Oxide Function in Coagulation – Part II
Curator and Author: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-function-in-coagulation/
Nitric Oxide, Platelets, Endothelium and Hemostasis
Curator and Author: Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2012/11/08/nitric-oxide-platelets-endothelium-and-hemostasis/
Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium
Curator and Author: Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2012/09/14/interaction-of-nitric-oxide-and-prostacyclin-in-vascular-endothelium/
Nitric Oxide and Immune Responses: Part 1
Curator and Author: Aviral Vatsa PhD, MBBS
http://pharmaceuticalintelligence.com/2012/10/18/nitric-oxide-and-immune-responses-part-1/
Nitric Oxide and Immune Responses: Part 2
Curator and Author: Aviral Vatsa PhD, MBBS
http://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/
Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II
Curator and Author: Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-and-inos-have-key-roles-in-kidney-diseases/
New Insights on Nitric Oxide donors – Part IV
Curator and Author: Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/
Crucial role of Nitric Oxide in Cancer
Curator and Author: Ritu Saxena, Ph.D.
http://pharmaceuticalintelligence.com/2012/10/16/crucial-role-of-nitric-oxide-in-cancer/
Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function
Curator and Author: Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/
Nitric Oxide and Immune Responses: Part 2
Author and Curator: Aviral Vatsa, PhD, MBBS
http://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/
Mitochondrial Damage and Repair under Oxidative Stress
Author and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/
Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?
Curator and Author: Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-century-view/
Targeting Mitochondrial-bound Hexokinase for Cancer Therapy
Curator and Author: Ziv Raviv, PhD, RN 04/06/2013
http://pharmaceuticalintelligence.com/2013/04/06/targeting-mitochondrial-bound-hexokinase-for-cancer-therapy/
Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis
Curator and Author: Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis/
Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III
Curator and Author: Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/
Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I
Curator and Author: Larry H Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2012/11/26/biochemistry-of-the-coagulation-cascade-and-platelet-aggregation/

Genomics, Transcriptomics, and Epigenetics

What is the meaning of so many RNAs?
Writer and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/06/what-is-the-meaning-of-so-many-rnas/
RNA and the transcription the genetic code
Larry H. Bernstein, MD, FCAP, Writer and Curator
http://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/
A Primer on DNA and DNA Replication
Writer and Curator: Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/07/29/a_primer_on_dna_and_dna_replication/
Pathology Emergence in the 21st Century
Author and Curator: Larry Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/03/pathology-emergence-in-the-21st-century/
RNA and the transcription the genetic code
Writer and Curator, Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/
Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H Bernstein, MD, FCAP
Author: Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/07/16/commentary-on-biomarkers-for-genetics-and-genomics-of-cardiovascular-disease-views-by-larry-h-bernstein-md-fcap/
Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies
Author an Curator: Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/05/18/observations-on-finding-the-genetic-links/
Silencing Cancers with Synthetic siRNAs
Larry H. Bernstein, MD, FCAP, Reviewer and Curator
http://pharmaceuticalintelligence.com/2013/12/09/silencing-cancers-with-synthetic-sirnas/
Cardiometabolic Syndrome and the Genetics of Hypertension: The Neuroendocrine Transcriptome Control Points
Reporter: Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/12/12/cardiometabolic-syndrome-and-the-genetics-of-hypertension-the-neuroendocrine-transcriptome-control-points/
Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets
Larry H. Bernstein, MD, FCAP, Reviewer and Curator
http://pharmaceuticalintelligence.com/2013/12/08/developments-in-the-genomics-and-proteomics-of-type-2-diabetes-mellitus-and-treatment-targets/
CT Angiography & TrueVision™ Metabolomics (Genomic Phenotyping) for new Therapeutic Targets to Atherosclerosis
Reporter: Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/11/15/ct-angiography-truevision-metabolomics-genomic-phenotyping-for-new-therapeutic-targets-to-atherosclerosis/
CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics
Genomics Curator, Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/08/30/cracking-the-code-of-human-life-the-birth-of-bioinformatics-computational-genomics/
Big Data in Genomic Medicine
Author and Curator, Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/12/17/big-data-in-genomic-medicine/
From Genomics of Microorganisms to Translational Medicine
Author and Curator: Demet Sag, PhD
http://pharmaceuticalintelligence.com/2014/03/20/without-the-past-no-future-but-learn-and-move-genomics-of-microorganisms-to-translational-medicine/
Summary of Genomics and Medicine: Role in Cardiovascular Diseases
Author and Curator, Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2014/01/06/summary-of-genomics-and-medicine-role-in-cardiovascular-diseases/

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Archive for the ‘Phosphorylation’ Category

Integrins, Cadherins, Signaling and the Cytoskeleton

Integrins, Cadherins, Signaling and the Cytoskeleton

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Oxidation and Synthesis of Fatty Acids

Oxidation and Synthesis of Fatty Acids

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Introduction to Lipid Metabolism

Introduction to Lipid Metabolism

Introduction to Lipid Metabolism

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Summary, Metabolic Pathways

Summary, Metabolic Pathways

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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

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Archive for the ‘Phosphorylation’ Category

Integrins, Cadherins, Signaling and the Cytoskeleton

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Introduction to Signaling

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Oxidation and Synthesis of Fatty Acids

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Introduction to Lipid Metabolism

Introduction to Lipid Metabolism

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Summary, Metabolic Pathways

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Metabolomics Summary and Perspective

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Mechanisms of Drug Resistance

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Metformin, Thyroid-Pituitary Axis, Diabetes Mellitus, and Metabolism

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Somatic, germ-cell, and whole sequence DNA in cell lineage and disease profiling

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