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Functional Correlates of Signaling Pathways

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

Functional Correlates of Signaling Pathways

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

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

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

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

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

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

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

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

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

the acute lung inflammation mediated by

neutrophils,
cytokines, and
oxidant stress.

Neutrophils are major effect cells at the frontier of

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

The tissue injury appears to be related to

proteases and toxic reactive oxygen radicals
released from activated neutrophils.

In addition, neutrophils can produce cytokines and chemokines that

enhance the acute inflammatory response.

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

mediated by a group of chemoattractants,
especially CXC chemokines.

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

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

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

Integrins and extracellular matrix in mechanotransduction

ligand binding of integrins

Integrins are a family of cell surface receptors which

mediate cell–matrix and cell–cell adhesions.

Among other functions they provide an important

mechanical link between the cells external and intracellular environments while

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

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

adhesion receptors cluster and when activated

the receptors bind to ligands in the extracellular matrix.

The extracellular matrix surrounds the cells of tissues and forms the

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

Cells attach to the extracellular matrix through

specific cell-surface receptors and molecules

including integrins and transmembrane proteoglycans.

The integrin family of αβ heterodimeric receptors act as

cell adhesion molecules

connecting the ECM to the actin cytoskeleton.

The actin cytoskeleton is involved in the regulation of

1.cell motility,

2.cell polarity,

3.cell growth, and

4.cell survival.

The combination of αβ subunits determines

binding specificity and

signaling properties.

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

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

the signaling functions of the receptor.

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

binding of cytoskeletal and signaling molecules,

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

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

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

binding of integrins depends on ECM divalent cations ch19

integrin coupled to F-actin via linker

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

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

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

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

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

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

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

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

Integrin recognition of ligands is not constitutive but

is regulated by alteration of integrin affinity for ligand binding.

For integrin binding to ligands to occur

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

Linking integrin conformation to function

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

Integrins work alongside other proteins such as

cadherins,

immunoglobulin superfamily

cell adhesion molecules,

selectins, and

syndecans

to mediate

cell–cell and

cell–matrix interactions and communication.

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

bound matrix components,

adhesion receptors,

and associated intracellular cytoskeletal and signaling molecules

form large functional, localized multiprotein complexes.

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

tissue properties including

1.embryonic development,

2.inflammatory responses,

3.wound healing,

4.and adult tissue homeostasis.

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

dynamic,

spatially, and

temporally regulated formation and

disassembly of multiprotein complexes that

form around the short cytoplasmic tails of integrins.

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

not only adhesion to the ECM

but are involved in complex signaling pathways

which include establishing

1.cell polarity,

2.directed cell migration, and

3.maintaining cell growth and survival.

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

to assemble the focal adhesion complex

which is capable of responding to environmental stimuli efficiently.

Mapping of the integrin

adhesome binding and signaling interactions

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

Genetic programming occurs with the binding of integrins to the ECM

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

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

Various different effects can arise depending on the

1.cell type,

2.matrix composition, and

3.integrins activated

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

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

During mechanical loading/stimulation of chondrocytes there is an

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

Using these strategies it was identified that

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

Osteoarthritic chondrocytes show a depolarization response to 0.33 Hz stimulation

in contrast to the hyperpolarization response of normal chondrocytes.

The mechanotransduction pathway in chondrocytes derived from normal and osteoarthritic cartilage

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

Normal and osteoarthritic chondrocytes show differences

at multiple stages of the mechanotransduction cascade.

Signaling pathways activated in chondrocytes

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

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

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

control cell proliferation,
survival,
differentiation,
matrix remodeling.

Integrins participate in development and maintenance of the tissue but also

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

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

Cells exhibited four types of mechanical responses:

(1) an immediate viscoelastic response;

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

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

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

Importantly, these adaptation responses differed biochemically.

The immediate and early responses were affected by

chemically dissipating cytoskeletal prestress (isometric tension), whereas

the later adaptive response was not.

The repositioning response was prevented by

inhibiting tension through interference with Rho signaling,

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

blocking mechanosensitive ion channels or

by inhibiting Src tyrosine kinases.

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

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

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

large protein families with diverse cellular functions.

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

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

This large number of motor proteins may reflect

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

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

mitosis,

motility, and

intracellular transport

Their involvement in a range of pathological processes

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

They are defined by their motor domains that contain both

the microtubule (MT) and
ATP binding sites.

Three ATP binding motifs—

the P-loop,
switch I,
switch II–

are highly conserved among

kinesins,
myosin motors, and

small GTPases.

They share a conserved mode of MT binding such that

MT binding,
ATP binding, and
hydrolysis

are functionally coupled for efficient MT-based work.

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

proteins and other items moving from one location to another.

A network of filaments called microtubules forms tracks

along which so-called motor proteins carry these items.

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

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

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

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

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

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

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

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

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

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

which lies at the tubulin intradimer interface.

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

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

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

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

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

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

determined by MT binding and motor nucleotide state.

Movement and extension of helix-α6 controls neck linker docking

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

its conformation alters in response to different nucleotide states.

Further,

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

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

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

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

ATP binding draws loop11 and loop9 closer together; causing

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

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

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

In both motors, microtubule binding promotes

ordered conformations of conserved loops that

stimulate ADP release,

enhance microtubule affinity and

prime the catalytic site for ATP binding.

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

large conformational changes elsewhere that

allow force generation and

neck linker docking towards the microtubule plus end.

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

mechanism for kinesins and

reveals the critical mechanistic contribution of the microtubule interface.

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

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

The vascular endothelium is the inner lining of blood vessels and

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

controlling the extravasation of fluids,

plasma proteins and leukocytes.

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

transient hyperpermeability

in response to stimulation with inflammatory mediators,

which takes place primarily in post-capillary venules

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

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

Dysregulated permeability is observed in various pathological conditions, such as

tumor-induced angiogenesis,
cerebrovascular accident and
atherosclerosis.

Two fundamentally different pathways regulate endothelial permeability,

the transcellular and
paracellular pathways.

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

vesicular transport systems,
fenestrae, and
biochemical transporters.

The paracellular route is controlled by

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

Endothelial cells are connected by

tight, gap and

adherens junctions,

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

vascular endothelial (VE)-cadherin,

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

Although multiple adhesion molecules are localized at endothelial junctions,

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

Like other cadherins, VE-cadherin mediates adhesion via

homophilic, calcium-dependent interactions.

This cell–cell adhesion

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

to the C-terminus of VE-cadherin.

VE-cadherin can directly bind

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

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

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

Numerous lines of evidence indicate that p120-catenin

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

Different models are proposed that describe how

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

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

through interactions with small GTPases.

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

decrease RhoA activity,

elevate active Rac1 and Cdc42, and thereby is thought

to regulate actin cytoskeleton organization and membrane trafficking.

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

Several mechanisms may be involved in the

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

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

The remainder of this review primarily focuses on the

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

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

It is a widely accepted concept that tyrosine phosphorylation of

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

A general idea has emerged that

tyrosine phosphorylation of the VE-cadherin complex

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

through dissociation of catenins from the cadherin.

However, tyrosine phosphorylation of VE-cadherin

is required for efficient transmigration of leukocytes.

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

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

2.a more active role in the transmigration process.

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

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

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

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

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

Specifically, our recent studies have provided evidence that

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

Here, we examined the details of how

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

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

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

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

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

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

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

Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle

MacKinnon. Fig 1 Ion channels exhibit three basic properties

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

depend on their highly evolved and specialized cytoskeletal apparatus.

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

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

In addition to the structural remodeling,

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

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

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

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

In LVAD-treated subjects,

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

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

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

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

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

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

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

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

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

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

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

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

1.plasma membrane,

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

ion channels are mainly localized.

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

Sarcomeric Proteins and Ion Channels

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

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

2.functional regulation by second messenger pathways,

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

Intense investigation of

the sarcomeric actin network,

the Z-line structure, and

chaperone molecules docking in the plasma membrane,

has shed new light on the molecular basis of

cytoskeletal interactions in regulating ion channels

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

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

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

Molecular interactions between the cytoskeleton and ion channels

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

sarcomere structure

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

the recent findings of structural and functional links between

the cytoskeleton and ion channels

could expand the therapeutic interventions in

arrhythmia management in structurally abnormal myocardium, where aberrant binding

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

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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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Development Of Super-Resolved Fluorescence Microscopy

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Development Of Super-Resolved Fluorescence Microscopy

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

CSO, Leaders in Pharmaceutical Business Intelligence

Article ID #153: Development Of Super-Resolved Fluorescence Microscopy. Published on 10/12/2014

WordCloud Image Produced by Adam Tubman

Development Of Super-Resolved Fluorescence Microscopy

Part I. Nobel Prize For Chemistry 2014: Eric Betzig, Stefan W. Hell
and William E. Moerner Honored For Development Of Super-
Resolved Fluorescence Microscopy

The 2014 Nobel Prize in Chemistry was awarded on 10/08/2014 to
Eric Betzig, Stefan W. Hell and William E. Moerner for
“the development of super-resolved fluorescence microscopy.”

The invention of the electron microscope by Max Knoll and Ernst Ruska at the
Berlin Technische Hochschule in 1931 finally overcame the barrier to higher
resolution that had been imposed by the limitations of visible light. Since then
resolution has defined the progress of the technology.

The ultimate goal was atomic resolution – the ability to see atoms – but this would
have to be approached incrementally over the course of decades. The earliest microscopes merely proved the concept: electron beams could, indeed, be tamed
to provide visible images of matter. By the late 1930s electron microscopes with theoretical resolutions of 10 nm were being designed and produced, and by 1944
this was further reduced to 2 nm. (The theoretical resolution of a an optical light microscope is 200 nm.)

Increases in the accelerating voltage of the electron beam accounted for much of
the improvement in resolution. But voltage was not everything. Improvements in electron lens technology minimized aberrations and provided a clearer picture,
which also contributed to improved resolution, as did better vacuum systems and brighter electron guns. So increasing the resolution of electron microscopes was a main driving force throughout the instrument’s development.

With nanoscopy, scientists could observe viruses, proteins and molecules there
are smaller than 0.0000002 metres.

Three researchers won the 2014 Nobel Prize in Chemistry on Wednesday,
October 8, for giving microscopes much sharper vision than was thought possible, letting scientists peer into living cells with unprecedented detail to seek the roots
of disease. It was awarded to U.S. researchers Eric Betzig and William Moerner
and German scientist Stefan Hell. They found ways to use molecules that glow on demand to overcome what was considered a fundamental limitation for optical microscopes.

Hell, 52, of Germany, is the director at the Max Planck Institute for Biophysical Chemistry and the division head at the German Cancer Research Center in
Heidelberg. He was honored for his work on fluorescence microscopy, a kind
of nano-flashlight where scientists use fluorescent molecules to see parts of a
cell. Later in his career, he developed the STED microscope, which collects light
from “a multitude of small volumes to create a whole.”

Moerner, a 61-year-old professor in chemistry and applied physics at Stanford University in California, is the recipient of the 2008 Wolf Prize in Chemistry, the
2009 Irving Langmuir Award and the 2013 Peter Debye Award. In 1989, he
was the first scientist to be able to measure the light absorption of a single molecule.
This inspired many chemists to begin focusing on single molecules, including Betzig.

Betzig, 54, the group leader at Janelia Farm Research campus at the Howard
Hughes Medical Institute in Virginia, developed new optical imaging tools for
biology. His work involved taking images of the same area multiple times, and illuminating just a few molecules each time. These images were then
superimposed to create a dense super image at the nano level,

The limitation of optical microscopy was thought to have been determined in a calculation published in 1873 that defined the limit of how tiny a detail could be revealed by optical microscopes. Based on experimental evidence and basic principles of physics, Ernst Abbe and Lord Rayleigh defined and formulated
this diffraction-limited resolution in the late 19th century (Abbe, 1873; Rayleigh,
1896). However, only cellular structure and objects that were at least 200 to
350 nm apart could be resolved by light microscopy because, the optical resolution
of light microscopy was limited to approximately half of the wavelength of the light used. Later key innovations—including fluorescence and confocal laser scanning microscopy (CLSM)—made optical microscopy one of the most powerful and
versatile diagnostic tools in modern cell biology. Using highly specific fluorescent labeling techniques such as immunocytochemistry, in situ hybridization, or
fluorescent protein tags, the spatial distribution and dynamics of virtually every subcellular structure, protein, or genomic sequence of interest can be analyzed in chemically fixed or living samples (Conchello and Lichtman, 2005; Giepmans et al., 2006).

The result of their advance is “really a window into the cell which we didn’t have before,” said Catherine Lewis, director of the cell biology and biophysics division
of the National Institute of General Medical Sciences in Bethesda, Maryland.

“You can observe the behavior of individual molecules in living cells in real time.
You can see … molecules moving around inside the cell. You can see them interacting with each other.”

The research of the three men has let scientists study diseases such as
Parkinson’s, Alzheimer’s and Huntington’s at a molecular level, the Royal
Swedish Academy of Sciences said.

Part II. Electron microscopy limitations

Manfred Von Ardenne in Berlin produced the earliest scanning-transmission
electron microscope in 1937. At the University of Toronto in Canada, Cecil Hall, James Hillier, and Albert Prebus, working under the direction of Eli Burton,
produced an advanced 1938 Toronto Model electron microscope that would
later become the basis for Radio Corporation of America’s Model B, the first commercial electron microscope in North America. Ruska at Siemens in
Germany produced the first commercial electron microscope in the world in 938.

Starting in 1939, scientists in Japan gathered to decide on the best way to build
an electron microscope. This group evolved into the Japan Electron Optics Laboratory (JEOL) that would eventually produce more models and varieties
of electron microscopes than any other company. Hitachi and Toshiba in Japan
also played a major role in the early development process.

The 1960s through the 1990s produced many innovative instruments and trends.
The introduction of the first commercial scanning electron microscopes (SEMs)
in 1965 opened up a new world of analysis for materials scientists. Ultrahigh
voltage TEM instruments (up to 3 MeV at CEMES-LOE/CNRS in Toulouse,
France, and at Hitachi in Tokyo, Japan), in the 1960s and 1970s gave electrons higher energy to penetrate more deeply into thick samples. The evolution and incorporation of other detectors (electron microprobes, electron energy loss spectroscopy (EELS), etc.) made the SEM into a true analytical electron
microscope (AEM) beginning in the 1970s. The development of brighter
electron sources, such as the lanthanum hexaboride filament (LAB₆) and the
field emission gun in the 1960s, and their commercialization in the 1970s
brought researchers a brighter source of electrons and with it better imaging
and resolution. Tilting specimen stages permitting examination of the specimen
from different angles aided significantly in the determination of crystal structure.
In the late 1980s and throughout the 1990s, the environmental electron
microscopes that allow scientists to examine samples under more natural
conditions of temperature and pressure have dramatically expanded the
types of samples that can be examined.

In medicine, the EM made a unique contribution to diagnostic anatomic
pathology in renal biopsy analysis. However, the small sample had to be
embedded, and in the early days one cut the specimen by breaking glass
for the cutting of the specimen. But even though EM ushered in a new era of molecular pathology, the contribution was limited, despite incremental
improvements.

In the past, the use of microscopes was limited by a physical restriction;
scientists could only see items that were larger than roughly half the
wavelength of light (.2 micrometers). However, the groundbreaking work
of the Nobel laureates bypassed the maximum resolution of traditional
microscopes and launched optical microscopy into the nanodimension.

Part III. Super resolution fluorescence microscopy

Bo Huang,^1,2 Mark Bates,³ and Xiaowei Zhuang^1,2,4Author information ► Copyright and License information ►
Annu Rev Biochem. 2009; 78: 993–1016.
http://dx.doi.org:/10.1146/annurev.biochem.77.061906.092014
PMCID: PMC2835776 NIHMSID: NIHMS179491

Achieving a spatial resolution that is not limited by the diffraction of
light, recent developments of super-resolution fluorescence microscopy
techniques allow the observation of many biological structures not
resolvable in conventional fluorescence microscopy. New advances
in these techniques now give them the ability to image three-dimensional
(3D) structures, measure interactions by multicolor colocalization, and
record dynamic processes in living cells at the nanometer scale. It is
anticipated that super-resolution fluorescence microscopy will become
a widely used tool for cell and tissue imaging to provide previously
unobserved details of biological structures and processes.

Keywords: Sub-diffraction limit, single-molecule, multicolor imaging,
three-dimensional imaging, live cell imaging, single-particle tracking,
photoswitchable probe

Among the various microscopy techniques, fluorescence microscopy is
one of the most widely used because of its two principal advantages:
Specific cellular components may be observed through molecule-specific
labeling, and light microscopy allows the observation of structures inside
a live sample in real time. Compared to other imaging techniques such
as electron microscopy (EM), however, conventional fluorescence
microscopy is limited by relatively low spatial resolution because of the
diffraction of light. This diffraction limit, about 200–300 nm in the lateral
direction and 500–700 nm in the axial direction, is comparable to or larger
than many subcellular structures, leaving them too small to be observed in
detail. In recent years, a number of “super-resolution” fluorescence microscopy techniques have been invented to overcome the diffraction barrier, including techniques that employ nonlinear effects to sharpen the point-spread function
of the microscope, such as stimulated emission depletion (STED) microscopy
(1, 2), related methods using other reversible saturable optically linear
fluorescence transitions (RESOLFTs) (3), and saturated structured-illumination microscopy (SSIM) (4), as well as techniques that are based on the localization
of individual fluorescent molecules, such as stochastic optical reconstruction microscopy (STORM) (5), photoactivated localization microscopy (PALM) (6),
and fluorescence photoactivation localization microscopy (FPALM) (7). These methods have yielded an order of magnitude improvement in spatial resolution
in all three dimensions over conventional light microscopy.

THE RESOLUTION LIMIT IN OPTICAL MICROSCOPY

Microscopes can be used to visualize fine structures in a sample by providing
a magnified image. However, even an arbitrarily high magnification does not
translate into the ability to see infinitely small details. Instead, the resolution
of light microscopy is limited because light is a wave and is subject to diffraction.

The diffraction limit

An optical microscope can be thought of as a lens system that produces a
magnified image of a small object. In this imaging process, light rays from
each point on the object converge to a single point at the image plane. However,
the diffraction of light prevents exact convergence of the rays, causing a sharp
point on the object to blur into a finite-sized spot in the image. The three-
dimensional (3D) intensity distribution of the image of a point object is called
the point spread function (PSF). The size of the PSF determines the resolution
of the microscope: Two points closer than the full width at half-maximum
(FWHM) of the PSF will be difficult to resolve because their images overlap substantially.

The FWHM of the PSF in the lateral directions (the x–y directions perpendicular
to the optical axis) can be approximated as Δxy ≈ 0.61λ / NA, where λ is the wavelength of the light, and NA is the numerical aperture of the objective
defined as NA = n sinα, with n being the refractive index of the medium and
α being the half-cone angle of the focused light produced by the objective.
The axial width of the PSF is about 2–3 times as large as the lateral width
for ordinary high NA objectives. When imaging with visible light (λ ≈ 550 nm),
the commonly used oil immersion objective with NA = 1.40 yields a PSF with
a lateral size of ~200 nm and an axial size of ~500 nm in a refractive index-
matched medium (Figure 1) (8).

Figure 1

The PSF of a common oil immersion objective with NA = 1.40, showing the
focal spot of 550 nm light in a medium with refractive index n = 1.515. The
intensity distribution in the x-z plane of the focus spot is computed numerically.

PFS of oil immersion microscope

Because the loss of high-frequency spatial information in optical microscopy
results from the diffraction of light when it propagates through a distance larger
than the wavelength of the light (far field), near-field microscopy is one of the
earliest approaches sought to achieve high spatial resolution. By exciting the fluorophores or detecting the signal through the nonpropagating light near the fluorophore, high-resolution information be retained. Near-field scanning optical microscopy (NSOM) acquires an image by scanning a sharp probe tip across
the sample, typically providing a resolution of 20–50 nm (9–11). Wide-field
imaging has also been recently demonstrated in the near-field regime using
a super lens with negative refractive index (12, 13). However, the short range
of the near-field region (tens of nanometers) compromises the ability of light microscopy to look into a sample, limiting the application of near-field microscopy
to near-surface features only. This limit highlights the need to develop far-field
high-resolution imaging methods.

Among far-field fluorescence microscopy techniques, confocal and multiphoton microscopy are among the most widely used to moderately enhance the spatial resolution (14, 15). By combining a focused laser for excitation and a pinhole for detection, confocal microscopy can, in principle, have a factor of √2 improvement
in the spatial resolution. In multiphoton microscopy, nonlinear absorption processes reduce the effective size of the excitation PSF. However, this gain in the PSF size
is counteracted by the increased wavelength of the excitation light. Thus, instead
of improving the resolution, the main advantage of confocal and multi-photon microscopy over wide-field microscopy is the reduction of out-of-focus fluorescence background, allowing optical sectioning in 3D imaging.

Two techniques, 4Pi and I5M microscopy, approach this ideal situation by using
two opposing objectives for excitation and/or detection (16, 17). By acquiring
multiple images with illumination patterns of different phases and orientations,
a high-resolution image can be reconstructed. Because the illumination pattern
itself is also limited by the diffraction of light, structured illumination microscopy
(SIM) is only capable of doubling the spatial resolution by combining two diffraction-limited sources of information. The best achievable result using these methods
would be an isotropic PSF with an additional factor of 2 in resolution improvement. This would correspond to ~100-nm image resolution in all three dimensions, as
has been demonstrated by the I5S technique, which combines I5M and SIM (22). Albeit a significant improvement, this resolution is still fundamentally limited by
the diffraction of light.

SUPER RESOLUTION FLUORESCENCE MICROSCOPY BY SPATIALLY PATTERNED EXCITATION

One approach to attain a resolution far beyond the limit of diffraction, i.e., to
realize super-resolution microscopy, is to introduce sub-diffraction-limit features
in the excitation pattern so that small-length-scale information can be read out.
We refer to this approach, including STED, RESOLFT, and SSIM, as super-
resolution microscopy by spatially patterned excitation or the “patterned excitation” approach.

The concept of STED microscopy was first proposed in 1994 (1) and subsequently demonstrated experimentally (2). Simply speaking, it uses a second laser (STED laser) to suppress the fluorescence emission from the fluorophores located off the center of the excitation. This suppression is achieved through stimulated emission: When an excited-state fluorophores encounters a photon that matches the energy difference between the excited and the ground state, it can be brought back to
the ground state through stimulated emission before spontaneous fluorescence emission occurs. This process effectively depletes excited-state fluorophores
capable of fluorescence emission (Figure 2a,b).

Figure 2

The principle of STED microscopy. (a) The process of stimulated emission. A
ground state (S₀) fluorophore can absorb a photon from the excitation light and
jump to the excited state (S₁).

STED microsopy

The pattern of the STED laser is typically generated by inserting a phase mask
into the light path to modulate its phase-spatial distribution (Figure 2b). One such phase mask generates a donut-shaped STED pattern in the xy plane (Figure 2c)
and has provided an xy resolution of ~30 nm (24). STED can also be employed
in 4Pi microscopy (STED-4Pi), resulting in an axial resolution of 30–40 nm (25). STED has been applied to biological samples either immuno-stained with
fluorophore labeled antibodies (26) or genetically tagged with fluorescent
proteins (FPs) (27). Dyes with high photostability under STED conditions and
large stimulated emission cross sections in the visible to near infrared (IR) range
are preferred. Atto 532 and Atto 647N are among the most often used dyes for
STED microscopy.

Stimulated emission is not the only mechanism capable of suppressing
undesired fluorescence emission. A more general scheme using saturable
depletion to achieve super resolution has been formalized with the name
RESOLFT microscopy (3). This scheme employs fluorescent probes that
can be reversibly photoswitched between a fluorescent on state and a dark
off state. The off state can be the ground state of a fluorophores as in the
case of STED, the triplet state as in ground-state-depletion microscopy
(28, 29), or the dark state of a reversibly photoswitchable fluorophore (30). RESOLFT has been demonstrated using a reversibly photoswitchable
fluorescent protein as FP595 which leads to a resolution better than 100 nm
at a depletion laser intensity of 600 W/cm²(30).

The same concept of employing saturable processes can also be applied
to SIM by introducing sub-diffraction-limit spatial features into the excitation
pattern. SSIM has been demonstrated using the saturation of fluorescence
emission, which occurs when a fluorophore is illuminated by a very high
intensity of excitation light (4). Under this strong excitation, it is immediately
pumped to the excited state each time it returns to the ground state. In SSIM,
where the sample is illuminated with a sinusoidal pattern of strong excitation
light, the peaks of the excitation pattern can be clipped by fluorescence
saturation and become flat, whereas fluorescence emission is still absent
from the zero points in the valleys (Figure 3a). These effects add higher order
spatial frequencies to the excitation pattern. Mixing this excitation pattern with
the high-frequency spatial features in the sample can effectively bring the sub-diffraction-limit spatial features into the detection range of the microscopy
(Figure 3b).

Figure 3

The principle of SSIM. (a) The generation of the illumination pattern. A
diffractive grating in the excitation path splits the light into two beams. Their interference after emerging from the objective and reaching the sample creates
a sinusoidal illumination

SSIM

Although the image of a single fluorophore, which resembles the PSF, is a
finite-sized spot, the precision of determining the fluorophores position from
its image can be much higher than the diffraction limit, as long as the image
results from multiple photons emitted from the fluorophore. Fitting an image
consisting of N photons can be viewed as N measurements of the fluorophore position, each with an uncertainty determined by the PSF (8), thus leading to
a localization precision approximated by:

Δloc≈ΔN−−√

where Δ_loc is the localization precision and Δ is the size of the PSF. This
scaling of the localization precision with the photon number allows super-
resolution microscopy with a resolution not limited by the diffraction of light.

High-precision localization of bright light has reached a precision as high
as ~1 Å (33). Taking advantage of single-molecule detection and imaging
(34, 35), nanometer localization precision has been achieved for single
fluorescent molecules (36).

Using fluorescent probes that can switch between a fluorescent and a dark
state, a recent invention overcomes this barrier by separating in the time
domain the otherwise spatially overlapping fluorescent images. In this approach, molecules within a diffraction limited region can be activated at different time
points so that they can be individually imaged, localized, and subsequently deactivated (Figure 4). Massively parallel localization is achieved through
wide-field imaging, so that the coordinates of many fluorophores can be
mapped and a super-resolution images subsequently reconstructed. This
concept has been independently conceived and implemented by three labs,
and it was given the names STORM (5), PALM (6), and FPALM (7), respectively.

Iterating the activation and imaging process allows the locations of many
fluorophores to be mapped and a super-resolution image to be constructed
from these fluorophore locations. In the following, we refer to this approach
as super-resolution microscopy by single-molecule localization.

Figure 4

The principle of stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), and fluorescence photo-
activation localization microscopy (FPALM). Different fluorescent probes
marking the sample structure are activated.

STORM

After capturing the images with a digital camera, the point-spread functions
of the individual molecules are localized with high precision based on the
photon output before the probes spontaneously photo-bleach or switch to
a dark state. The positions of localized molecular centers are indicated with
black crosses. The process is repeated in Figures (c) through (e) until all of
the fluorescent probes are exhausted due to photo-bleaching or because the background fluorescence becomes too high. The final super-resolution image
(Figure (f)) is constructed by plotting the measured positions of the fluorescent probes.
http://microscopyu.com/tutorials/flash/superresolution/storm/index.html

The resolution of this technique is limited by the number of photons detected
per photoactivation event, which varies from several hundred for FPs (6) to
several thousand for cyanine dyes such as Cy5 (5, 46). These numbers
theoretically allow more than an order of magnitude improvement in spatial
resolution according to the √N scaling rule. In practice, a lateral resolution
of ~20 nm has been established experimentally using the photoswitchable
cyanine dyes (5, 46). Super-resolution images of biological samples have
been reported with directly labeled DNA structures and immunostained DNA-
protein complexes in vitro (5) as well as with FPtagged or immunostained
cellular structures (6, 44, 46).

Table 1 Photoswitchable fluorophores used in super resolution
fluorescence microscopy

Photoswitchable fluorophores

Recent advances in super-resolution fluorescence microscopy
(including the capability for 3D, multicolor, live-cell imaging) enable
new applications in biological samples. These technical advances
were made possible through the development of both imaging optics
and fluorescent probes.

3D imaging using the single-molecule localization approach
3D imaging using the patterned excitation approach
Multicolor imaging
Multicolor imaging using the patterned excitation approach
Multicolor imaging using the single-molecule localization approach

Live cell imaging

Fluorescence imaging of a live cell has two requirements: specific labeling
of the cell and a time resolution that is high enough to record relevant
dynamics in the cell. Many fluorescent proteins and organic dyes, including
cyanine dyes (46) and caged dyes, have been shown switchable in live cells.

Because STED has a much smaller PSF than scanning confocal microscopy,
STED would inherently take more time to scan though the same size of image
field. By increasing the scanning speed and limiting the field of view to a few µm, Westphal and coworkers have observed Brownian motion of a dense suspension
of nanoparticles with an impressive rate of 80 frames per second (fps) using
STED microscopy (63). More recently, they have demonstrated video-rate
(28 fps) imaging of live hippocampal neurons and observed the movement of individual synaptic vesicles with 60–80-nm resolution (64).

Sub-diffraction-limit imaging of focal adhesion proteins in live cells has recently
been demonstrated (65). Photoswitchable fluorescent protein, EosFP, was used
to label the focal adhesion protein paxillin. A time resolution of ~25–60 seconds
per frame was obtained, and during this time interval, approximately 103
fluorophores were activated and localized per square micrometer, providing
an effective resolution of 60–70 nm by the Nyquist criterion (65). More recently, super-resolution imaging has also been demonstrated in live bacteria with photoswitchable enhanced yellow fluorescent protein (EYFP), allowing the
MreB structure in the cell to be traced (66).

The optical resolution

Optical resolution is the intrinsic ability of a given method to resolve a structure
and can be defined as the ability to distinguish two point sources in proximity.
For the patterned excitation approaches, such as STED, SSIM, and RESOLFT,
the optical resolution is represented by the size of the effective PSF. For the
single-molecule localization approach, such as STORM/PALM/FPALM, the
precision of determining the positions of individual fluorescent probes is the
principal measure of optical resolution.

By using a spatially patterned excitation profile, this approach achieves super resolution by generating an effective excitation volume with dimensions far
below the diffraction limit. Taking STED as an example, the sharpness of the
PSF results from the saturation of depletion of excited-state fluorophores in
the region neighboring the zero point of the STED laser (which coincide with
the focal point of the excitation laser). With an increasing STED laser power,
the saturated region expands toward the zero point, but fluorophores at the
zero point are not affected by the STED laser if the zero point is strictly kept
at zero intensity. Therefore, a theoretically unlimited gain in spatial resolution
may be achieved if the zero point in the depletion pattern is ideal.

The single-molecule localization approach achieves super resolution through
high precision localization of individual fluorophores. The number of photons
collected from a fluorophore is a principal factor limiting the localization
precision and hence the resolution of the final image.

Several photoswitchable fluorophores have been reported to give thousands
of photons detected per activation event [e.g., 6000 from Cy5 (46)].With the
PSF fitting procedure and the mechanical stability of the system optimized,
the background signal suppressed, and the nonuniformity of camera pixels
corrected, optical resolution of just a few nanometers could potentially be
achieved, reaching the molecular scale. As in the case of the patterned
excitation approach, the optical resolution here is also unlimited, in principle,
given a sufficient number of photons detected from the fluorescent probes.

Part III. A guide to super-resolution fluorescence microscopy

L Schermelleh 1, R Heintzmann 2,3,4, and H Leonhardt 1
JCB Jul 19, 2010 // 190(2): 165-175
The Rockefeller University Press,
http://dx.doi.org:/10.1083/jcb.201002018

Based on experimental evidence and basic principles of physics, Ernst Abbe
and Lord Rayleigh defined and formulated this diffraction-limited resolution in
the late 19th century (Abbe, 1873; Rayleigh, 1896). Later key innovations—including fluorescence and confocal laser scanning microscopy (CLSM)—made optical microscopy one of the most powerful and versatile diagnostic
tools in modern cell biology.

The optical resolution defines the physical limit of the smallest structure it
can resolve. When imaging a biological sample, the effective resolution is
also affected by several sample-specific factors, including the labeling density,
probe size, and how well the ultrastructures are preserved during sample
preparation.

The diffraction (Abbe) limit of detection

Resolution is often defined as the largest distance at which the image of
two point-like objects seems to amalgamate. Thus, most resolution criteria
(Rayleigh limit,Sparrow limit, full width at half maximum of the PSF) directly
relate to properties of the PSF. These are useful resolution criteria for visible
observation of specimen, but there are several shortcomings of such a definition
of resolution: (1) Knowing that the image is an image of two particles, these
can in fact be discriminated with the help of a computer down to arbitrary
smaller distances. Determining the positions of two adjacent particles thus
becomes a question of experimental precision and most notably photon statistics
rather than being described by the Rayleigh limit. (2) These limits do not
necessarily correspond well to what level of detail can be seen in images or
real world objects; e.g., the Rayleigh limit is defined as the distance from the
center to the first minimum of the point spread function, which can be made
arbitrarily small with the help of ordinary linear optics (e.g., Toraldo-filters),
albeit at the expense of the side lobes becoming much higher than the central
maximum. (3)

Abbe’s formulation of a resolution limit avoids all of the above shortcomings
at the expense of a less direct interpretation. The process of imaging can be
described by a convolution operation. With the help of a Fourier transformation,
every object (whether periodic or not) can uniquely be described as a sum of
sinusoidal curves with different spatial frequencies (where higher frequencies
represent fine object details and lower frequencies represent coarse details).
The rather complex process of convolution can be greatly simplified by looking
at the equivalent operation in Fourier space: The Fourier-transformed object
just needs to be multiplied with the
Fourier-transformed PSF to yield the Fourier-transformed ideal image (without
the noise). Because the Fourier-transformed PSF now describes how well each
spatial frequency of the Fourier-transformed object gets transferred to appear in the
image, this Fourier-transformed PSF is called the optical transfer function, OTF
(right panel). Its strength at each spatial frequency (e.g., measured in oscillations
per meter) conveniently describes the contrast that a sinusoidal object would
achieve in an image.

Abbe limit

Interestingly, the detection OTF of a microscope has a fixed frequency
border (Abbe limit frequency, right panel). The maximum-to-maximum
distance Λmin of the corresponding sine curve is commonly referred to
as Abbe’s limit (left panel). In other words: The Abbe limit is the smallest
periodicity in a structure, which can be discriminated in its image. As a
point object contains all spatial frequencies, this Abbe limit sine curve
needs to also be present in the PSF. A standard wide-field microscope
creates an image of a point object (e.g., an emitting molecule) by capturing
the light from that molecule at various places of the objective lens, and
processing it with further lenses to then interfere at the image plane.
Conveniently due to the reciprocity principle in optics, the Abbe limit Λmin
along an in-plane direction in fluorescence imaging corresponds to the
maximum-to-maximum distance of the intensity structure one would get by
interfering two waves at extreme angles captured by the objective lens:
where λ/n is the wavelength of light in the medium of refractive index n.
The term NA = n sin(α) conveniently combines the half opening angle α
of the objective and the refractive index n of the embedding medium.

Abbe’s famous resolution limit is so attractive because it simply depends
on the maximal relative angle between different waves leaving the
object and being captured by the objective lens to be sent to the image.
It describes the smallest level of detail that can possibly be imaged with
this PSF “brush”. No periodic object detail smaller than this shortest
wavelength can possibly be transferred to the image.

Confocal laser scanning microscopy employs a redesigned optical
path and specialized hardware. A tightly focused spot of laser light is
used to scan the sample and a small aperture (or pinhole) in the
confocal image plane of the light path allows only light originating
from the nominal focus to pass (Cremer and Cremer, 1978; Sheppard
and Wilson, 1981; Brakenhoff et al., 1985). The emitted light is
detected by a photomultiplier tube (PMT) or an avalanche photodiode
(APD) and the image is then constructed by mapping the detected
light in dependence of the position of the scanning spot. CLSM can
achieve a better resolution than wide-field fluorescence microscopy
but, to obtain a significant practical advantage, the pinhole needs to
be closed to an extent where most of the light is discarded
(Heintzmann et al., 2003).

Wide-field deconvolution and CLSM have long been the gold standards
in optical bioimaging, but we are now witnessing a revolution in light
microscopy that will fundamentally expand our perception of the cell.
Recently, several new technologies,collectively termed super-resolution
microscopy or nanoscopy, have been developed that break or bypass
the classical diffraction limit and shift the optical resolution down to
macromolecular or even molecular levels (Table I).

Super-resolution light microscopy methods

super resolution microscopy

http://zeiss-campus.magnet.fsu.edu/articles/superresolution/introduction.html

Conceptually, one can discern near-field from far-field methods and
whether the subdiffraction resolution is based on a linear or nonlinear
response of the sample to its locally illuminating (exciting or depleting) irradiance. The required nonlinearity is currently achieved by using reversible saturable optical fluorescence transitions (RESOLFT) between molecular states (Hofmann et al., 2005; Hell, 2007).

Besides these saturable optical fluorescence transitions also other
approaches, e.g., Rabi oscillations, could be used to generate the
required nonlinear response.

Note that each of the novel imaging modes has its individual signal-
to-noise consideration depending on various factors. A full
discussion of this issue is beyond the scope of this review, but as a
general rule, single-point scanning systems, albeit fundamentally limited
in speed by fluorescence saturation effects, can have better signal-
to-noise performance for thicker samples.

With three-dimensional SIM (3D-SIM), an additional twofold increase
in the axial resolution can be achieved by generating an excitation
light modulation along the z-axis using three-beam interference
(Gustafsson et al., 2008; Schermelleh et al.,2008) and processing a
z-stack of images accordingly. Thus, with 3D-SIM an approximately eightfold smaller volume can be resolved in comparison to conventional microscopy (Fig. 2). To computationally reconstruct a three-dimensional dataset of a typical mammalian cell of 8-µm height with a
z-spacing of 125 nm, roughly 1,000 raw images (512 × 512 pixels) are
recorded. Because no special photophysics is needed, virtually all modern fluorescent labels can be used provided they are sufficiently photostable
to accommodate the additional exposure cycles.

Resolvable volumes obtained with current commercial super-resolution microscopes.

A schematic 3D representation of focal volumes is shown for the indicated
emission maxima. The approximate lateral (x,y) and axial (z) resolution
and resolvable volumes are listed. Note that STED/CW-STED and 3D-SIM
can reach up to 20 µm into the sample, whereas PALM/STORM is usually
confined to the evanescent wave field near the sample bottom. It should be
noted that deconvolution approaches can further improve STED resolution.
For comparison the “focal volume” for PALM/STORM was estimated based
on the localization precision in combination with the z-range of TIRF.

Resolvable volumes obtained

Super-resolution microscopy of biological samples.

(A) Conventional wide-field image (left) and 3D-SIM image of a mouse
C2C12 prometaphase cell stained with primary antibodies against
lamin B and tubulin, and secondary antibodies conjugated to Alexa 488
(green) and Alexa 594 (red), respectively. Nuclear chromatin was stained
with DAPI (blue). 3D image stacks were acquired with a DeltaVision OMX
prototype system (Applied Precision). The bottom panel shows the
respective orthogonal cross sections. (B) HeLa cell stained with primary
antibodies against the nuclear pore complex protein Nup153 and
secondary antibodies conjugated with ATTO647N. The image was
acquired with a TCS STED confocal microscope (Leica). (C) TdEosFP-
paxillin expressed in a Hep G2 cell to label adhesion complexes at
the lower surface. The image was acquired on an ELYRA P.1
prototype system (Carl Zeiss, Inc.) using TIRF illumination. Single
molecule positional information was projected from 10,000 frames
recorded at 30 frames per second. On the left, signals were summed
up to generate a TIRF image with conventional wide-field lateral
resolution. Bars: 5 µm (insets, 0.5 µm).

biological images

APPLICATIONS IN BIOLOGICAL SYSTEMS

The cytoskeleton of mammalian cells, especially microtubules
(Figure 5a) (29, 44, 46, 52), is the most commonly used benchmark
structure for super-resolution imaging. Other cytoskeletal structures
imaged so far include actin filaments in the lamellipodium (6),
keratin intermediate filaments (59), neurofilaments (26, 83) and
MreB in Caulobacter (66).

Figure 5

cytoskeleton. f5.

Examples of super-resolution images of biological samples.
(a) Two-color STORM imaging of immunostained microtubule (green)
and clathrin-coated pits (red) (From Reference 46. Reprinted with
permission from AAAS).

Organelles, such as the endoplasmic reticulum (27), lysosome (6),
endocytic and exocytic vesicles (46, 52, 64), and mitochondria
(6, 53, 56), have also been imaged. For example, using the single-molecule localization approach, 3D STORM imaging has clearly
resolved the ~150-nm diameter, hemispherical cage shape of clathrin-coated pits (46, 52), which only appear as diffraction-limited spots
without any feature in conventional fluorescence microscopy (Figure 5a,b).
Two-color 3D STED has resolved the hollow shape of the mitochondrial
outer membrane (marked by the translocase protein Tom20), enclosing
a matrix protein Hsp60 (56), even though the diameter of mitochondria is
only about 300–500 nm (Figure 5c). The outer membrane structure of
mitochondria and their interactions with microtubules have been resolved
by two-color 3D STORM (53). The transport of synaptic vesicles
has been recorded at video rate using 2D STED (Figure 5d ) (64).

Many plasma membrane proteins or membrane associated protein
complexes have also been studied by super-resolution fluorescence
microscopy. For example, synaptotagmin clusters after exocytosis in
primary cultured hippocampal neurons (84), the donut-shaped
clusters of Drosophila protein Bruchpilot at the neuromuscular
synaptic active zone (85), and the size distribution of syntaxin clusters
have all been imaged (86, 87). Photoactivation has enabled the tracking
of the influenza protein hemagglutinin and the retroviral protein Gag in
live cells, revealing the membrane microdomains (67) and the spatial
heterogeneity of membrane diffusion (68). The morphology and transport
of the focal adhension complex has also been observed using live-cell
PALM (Figure 5e) (65).

Summary points

Super resolution fluorescence microscopy with a spatial resolution not limited by the diffraction of
light has been implemented using saturated depletion/excitation or single-molecule localization
of switchable fluorophores.
Three-dimensional imaging with an optical resolution as high as ~20 nm in the lateral direction
and 40–50 nm in axial dimension has been achieved.
The resolution of these super-resolution fluorescence microscopy techniques can in principle
reach molecular scale.
In practice, the resolution of the images are not only limited by the intrinsic optical resolution,
but also by sample specific factors including the labeling density, probe size and sample preservation.
Multicolor super resolution imaging has been implemented, allowing colocalization measurements
to be performed at nanometer scale resolution and molecular interaction to be more précisely
identified in cells.
Super-resolution fluorescence imaging allows dynamic processes to be investigated at the tens of
nanometer resolution in living cells.
Many cellular structures have been imaged at sub-diffraction-limit resolution.

Future issues

Achieving molecular scale resolution (a few nanometers or less).
Fast super resolution imaging of a large view field by multi-point scanning or high-speed single-molecule switching/localization.
Developing new fluorescent probes that are brighter, more photostable and switchable fluorophores
that have high on-off contrast and fast switching rate.
Developing fluorescent labeling methods that can stain the target with small molecules at high specificity,
high density and good ultrastructure preservation.
Application of super resolution microscopy to provide novel biological insights

Acronyms

Fluorescent Protein

FPALM

Fluorescence PhotoActivation Localization Microscopy

I5M

Combination of I2M (Illumination Interference Microscopy) and I3M
(Incoherent Imaging Interference Microscopy)

PALM

PhotoActivated Localization Microscopy

PSF

Point Spread Function

RESOLFT

REversible Saturable Optically Linear Fluorescence Transition

SIM

Structured Illumination Microscopy

SSIM

Saturated Structured Illumination Microscopy

STED

STimulated Emission Depletion

STORM

STochastic Optical Reconstruction Microscopy

glossary

Numerical aperture (NA)

The numerical aperture of an objective characterizes the solid angle
of light collected from a point light source at the focus of the objective.

Stimulated emission

The process that an excited state molecule or atom jumps to the
ground state by emitting another photon that is identical to the incoming
photon. It is the basis of laser.

Fluorescence saturation

At high excitation intensity, the fluorescence lifetime instead of the excitation
rate becomes the rate limiting step of fluorescence emission, causing the
fluorescence signal not to increase proportionally with the excitation intensity.

Nyquist criterion

To determine a structure, the sampling interval needs to be no larger than
half of the feature size.

Mitochondria

Organelles in eukaryotic cells for APT generation, consisting of two
membrane (inner and outer) enclosing the inter membrane space and
the matrix inside the inner membrane.

Clathrin-coated pit

Vesicle forming machinery involved in endocytosis and intracellular
vesicle transport, consisting of clathrin coats, adapter proteins, and
other regulatory proteins.

Focal adhesion

The macromolecular complex serving as the mechanical connection
and signaling hub between a cell and the extracellular matrix or other cells.

Selected references with abstract

Near-Field Optics: Microscopy, Spectroscopy, and Surface
Modification Beyond the Diffraction Limit
Eric Betzig, Jay K. Trautman
AT&T Bell Laboratories, Murray Hill, NJ 07974
Science 10 Jul 1992; 257(5067) pp. 189-195
http://dx.doi.org:/0.1126/science.257.5067.189

The near-field optical interaction between a sharp probe and a sample
of interest can be exploited to image, spectroscopically probe, or modify
surfaces at a resolution (down to ∼12 nm) inaccessible by traditional far-field
techniques. Many of the attractive features of conventional optics are
retained, including noninvasiveness, reliability, and low cost. In addition, most
optical contrast mechanisms can be extended to the near-field regime,
resulting in a technique of considerable versatility. This versatility
is demonstrated by several examples, such as the imaging of nanometric-scale features in mammalian tissue sections and the creation of ultrasmall,
magneto-optic domains having implications for high density data storage.
Although the technique may find uses in many diverse fields, two of the
most exciting possibilities are localized optical spectroscopy of semiconductors
and the fluorescence imaging of living cells.

Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

E Betzig1,2,*,†, GH. Patterson3, R Sougrat3, O.W Lindwasser3,
S Olenych4, JS. Bonifacino3, MW. Davidson4, JL Schwartz3, HF. Hess5,* 1 Howard Hughes Medical Institute, Janelia Farm Research Campus,
Ashburn, VA 2 New Millennium Research, LLC, Okemos, MI. 3 Cell Biology and Metabolism Branch, National Institute of Child Health
and Human Development (NICHD), Bethesda, MD. 4 National High
Magnetic Field Laboratory, Florida State University, Tallahassee, FL.
5 NuQuest Research, LLC, La Jolla, CA.
Science 15 Sep 2006; 313(5793): pp. 1642-1645
http://dx.doi.org:/10.1126/science.1127344

We introduce a method for optically imaging intracellular proteins at
nanometer spatial resolution. Numerous sparse subsets of photo-activatable fluorescent protein molecules were activated, localized
(to ∼2 to 25 nanometers), and then bleached. The
aggregate position information from all subsets was then assembled
into a super-resolution image. We used this method—termed photo-
activated localization microscopy to image specific target proteins
in thin sections of lysosomes and mitochondria; in fixed whole cells,
we imaged vinculin at focal adhesions, actin within a lamellipodium,
and the distribution of the retroviral protein Gag at the plasma
membrane.

Toward fluorescence nanoscopy.

Hell SW. Author information
Nat Biotechnol. 2003 Nov; 21(11):1347-55.
http://www.ncbi.nlm.nih.gov/pubmed/14595362

For more than a century, the resolution of focusing light microscopy
has been limited by diffraction to 180 nm in the focal plane and to
500 nm along the optic axis. Recently, microscopes have been
reported that provide three- to seven-fold improved axial
resolution in live cells. Moreover, a family of concepts has emerged
that overcomes the diffraction barrier altogether. Its first exponent,
stimulated emission depletion microscopy, has so far displayed a
resolution down to 28 nm. Relying on saturated optical transitions,
these concepts are limited only by the attainable saturation level.
As strong saturation should be feasible at low light intensities,
nanoscale imaging with focused light may be closer than ever.
PMID: 14595362

Far-field optical nanoscopy.

Hell SW. Author information
Science. 2007 May 25;316(5828):1153-8.
http://www.ncbi.nlm.nih.gov/pubmed/17525330

In 1873, Ernst Abbe discovered what was to become a well-known
paradigm: the inability of a lens-based optical microscope to
discern details that are closer together than half of the wavelength
for its most popular imaging mode, fluorescence microscopy, the
diffraction barrier is crumbling. Here, I discuss the physical concepts
that have pushed fluorescence microscopy to the nanoscale, once
the prerogative of electron and scanning probe microscopes. Initial
applications indicate that emergent far-field optical nanoscopy will
have a strong impact in the life sciences and in other areas benefiting
from nanoscale visualization.
PMID: 17525330

Imaging intracellular fluorescent proteins at nanometer resolution.

Betzig E1, Patterson GH, Sougrat R, Lindwasser OW, Olenych S,
Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF.
Author information
Science. 2006 Sep 15;313(5793):1642-5. Epub 2006 Aug 10
http://www.ncbi.nlm.nih.gov/pubmed/16902090

We introduce a method for optically imaging intracellular proteins at
nanometer spatial resolution. Numerous sparse subsets of photo-ctivatable fluorescent protein molecules were activated, localized
(to approximately 2 to 25 nanometers), and then bleached. The
aggregate position information from all subsets was then assembled
into a super-resolution image. We used this method–termed photo-activated localization microscopy–to image specific target proteins in
thin sections of lysosomes and mitochondria; in fixed whole cells,
we imaged vinculin at focal adhesions, actin within a lamellipodium,
and the distribution of the retroviral protein Gag at the plasma
membrane.

Comment in

The limits of light.[Nat Rev Mol Cell Biol. 2010]

PMID: 16902090 [PubMed – indexed for MEDLINE]

Illuminating single molecules in condensed matter.

Moerner WE1, Orrit M. Author information
Science. 1999 Mar 12;283(5408):1670-6.
http://www.ncbi.nlm.nih.gov/pubmed/10073924

Efficient collection and detection of fluorescence coupled with careful
minimization of background from impurities and Raman scattering
now enable routine optical microscopy and study of single molecules
in complex condensed matter environments. This ultimate method
for unraveling ensemble averages leads to the observation of
new effects and to direct measurements of stochastic fluctuations.
Experiments at cryogenic temperatures open new directions in
molecular spectroscopy, quantum optics, and solid-state dynamics.
Room-emperature investigations apply several techniques
(polarization microscopy, single-molecule imaging, emission time
dependence, energy transfer, lifetime studies, and the like) to a
growing array of biophysical problems where new insight may be
gained from direct observations of hidden static and dynamic
inhomogeneity. PMID: 10073924

Fluorescence microscopy with super-resolved optical sections.

Egner A1, Hell SW. Author information
Trends Cell Biol. 2005 Apr;15(4):207-15.
http://www.ncbi.nlm.nih.gov/pubmed/15817377

The fluorescence microscope, especially its confocal variant, has
become a standard tool in cell biology research for delivering
3D-images of intact cells. However, the resolution of any standard
optical microscope is atleast 3 times poorer along the axis of the
lens that in its focal plane. Here, we review principles and applications
of an emerging family of fluorescence microscopes, such as 4Pi
microscopes, which improve axial resolution by a factor of seven by
employing two opposing lenses. Noninvasive axial sections of 80-160 nm
thickness deliver more faithful 3D-images of subcellular features,
providing a new opportunity to significantly enhance our understanding
of cellular structure and function. PMID: 15817377

4Pi-confocal microscopy provides three-dimensional images of the
microtubule network with 100- to 150-nm resolution.

Nagorni M1, Hell SW. Author information
J Struct Biol. 1998 Nov;123(3):236-47.

We show the applicability of 4Pi-confocal microscopy to three-dimensional imaging of the microtubule network in a fixed mouse
fibroblast cell.Comparison with two-photon confocal resolution
reveals a fourfold better axial resolution in the 4Pi-confocal case.
By combining 4Pi-confocal microscopy with Richardson-Lucy
image restoration a further resolution increase is achieved.
Featuring a three-dimensional resolution in the range 100-150 nm,
the 4Pi-confocal (restored) images are intrinsically more detailed
than their confocal counterparts. Our images constitute what
to our knowledge are the best-resolved three-dimensional
images of entangled cellular microtubules obtained with light
to date. PMID: 9878578

Part IV. Super-resolution microscopy

Super-resolution microscopy is a form of light microscopy. Due
to the diffraction of light, the resolution of conventional light
microscopy is limited as stated by Ernst Abbe in 1873.[1]
A good approximation of the resolution attainable is the full
width at half maximum (FWHM) of the point spread function,
and a precise wide-field microscope with high numerical
aperture and visible light usually reaches a resolution of ~250 nm.

Super-resolution techniques allow the capture of images with
a higher resolution than the diffraction limit. They fall into
two broad categories,
“true” super-resolution techniques, which capture information
contained in evanescent waves, and “functional” super-
resolution techniques, which use clever experimental
techniques and known limitations on the matter being
imaged to reconstruct a super-resolution image.[2]

True subwavelength imaging techniques include those that
utilize the Pendry Superlens and near field scanning optical
microscopy, the 4Pi Microscope and structured illumination
microscopy technologies like SIM and SMI. However, the
majority of techniques of importance in biological imaging
fall into the functional category.

Groups of methods for functional super-resolution microscopy:

Deterministic super-resolution: The most commonly used emitters in biological
microscopy, fluorophores, show a nonlinear response to excitation, and this
nonlinear response can be exploited to enhance resolution. These
methods include STED, GSD, RESOLFTand SSIM.
Stochastic super-resolution: The chemical complexity of many molecular
light sources gives them a complex temporal behaviour, which can be used
to make several close-by fluorophores emit light at separate times and
thereby become resolvable in time. These methods include SOFI and all
single-molecule localization methods (SMLM) such as SPDM,
SPDMphymod, PALM, FPALM, STORM and dSTORM.

Part V. HIV-1

Conformational dynamics of single HIV-1 envelope
trimers on the surface of native virions

James B. Munro 1,*,‡, Jason Gorman 2, Xiaochu Ma 1,
Zhou Zhou 3, James Arthos 4,
Dennis R. Burton 5,6, et al.
1Department of Microbial Pathogenesis, Yale University
School of Medicine, New Haven, CT. 2Vaccine Research
Center, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD .
3Department of Physiology and Biophysics, Weill
Cornell Medical College of Cornell University, New York, NY .
4Laboratory of Immunoregulation, National Institute of Allergy
and Infectious Diseases, National Institutes of Health, Bethesda,
MD . 5Department of Immunology and Microbial Science, and
IAVI Neutralizing Antibody Center, The Scripps Research
Institute, La Jolla, CA . 6Ragon Institute of MGH, MIT, and
Harvard, Cambridge, MA. 7International AIDS Vaccine Initiative
(IAVI), New York, NY . 8Department of
Chemistry, University of Pennsylvania, Philadelphia, PA.

The HIV-1 envelope (Env) mediates viral entry into host cells.
To enable the direct imaging of conformational dynamics
within Env we introduced fluorophores into variable
regions of the gp120 subunit and measured single-molecule
fluorescence resonance energy transfer (smFRET) within
the context of native trimers on the surface of HIV-1 virions.
Our observations revealed unliganded HIV-1 Env to be
intrinsically dynamic, transitioning between three distinct
pre-fusion conformations, whose relative occupancies
were remodeled by receptor CD4 and antibody binding.
The distinct properties of neutralization-sensitive and
neutralization-resistant HIV-1 isolates support a dynamics-based mechanism of immune evasion and ligand recognition.

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

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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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Lipid Metabolism

Posted in Atherogenic Processes & Pathology, Cachexia, Cardiovascular Research, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Curation, Cytoskeleton, Diabetes Mellitus, Dialectic, Discovery process, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Endocrine Diseases, Enzyme Induction, Epigenetics and Cardiovascular Risks, Experimental validation, Explanatory, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Metabolomics, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Na-K transport, Na-K-ATPase, Nephrology, Nutrigenomics, Nutrition, Nutritional Supplements: Atherogenesis, lipid metabolism, Origins of Cardiovascular Disease, Oxidative phosphorylation, Personalized and Precision Medicine & Genomic Research, Phosphorylation, Population Health Management, Nutrition and Phytochemistry, Pre-Clinical Animal Model Development, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Signaling, Synaptic vesicle, Systemic Inflammatory Response Related Disorders, Translational Effectiveness, Translational Research, Translational Science, Warburg effect, Water Transporters, tagged Atherosclerosis, Cytokines, diet and cancer, eicosenoids, fatty acid deficiency, Fatty acids, insulin and glucagon, islet cells, Lipid metabolism, Lipids, liver metabolism, oinflammatoty, omega 3 FA, omega 6 FA, omega 6 ftty acid excess, omega-6/omega-3, PPAR gamma, PUFA, total parenteral nutrition, triene/tetraene ratio on August 15, 2014| Leave a Comment »

Lipid Metabolism

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

This is fourth of a series of articles, lipid metabolism, that began with signaling and signaling pathways. These discussion lay the groundwork to proceed in later discussions that will take on a somewhat different approach. These are critical to develop a more complete point of view of life processes. I have indicated that many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different. 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.

Signaling and signaling pathways
Signaling transduction tutorial.
Carbohydrate metabolism

3.1 Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

Lipid metabolism
Protein synthesis and degradation
Subcellular structure
Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

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 Oxidationto 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

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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

SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver

Jay D. Horton^1,2, Joseph L. Goldstein¹ and Michael S. Brown¹

¹Department of Molecular Genetics, and
²Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

J Clin Invest. 2002;109(9):1125–1131.
http://dx.doi.org:/10.1172/JCI15593
Lipid homeostasis in vertebrate cells is regulated by a family of membrane-bound transcription factors designated sterol regulatory element–binding proteins (SREBPs). SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the NADPH cofactor required to synthesize these molecules (1–4). In the liver, three SREBPs regulate the production of lipids for export into the plasma as lipoproteins and into the bile as micelles. The complex, interdigitated roles of these three SREBPs have been dissected through the study of ten different lines of gene-manipulated mice. These studies form the subject of this review.

SREBPs: activation through proteolytic processing

SREBPs belong to the basic helix-loop-helix–leucine zipper (bHLH-Zip) family of transcription factors, but they differ from other bHLH-Zip proteins in that they are synthesized as inactive precursors bound to the endoplasmic reticulum (ER) (1, 5). Each SREBP precursor of about 1150 amino acids is organized into three domains: (a) an NH₂-terminal domain of about 480 amino acids that contains the bHLH-Zip region for binding DNA; (b) two hydrophobic transmembrane–spanning segments interrupted by a short loop of about 30 amino acids that projects into the lumen of the ER; and (c) a COOH-terminal domain of about 590 amino acids that performs the essential regulatory function described below.

In order to reach the nucleus and act as a transcription factor, the NH₂-terminal domain of each SREBP must be released from the membrane proteolytically (Figure 1). Three proteins required for SREBP processing have been delineated in cultured cells, using the tools of somatic cell genetics (see ref. 5for review). One is an escort protein designated SREBP cleavage–activating protein (SCAP). The other two are proteases, designated Site-1 protease (S1P) and Site-2 protease (S2P). Newly synthesized SREBP is inserted into the membranes of the ER, where its COOH-terminal regulatory domain binds to the COOH-terminal domain of SCAP (Figure 1).

Figure 1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1

Model for the sterol-mediated proteolytic release of SREBPs from membranes. SCAP is a sensor of sterols and an escort of SREBPs. When cells are depleted of sterols, SCAP transports SREBPs from the ER to the Golgi apparatus, where two proteases, Site-1 protease (S1P) and Site-2 protease (S2P), act sequentially to release the NH₂-terminal bHLH-Zip domain from the membrane. The bHLH-Zip domain enters the nucleus and binds to a sterol response element (SRE) in the enhancer/promoter region of target genes, activating their transcription. When cellular cholesterol rises, the SCAP/SREBP complex is no longer incorporated into ER transport vesicles, SREBPs no longer reach the Golgi apparatus, and the bHLH-Zip domain cannot be released from the membrane. As a result, transcription of all target genes declines. Reprinted from ref. 5 with permission.

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SCAP is both an escort for SREBPs and a sensor of sterols. When cells become depleted in cholesterol, SCAP escorts the SREBP from the ER to the Golgi apparatus, where the two proteases reside. In the Golgi apparatus, S1P, a membrane-bound serine protease, cleaves the SREBP in the luminal loop between its two membrane-spanning segments, dividing the SREBP molecule in half (Figure 1). The NH₂-terminal bHLH-Zip domain is then released from the membrane via a second cleavage mediated by S2P, a membrane-bound zinc metalloproteinase. The NH₂-terminal domain, designated nuclear SREBP (nSREBP), translocates to the nucleus, where it activates transcription by binding to nonpalindromic sterol response elements (SREs) in the promoter/enhancer regions of multiple target genes.

Figure 1

When the cholesterol content of cells rises, SCAP senses the excess cholesterol through its membranous sterol-sensing domain, changing its conformation in such a way that the SCAP/SREBP complex is no longer incorporated into ER transport vesicles. The net result is that SREBPs lose their access to S1P and S2P in the Golgi apparatus, so their bHLH-Zip domains cannot be released from the ER membrane, and the transcription of target genes ceases (1, 5). The biophysical mechanism by which SCAP senses sterol levels in the ER membrane and regulates its movement to the Golgi apparatus is not yet understood. Elucidating this mechanism will be fundamental to understanding the molecular basis of cholesterol feedback inhibition of gene expression.

SREBPs: two genes, three proteins

The mammalian genome encodes three SREBP isoforms, designated SREBP-1a, SREBP-1c, and SREBP-2. SREBP-2 is encoded by a gene on human chromosome 22q13. Both SREBP-1a and -1c are derived from a single gene on human chromosome 17p11.2 through the use of alternative transcription start sites that produce alternate forms of exon 1, designated 1a and 1c (1). SREBP-1a is a potent activator of all SREBP-responsive genes, including those that mediate the synthesis of cholesterol, fatty acids, and triglycerides. High-level transcriptional activation is dependent on exon 1a, which encodes a longer acidic transactivation segment than does the first exon of SREBP-1c. The roles of SREBP-1c and SREBP-2 are more restricted than that of SREBP-1a. SREBP-1c preferentially enhances transcription of genes required for fatty acid synthesis but not cholesterol synthesis. Like SREBP-1a, SREBP-2 has a long transcriptional activation domain, but it preferentially activates cholesterol synthesis (1). SREBP-1a and SREBP-2 are the predominant isoforms of SREBP in most cultured cell lines, whereas SREBP-1c and SREBP-2 predominate in the liver and most other intact tissues (6).

When expressed at higher than physiologic levels, each of the three SREBP isoforms can activate all enzymes indicated in Figure 2, which shows the biosynthetic pathways used to generate cholesterol and fatty acids. However, at normal levels of expression, SREBP-1c favors the fatty acid biosynthetic pathway and SREBP-2 favors cholesterologenesis. SREBP-2–responsive genes in the cholesterol biosynthetic pathway include those for the enzymes HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase. SREBP-1c–responsive genes include those for ATP citrate lyase (which produces acetyl-CoA) and acetyl-CoA carboxylase and fatty acid synthase (which together produce palmitate [C16:0]). Other SREBP-1c target genes encode a rate-limiting enzyme of the fatty acid elongase complex, which converts palmitate to stearate (C18:0) (ref.7); stearoyl-CoA desaturase, which converts stearate to oleate (C18:1); and glycerol-3-phosphate acyltransferase, the first committed enzyme in triglyceride and phospholipid synthesis (3). Finally, SREBP-1c and SREBP-2 activate three genes required to generate NADPH, which is consumed at multiple stages in these lipid biosynthetic pathways (8) (Figure 2).

Figure 2

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2

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Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Knockout and transgenic mice

Ten different genetically manipulated mouse models that either lack or overexpress a single component of the SREBP pathway have been generated in the last 6 years (9–16). The key molecular and metabolic alterations observed in these mice are summarized in Table 1.

Table 1
Alterations in hepatic lipid metabolism in gene-manipulated mice overexpressing or lacking SREBPs

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Knockout mice that lack all nSREBPs die early in embryonic development. For instance, a germline deletion of S1p, which prevents the processing of all SREBP isoforms, results in death before day 4 of development (15, 17). Germline deletion of Srebp2 leads to 100% lethality at a later stage of embryonic development than does deletion of S1p (embryonic day 7–8). In contrast, germline deletion of Srebp1, which eliminates both the 1a and the 1c transcripts, leads to partial lethality, in that about 15–45% of Srebp1^–/– mice survive (13). The surviving homozygotes manifest elevated levels of SREBP-2 mRNA and protein (Table 1), which presumably compensates for the loss of SREBP-1a and -1c. When the SREBP-1c transcript is selectively eliminated, no embryonic lethality is observed, suggesting that the partial embryonic lethality in the Srebp1^–/– mice is due to the loss of the SREBP-1a transcript (16).

To bypass embryonic lethality, we have produced mice in which all SREBP function can be disrupted in adulthood through induction of Cre recombinase. For this purpose, loxP recombination sites were inserted into genomic regions that flank crucial exons in the Scap or S1p genes (so-called floxed alleles) (14, 15). Mice homozygous for the floxed gene and heterozygous for a Cre recombinase transgene, which is under control of an IFN-inducible promoter (MX1-Cre), can be induced to delete Scap or S1p by stimulating IFN expression. Thus, following injection with polyinosinic acid–polycytidylic acid, a double-stranded RNA that provokes antiviral responses, the Cre recombinase is produced in liver and disrupts the floxed gene by recombination between the loxP sites.

Cre-mediated disruption of Scap or S1p dramatically reduces nSREBP-1 and nSREBP-2 levels in liver and diminishes expression of all SREBP target genes in both the cholesterol and the fatty acid synthetic pathways (Table 1). As a result, the rates of synthesis of cholesterol and fatty acids fall by 70–80% in Scap- and S1p-deficient livers.

In cultured cells, the processing of SREBP is inhibited by sterols, and the sensor for this inhibition is SCAP (5). To learn whether SCAP performs the same function in liver, we have produced transgenic mice that express a mutant SCAP with a single amino acid substitution in the sterol-sensing domain (D443N) (12). Studies in tissue culture show that SCAP(D443N) is resistant to inhibition by sterols. Cells that express a single copy of this mutant gene overproduce cholesterol (18). Transgenic mice that express this mutant version of SCAP in the liver exhibit a similar phenotype (12). These livers manifest elevated levels of nSREBP-1 and nSREBP-2, owing to constitutive SREBP processing, which is not suppressed when the animals are fed a cholesterol-rich diet. nSREBP-1 and -2 increase the expression of all SREBP target genes shown in Figure 2, thus stimulating cholesterol and fatty acid synthesis and causing a marked accumulation of hepatic cholesterol and triglycerides (Table 1). This transgenic model provides strong in vivo evidence that SCAP activity is normally under partial inhibition by endogenous sterols, which keeps the synthesis of cholesterol and fatty acids in a partially repressed state in the liver.

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Function of individual SREBP isoforms in vivo

To study the functions of individual SREBPs in the liver, we have produced transgenic mice that overexpress truncated versions of SREBPs (nSREBPs) that terminate prior to the membrane attachment domain. These nSREBPs enter the nucleus directly, bypassing the sterol-regulated cleavage step. By studying each nSREBP isoform separately, we could determine their distinct activating properties, albeit when overexpressed at nonphysiologic levels.

Overexpression of nSREBP-1c in the liver of transgenic mice produces a triglyceride-enriched fatty liver with no increase in cholesterol (10). mRNAs for fatty acid synthetic enzymes and rates of fatty acid synthesis are elevated fourfold in this tissue, whereas the mRNAs for cholesterol synthetic enzymes and the rate of cholesterol synthesis are not increased (8). Conversely, overexpression of nSREBP-2 in the liver increases the mRNAs only fourfold. This increase in cholesterol synthesis is even more remarkable when encoding all cholesterol biosynthetic enzymes; the most dramatic is a 75-fold increase in HMG-CoA reductase mRNA (11). mRNAs for fatty acid synthesis enzymes are increased to a lesser extent, consistent with the in vivo observation that the rate of cholesterol synthesis increases 28-fold in these transgenic nSREBP-2 livers, while fatty acid synthesis increases one considers the extent of cholesterol overload in this tissue, which would ordinarily reduce SREBP processing and essentially abolish cholesterol synthesis (Table 1).

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We have also studied the consequences of overexpressing SREBP-1a, which is expressed only at low levels in the livers of adult mice, rats, hamsters, and humans (6). nSREBP-1a transgenic mice develop a massive fatty liver engorged with both cholesterol and triglycerides (9), with heightened expression of genes controlling cholesterol biosynthesis and, still more dramatically, fatty acid synthesis (Table 1). The preferential activation of fatty acid synthesis (26-fold increase) relative to cholesterol synthesis (fivefold increase) explains the greater accumulation of triglycerides in their livers. The relative representation of the various fatty acids accumulating in this tissue is also unusual. Transgenic nSREBP-1a livers contain about 65% oleate (C18:1), markedly higher levels than the 15–20% found in typical wild-type livers (8) — a result of the induction of fatty acid elongase and stearoyl-CoA desaturase-1 (7). Considered together, the overexpression studies indicate that both SREBP-1 isoforms show a relative preference for activating fatty acid synthesis, whereas SREBP-2 favors cholesterol.

The phenotype of animals lacking the Srebp1 gene, which encodes both the SREBP-1a and -1c transcripts, also supports the notion of distinct hepatic functions for SREBP-1 and SREBP-2 (13). Most homozygous SREBP-1 knockout mice die in utero. The surviving Srebp1^–/– mice show reduced synthesis of fatty acids, owing to reduced expression of mRNAs for fatty acid synthetic enzymes (Table 1). Hepatic nSREBP-2 levels increase in these mice, presumably in compensation for the loss of nSREBP-1. As a result, transcription of cholesterol biosynthetic genes increases, producing a threefold increase in hepatic cholesterol synthesis (Table 1).

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The studies in genetically manipulated mice clearly show that, as in cultured cells, SCAP and S1P are required for normal SREBP processing in the liver. SCAP, acting through its sterol-sensing domain, mediates feedback regulation of cholesterol synthesis. The SREBPs play related but distinct roles: SREBP-1c, the predominant SREBP-1 isoform in adult liver, preferentially activates genes required for fatty acid synthesis, while SREBP-2 preferentially activates the LDL receptor gene and various genes required for cholesterol synthesis. SREBP-1a and SREBP-2, but not SREBP-1c, are required for normal embryogenesis.

Transcriptional regulation of SREBP genes

Regulation of SREBPs occurs at two levels — transcriptional and posttranscriptional. The posttranscriptional regulation discussed above involves the sterol-mediated suppression of SREBP cleavage, which results from sterol-mediated suppression of the movement of the SCAP/SREBP complex from the ER to the Golgi apparatus (Figure 1). This form of regulation is manifest not only in cultured cells (1), but also in the livers of rodents fed cholesterol-enriched diets (19).

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The transcriptional regulation of the SREBPs is more complex. SREBP-1c and SREBP-2 are subject to distinct forms of transcriptional regulation, whereas SREBP-1a appears to be constitutively expressed at low levels in liver and most other tissues of adult animals (6). One mechanism of regulation shared by SREBP-1c and SREBP-2 involves a feed-forward regulation mediated by SREs present in the enhancer/promoters of each gene (20, 21). Through this feed-forward loop, nSREBPs activate the transcription of their own genes. In contrast, when nSREBPs decline, as in Scap or S1p knockout mice, there is a secondary decline in the mRNAs encoding SREBP-1c and SREBP-2 (14, 15).

Three factors selectively regulate the transcription of SREBP-1c: liver X-activated receptors (LXRs), insulin, and glucagon. LXRα and LXRβ, nuclear receptors that form heterodimers with retinoid X receptors, are activated by a variety of sterols, including oxysterol intermediates that form during cholesterol biosynthesis (22–24). An LXR-binding site in the SREBP-1c promoter activates SREBP-1c transcription in the presence of LXR agonists (23). The functional significance of LXR-mediated SREBP-1c regulation has been confirmed in two animal models. Mice that lack both LXRα and LXRβ express reduced levels of SREBP-1c and its lipogenic target enzymes in liver and respond relatively weakly to treatment with a synthetic LXR agonist (23). Because a similar blunted response is found in mice that lack SREBP-1c, it appears that LXR increases fatty acid synthesis largely by inducing SREBP-1c (16). LXR-mediated activation of SREBP-1c transcription provides a mechanism for the cell to induce the synthesis of oleate when sterols are in excess (23). Oleate is the preferred fatty acid for the synthesis of cholesteryl esters, which are necessary for both the transport and the storage of cholesterol.

LXR-mediated regulation of SREBP-1c appears also to be one mechanism by which unsaturated fatty acids suppress SREBP-1c transcription and thus fatty acid synthesis. Rodents fed diets enriched in polyunsaturated fatty acids manifest reduced SREBP-1c mRNA expression and low rates of lipogenesis in liver (25). In vitro, unsaturated fatty acids competitively block LXR activation of SREBP-1c expression by antagonizing the activation of LXR by its endogenous ligands (26). In addition to LXR-mediated transcriptional inhibition, polyunsaturated fatty acids lower SREBP-1c levels by accelerating degradation of its mRNA (27). These combined effects may contribute to the long-recognized ability of polyunsaturated fatty acids to lower plasma triglyceride levels.

SREBP-1c and the insulin/glucagon ratio

The liver is the organ responsible for the conversion of excess carbohydrates to fatty acids to be stored as triglycerides or burned in muscle. A classic action of insulin is to stimulate fatty acid synthesis in liver during times of carbohydrate excess. The action of insulin is opposed by glucagon, which acts by raising cAMP. Multiple lines of evidence suggest that insulin’s stimulatory effect on fatty acid synthesis is mediated by an increase in SREBP-1c. In isolated rat hepatocytes, insulin treatment increases the amount of mRNA for SREBP-1c in parallel with the mRNAs of its target genes (28, 29). The induction of the target genes can be blocked if a dominant negative form of SREBP-1c is expressed (30). Conversely, incubating primary hepatocytes with glucagon or dibutyryl cAMP decreases the mRNAs for SREBP-1c and its associated lipogenic target genes (30, 31).

In vivo, the total amount of SREBP-1c in liver and adipose tissue is reduced by fasting, which suppresses insulin and increases glucagon levels, and is elevated by refeeding (32, 33). The levels of mRNA for SREBP-1c target genes parallel the changes in SREBP-1c expression. Similarly, SREBP-1c mRNA levels fall when rats are treated with streptozotocin, which abolishes insulin secretion, and rise after insulin injection (29). Overexpression of nSREBP-1c in livers of transgenic mice prevents the reduction in lipogenic mRNAs that normally follows a fall in plasma insulin levels (32). Conversely, in livers of Scap knockout mice that lack all nSREBPs in the liver (14) or knockout mice lacking either nSREBP-1c (16) or both SREBP-1 isoforms (34), there is a marked decrease in the insulin-induced stimulation of lipogenic gene expression that normally occurs after fasting/refeeding. It should be noted that insulin and glucagon also exert a posttranslational control of fatty acid synthesis though changes in the phosphorylation and activation of acetyl-CoA carboxylase. The posttranslational regulation of fatty acid synthesis persists in transgenic mice that overexpress nSREBP-1c (10). In these mice, the rates of fatty acid synthesis, as measured by [³H]water incorporation, decline after fasting even though the levels of the lipogenic mRNAs remain high (our unpublished observations).

Taken together, the above evidence suggests that SREBP-1c mediates insulin’s lipogenic actions in liver. Recent in vitro and in vivo studies involving adenoviral gene transfer suggest that SREBP-1c may also contribute to the regulation of glucose uptake and glucose synthesis. When overexpressed in hepatocytes, nSREBP-1c induces expression of glucokinase, a key enzyme in glucose utilization. It also suppresses phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme (35, 36).

SREBPs in disease

Many individuals with obesity and insulin resistance also have fatty livers, one of the most commonly encountered liver abnormalities in the US (37). A subset of individuals with fatty liver go on to develop fibrosis, cirrhosis, and liver failure. Evidence indicates that the fatty liver of insulin resistance is caused by SREBP-1c, which is elevated in response to the high insulin levels. Thus, SREBP-1c levels are elevated in the fatty livers of obese (ob/ob) mice with insulin resistance and hyperinsulinemia caused by leptin deficiency (38, 39). Despite the presence of insulin resistance in peripheral tissues, insulin continues to activate SREBP-1c transcription and cleavage in the livers of these insulin-resistant mice. The elevated nSREBP-1c increases lipogenic gene expression, enhances fatty acid synthesis, and accelerates triglyceride accumulation (31, 39). These metabolic abnormalities are reversed with the administration of leptin, which corrects the insulin resistance and lowers the insulin levels (38).

Metformin, a biguanide drug used to treat insulin-resistant diabetes, reduces hepatic nSREBP-1 levels and dramatically lowers the lipid accumulation in livers of insulin-resistant ob/ob mice (40). Metformin stimulates AMP-activated protein kinase (AMPK), an enzyme that inhibits lipid synthesis through phosphorylation and inactivation of key lipogenic enzymes (41). In rat hepatocytes, metformin-induced activation of AMPK also leads to decreased mRNA expression of SREBP-1c and its lipogenic target genes (41), but the basis of this effect is not understood.

The incidence of coronary artery disease increases with increasing plasma LDL-cholesterol levels, which in turn are inversely proportional to the levels of hepatic LDL receptors. SREBPs stimulate LDL receptor expression, but they also enhance lipid synthesis (1), so their net effect on plasma lipoprotein levels depends on a balance between opposing effects. In mice, the plasma levels of lipoproteins tend to fall when SREBPs are either overexpressed or underexpressed. In transgenic mice that overexpress nSREBPs in liver, plasma cholesterol and triglycerides are generally lower than in control mice (Table 1), even though these mice massively overproduce fatty acids, cholesterol, or both. Hepatocytes of nSREBP-1a transgenic mice overproduce VLDL, but these particles are rapidly removed through the action of LDL receptors, and they do not accumulate in the plasma. Indeed, some nascent VLDL particles are degraded even before secretion by a process that is mediated by LDL receptors (42). The high levels of nSREBP-1a in these animals support continued expression of the LDL receptor, even in cells whose cholesterol concentration is elevated. In LDL receptor–deficient mice carrying the nSREBP-1a transgene, plasma cholesterol and triglyceride levels rise tenfold (43).

Mice that lack all SREBPs in liver as a result of disruption of Scap or S1p also manifest lower plasma cholesterol and triglyceride levels (Table 1).

http://dm5migu4zj3pb.cloudfront.net/manuscripts/15000/15593/small/JCI0215593.t1.gif

In these mice, hepatic cholesterol and triglyceride synthesis is markedly reduced, and this likely causes a decrease in VLDL production and secretion. LDL receptor mRNA and LDL clearance from plasma is also significantly reduced in these mice, but the reduction in LDL clearance is less than the overall reduction in VLDL secretion, the net result being a decrease in plasma lipid levels (15). However, because

humans and mice differ substantially with regard to LDL receptor expression, LDL levels, and other aspects of lipoprotein metabolism,

it is difficult to predict whether human plasma lipids will rise or fall when the SREBP pathway is blocked or activated.

SREBPs in liver: unanswered questions

The studies of SREBPs in liver have exposed a complex regulatory system whose individual parts are coming into focus. Major unanswered questions relate to the ways in which the transcriptional and posttranscriptional controls on SREBP activity are integrated so as to permit independent regulation of cholesterol and fatty acid synthesis in specific nutritional states. A few clues regarding these integration mechanisms are discussed below.

Whereas cholesterol synthesis depends almost entirely on SREBPs, fatty acid synthesis is only partially dependent on these proteins. This has been shown most clearly in cultured nonhepatic cells such as Chinese hamster ovary cells. In the absence of SREBP processing, as when the Site-2 protease is defective, the levels of mRNAs encoding cholesterol biosynthetic enzymes and the rates of cholesterol synthesis decline nearly to undetectable levels, whereas the rate of fatty acid synthesis is reduced by only 30% (44). Under these conditions, transcription of the fatty acid biosynthetic genes must be maintained by factors other than SREBPs. In liver, the gene encoding fatty acid synthase (FASN) can be activated transcriptionally by upstream stimulatory factor, which acts in concert with SREBPs (45). The FASN promoter also contains an LXR element that permits a low-level response to LXR ligands even when SREBPs are suppressed (46). These two transcription factors may help to maintain fatty acid synthesis in liver when nSREBP-1c is low.

Another mechanism of differential regulation is seen in the ability of cholesterol to block the processing of SREBP-2, but not SREBP-1, under certain metabolic conditions. This differential regulation has been studied most thoroughly in cultured cells such as human embryonic kidney (HEK-293) cells. When these cells are incubated in the absence of fatty acids and cholesterol, the addition of sterols blocks processing of SREBP-2, but not SREBP-1, which is largely produced as SREBP-1a in these cells (47). Inhibition of SREBP-1 processing requires an unsaturated fatty acid, such as oleate or arachidonate, in addition to sterols (47). In the absence of fatty acids and in the presence of sterols, SCAP may be able to carry SREBP-1 proteins, but not SREBP-2, to the Golgi apparatus. Further studies are necessary to document this apparent independent regulation of SREBP-1 and SREBP-2 processing and to determine its mechanism.

Acknowledgments

Support for the research cited from the authors’ laboratories was provided by grants from the NIH (HL-20948), the Moss Heart Foundation, the Keck Foundation, and the Perot Family Foundation. J.D. Horton is a Pew Scholar in the Biomedical Sciences and is the recipient of an Established Investigator Grant from the American Heart Association and a Research Scholar Award from the American Digestive Health Industry.

References

Brown, MS, Goldstein, JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997. 89:331-340.

View this article via: PubMed

Horton, JD, Shimomura, I. Sterol regulatory element-binding proteins: activators of cholesterol and fatty acid biosynthesis. Curr Opin Lipidol 1999. 10:143-150.

View this article via: PubMed

Edwards, PA, Tabor, D, Kast, HR, Venkateswaran, A. Regulation of gene expression by SREBP and SCAP. Biochim Biophys Acta 2000. 1529:103-113.

View this article via: PubMed

Sakakura, Y, et al. Sterol regulatory element-binding proteins induce an entire pathway of cholesterol synthesis. Biochem Biophys Res Commun 2001. 286:176-183.

View this article via: PubMed

Goldstein, JL, Rawson, RB, Brown, MS. Mutant mammalian cells as tools to delineate the sterol regulatory element-binding protein pathway for feedback regulation of lipid synthesis. Arch Biochem Biophys 2002. 397:139-148.

View this article via: PubMed

Shimomura, I, Shimano, H, Horton, JD, Goldstein, JL, Brown, MS. Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells. J Clin Invest 1997. 99:838-845.

View this article via: JCI.org PubMed

Moon, Y-A, Shah, NA, Mohapatra, S, Warrington, JA, Horton, JD. Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins. J Biol Chem 2001. 276:45358-45366.

View this article via: PubMed

Shimomura, I, Shimano, H, Korn, BS, Bashmakov, Y, Horton, JD. Nuclear sterol regulatory element binding proteins activate genes responsible for entire program of unsaturated fatty acid biosynthesis in transgenic mouse liver. J Biol Chem 1998. 273:35299-35306.

View this article via: PubMed

Shimano, H, et al. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J Clin Invest 1996. 98:1575-1584.

View this article via: JCI.org PubMed

Shimano, H, et al. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J Clin Invest 1997. 99:846-854.

View this article via: JCI.org PubMed

Horton, JD, et al. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2. J Clin Invest 1998. 101:2331-2339.

View this article via: JCI.org PubMed

Korn, BS, et al. Blunted feedback suppression of SREBP processing by dietary cholesterol in transgenic mice expressing sterol-resistant SCAP(D443N). J Clin Invest 1998. 102:2050-2060.

View this article via: JCI.org PubMed

Shimano, H, et al. Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene. J Clin Invest 1997. 100:2115-2124.

View this article via: JCI.org PubMed

Matsuda, M, et al. SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation. Genes Dev 2001. 15:1206-1216.

View this article via: PubMed

Yang, J, et al. Decreased lipid synthesis in livers of mice with disrupted Site-1 protease gene. Proc Natl Acad Sci USA 2001. 98:13607-13612.

View this article via: PubMed

Liang, G, et al. Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. J Biol Chem 2002. 277:9520-9528.

http://www.jci.org/articles/view/15593

Structural Biochemistry/Lipids/Membrane Lipids

< Structural Biochemistry‎ | Lipids

Membrane proteins rely on their interaction with membrane lipids to uphold its structure and maintain its functions as a protein. For membrane proteins to purify and crystallize, it is essential for the membrane protein to be in the appropriate lipid environment. Lipids assist in crystallization and stabilize the protein and provide lattice contacts. Lipids can also help obtain membrane protein structures in a native conformation. Membrane protein structures contain bound lipid molecules. Biological membranes are important in life, providing permeable barriers for cells and their organelles. The interaction between membrane proteins and lipids facilitates basic processes such as respiration, photosynthesis, transport, signal transduction and motility. These basic processes require a diverse group of proteins, which are encoded by 20-30% of an organism’s annotated genes.

There exist a great number of membrane lipids. Specifically, eukaryotic cells have a very complex collection of lipids that rely on many of the cell’s resources for its synthesis. Interactions between proteins and lipids can be very specific. Specific types of lipids can make a structure stable, provide control in insertion and folding processes, and help to assemble multisubunit complexes or supercomplexes, and most importantly, can significantly affect a membrane protein’s functions. Protein and lipid interactions are not sufficiently tight, meaning that lipids are retained during membrane protein purification. Since cellular membranes are fluid arrangements of lipids, some lipids affect interesting changes to membrane due to their characteristics. Glycosphigolipids and cholesterol tend to form small islands within the membranes, called lipid rafts, due to their physical properties. Some proteins also tend to cluster in lipid raft, while others avoid being in lipid rafts. However, the existence of lipid rafts in cells seems to be transitory.

Recent progress in determining membrane protein structure has brought attention to the importance of maintaining a favorable lipid environment so proteins to crystallize and purify successfully. Lipids assist in crystallization by stabilizing the protein fold and the relationships between subunits or monomers. The lipid content in protein-lipid detergent complexes can be altered by adjusting solubilisation and purification protocols, also by adding native or non-native lipids.

There are three type of membrane lipids: 1. Phospholipids: major class of membrane lipids. 2. glycolipids. 3. Cholesterols. Membrane lipids were started with eukaryotes and bacteria.

http://en.wikibooks.org/wiki/Structural_Biochemistry/Lipids/Membrane_Lipids

Types of Membrane Lipids

Lipids are often used as membrane constituents. The three major classes that membrane lipids are divided into are phospholipids, glycolipids, and cholesterol. Lipids are found in eukaryotes and bacteria. Although the lipids in archaea have many features that are related to the membrane formation that is similar with lipids of other organisms, they are still distinct from one another. The membranes of archaea differ in composition in three major ways. Firstly, the nonpolar chains are joined to a glycerol backbone by ether instead of esters, allowing for more resistance to hydrolysis. Second, the alkyl chains are not linear, but branched and make them more resistant to oxidation. The ability of archaeal lipids to resist hydrolysis and oxidation help these types of organisms to withstand the extreme conditions of high temperature, low pH, or high salt concentration. Lastly, the stereochemistry of the central glycerol is inverted. Membrane lipids have an extensive repertoire, but they possess a critical common structural theme in which they are amphipathic molecules, meaning they contain both a hydrophilic and hydrophobic moiety.

Membrane lipids are all closed bodies or boundaries separating substituent parts of the cell. The thickness of membranes is usually between 60 and 100 angstroms. These bodies are constructed from non-covalent assemblies. Their polar heads align with each other and their non-polar hydrocarbon tails align as well. The resulting stability is credited to hydrophobic interaction which proves to be quite stable due to the length of their hydrocarbon tails.

Membrane Lipids

Lipid Vesicles

Lipid vesicles, also known as liposomes, are vesicles that are essentially aqueous vesicles that are surrounded by a circular phospholipid bilayer. Like the other phospholipid structures, they have the hydrocarbon/hydrophobic tails facing inward, away from the aqueous solution, and the hydrophilic heads facing towards the aqueous solution. These vesicles are structures that form enclosed compartments of ions and solutes, and can be utilized to study the permeability of certain membranes, or to transfer these ions or solutes to certain cells found elsewhere.

Liposomes as vesicles can serve various clinical uses. Injecting liposomes containing medicine or DNA (for gene therapy) into patients is a possible method of drug delivery. The liposomes fuse with other cells’ membranes and therefore combine their contents with that of the patient’s cell. This method of drug delivery is less toxic than direct exposure because the liposomes carry the drug directly to cells without any unnecessary intermediate steps.

Because of the hydrophobic interactions among several phospholipids and glycolipids, a certain structure called the lipid bilayer or bimolecular sheet is favored. As mentioned earlier, phospholipids and glycolipids have both hydrophilic and hydrophobic moieties; thus, when several phospholipids or glycolipids come together in an aqueous solution, the hydrophobic tails interact with each other to form a hydrophobic center, while the hydrophilic heads interact with each other forming a hydrophilic coating on each side of the bilayer.

http://upload.wikimedia.org/wikibooks/en/b/ba/Liposome_final%2A.png

http://upload.wikimedia.org/wikibooks/en/f/fa/Membrane_bilayer.jpg

Liposome_

Membrane_bilayer

Evidence Report/Technology Assessment Number 89

Effects of Omega-3 Fatty Acids on Lipids and Glycemic Control in Type II Diabetes and the Metabolic Syndrome and on Inflammatory Bowel Disease, Rheumatoid Arthritis, Renal Disease, Systemic Lupus Erythematosus, and Osteoporosis

Prepared for:

Agency for Healthcare Research and Quality

U.S. Department of Health and Human Services

540 Gaither Road

Rockville, MD 20850

http://www.ahrq.gov

Contract No. 290-02-0003

Chapter 1. Introduction

This report is one of a group of evidence reports prepared by three Agency for Healthcare Research and Quality (AHRQ)-funded Evidence-Based Practice Centers (EPCs) on the role of omega-3 fatty acids (both from food sources and from dietary supplements) in the prevention or treatment of a variety of diseases. These reports were requested and funded by the Office of Dietary Supplements, National Institutes of Health. The three EPCs – the Southern California EPC (SCEPC, based at RAND), the Tufts-New England Medical Center (NEMC) EPC, and the University of Ottawa EPC – have each produced evidence reports. To ensure consistency of approach, the three EPCs collaborated on selected methodological elements, including literature search strategies, rating of evidence, and data table design.

The aim of these reports is to summarize the current evidence on the effects of omega-3 fatty acids on prevention and treatment of cardiovascular diseases, cancer, child and maternal health, eye health, gastrointestinal/renal diseases, asthma, immune- mediated diseases, tissue/organ transplantation, mental health, and neurological diseases and conditions. In addition to informing the research community and the public on the effects of omega-3 fatty acids on various health conditions, it is anticipated that the findings of the reports will also be used to help define the agenda for future research.

This report focuses on the effects of omega-3 fatty acids on immune- mediated diseases, bone metabolism, and gastrointestinal/renal diseases. Subsequent reports from the SCEPC will focus on cancer and neurological diseases and conditions.

This chapter provides a brief review of the current state of knowledge about the metabolism, physiological functions, and sources of omega-3 fatty acids.

The Recognition of Essential Fatty Acids

Dietary fat has long been recognized as an important source of energy for mammals, but in the late 1920s, researchers demonstrated the dietary requirement for particular fatty acids, which came to be called essential fatty acids. It was not until the advent of intravenous feeding, however, that the importance of essential fatty acids was widely accepted: Clinical signs of essential fatty acid deficiency are generally observed only in patients on total parenteral nutrition who received mixtures devoid of essential fatty acids or in those with malabsorption syndromes.

These signs include dermatitis and changes in visual and neural function. Over the past 40 years, an increasing number of physiological functions, such as immunomodulation, have been attributed to the essential fatty acids and their metabolites, and this area of research remains quite active.1, 2

Fatty Acid Nomenclature

The fat found in foods consists largely of a heterogeneous mixture of triacylglycerols (triglycerides)–glycerol molecules that are each combined with three fatty acids. The fatty acids can be divided into two categories, based on chemical properties: saturated fatty acids, which are usually solid at room temperature, and unsaturated fatty acids, which are liquid at room temperature. The term “saturation” refers to a chemical structure in which each carbon atom in the fatty acyl chain is bound to (saturated with) four other atoms, these carbons are linked by single bonds, and no other atoms or molecules can attach; unsaturated fatty acids contain at least one pair of carbon atoms linked by a double bond, which allows the attachment of additional atoms to those carbons (resulting in saturation). Despite their differences in structure, all fats contain approximately the same amount of energy (37 kilojoules/gram, or 9 kilocalories/gram).

The class of unsaturated fatty acids can be further divided into monounsaturated and polyunsaturated fatty acids. Monounsaturated fatty acids (the primary constituents of olive and canola oils) contain only one double bond. Polyunsaturated fatty acids (PUFAs) (the primary constituents of corn, sunflower, flax seed and many other vegetable oils) contain more than one double bond. Fatty acids are often referred to using the number of carbon atoms in the acyl chain, followed by a colon, followed by the number of double bonds in the chain (e.g., 18:1 refers to the 18-carbon monounsaturated fatty acid, oleic acid; 18:3 refers to any 18-carbon PUFA with three double bonds).

PUFAs are further categorized on the basis of the location of their double bonds. An omega or n notation indicates the number of carbon atoms from the methyl end of the acyl chain to the first double bond. Thus, for example, in the omega-3 (n-3) family of PUFAs, the first double bond is 3 carbons from the methyl end of the molecule. The trivial names, chemical names and abbreviations for the omega-3 fatty acids are detailed in Table 1.1. Finally, PUFAs can be categorized according to their chain length. The 18-carbon n-3 and n-6 short-chain PUFAs are precursors to the longer 20- and 22-carbon PUFAs, called long-chain PUFAs (LCPUFAs).

Fatty Acid Metabolism

Mammalian cells can introduce double bonds into all positions on the fatty acid chain except the n-3 and n-6 position. Thus, the short-chain alpha- linolenic acid (ALA, chemical abbreviation: 18:3n-3) and linoleic acid (LA, chemical abbreviation: 18:2n-6) are essential fatty acids.

No other fatty acids found in food are considered ‘essential’ for humans, because they can all be synthesized from the short chain fatty acids.

Following ingestion, ALA and LA can be converted in the liver to the long chain, more unsaturated n-3 and n-6 LCPUFAs by a complex set of synthetic pathways that share several enzymes (Figure 1). LC PUFAs retain the original sites of desaturation (including n-3 or n-6). The omega-6 fatty acid LA is converted to gamma-linolenic acid (GLA, 18:3n-6), an omega- 6 fatty acid that is a positional isomer of ALA. GLA, in turn, can be converted to the longerchain omega-6 fatty acid, arachidonic acid (AA, 20:4n-6). AA is the precursor for certain classes of an important family of hormone- like substances called the eicosanoids (see below).

The omega-3 fatty acid ALA (18:3n-3) can be converted to the long-chain omega-3 fatty acid, eicosapentaenoic acid (EPA; 20:5n-3). EPA can be elongated to docosapentaenoic acid (DPA 22:5n-3), which is further desaturated to docosahexaenoic acid (DHA; 22:6n-3). EPA and DHA are also precursors of several classes of eicosanoids and are known to play several other critical roles, some of which are discussed further below.

The conversion from parent fatty acids into the LC PUFAs – EPA, DHA, and AA – appears to occur slowly in humans. In addition, the regulation of conversion is not well understood, although it is known that ALA and LA compete for entry into the metabolic pathways.

Physiological Functions of EPA and AA

As stated earlier, fatty acids play a variety of physiological roles. The specific biological functions of a fatty acid are determined by the number and position of double bonds and the length of the acyl chain.

Both EPA (20:5n-3) and AA (20:4n-6) are precursors for the formation of a family of hormone- like agents called eicosanoids. Eicosanoids are rudimentary hormones or regulating – molecules that appear to occur in most forms of life. However, unlike endocrine hormones, which travel in the blood stream to exert their effects at distant sites, the eicosanoids are autocrine or paracrine factors, which exert their effects locally – in the cells that synthesize them or adjacent cells. Processes affected include the movement of calcium and other substances into and out of cells, relaxation and contraction of muscles, inhibition and promotion of clotting, regulation of secretions including digestive juices and hormones, and control of fertility, cell division, and growth.3

The eicosanoid family includes subgroups of substances known as prostaglandins, leukotrienes, and thromboxanes, among others. As shown in Figure 1.1, the long-chain omega-6 fatty acid, AA (20:4n-6), is the precursor of a group of eicosanoids that include series-2 prostaglandins and series-4 leukotrienes. The omega-3 fatty acid, EPA (20:5n-3), is the precursor to a group of eicosanoids that includes series-3 prostaglandins and series-5 leukotrienes. The AA-derived series-2 prostaglandins and series-4 leukotrienes are often synthesized in response to some emergency such as injury or stress, whereas the EPA-derived series-3 prostaglandins and series-5 leukotrienes appear to modulate the effects of the series-2 prostaglandins and series-4 leukotrienes (usually on the same target cells). More specifically, the series-3 prostaglandins are formed at a slower rate and work to attenuate the effects of excessive levels of series-2 prostaglandins. Thus, adequate production of the series-3 prostaglandins seems to protect against heart attack and stroke as well as certain inflammatory diseases like arthritis, lupus, and asthma.3.

EPA (22:6 n-3) also affects lipoprotein metabolism and decreases the production of substances – including cytokines, interleukin 1ß (IL-1ß), and tumor necrosis factor a (TNF-a) – that have pro-inflammatory effects (such as stimulation of collagenase synthesis and the expression of adhesion molecules necessary for leukocyte extravasation [movement from the circulatory system into tissues]).2 The mechanism responsible for the suppression of cytokine production by omega-3 LC PUFAs remains unknown, although suppression of omega-6-derived eicosanoid production by omega-3 fatty acids may be involved, because the omega-3 and omega-6 fatty acids compete for a common enzyme in the eicosanoid synthetic pathway, delta-6 desaturase.

DPA (22:5n-3) (the elongation product of EPA) and its metabolite DHA (22:6n-3) are frequently referred to as very long chain n-3 fatty acids (VLCFA). Along with AA, DHA is the major PUFA found in the brain and is thought to be important for brain development and function. Recent research has focused on this role and the effect of supplementing infant formula with DHA (since DHA is naturally present in breast milk but not in formula).

Dietary Sources and Requirements

Both ALA and LA are present in a variety of foods. LA is present in high concentrations in many commonly used oils, including safflower, sunflower, soy, and corn oil. ALA is present in some commonly used oils, including canola and soybean oil, and in some leafy green vegetables. Thus, the major dietary sources of ALA and LA are PUFA-rich vegetable oils. The proportion of LA to ALA as well as the proportion of those PUFAs to others varies considerably by the type of oil. With the exception of flaxseed, canola, and soybean oil, the ratio of LA to ALA in vegetable oils is at least 10 to 1. The ratios of LA to ALA for flaxseed, canola, and soy are approximately 1: 3.5, 2:1, and 8:1, respectively; however, flaxseed oil is not typically consumed in the North American diet. It is estimated that on average in the U.S., LA accounts for 89% of the total PUFAs consumed, and ALA accounts for 9%. Another estimate suggests that Americans consume 10 times more omega-6 than omega-3 fatty acids.4 Table 1.2 shows the proportion of omega 3 fatty acids for a number of foods.

Syntheis and Degradation

Source of Acetyl CoA for Fatty Acid Synthesis

step 1

ACP (acyl carrier protein)

synthesis requires acetyl CoA from citrate shuttle

conversion to fatty acyl co A in cytoplasm

ACP (acyl carrier protein)

FA synthesis not exactly reverse of catabolism

Fatty Acid Synthase

complete FA synthesis

Desaturation

Elongation and Desaturation of Fatty Acids

release of FAs from adiposites

Fatty acid beta oxidation and Krebs cycle produce NAD, NADH, FADH2

ketone bodies

metabolism of ketone bodies

Arachidonoyl-mimicking

Arachidonate pathways

arachidonic acid derivatives

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides

Model for the sterol-mediated proteolytic release of SREBPs from membrane

hormone regulation

insulin receptor and and insulin receptor signaling pathway (IRS)

islet brain glucose signaling

Fish source

omega FAs

Excessive omega 6s

omega 6s

diet and cancer

Patients at risk of FA deficiency

PPAR role

Omega 6_3 pathways

n3 vs n6 PUFAs

triene-teraene ratio

arachidonic acid, leukotrienes, PG and thromboxanes

Cox 2 and cancer

Lipidomics of atherosclerotic plaques

Effect of TPN on EFAD

benefits of omega 3s

food consumption

Read Full Post »

Signaling transduction tutorial

Posted in Anaerobic Glycolysis, Biological Networks, Gene Regulation and Evolution, Ca2+ triggered activation, Ca2+ triggered activation, Calcium Signaling, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Computational Biology/Systems and Bioinformatics, Curation methodology, Cytoskeleton, Developmental biology, Diabetes Mellitus, Discovery process, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Electrophysiology, Endocrine Diseases, Enzyme Induction, Explanatory, Gene Regulation, Genomic Expression, Human Immune System in Health and in Disease, International Global Work in Pharmaceutical, Ionic Transporters Na+, Metabolomics, Mg++, Molecular Genetics & Pharmaceutical, Na-K transport, Na-K-ATPase, Neurohumoral Transmission, Open Access Journals, Proteomics, Synaptic vesicle, Technology Advance Assessment of, Warburg effect, tagged cell signaling, Signal transduction, signal transduction pathways, signaling cascades on August 12, 2014| Leave a Comment »

Signaling transduction tutorial

Larry H. Bernstein, MD, FCAP, Reporter and Curator
Leaders in Pharmaceutical Intelligence

http://pharmaceuticalintelligence.com/8-10-2014/Signaling transduction tutorial

This portion of the discussion is a series of articles on signaling and signaling pathways. Many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different. I considered placing this after the discussion of proteins and how they play out their essential role, but this is quite a suitable place for a progression to what follows. This is introduced by material taken from Wikipedia, which will be followed by a series of mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, with the later goal of separating views introduced by molecular biology and genomics from functional cellular dynamics that are not dependent on the classic view. The work is vast, and this discussion does not attempt to cover it in great depth. It is the first in a series. This discussion, in particular is a tutorial on signaling transduction that was already available, and relevant. One may note that all of the slides used herein were also used in the previous blog, but in a different construction. I shall tweak the contents, as I find helpful.

Signaling and signaling pathways
Signaling transduction tutorial.
Carbohydrate metabolism
Lipid metabolism
Protein synthesis and degradation
Subcellular structure
Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Signal Transduction Tutorial

The goal of this tutorial is for you to gain an understanding of how cell signaling occurs in a cell. Upon completion of the tutorial,

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

You will also have detailed knowledge of

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

Description of Signal Transduction

As living organisms

we are constantly receiving and interpreting signals from our environment.

These signals can come

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

The cells of our bodies are also

constantly receiving signals from other cells.

These signals are important to

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

Signals are most often chemicals that can be found

in the extracellular fluid around cells.

These chemicals can come

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

intercellular signaling

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

Signaling molecules may trigger any number of cellular responses, including

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

Overview of Cell Signaling

Cell signaling can be divided into 3 stages.

Reception: A cell detects a signaling molecule from the outside of the cell. A signal is detected when the chemical signal (also known as a ligand) binds to a receptor protein on the surface of the cell or inside the cell.
Transduction: When the signaling molecule binds the receptor it changes the receptor protein in some way. This change initiates the process of transduction. Signal transduction is usually a pathway of several steps. Each relay molecule in the signal transduction pathway changes the next molecule in the pathway.
Response: Finally, the signal triggers a specific cellular response.

signal transduction_simple

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

Reception

Signal Transduction – ligand binds to surface receptor

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

by changing shape or

conformational-rearrangements

Enzyme_Model allosterism

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

Examples of membrane receptors include

G Protein-Coupled Receptors and

membrane_receptor_g protein

Receptor Tyrosine Kinases.

activation of receptor Tyrosine Kinase

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

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

Chemical messengers that are hydrophobic or very small (steroid hormones for example) can

pass through the plasma membrane without assistance and
bind these intracellular receptors.

Once bound and activated by the signal molecule,

the activated receptor can initiate a cellular response, such as a
change in gene expression.

Note that this is the first time that change in gene expression is stated. Is the change in gene expression implication of a change in the genetic information – such as – mutation? That does not have to be the case in the normal homeostatic case. This might only be

a change in the rate of a transcription or a suppression of expression through RNA.

intracellular_receptor_steroid

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

Transduction

Since signaling systems need to be

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

Signal transduction cascades amplify the signal output

Steps in the signal transduction pathway often involve

the addition or removal of phosphate groups which results in the activation of proteins.
Enzymes that transfer phosphate groups from ATP to a protein are called protein kinases.

Many of the relay molecules in a signal transduction pathway are protein kinases and

often act on other protein kinases in the pathway. Often
this creates a phosphorylation cascade, where
one enzyme phosphorylates another, which then phosphorylates another protein, causing a chain reaction.

phosphorylation-cascade

Also important to the phosphorylation cascade are

a group of proteins known as protein phosphatases.

Protein phosphatases are enzymes that can rapidly remove phosphate groups from proteins (dephosphorylation) and thus inactivate protein kinases. Protein phosphatases are

the “off switch” in the signal transduction pathway.

Turning the signal transduction pathway off when the signal is no longer present is important

to ensure that the cellular response is regulated appropriately.

Dephosphorylation also makes protein kinases

available for reuse and
enables the cell to respond again when another signal is received.

Kinases are not the only tools used by cells in signal transduction. Small, nonprotein, water-soluble molecules or ions called second messengers (the ligand that binds the receptor is the first messenger) can also

relay signals received by receptors on the cell surface
to target molecules in the cytoplasm or the nucleus.

membrane protein receptor binds with hormone

insulin receptor and and insulin receptor signaling pathway (IRS)

binding-proteins-and-bioavailable-25-hydroxyvitamin-d

Examples of second messengers include cyclic AMP (cAMP) and calcium ions.

membrane_receptor_g protein

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

Response

Cell signaling ultimately leads to the regulation of one or more cellular activities. Regulation of gene expression (turning transcription of specific genes on or off) is a common outcome of cell signaling. A signaling pathway may also

regulate the activity of a protein, for example

ion-transporters-and-channels-in-mammalian-choroidal-epithelium

Ca(2+) and contraction

transepithelial-electrogenic-ion-transport

calcium release flux

opening or closing an ion channel in the plasma membrane or
promoting a change in cell metabolism such as catalyzing the breakdown of glycogen.

Signaling pathways can also lead to important cellular events such as

cell division or apoptosis (programmed cell death).

ubiquitylation-is-a-multistep-reaction.

Involvement of VSMCs apoptosis in fibrous plaque rupture.

G- Protein-Coupled Receptor

membrane_receptor_g protein

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

Signal Transduction Tutorial by Dr. Katherine Harris is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

Funded by the U.S. Department of Education, College Cost Reduction and Access (CCRAA) grant award # P031C080096.

http://creativecommons.org/licenses/by-nc-sa/3.0/

NonCommercial — You may not use the material for commercial purposes.
ShareAlike — If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original.

Adapt — remix, transform, and build upon the material

hormone + receptor signaling

http://home.earthlink.net/~dayvdanls/SignalTrans.gif

http://pi-silico.hkbu.edu.hk/wp-content/uploads/2012/12/Signal-Transduction-Pathway.png

http://upload.wikimedia.org/wikipedia/commons/a/a4/1Signal_Transduction_Pathways_Model.jpg

Akt mTOR pathway

http://cc.scu.edu.cn/G2S/eWebEditor/uploadfile/20120810155043970.jpg

Quia – 9AP Chapter 11 – Cell Commun

http://www.quia.com/files/quia/users/lmcgee/membranetransport/cell_communication/reception_transduction_resp.gif

http://cc.scu.edu.cn/G2S/eWebEditor/uploadfile/20120810155043970.jpg

HER2 in Breast Cancer–What Does it Mean?

http://img.medscape.com/fullsize/migrated/editorial/clinupdates/2000/681/tu02.fig2.jpg

Protease signalling: the cutting edge

http://emboj.embopress.org/content/embojnl/31/7/1630/F5.large.jpg

Quia – 9AP Chapter 11 – Cell Commun

http://www.quia.com/files/quia/users/lmcgee/membranetransport/cell_communication/phosphorylation-cascade.gif

Signal Transduction in Autism

http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-14/1403.jpg

The multiple protein-dependent steps in signal transduction

http://www.nature.com/nrm/journal/v1/n2/images/nrm1100_145a_i2.gif

CONVERSING AT THE CELLULAR LEVEL: AN INTRODUCTION TO SIGNAL …

scq.ubc.ca

http://www.scq.ubc.ca/wp-content/uploads/2006/07/transduction.gif

Biology 1710 > Davis > Flashcards > exam 1 | StudyBlue

studyblue.com

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Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1

Posted in Academic Publishing, Acute Myocardial Infarction, Advanced Drug Manufacturing Technology, Alzheimer's Disease, Atherogenic Processes & Pathology, Autologous Cell Therapy, Automated Cell Processing, Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Biomedical Measurement Science, Bone Disease and Musculoskeletal Disease, Bone marrow derived cells, Ca2+ triggered activation, Ca2+ triggered activation, Cachexia, Calcium Signaling, Calmodulin, Calmodulin Kinase and Contraction, Cardiac & Vascular Repair Tools Subsegment, Cardiac and Cardiovascular Surgical Procedures, Cardiac muscle regeneration, Cardiomyopathy, Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Pulmonary Arterial Hypertension (PAH), Circulating Progenitor Cells, Coagulation Therapy and Internal Bleeding, Competition in regenerative stem cell science, Computational Biology/Systems and Bioinformatics, Curation, Cytoskeleton, Diabetes Mellitus, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Delivery Platform Technology, Drug Toxicity, Electrophysiology, Endothelial cells, Epigenetics and Cardiovascular Risks, Etiology, Evolutionary cognition, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Genomic Expression, Health Economics and Outcomes Research, Human Immune System in Health and in Disease, International Global Work in Pharmaceutical, Interviews with Scientific Leaders, Medical and Population Genetics, Medical Device Therapies for Altzheimer’s Disease, Medical Devices R&D Investment, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Metabolomics, Molecular Genetics & Pharmaceutical, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Na-K transport, Na-K-ATPase, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Nutrition, Nutritional Supplements: Atherogenesis, lipid metabolism, Origins of Cardiovascular Disease, Oxidative phosphorylation, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmacologic toxicities, Pharmacotherapy of Cardiovascular Disease, Phosphorylation, PKC, Population Health Management, Genetics & Pharmaceutical, Population Health Management, Nutrition and Phytochemistry, Proteomics, Regenerative Biology and Medicine, S-nitrosylation, Signaling, Skeletal muscle regeneration, Statistical Methods for Research Evaluation, Stem Cells for Regenerative Medicine, Stents & Tools, Synaptic vesicle, Systemic Inflammatory Response Related Disorders, Technology Advance Assessment of, Translational Effectiveness, Translational Science, Ubiquitinylation, Water Transporters, tagged bioengineering, Cardiovascular Disorders, computational biology, Coronary artery disease, DNA Sequencing, functional proteomics, gene expression, gene silencing, genetic engineering, Heart Failure, Human Genome Project, Hypertension, MicroRNA, mtRNA, myocardial infarction, nitric oxide, Nitric oxide synthase, Personalized medicine, protein modifications, Proteomics, reverse engineering, RNA, signaling pathways, Stem cell, synbiology on April 28, 2014| Leave a Comment »

Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1

Author and Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

Article ID #135: Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1. Published on 4/28/2014

WordCloud Image Produced by Adam Tubman

Part 1 of Volume 4 in the e-series A: Cardiovascular Diseases and Translational Medicine, provides a foundation for grasping a rapidly developing surging scientific endeavor that is transcending laboratory hypothesis testing and providing guidelines to:

Target genomes and multiple nucleotide sequences involved in either coding or in regulation that might have an impact on complex diseases, not necessarily genetic in nature.
Target signaling pathways that are demonstrably maladjusted, activated or suppressed in many common and complex diseases, or in their progression.
Enable a reduction in failure due to toxicities in the later stages of clinical drug trials as a result of this science-based understanding.
Enable a reduction in complications from the improvement of machanical devices that have already had an impact on the practice of interventional procedures in cardiology, cardiac surgery, and radiological imaging, as well as improving laboratory diagnostics at the molecular level.
Enable the discovery of new drugs in the continuing emergence of drug resistance.
Enable the construction of critical pathways and better guidelines for patient management based on population outcomes data, that will be critically dependent on computational methods and large data-bases.

What has been presented can be essentially viewed in the following Table:

Summary Table for TM – Part 1

There are some developments that deserve additional development:

1. The importance of mitochondrial function in the activity state of the mitochondria in cellular work (combustion) is understood, and impairments of function are identified in diseases of muscle, cardiac contraction, nerve conduction, ion transport, water balance, and the cytoskeleton – beyond the disordered metabolism in cancer. A more detailed explanation of the energetics that was elucidated based on the electron transport chain might also be in order.

2. The processes that are enabling a more full application of technology to a host of problems in the environment we live in and in disease modification is growing rapidly, and will change the face of medicine and its allied health sciences.

Electron Transport and Bioenergetics

Deferred for metabolomics topic

Synthetic Biology

Introduction to Synthetic Biology and Metabolic Engineering

Kristala L. J. Prather: Part-1 <iBiology > iBioSeminars > Biophysics & Chemical Biology >

http://www.ibiology.org Lecturers generously donate their time to prepare these lectures. The project is funded by NSF and NIGMS, and is supported by the ASCB and HHMI.
Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”.

Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching. Learn more about how Kris became a scientist at

http://science360.gov/obj/video/753bb4fa-aafb-4315-848e-51066ed9799a/finding-way-kristala-l-jones-prather-phd

Prather 1: Synthetic Biology and Metabolic Engineering 2/6/14IntroductionLecture Overview In the first part of her lecture, Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”. The key material in building these machines is synthetic DNA. Synthetic DNA can be added in different combinations to biological hosts, such as bacteria, turning them into chemical factories that can produce small molecules of choice. In Part 2, Prather describes how her lab used design principles to engineer E. coli that produce glucaric acid from glucose. Glucaric acid is not naturally produced in bacteria, so Prather and her colleagues “bioprospected” enzymes from other organisms and expressed them in E. coli to build the needed enzymatic pathway. Prather walks us through the many steps of optimizing the timing, localization and levels of enzyme expression to produce the greatest yield. Speaker Bio: Kristala Jones Prather received her S.B. degree from the Massachusetts Institute of Technology and her PhD at the University of California, Berkeley both in chemical engineering. Upon graduation, Prather joined the Merck Research Labs for 4 years before returning to academia. Prather is now an Associate Professor of Chemical Engineering at MIT and an investigator with the multi-university Synthetic Biology Engineering Reseach Center (SynBERC). Her lab designs and constructs novel synthetic pathways in microorganisms converting them into tiny factories for the production of small molecules. Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.

VIEW VIDEOS

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=0

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=12

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=74

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=129

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=168

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk

David Bartel: micro-RNAs

I. Introduction to microRNAs

https://www.youtube.com/watch?feature=player_embedded&v=dupzE66J8u4#t=0

– See more at: http://www.ibiology.org/ibioseminars/genetics-gene-regulation/david-bartel-part-1.html#sthash.nGGr1tIt.dpuf

II. Regulatory Effects of Mammalian microRNAs

https://www.youtube.com/watch?feature=player_embedded&v=TcZK_A_wcWQ#t=0

https://www.youtube.com/watch?feature=player_embedded&v=TcZK_A_wcWQ

Calcium Cycling in Synthetic and Contractile Phasic or Tonic Vascular Smooth Muscle Cells

in INTECH
Current Basic and Pathological Approaches to
the Function of Muscle Cells and Tissues – From Molecules to HumansLarissa Lipskaia, Isabelle Limon, Regis Bobe and Roger Hajjar
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/48240

1. Introduction
Calcium ions (Ca ) are present in low concentrations in the cytosol (~100 nM) and in high concentrations (in mM range) in both the extracellular medium and intracellular stores (mainly sarco/endo/plasmic reticulum, SR). This differential allows the calcium ion messenger that carries information
as diverse as contraction, metabolism, apoptosis, proliferation and/or hypertrophic growth. The mechanisms responsible for generating a Ca signal greatly differ from one cell type to another.

In the different types of vascular smooth muscle cells (VSMC), enormous variations do exist with regard to the mechanisms responsible for generating Ca signal. In each VSMC phenotype (synthetic/proliferating and contractile [1], tonic or phasic), the Ca signaling system is adapted to its particular function and is due to the specific patterns of expression and regulation of Ca.
For instance, in contractile VSMCs, the initiation of contractile events is driven by mem- brane depolarization; and the principal entry-point for extracellular Ca is the voltage-operated L-type calcium channel (LTCC). In contrast, in synthetic/proliferating VSMCs, the principal way-in for extracellular Ca is the store-operated calcium (SOC) channel.
Whatever the cell type, the calcium signal consists of limited elevations of cytosolic free calcium ions in time and space. The calcium pump, sarco/endoplasmic reticulum Ca ATPase (SERCA), has a critical role in determining the frequency of SR Ca release by upload into the sarcoplasmic
sensitivity of SR calcium channels, Ryanodin Receptor, RyR and Inositol tri-Phosphate Receptor, IP3R.
Synthetic VSMCs have a fibroblast appearance, proliferate readily, and synthesize increased levels of various extracellular matrix components, particularly fibronectin, collagen types I and III, and tropoelastin [1].
Contractile VSMCs have a muscle-like or spindle-shaped appearance and well-developed contractile apparatus resulting from the expression and intracellular accumulation of thick and thin muscle filaments [1].

Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs

Figure 1. Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs.

Left panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Contractile re-sponse is initiated by extracellular Ca influx due to activation of Receptor Operated Ca (through phosphoinositol-coupled receptor) or to activation of L-Type Calcium channels (through an increase in luminal pressure). Small increase of cytosolic due IP3 binding to IP3R (puff) or RyR activation by LTCC or ROC-dependent Ca influx leads to large SR Ca IP3R or RyR clusters (“Ca -induced Ca SR calcium pumps (both SERCA2a and SERCA2b are expressed in quiescent VSMCs), maintaining high concentration of cytosolic Ca and setting the sensitivity of RyR or IP3R for the next spike.
Contraction of VSMCs occurs during oscillatory Ca transient.
Middle panel: schematic representa tion of atherosclerotic vessel wall. Contractile VSMC are located in the media layer, synthetic VSMC are located in sub-endothelial intima.
Right panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Agonist binding to phosphoinositol-coupled receptor leads to the activation of IP3R resulting in large increase in cytosolic Ca calcium pumps (only SERCA2b, having low turnover and low affinity to Ca depletion leads to translocation of SR Ca sensor STIM1 towards PM, resulting in extracellular Ca influx though opening of Store Operated Channel (CRAC). Resulted steady state Ca transient is critical for activation of proliferation-related transcription factors ‘NFAT).
Abbreviations: PLC – phospholipase C; PM – plasma membrane; PP2B – Ca /calmodulin-activated protein phosphatase 2B (calcineurin); ROC- receptor activated channel; IP3 – inositol-1,4,5-trisphosphate, IP3R – inositol-1,4,5- trisphosphate receptor; RyR – ryanodine receptor; NFAT – nuclear factor of activated T-lymphocytes; VSMC – vascular smooth muscle cells; SERCA – sarco(endo)plasmic reticulum Ca sarcoplasmic reticulum.

Time for New DNA Synthesis and Sequencing Cost Curves

By Rob Carlson

I’ll start with the productivity plot, as this one isn’t new. For a discussion of the substantial performance increase in sequencing compared to Moore’s Law, as well as the difficulty of finding this data, please see this post. If nothing else, keep two features of the plot in mind: 1) the consistency of the pace of Moore’s Law and 2) the inconsistency and pace of sequencing productivity. Illumina appears to be the primary driver, and beneficiary, of improvements in productivity at the moment, especially if you are looking at share prices. It looks like the recently announced NextSeq and Hiseq instruments will provide substantially higher productivities (hand waving, I would say the next datum will come in another order of magnitude higher), but I think I need a bit more data before officially putting another point on the plot.

cost-of-oligo-and-gene-synthesis

Illumina’s instruments are now responsible for such a high percentage of sequencing output that the company is effectively setting prices for the entire industry. Illumina is being pushed by competition to increase performance, but this does not necessarily translate into lower prices. It doesn’t behoove Illumina to drop prices at this point, and we won’t see any substantial decrease until a serious competitor shows up and starts threatening Illumina’s market share. The absence of real competition is the primary reason sequencing prices have flattened out over the last couple of data points.

Note that the oligo prices above are for column-based synthesis, and that oligos synthesized on arrays are much less expensive. However, array synthesis comes with the usual caveat that the quality is generally lower, unless you are getting your DNA from Agilent, which probably means you are getting your dsDNA from Gen9.

Note also that the distinction between the price of oligos and the price of double-stranded sDNA is becoming less useful. Whether you are ordering from Life/Thermo or from your local academic facility, the cost of producing oligos is now, in most cases, independent of their length. That’s because the cost of capital (including rent, insurance, labor, etc) is now more significant than the cost of goods. Consequently, the price reflects the cost of capital rather than the cost of goods. Moreover, the cost of the columns, reagents, and shipping tubes is certainly more than the cost of the atoms in the sDNA you are ostensibly paying for. Once you get into longer oligos (substantially larger than 50-mers) this relationship breaks down and the sDNA is more expensive. But, at this point in time, most people aren’t going to use longer oligos to assemble genes unless they have a tricky job that doesn’t work using short oligos.

Looking forward, I suspect oligos aren’t going to get much cheaper unless someone sorts out how to either 1) replace the requisite human labor and thereby reduce the cost of capital, or 2) finally replace the phosphoramidite chemistry that the industry relies upon.

IDT’s gBlocks come at prices that are constant across quite substantial ranges in length. Moreover, part of the decrease in price for these products is embedded in the fact that you are buying smaller chunks of DNA that you then must assemble and integrate into your organism of choice.

Someone who has purchased and assembled an absolutely enormous amount of sDNA over the last decade, suggested that if prices fell by another order of magnitude, he could switch completely to outsourced assembly. This is a potentially interesting “tipping point”. However, what this person really needs is sDNA integrated in a particular way into a particular genome operating in a particular host. The integration and testing of the new genome in the host organism is where most of the cost is. Given the wide variety of emerging applications, and the growing array of hosts/chassis, it isn’t clear that any given technology or firm will be able to provide arbitrary synthetic sequences incorporated into arbitrary hosts.

TrackBack URL: http://www.synthesis.cc/cgi-bin/mt/mt-t.cgi/397

Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products

28 Nov 2013 | PR Web

Dr. Jon Rowley and Dr. Uplaksh Kumar, Co-Founders of RoosterBio, Inc., a newly formed biotech startup located in Frederick, are paving the way for even more innovation in the rapidly growing fields of Synthetic Biology and Regenerative Medicine. Synthetic Biology combines engineering principles with basic science to build biological products, including regenerative medicines and cellular therapies. Regenerative medicine is a broad definition for innovative medical therapies that will enable the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. Regenerative therapies that are in clinical trials today may enable repair of damaged heart muscle following heart attack, replacement of skin for burn victims, restoration of movement after spinal cord injury, regeneration of pancreatic tissue for insulin production in diabetics and provide new treatments for Parkinson’s and Alzheimer’s diseases, to name just a few applications.

While the potential of the field is promising, the pace of development has been slow. One main reason for this is that the living cells required for these therapies are cost-prohibitive and not supplied at volumes that support many research and product development efforts. RoosterBio will manufacture large quantities of standardized primary cells at high quality and low cost, which will quicken the pace of scientific discovery and translation to the clinic. “Our goal is to accelerate the development of products that incorporate living cells by providing abundant, affordable and high quality materials to researchers that are developing and commercializing these regenerative technologies” says Dr. Rowley

Life at the Speed of Light

http://kcpw.org/?powerpress_pinw=92027-podcast

NHMU Lecture featuring – J. Craig Venter, Ph.D.
Founder, Chairman, and CEO – J. Craig Venter Institute; Co-Founder and CEO, Synthetic Genomics Inc.

J. Craig Venter, Ph.D., is Founder, Chairman, and CEO of the J. Craig Venter Institute (JVCI), a not-for-profit, research organization dedicated to human, microbial, plant, synthetic and environmental research. He is also Co-Founder and CEO of Synthetic Genomics Inc. (SGI), a privately-held company dedicated to commercializing genomic-driven solutions to address global needs.

In 1998, Dr. Venter founded Celera Genomics to sequence the human genome using new tools and techniques he and his team developed. This research culminated with the February 2001 publication of the human genome in the journal, Science. Dr. Venter and his team at JVCI continue to blaze new trails in genomics. They have sequenced and a created a bacterial cell constructed with synthetic DNA, putting humankind at the threshold of a new phase of biological research. Whereas, we could previously read the genetic code (sequencing genomes), we can now write the genetic code for designing new species.

The science of synthetic genomics will have a profound impact on society, including new methods for chemical and energy production, human health and medical advances, clean water, and new food and nutritional products. One of the most prolific scientists of the 21st century for his numerous pioneering advances in genomics, he guides us through this emerging field, detailing its origins, current challenges, and the potential positive advances.

His work on synthetic biology truly embodies the theme of “pushing the boundaries of life.” Essentially, Venter is seeking to “write the software of life” to create microbes designed by humans rather than only through evolution. The potential benefits and risks of this new technology are enormous. It also requires us to examine, both scientifically and philosophically, the question of “What is life?”

J Craig Venter wants to digitize DNA and transmit the signal to teleport organisms

http://pharmaceuticalintelligence.com/2013/11/01/j-craig-venter-wants-to-digitize-dna-and-transmit-the-signal-to-teleport-organisms/

2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

http://pharmaceuticalintelligence.com/2013/02/11/2013-genomics-the-era-beyond-the-sequencing-human-genome-francis-collins-craig-venter-eric-lander-et-al/

Human Longevity Inc (HLI) – $70M in Financing of Venter’s New Integrative Omics and Clinical Bioinformatics

http://pharmaceuticalintelligence.com/2014/03/05/human-longevity-inc-hli-70m-in-financing-of-venters-new-integrative-omics-and-clinical-bioinformatics/

Where Will the Century of Biology Lead Us?

By Randall Mayes

A technology trend analyst offers an overview of synthetic biology, its potential applications, obstacles to its development, and prospects for public approval.

In addition to boosting the economy, synthetic biology projects currently in development could have profound implications for the future of manufacturing, sustainability, and medicine.
Before society can fully reap the benefits of synthetic biology, however, the field requires development and faces a series of hurdles in the process. Do researchers have the scientific know-how and technical capabilities to develop the field?

Biology + Engineering = Synthetic Biology

Bioengineers aim to build synthetic biological systems using compatible standardized parts that behave predictably. Bioengineers synthesize DNA parts—oligonucleotides composed of 50–100 base pairs—which make specialized components that ultimately make a biological system. As biology becomes a true engineering discipline, bioengineers will create genomes using mass-produced modular units similar to the microelectronics and computer industries.

Currently, bioengineering projects cost millions of dollars and take years to develop products. For synthetic biology to become a Schumpeterian revolution, smaller companies will need to be able to afford to use bioengineering concepts for industrial applications. This will require standardized and automated processes.

A major challenge to developing synthetic biology is the complexity of biological systems. When bioengineers assemble synthetic parts, they must prevent cross talk between signals in other biological pathways. Until researchers better understand these undesired interactions that nature has already worked out, applications such as gene therapy will have unwanted side effects. Scientists do not fully understand the effects of environmental and developmental interaction on gene expression. Currently, bioengineers must repeatedly use trial and error to create predictable systems.

Similar to physics, synthetic biology requires the ability to model systems and quantify relationships between variables in biological systems at the molecular level.

The second major challenge to ensuring the success of synthetic biology is the development of enabling technologies. With genomes having billions of nucleotides, this requires fast, powerful, and cost-efficient computers. Moore’s law, named for Intel co-founder Gordon Moore, posits that computing power progresses at a predictable rate and that the number of components in integrated circuits doubles each year until its limits are reached. Since Moore’s prediction, computer power has increased at an exponential rate while pricing has declined.

DNA sequencers and synthesizers are necessary to identify genes and make synthetic DNA sequences. Bioengineer Robert Carlson calculated that the capabilities of DNA sequencers and synthesizers have followed a pattern similar to computing. This pattern, referred to as the Carlson Curve, projects that scientists are approaching the ability to sequence a human genome for $1,000, perhaps in 2020. Carlson calculated that the costs of reading and writing new genes and genomes are falling by a factor of two every 18–24 months. (see recent Carlson comment on requirement to read and write for a variety of limiting conditions).

Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products

http://pharmaceuticalintelligence.com/2013/11/28/startup-to-strengthen-synthetic-biology-and-regenerative-medicine-industries-with-cutting-edge-cell-products/

Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

http://pharmaceuticalintelligence.com/2013/05/17/synthetic-biology-on-advanced-genome-interpretation-for-gene-variants-and-pathways-what-is-the-genetic-base-of-atherosclerosis-and-loss-of-arterial-elasticity-with-aging/

Synthesizing Synthetic Biology: PLOS Collections

http://pharmaceuticalintelligence.com/2012/08/17/synthesizing-synthetic-biology-plos-collections/

Capturing ten-color ultrasharp images of synthetic DNA structures resembling numerals 0 to 9

http://pharmaceuticalintelligence.com/2014/02/05/capturing-ten-color-ultrasharp-images-of-synthetic-dna-structures-resembling-numerals-0-to-9/

Silencing Cancers with Synthetic siRNAs

http://pharmaceuticalintelligence.com/2013/12/09/silencing-cancers-with-synthetic-sirnas/

Genomics Now—and Beyond the Bubble

Futurists have touted the twenty-first century as the century of biology based primarily on the promise of genomics. Medical researchers aim to use variations within genes as biomarkers for diseases, personalized treatments, and drug responses. Currently, we are experiencing a genomics bubble, but with advances in understanding biological complexity and the development of enabling technologies, synthetic biology is reviving optimism in many fields, particularly medicine.

BY MICHAEL BROOKS 17 APR, 2014 http://www.newstatesman.com/

Michael Brooks holds a PhD in quantum physics. He writes a weekly science column for the New Statesman, and his most recent book is The Secret Anarchy of Science.

The basic idea is that we take an organism – a bacterium, say – and re-engineer its genome so that it does something different. You might, for instance, make it ingest carbon dioxide from the atmosphere, process it and excrete crude oil.

That project is still under construction, but others, such as using synthesised DNA for data storage, have already been achieved. As evolution has proved, DNA is an extraordinarily stable medium that can preserve information for millions of years. In 2012, the Harvard geneticist George Church proved its potential by taking a book he had written, encoding it in a synthesised strand of DNA, and then making DNA sequencing machines read it back to him.

When we first started achieving such things it was costly and time-consuming and demanded extraordinary resources, such as those available to the millionaire biologist Craig Venter. Venter’s team spent most of the past two decades and tens of millions of dollars creating the first artificial organism, nicknamed “Synthia”. Using computer programs and robots that process the necessary chemicals, the team rebuilt the genome of the bacterium Mycoplasma mycoides from scratch. They also inserted a few watermarks and puzzles into the DNA sequence, partly as an identifying measure for safety’s sake, but mostly as a publicity stunt.

What they didn’t do was redesign the genome to do anything interesting. When the synthetic genome was inserted into an eviscerated bacterial cell, the new organism behaved exactly the same as its natural counterpart. Nevertheless, that Synthia, as Venter put it at the press conference to announce the research in 2010, was “the first self-replicating species we’ve had on the planet whose parent is a computer” made it a standout achievement.

Today, however, we have entered another era in synthetic biology and Venter faces stiff competition. The Steve Jobs to Venter’s Bill Gates is Jef Boeke, who researches yeast genetics at New York University.

Boeke wanted to redesign the yeast genome so that he could strip out various parts to see what they did. Because it took a private company a year to complete just a small part of the task, at a cost of $50,000, he realised he should go open-source. By teaching an undergraduate course on how to build a genome and teaming up with institutions all over the world, he has assembled a skilled workforce that, tinkering together, has made a synthetic chromosome for baker’s yeast.

Stepping into DIYbio and Synthetic Biology at ScienceHack

Posted April 22, 2014 by Heather McGaw and Kyrie Vala-Webb

We got a crash course on genetics and protein pathways, and then set out to design and build our own pathways using both the “Genomikon: Violacein Factory” kit and Synbiota platform. With Synbiota’s software, we dragged and dropped the enzymes to create the sequence that we were then going to build out. After a process of sketching ideas, mocking up pathways, and writing hypotheses, we were ready to start building!

The night stretched long, and at midnight we were forced to vacate the school. Not quite finished, we loaded our delicate bacteria, incubator, and boxes of gloves onto the bus and headed back to complete our bacterial transformation in one of our hotel rooms. Jammed in between the beds and the mini-fridge, we heat-shocked our bacteria in the hotel ice bucket. It was a surreal moment.

While waiting for our bacteria, we held an “unconference” where we explored bioethics, security and risk related to synthetic biology, 3D printing on Mars, patterns in juggling (with live demonstration!), and even did a Google Hangout with Rob Carlson. Every few hours, we would excitedly check in on our bacteria, looking for bacterial colonies and the purple hue characteristic of violacein.

Most impressive was the wildly successful and seamless integration of a diverse set of people: in a matter of hours, we were transformed from individual experts and practitioners in assorted fields into cohesive and passionate teams of DIY biologists and science hackers. The ability of everyone to connect and learn was a powerful experience, and over the course of just one weekend we were able to challenge each other and grow.

Returning to work on Monday, we were hungry for more. We wanted to find a way to bring the excitement and energy from the weekend into the studio and into the projects we’re working on. It struck us that there are strong parallels between design and DIYbio, and we knew there was an opportunity to bring some of the scientific approaches and curiosity into our studio.

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Curation, HealthCare System in the US, and Calcium Signaling Effects on Cardiac Contraction, Heart Failure, and Atrial Fibrillation, and the Relationship of Calcium Release at the Myoneural Junction to Beta Adrenergic Release

Posted in Accountable Care Organizations, Affordable Care Act, Atherogenic Processes & Pathology, Best evidence, Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, Ca2+ triggered activation, Ca2+ triggered activation, Calcium Signaling, Calmodulin, Calmodulin Kinase and Contraction, Cardiac muscle regeneration, Cardiomyopathy, Cardiovascular Pharmacogenomics, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Congenital Heart Disease, Curation, Curation methodology, Cytoskeleton, De novo synthesis, Dialectic, Discovery process, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Evolutionary cognition, Experimental validation, Explanatory, Federal Budget Appropriations, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Health Economics and Outcomes Research, Healthcare costs and reimbursement, HealthCare IT, Healthcare Reform, Historical relevance, HTN, Indigent Nutrition, Ionic Transporters Na+, K, Medical Devices R&D Investment, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Medicare and Medicaid, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Na-K transport, Na-K-ATPase, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Nutritional Supplements: Atherogenesis, lipid metabolism, Origins of Cardiovascular Disease, Pharmacotherapy of Cardiovascular Disease, PKC, Population Health Management, Genetics & Pharmaceutical, Pre-Clinical Animal Model Development, Regenerative Biology and Medicine, Skilled Nursing Facilities, Stem Cells for Regenerative Medicine, Synaptic vesicle, Technology Advance Assessment of, Technology Capital Expenses, Technology Transfer: Biotech and Pharmaceutical, Theoretic convergence, Translational Effectiveness, Translational Research, Translational Science, Uninsured and Underinsured, tagged 249 SNPs associated with a number of heart-related conditions and treatments., Accountable Care Organization, acute coronary syndromes, Acute decompensated heart failure, Affordable Care Act, atherogenesis, Atherosclerosis, Atrial Fibrillation: IL6R Polymorphism in Whites and African Americans, calcium calmodulin kinase, calcium efflux, Cardiac & Vascular Repair, cardiac arrhythmias, cardiac biomarkers, Cardiogenesis, cardiomyocyte, cardiomyocyte hypertrophy, Content Curation, Coronary artery disease, Coronary circulation, Curation, efflux transporter, financial cost accounting, ion channels, ion transport, Na-K-Cl cotransporter, PKC, Pyruvate Kinase, seven CYP2C19 SNPs linked to Plavix response, SNPs, Synaptic vesicle on January 2, 2014| Leave a Comment »

Curation, HealthCare System in the US, and Calcium Signaling Effects on Cardiac Contraction, Heart Failure, and Atrial Fibrillation, and the Relationship of Calcium Release at the Myoneural Junction to Beta Adrenergic Release

Curator and e-book Contributor: Larry H. Bernstein, MD, FCAP
Curator and BioMedicine e-Series Editor-in-Chief: Aviva Lev Ari, PhD, RN

and

Content Consultant to Six-Volume e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

This portion summarises what we have covered and is now familiar to the reader. There are three related topics, and an extension of this embraces other volumes and chapters before and after this reading. This approach to the document has advantages over the multiple authored textbooks that are and have been pervasive as a result of the traditional publication technology. It has been stated by the founder of ScoopIt, that amount of time involved is considerably less than required for the original publications used, but the organization and construction is a separate creative process. In these curations we amassed on average five articles in one curation, to which, two or three curators contributed their views. There were surprises, and there were unfulfilled answers along the way. The greatest problem that is being envisioned is the building a vision that bridges and unmasks the hidden “dark matter” between the now declared “OMICS”, to get a more real perspective on what is conjecture and what is actionable. This is in some respects unavoidable because the genome is an alphabet that is matched to the mino acid sequences of proteins, which themselves are three dimensional drivers of sequences of metabolic reactions that can be altered by the accumulation of substrates in critical placements, and in addition, the proteome has functional proteins whose activity is a regulatory function and not easily identified. In the end, we have to have a practical conception, recognizing the breadth of evolutionary change, and make sense of what we have, while searching for more.

We introduced the content as follows:

1. We introduce the concept of curation in the digital context, and it’s application to medicine and related scientific discovery.

Topics were chosen were used to illustrate this process in the form of a pattern, which is mostly curation, but is significantly creative, as it emerges in the context of this e-book.

Alternative solutions in Treatment of Heart Failure (HF), medical devices, biomarkers and agent efficacy is handled all in one chapter.
PCI for valves vs Open heart Valve replacement
PDA and Complications of Surgery — only curation could create the picture of this unique combination of debate, as exemplified of Endarterectomy (CEA) vs Stenting the Carotid Artery (CAS), ischemic leg, renal artery stenosis.

2. The etiology, or causes, of cardiovascular diseases consist of mechanistic explanations for dysfunction relating to the heart or vascular system. Every one of a long list of abnormalities has a path that explains the deviation from normal. With the completion of the analysis of the human genome, in principle all of the genetic basis for function and dysfunction are delineated. While all genes are identified, and the genes code for all the gene products that constitute body functions, there remains more unknown than known.

3. Human genome, and in combination with improved imaging methods, genomics offers great promise in changing the course of disease and aging.

4. If we tie together Part 1 and Part 2, there is ample room for considering clinical outcomes based on individual and organizational factors for best performance. This can really only be realized with considerable improvement in information infrastructure, which has miles to go.

Curation

Curation is an active filtering of the web’s and peer reviewed literature found by such means – immense amount of relevant and irrelevant content. As a result content may be disruptive. However, in doing good curation, one does more than simply assign value by presentation of creative work in any category. Great curators comment and share experience across content, authors and themes.
Great curators may see patterns others don’t, or may challenge or debate complex and apparently conflicting points of view. Answers to specifically focused questions comes from the hard work of many in laboratory settings creatively establishing answers to definitive questions, each a part of the larger knowledge-base of reference. There are those rare “Einstein’s” who imagine a whole universe, unlike the three blindmen of the Sufi tale. One held the tail, the other the trunk, the other the ear, and they all said this is an elephant!
In my reading, I learn that the optimal ratio of curation to creation may be as high as 90% curation to 10% creation. Creating content is expensive. Curation, by comparison, is much less expensive. The same source says “Scoop.it is my content marketing testing “sandbox”. In sharing, he says that comments provide the framework for what and how content is shared.

Healthcare and Affordable Care Act

We enter year 2014 with the Affordable Care Act off to a slow start because of the implementation of the internet signup requiring a major repair, which is, unfortunately, as expected for such as complex job across the US, and with many states unwilling to participate. But several states – California, Connecticut, and Kentucky – had very effective state designed signups, separate from the federal system. There has been a very large rush and an extension to sign up. There are many features that we can take note of:

1. The healthcare system needed changes because we have the most costly system, are endowed with advanced technology, and we have inexcusable outcomes in several domains of care, including, infant mortality, and prenatal care – but not in cardiology.

2. These changes that are notable are:

The disparities in outcome are magnified by a large disparity in highest to lowest income bracket.
This is also reflected in educational status, and which plays out in childhood school lunches, and is also affected by larger class size and cutbacks in school programs.
This is not helped by a large paralysis in the two party political system and the three legs of government unable to deal with work and distraction.
Unemployment is high, and the banking and home construction, home buying, and rental are in realignment, but interest rates are problematic.

3. The medical care system is affected by the issues above, but the complexity is not to be discounted.

The medical schools are unable at this time to provide the influx of new physicians needed, so we depend on a major influx of physicians from other countries
The technology for laboratories, proteomic and genomic as well as applied medical research is rejuvenating the practice in cardiology more rapidly than any other field.
In fields that are imaging related the life cycle of instruments is shorter than the actual lifetime use of the instruments, which introduces a shortening of ROI.
Hospitals are consolidating into large consortia in order to maintain a more viable system for referral of specialty cases, and also is centralizing all terms of business related to billing.
There is reduction in independent physician practices that are being incorporated into the hospital enterprise with Part B billing under the Physician Organization – as in Partners in Greater Boston, with the exception of “concierge” medical practices.
There is consolidation of specialty laboratory services within state, with only the most specialized testing going out of state (Quest, LabCorp, etc.)
Medicaid is expanded substantially under the new ACA.
The federal government as provider of services is reducing the number of contractors for – medical devices, diabetes self-testing, etc.
The current rearrangements seeks to provide a balance between capital expenses and fixed labor costs that it can control, reduce variable costs (reagents, pharmaceutical), and to take in more patients with less delay and better performance – defined by outside agencies.

Cardiology, Genomics, and calcium ion signaling and ion-channels in cardiomyocyte function in health and disease – including heart failure, rhythm abnormalities, and the myoneural release of neurotransmitter at the vesicle junction.

This portion is outlined as follows:

2.1 Human Genome: Congenital Etiological Sources of Cardiovascular Disease	2.2 The Role of Calcium in Health and Disease	2.3 Vasculature and Myocardium: Diagnosing the Conditions of Disease
Genomics & Genetics of Cardiovascular Disease Diagnoses	actin cytoskeleton	wall stress, ventricular workload, contractile reserve
Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging	calcium and actin skeleton, signaling, cell motility	hypertension & vascular compliance
Genetics of Conduction Disease	Ca+ stimulated exostosis: calmodulin & PKC (neurotransmitter)	complications & MVR
	disruption of Ca2+ homeostasis cardiac & vascular smooth muscle
	synaptotagmin as Ca2+ sensor & vesicles
	atherosclerosis & ion channels

It is increasingly clear that there are mutations that underlie many human diseases, and this is true of the cardiovascular system. The mutations are mistakes in the insertion of a purine nucleotide, which may or may not have any consequence. This is why the associations that are being discovered in research require careful validation, and even require demonstration in “models” before pursuing the design of pharmacological “target therapy”. The genomics in cardiovascular disease involves very serious congenital disorders that are asserted early in life, but the effects of and development of atherosclerosis involving large and medium size arteries has a slow progression and is not dominated by genomic expression. This is characterized by loss of arterial elasticity. In addition there is the development of heart failure, which involves the cardiomyocyte specifically. The emergence of regenerative medical interventions, based on pleuripotent inducible stem cell therapy is developing rapidly as an intervention in this sector.

Finally, it is incumbent on me to call attention to the huge contribution that research on calcium (Ca2+) signaling has made toward the understanding of cardiac contraction and to the maintenance of the heart rhythm. The heart is a syncytium, different than skeletal and smooth muscle, and the innervation is by the vagus nerve, which has terminal endings at vesicles which discharge at the myocyte junction. The heart specifically has calmodulin kinase CaMK II, and it has been established that calmodulin is involved in the calcium spark that triggers contraction. That is only part of the story. Ion transport occurs into or out of the cell, the latter termed exostosis. Exostosis involves CaMK II and pyruvate kinase (PKC), and they have independent roles. This also involves K+-Na+-ATPase. The cytoskeleton is also discussed, but the role of aquaporin in water transport appears elsewhere, as the transport of water between cells. When we consider the Gibbs-Donnan equilibrium, which precedes the current work by a century, we recall that there is an essential balance between extracellular Na+ + Ca2+ and the intracellular K+ + Mg2+, and this has been superceded by an incompletely defined relationship between ions that are cytoplasmic and those that are mitochondrial. The glass is half full!

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The Cost to Value Conundrum in Cardiovascular Healthcare Provision

Posted in Accountable Care Organizations, Affordable Care Act, Anemia, Atherogenic Processes & Pathology, Best evidence, Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, Biomedical Measurement Science, Ca2+ triggered activation, Ca2+ triggered activation, Calcium Signaling, Cardiac & Vascular Repair Tools Subsegment, Cardiac and Cardiovascular Surgical Procedures, Cardiac muscle regeneration, Cardiomyopathy, Cardiovascular Pharmacogenomics, Coagulation Therapy and Internal Bleeding, Computational Biology/Systems and Bioinformatics, Curation, Curation methodology, Cytoskeleton, Diabetes Mellitus, Electrophysiology, Evidence-based decision-making, Experimental validation, Federal Budget Appropriations, Frontiers in Cardiology and Cardiovascular Disorders, Health Economics and Outcomes Research, Health Law & Patient Safety, Healthcare costs and reimbursement, Healthcare Reform, Historical relevance, HTN, HTN in Youth, Indigent Nutrition, Interviews with Scientific Leaders, Ionic Transporters Na+, K, Medical Devices R&D and Inventions, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Medicare and Medicaid, Na-K transport, Na-K-ATPase, Nephrology, Nitric Oxide in Health and Disease, Nutrition, Nutritional Supplements: Atherogenesis, lipid metabolism, Origins of Cardiovascular Disease, Pharmaceutical Analytics, Pharmacogenomics, Pharmacologic toxicities, Pharmacotherapy of Cardiovascular Disease, Population Health Management, Genetics & Pharmaceutical, Population Health Management, Nutrition and Phytochemistry, Prescription Drugs Costs, Regenerative Biology and Medicine, Resident-cell-based, Skilled Nursing Facilities, Stem Cells for Regenerative Medicine, Stents & Tools, Synaptic vesicle, Systematic Error (Bias), Systemic Inflammatory Response Related Disorders, Technology Advance Assessment of, Technology Capital Expenses, Technology Transfer: Biotech and Pharmaceutical, Translational Effectiveness, Translational Science, Uninsured and Underinsured, Valves & Tools, tagged acute myocardial infarct, American College of Cardiology ACC, Antiarrhythmic drugs, arrythmias, Artificial Biosensor, Artificial cardiac pacemaker, Artificial heart, Bare-metal stent, Cardiac & Vascular Repair, cardiac myocyte regeneration, Cardiogenic Shock, Cardiology, Cardiometabolic Syndrome, CHF, Content Curation, Coronary artery disease, Coronary stent, Curation methodology, Drug-eluting stents, gene expression, Hypertension, myocardial hypertrophy, nitric oxide, Nitric oxide synthase, RNA, Stem cell on January 1, 2014| Leave a Comment »

The Cost to Value Conundrum in Cardiovascular Healthcare Provision

Author: Larry H. Bernstein, MD, FCAP

Article ID #98: The Cost to Value Conundrum in Cardiovascular Healthcare Provision. Published on 1/1/2014

WordCloud Image Produced by Adam Tubman

I write this introduction to Volume 2 of the e-series on Cardiovascular Diseases, which curates the basic structure and physiology of the heart, the vasculature, and related structures, e.g., the kidney, with respect to:

1. Pathogenesis
2. Diagnosis
3. Treatment

Curation is an introductory portion to Volume Two, which is necessary to introduce the methodological design used to create the following articles. More needs not to be discussed about the methodology, which will become clear, if only that the content curated is changing based on success or failure of both diagnostic and treatment technology availability, as well as the systems needed to support the ongoing advances. Curation requires:

meaningful selection,
enrichment, and
sharing combining sources and
creation of new synnthesis

Curators have to create a new perspective or idea on top of the existing media which supports the content in the original. The curator has to select from the myriad upon myriad options available, to re-share and critically view the work. A search can be overwhelming in size of the output, but the curator has to successfully pluck the best material straight out of that noise.

Part 1 is a highly important treatment that is not technological, but about the system now outdated to support our healthcare system, the most technolog-ically advanced in the world, with major problems in the availability of care related to economic disparities. It is not about technology, per se, but about how we allocate healthcare resources, about individuals’ roles in a not full list of lifestyle maintenance options for self-care, and about the important advances emerging out of the Affordable Care Act (ACA), impacting enormously on Medicaid, which depends on state-level acceptance, on community hospital, ambulatory, and home-care or hospice restructuring, which includes the reduction of management overhead by the formation of regional healthcare alliances, the incorporation of physicians into hospital-based practices (with the hospital collecting and distributing the Part B reimbursement to the physician, with “performance-based” targets for privileges and payment – essential to the success of an Accountable Care Organization (AC)). One problem that ACA has definitively address is the elimination of the exclusion of patients based on preconditions. One problem that has been left unresolved is the continuing existence of private policies that meet financial capabilities of the contract to provide, but which provide little value to the “purchaser” of care. This is a holdout that persists in for-profit managed care as an option. A physician response to the new system of care, largely fostered by a refusal to accept Medicaid, is the formation of direct physician-patient contracted care without an intermediary.

In this respect, the problem is not simple, but is resolvable. A proposal for improved economic stability has been prepared by Edward Ingram. A concern for American families and businesses is substantially addressed in a macroeconomic design concept, so that financial services like housing, government, and business finance, savings and pensions, boosting confidence at every level giving everyone a better chance of success in planning their personal savings and lifetime and business finances.

http://macro-economic-design.blogspot.com/p/book.html

Part 2 is a collection of scientific articles on the current advances in cardiac care by the best trained physicians the world has known, with mastery of the most advanced vascular instrumentation for medical or surgical interventions, the latest diagnostic ultrasound and imaging tools that are becoming outdated before the useful lifetime of the capital investment has been completed. If we tie together Part 1 and Part 2, there is ample room for considering clinical outcomes based on individual and organizational factors for best performance. This can really only be realized with considerable improvement in information infrastructure, which has miles to go. Why should this be? Because for generations of IT support systems, they are historically focused on billing and have made insignificant inroads into the front-end needs of the clinical staff.

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Ca2+-Stimulated Exocytosis: The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Posted in Ca2+ triggered activation, Ca2+ triggered activation, Calcium Signaling, Calmodulin, Cell Biology, Signaling & Cell Circuits, Cytoskeleton, Innovations in Neurophysiology & Neuropsychology, Neurohumoral Transmission, PKC, Proteomics, Synaptic vesicle, tagged Ca2+-regulation, Ca2+/calmodulin-dependent protein kinase, Calmodulin, calmodulin-dependent pathway, calmodulin-independent pathway, CaMK, exocytosis, Howard Hughes Medical Institute, PKC, Protein kinase C, SNARE, Stanford University School of Medicine, Synaptic vesicle on December 23, 2013| Leave a Comment »

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

This article is Part V in a series of TWELVE articles, listed at the end of this article, on the

cytoskeleton,
calcium calmodulin kinase signaling,
muscle and nerve transduction, and
calcium,
Na+-K+-ATPase,
neurohumoral activity and vesicles vital and essential for all functions related to

cell movement,
migration, and
contraction.

Calmodulin and Protein Kinase C Increase Ca–stimulated Secretion by Modulating
Membrane-attached Exocytic Machinery

YA Chen, V Duvvuri, H Schulmani, and RH.Scheller‡

From the ‡Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology,
and the Department of Neurobiology, Stanford University School of Medicine, Stanford, CA

J Biol Chem 1999; 274(37): 25469-26476.

The molecular mechanisms underlying the Ca2+ regulation of hormone and neurotransmitter release
are largely unknown.

Using a reconstituted [3H]norepinephrine release assay in permeabilized PC12 cells, we found

essential proteins that support the triggering stage of Ca2+-stimulated exocytosis
are enriched in an EGTA extract of brain membranes.

Fractionation of this extract allowed purification of two factors that stimulate secretion

in the absence of any other cytosolic proteins.

These are calmodulin and protein kinase Ca (PKCa). Their effects on secretion were

confirmed using commercial and recombinant proteins.

Calmodulin enhances secretion

in the absence of ATP, whereas
PKC requires ATP to increase secretion, suggesting that
phosphorylation is involved in PKC-mediated stimulation
but not calmodulin mediated stimulation.
Both proteins modulate
- The half-maximal increase was elicited by
  3 nM PKC and 75 nM calmodulin.

These results suggest that calmodulin and PKC increase Ca2+-activated exocytosis by

directly modulating the membrane- or cytoskeleton-attached exocytic machinery
downstream of Ca2+ elevation.

The abbreviations used are:

NE, norepinephrine; PKC, protein kinase C; CaM, calmodulin; SNAP-25, synaptosome-associated protein of 25 kDa; CAPS, calcium-dependent activator protein for secretion; SNARE, SNAP (soluble N-ethylmaleimide-sensitive factor attachment proteins) receptor; CaMK, Ca2+/calmodulin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; AMP-PNP, adenosine 59-(b,g- imido) triphosphate; HA, hydroxyapatite

*This work was supported in part by Conte Center Grant MH48108. The costs of publication of
this article were defrayed in part by the payment of page charges. This article has been marked
“advertisement” in accordance with 18 U.S.C. Section 1734.

The molecular mechanisms of presynaptic vesicle release have been extensively examined
by a combination of

biochemical,
genetic, and
electrophysiological techniques.

A series of protein-protein interaction cascades have been proposed to lead to vesicle
docking and fusion (1–3). The SNARE protein family, including

syntaxin, SNAP-25, and vesicle-associated membrane protein
(VAMP, also called synaptobrevin),
plays an essential role in promoting membrane fusion, and
is thought to comprise the basic fusion machinery (4, 5).

In Ca2+-stimulated exocytosis, many additional proteins are important in the Ca2+ regulation
of the basic membrane trafficking apparatus. Calcium

not only triggers rapid fusion of release-competent vesicles, but is also involved in
earlier processes which replenish the pool of readily releasable vesicles (6).

Furthermore, it appears to be critical in initiating several forms of synaptic plasticity including

post-tetanic potentiation (7).

The molecular mechanisms by which Ca2+ regulates these processes is not well understood.

PC12 cells have often been utilized to study Ca2+-activated exocytosis, as

they offer a homogeneous cell population that possesses the same basic exocytic machinery as neurons (8).

In this study, we used an established cracked cell assay, in which

[3H]norepinephrine (NE)1 labeled PC12 cells are
permeabilized by mechanical “cracking” and
then reconstituted for secretion of NE in the presence of test proteins (9).

Transmitter-filled vesicles and intracellular cytoskeletal structures

remain intact in these cells,
while cytosolic proteins leak out (10).

These cracked cells readily release NE upon addition of

ATP,
brain cytosol, and
1 mM free Ca2+
- at an elevated temperature.

We term this a “composite assay,” as

all essential components are added into one reaction mixture.

Alternatively, cracked cells can be

first primed with cytosol and ATP, washed, then
reconstituted for NE release with cytosol and Ca2+ (11).

This sequential priming-triggering protocol is useful

for determining whether a protein acts early or late in the exocytic pathway, and
whether its effect is dependent on Ca2+ or ATP.

This semi-intact cell system serves as

a bridge between an in vitro system comprised of purified components, and
electro-physiological systems that monitor release in vivo.
It provides information on protein functions in a cell with an intact membrane infrastructure while being easily manipulatable.

Ca2+ regulation by membrane depolarization is no longer a concern, as
intra-cellular Ca2+ concentration can be controlled by a buffered solution.

Indirect readout of neurotransmitter release using a postsynaptic cell is replaced by
direct readout of [3H]NE released into the buffer.

Complications associated with interpreting overlapping

exo- and endocytotic signals are also eliminated as only one round of exocytosis is measured.

Finally, concentration estimates are likely to be accurate, since

added compounds do not need to diffuse long distances along axons and dendrites to their sites of action.

Using this assay, several proteins required for NE release have been purified from rat brain cytosol, including

phosphatidyl-inositol transfer protein (12),
phosphatidylinositol-4-phosphate 5-kinase (13), and
calcium-dependent activator protein for secretion (CAPS) (9).

The validity of the cracked cell system is confirmed by the finding that

phosphatidylinositol transfer protein and CAPS are mammalian homologues of
- yeast SEC14p (12) and
- nematode UNC31p, respectively (14),
both proteins involved in membrane trafficking (15, 16).

Calmodulin is the most ubiquitous calcium mediator in eukaryotic cells, yet its involvement in membrane trafficking has not been well established. Some early studies showed

that calmodulin inhibitors (17–19), anti-calmodulin antibodies (20,21),

calmodulin binding inhibitory peptides (22) inhibited Ca2+-activated exocytosis.

However, in other studies, calmodulin-binding peptides and an anti-calmodulin antibody led to the conclusion that

calmodulin is only involved in endocytosis,
not exocytosis (23).

More recently, it was reported that

Ca2+/ calmodulin signals the completion of docking and
triggers a late step of homotypic vacuole fusion in yeast,

thus suggesting an essential role for Ca2+/calmodulin in constitutive intracellular membrane fusion (24).

If calmodulin indeed plays an important role in exocytosis,

a likely target of calmodulin is
Ca2+/calmodulin-dependent protein kinase II (CaMKII),
- a multifunctional kinase that is found on synaptic vesicles (25) and
- has been shown to potentiate neurotransmitter release (26, 27).

Another Ca2+ signaling molecule, PKC, has also been implicated in regulated exocytosis.
In various cell systems, it has been shown that

the phorbol esters stimulate secretion (28, 29).

It is usually assumed that phorbol esters effect on exocytosis is

through activation of PKC,
but Munc13-1 was recently shown to be a presynaptic phorbol ester receptor that enhances neurotransmitter release (30, 31),

which complicates the interpretation of some earlier reports. The mode of action of PKC remains controversial. There is evidence

that PKC increases the intracellular Ca2+ levels by modulating plasma membrane Ca2+ channels (32, 33),
that it increases the size of the release competent vesicle pool (34, 35), or
that it increases the Ca2+ sensitivity of the membrane trafficking apparatus (36).

no consensus on these issues has been reached.

PKC substrates that have been implicated in exocytosis include

SNAP-25 (37),
synaptotagmin (28),
CAPS (38), and
nsec1 (39).

It is believed that upon phosphorylation, these PKC substrates might

interact differently with their binding partners, which, in turn,
leads to the enhancement of exocytosis.

In addition, evidence is accumulating that PKC and calmodulin interfere with each others actions, as

PKC phosphorylation sites are embedded in the calmodulin-binding domains of substrates such as
neuromodulin and
neurogranin (40).

It is therefore possible that PKC could modulate exocytosis via

a calmodulin-dependent pathway by synchronously releasing calmodulin from storage proteins.

In this study, we fractionated an EGTA extract of brain membranes in order to identify active components that could reconstitute release in the cracked cell assay system. We identified calmodulin and PKC as two active factors. Thus, we demonstrate that

calmodulin and PKC play a role in the Ca2+ regulation of exocytosis, and provide further insight into the mechanisms of their action.

DISCUSSION

In this study, we first identified an EGTA extract of brain membranes as a protein source

capable of reconstituting Ca2+- activated exocytosis in cracked PC12 cells.

EGTA only extracts a small pool of Ca2+-dependent membrane-associating proteins,

it served as an efficient initial purification step.

Further protein chromatography led to the identification of two active factors in the starting extract,

calmodulin and PKC,
which together accounted for about half of the starting activity.

Upon confirmation with commercially obtained proteins, this result unambiguously demonstrated

that calmodulin and PKC mediate aspects of Ca2+-dependent processes in exocytosis.

The finding that brain membrane EGTA extract alone is able

to replace cytosol in supporting Ca2+-triggered NE secretion

in PC12 cells is somewhat surprising. We suggest that the likely explanation is 2-fold.

some cytosolic proteins essential for exocytosis have a membrane-bound pool
within permeabilized cells, whose activity might be sufficient for a normal level of exocytosis.
although the 100,000 3 g membrane pellet was washed to remove as many cytosolic proteins as possible,

some cytosolic proteins that associate with membranes in a
- Ca2+-independent manner are probably present in the membrane EGTA extract.
these proteins likely constitute only a small percentage of the proteins in the extract, as
- the characteristics of the activity triggered by the membrane extract
- are quite different to that of cytosol (Fig. 2).

Using an unbiased biochemical purification method, we demonstrated that

calmodulin and PKC directly modulate the exocytotic machinery downstream of Ca2+ entry
they signal through membrane-attached molecules to increase exocytosis.

These targets include integral and peripheral membrane proteins, and cytosolic proteins that have a significant
membrane-bound pool. The modest stimulation by calmodulin and PKC on secretion might suggest a regulatory

role. However, it is also possible that some intermediates in their signaling pathways are in limiting amounts in the
cell ghosts, so that their full effects were not observed. Half-maximal stimulation was obtained at

about 3 nM for PKC and
at about 75 nM for calmodulin.

This is consistent with an enzymatic role for PKC, and predicts a high-affinity interaction between

calmodulin and its substrate protein.

Ca2+ regulates exocytosis at many different levels. Prior studies indicated that Ca2+ signaling occurs in

the priming steps as well
as in triggering steps (49, 50).

Our priming triggering protocol

does not allow Ca2+-dependent priming events to be assayed, as EGTA is present in the priming reaction.
a different approach revealed the existence of both high and low Ca2+-dependent processes (Fig. 2).
this analysis indicated that late triggering events require high [Ca2+], whereas
early priming events require low [Ca2+]. If, as proposed, there is

a pronounced intracellular spatial and temporal [Ca2+] gradient from

the point of Ca2+ entry during depolarization (51),
perhaps triggered events occur closer to the point of Ca2+ entry,
while Ca2+-dependent priming events occur further away from the point of Ca2+ entry.

Fig 2A. measurements of range of [Ca2+]total - average [Ca2+]free values._page_004

Fig. 2B. measurements of range of [Ca2+] total - average [Ca2+]free values_edited-1

Distinct Ca2+ sensors at these stages might be appropriately tuned to different [Ca2+] to handle different tasks.

By analyzing the Ca2+ sensitivity of calmodulin-and PKC-stimulated release, we addressed the question of

whether calmodulin and PKC plays an early or a late role in vesicle release.
they both require relatively high [Ca2+] (Fig. 8B),
implying that calmodulin and PKC both mediate late triggering events, consistent with some earlier reports
(34, 52, 53).

In addition, it is interesting to note that PKC does not alter the calcium sensitivity of release in cracked cells, in contrast

to observations from the chick ciliary ganglion (36). Therefore, in contrast to previous electrophysiological studies (28),
we are able to limit the possible modes of PKC action in our system to an increase in the readily releasable vesicle pool or
release sites, or an enhancement of the probability of release of individual vesicles upon Ca2+ influx.

The experiments assaying the calcium sensitivity of release (Figs. 2, 5, and 8) demonstrated

a drop in release at very high [Ca2+].

FIG. 8. PKC and calmodulin stimulate... the late triggering reaction_page_006

This decline in release at high [Ca2+] has been previously reported (49, 51), and may represent

the true Ca2+ sensitivity of the Ca2+-sensing mechanism inside cells.

However, in our system, it could also be due to the activation of a variety of Ca2+ -activated proteases, as experiments are usually performed in the presence of crude extracts, which include unsequestered proteases.

What might the molecular targets of PKC and calmodulin be? An obvious calmodulin target molecule is CaMKII.

but calmodulin’s effect on exocytosis is ATP-independent, rendering the involvement of a kinase unlikely.

Calmodulin has also been shown to associate with

synaptic vesicles in a Ca2+-dependent fashion through synaptotagmin (54),
probably by binding to its C-terminal tail (55), and to promote Rab3A dissociation from synaptic vesicles (56).
However, there was little calcium-dependent binding of calmodulin to synaptotagmin
- either on synaptic vesicles, in a bead binding assay with recombinant proteins,
- or in a calmodulin overlay (data not shown).

In addition, using immobilized calmodulin, we did not see

significant Ca2+-dependent pull-down of synaptotagmin or Rab3A from rat brain extract (data not shown).

Recent work has suggested three other candidate targets for calmodulin, Munc13, Pollux, and CRAG (57).

Pollux has similarity to a portion of a yeast Rab GTPase-activating protein, while
CRAG is related to Rab3 GTPase exchange proteins.

Further work is required to investigate the role of their interactions with calmodulin in vivo.

The recent report that calmodulin mediates yeast vacuole fusion (24) is intriguing, as it raises the possibility that

calmodulin, a highly conserved ubiquitous molecule,
- may mediate many membrane trafficking events.

It is not yet known if

the effector molecule of calmodulin is conserved or variable across species and different trafficking steps.

It is enticing to propose a model for Ca2+ sensing whereby

calmodulin is a high affinity Ca2+ sensor for both constitutive and regulated membrane fusion.

In the case of constitutive fusion, calmodulin may be the predominant Ca2+ sensor.
In the case of slow, non-local exocytosis of large dense core granules, an additional requirement for
the concerted actions of other molecule(s) that are better tuned to intermediate rises in [Ca2+] might exist.

At the highly localized sites of fast exocytosis of small clear vesicles where high [Ca2+] is reached,

specialized low affinity sensor(s) are likely required
in addition to calmodulin to achieve membrane fusion.

Therefore, although calmodulin participates in multiple types of vesicle fusion,

the impact of Ca2+ sensing by calmodulin on vesicle release likely varies.

Due to the fact that calmodulin binding to some proteins can be modulated by PKC phosphorylation, one might suspect

PKC action on exocytosis proceeds through a calmodulin-dependent pathway.
but the effects of calmodulin and PKC are additive within our system,
- suggesting that PKC does not act by releasing calmodulin from a substrate
  - that functions as a calmodulin storage protein.

How Ca2+ regulates presynaptic vesicle release has been an open question for many years. By

identifying calmodulin and PKC as modulators of Ca21-regulated exocytosis and clarifying their functions,
we have extended our knowledge of the release process.

While the basic machinery of membrane fusion is becoming better understood,

the multiple effects of Ca2+ on exocytosis remain to be elucidated at the molecular level.

In addition, the ways that Ca2+ regulation may be important to

the mechanisms of synaptic plasticity in the central nervous system

EXPERIMENTAL PROCEDURES

Materials
Rat Brain Cytosol Preparation
Membrane EGTA Extract Preparation

Cracked Cell Assay

PC12 cells were maintained and [3H]NE labeled as described previously (11). Labeled cells were harvested by pipetting with ice-cold potassium glutamate buffer (50 mM Hepes, pH 7.2, 105 mM potassium glutamate, 20 mM potassium acetate, 2 mM EGTA) containing 0.1% bovine serum albumin. Subsequent manipulations were carried out at 0–4 °C. Labeled cells (1–1.5 ml/dish) were mechanically permeabilized passage through a stainless steel homogenizer. The cracked cells were adjusted to 11 mM EGTA and

incubated on ice for 0.5–3 h, followed by three washes in which
the cells were centrifuged at 800 3 g for 5 min and
resuspended in potassium glutamate buffer containing 0.1% bovine serum albumin.

Composite Assay

Each release reaction contains 0.5–1 million cracked cells, 1.5 mM free Ca2+, 2 mM MgATP,
and the protein solution to be tested in potassium glutamate buffer. Release reactions were initiated
by incubation at 30 °C and terminated by returning to ice. The supernatant of each reaction was
isolated by centrifugation at 2,500 3 g for 30 min at 4 °C, and the

released [3H]NE was quantified by scintillation counting (Beckman LS6000IC).

Cell pellets were dissolved in 1% Triton X-100, 0.02% azide and similarly counted. NE release

was calculated as a percentage of total [3H] in the supernatant.

Priming Assay

A priming reaction contains about

1–2 million cracked cells,
2 mM MgATP, and
the protein solution to be tested.
Ca2+ is omitted.

The primed cells were spun down, washed once with fresh potassium glutamate buffer, and

distributed into two triggering reactions, each containing
rat brain cytosol and free Ca2+
The triggering reaction was performed at 30 °C for 3 min, and
the NE release was measured
- as in a composite assay.

Triggering Assay

Cracked cells were primed …, centrifuged, washed …, and

distributed into triggering reactions containing

1.5 mM free Ca2+ and the protein solution

To inhibit any ATP dependent activity in the triggering reaction, an

ATP depletion system of

1. hexokinase
2. MgCl2,
3. glucose or

a non-hydrolyzable ATP analogue AMPPNP

was added into the triggering reaction. NE release was measured as above.

Free Ca2+ Concentration Determination

The range of Ca2+free in the release reaction (Fig. 2B) was achieved

by adding Ca2+ into potassium glutamate buffer to reach final [Ca2+] total values of
- 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, and 2.0 mM.
The pH of the reaction was 7.24 when no Ca2+ was added and
7.04 when 2.0 mM Ca2+ was added,
- in the absence of protein extracts or cracked cells.

Fig. 2B. The range of [Ca21]free in the release reaction (Fig. 2B)

Free Ca2+ concentrations were determined using video microscopic
measurements of fura-2 fluorescence (41). [Ca2+]free was calculated from the equation

[Ca2+]free 5 Kd*3 (R 2 Rmin)/(Rmax 2 R) (42).

The values of Rmin, Rmax, and Kd* were determined in the following solutions:

potassium glutamate buffer (PGB) containing

8 x 3 10^6 cracked cells/ml, 2 mM MgATP (PGB+CC)

1) Rmin: PGB+CC and 10 mM additional EGTA;

2) Rmax: PBG+CC, and 10 mM total Ca2+;

3) Kd*: PGB+CC, 28 mM additional EGTA, and 18 mM total Ca2+, pH 7.2
([Ca2+]free 5 = 169 nM, determined in the absence of cells and MgATP

based on fura-2 calibration in cell-free solutions).

These solutions were

incubated at 37 °C ,
mixed with fura-2 pentapotassium salt
(100 mM; Molecular Probes, Eugene, OR), and
imaged.

This procedure allowed us to take into account

changes in fura-2 properties
caused by the presence of
- permeabilized cells.

Duplicate measurements of the above range of [Ca2+] total gave

the following average [Ca2+] free values:
106, 146, 277, 462, 971, 1468, 1847, and 2484 nM.

Purification of Active Proteins

All procedures were carried out at 4 °C or on ice. Membrane
EGTA extract of one or two bovine brain(s) was

filtered through cheesecloth and
loaded overnight onto a column packed with DEAE-Sepharose
CL-6B beads (Amersham Pharmacia Biotech).

The column was then

washed with
(20 mM Hepes, pH 7.5, 0.25 mM sucrose, 2 mM EGTA, 1 mM dithiothreitol) and
step eluted with 10 column volumes of elution buffer
(20 mM Hepes, pH 7.5, 2 mM EGTA, 400 mM KCl, 1 mM dithiothreitol).
100 ml of every other fraction was
dialyzed overnight into PGB, and
tested in a composite release assay for activity.

The active fractions were pooled and dialyzed into zero salt buffer
(20 mM Hepes, pH 7.5, 2 mM EGTA) and
batch bound to 10 ml of Affi-Gel Blue beads (Bio-Rad) or DyeMatrex-Green A beads (Amicon)

Blue beads were used in earlier experiments, and Green beads were used later to

specifically deplete CAPS, which was known to bind to Green beads (9).

The unbound material was

collected,
concentrated to about 2 ml using a Centriprep-10 (Amicon), and
loaded onto a 120-ml HiPrep Sephacryl S-200 gel filtration column
(Amersham Pharmacia Biotech).

Samples were run on the S-200 column in PGB at a flow rate of 7 ml/h.

10–50 ml of every other fraction was tested for
- activity in the cracked cell composite assay, and
two peaks of activity were observed (Fig. 3).

The first peak of activity had a predicted molecular mass of 85 kDa.
The corresponding material was

adjusted to 10 mM potassium phosphate concentration (pH 7.2) and
loaded onto a 1-ml column packed with hydroxyapatite Bio-Gel HT
(Bio-Rad).

The bound material was

eluted with a linear K-PO4 gradient from 10 to 500 mM (pH 7.2)
at a flow rate of about 0.1 ml/min, and
0.4–0.5-ml fractions were collected.
each fraction was dialyzed into PGB and
tested for activity.

The fractions were also analyzed by

SDS-PAGE and silver staining (Sigma silver stain kit).

The active material was concentrated and resolved

on an 8% poly-acrylamide gel.

Two Coomassie-stained protein bands that matched the activity profile (Fig. 6)

were excised from the gel,
sequenced by the Stanford PAN facility.

The two polypeptide sequences obtained from the upper band were:

LLNQEEGEYYNVPIXEGD
IRSTLNPRWDESFT.

The only bovine protein that contains both polypeptides is PKCa.
The four polypeptide sequences obtained from the lower band were:

YELTGKFERLIVGLMRPPAY,
LIEILASRTNEQIHQLVAA,
MLVVLLQGTREEDDVVSEDL, and
EMSGDVRDVFVAIVQSVK.

Based on these sequences, the protein band was

unambiguously identified to be bovine annexin VI.

The second S-200 peak has a predicted molecular mass of 25 kDa.
The corresponding material was

dialyzed into zero salt buffer
(20 mM Tris, pH 7.5, 1 mM EGTA) and
injected onto a Mono-Q HR 5/5 FPLC column
(Pharmacia).

The FPLC run was performed at 18 °C at 1 ml/min and

1-ml fractions were collected
with a linear salt gradient from 0 to 1 M KCl over 71 ml.

The fractions containing proteins (determined by A280) were

dialyzed into PGB and
tested in the cracked cell assay.

Western Blot

Anti-calmodulin antibody and anti-PKC antibody were used, and

ECL (Amersham) was used for detection.

RESULTS

A Membrane EGTA Extract Supports NE Release

Brain cytosol, prepared as the supernatant of the brain homogenate,

effectively stimulates NE release
in the cracked cell assay (Fig. 1)
as previously shown (9).

We wondered whether crude extracts other than cytosol

could support NE release, and we focused on
extractable peripheral membrane proteins.

We found that a salt or EGTA extract of brain membranes,
membranes defined as the

100,000 3 g pellet of the crude homogenate,
reconstituted secretion in the absence of cytosol.
the salt extract only slightly enhanced NE release
above background (data not shown), the

EGTA extract not only stimulated NE release to a high level,

similar to that supported by cytosol, but also
had a higher specific activity than cytosol (Fig. 1).

Fig. 1 EGTA extract can support NE release_page_003_edited-3

FIG. 1. The EGTA extract of brain membranes can support NE release in the absence of cytosol. Rat brain membrane EGTA extract (closed triangles) and rat brain cytosol (closed squares) were prepared as described under “Experimental Procedures.” NE release was measured in a composite reaction mixture of cracked cells, MgATP, Ca2+, and the indicated amount of crude extracts.

The ability of the membrane EGTA extract to support secretion is consistent with the fact that

following cracking, the cells are immediately extracted with EGTA, and are presumably
devoid of most membrane EGTA-extractable factors.

This also suggests that these factors, some of which are probably

Ca2+-dependent membrane-associating proteins,
participate in Ca2+- triggered exocytosis.

The Membrane EGTA Extract Is Enriched in Triggering Fators

NE release in cracked cells can be resolved into two sequential stages,

an ATP-dependent priming stage and
an ATP-independent Ca21-dependent triggering stage (11), and
proteins can be tested for activity in either stage.

An effect in priming indicates

an early role for the protein, and
an effect in triggering a late ATP-independent role.

Since the protein composition of the

membrane EGTA extract and cytosol are different,

we tested whether they had different activities

in the priming stage versus the triggering stage.

We found that the membrane EGTA extract is enriched in factors that

act during triggering stage of NE release, as
the same amount of protein from the membrane EGTA extract as cytosol
gave a higher stimulation in the triggering assay, but
not in the priming assay (Fig. 2A).

Regular cytosol is prepared in a buffer containing 2 mM EGTA, and thus

presumably contains some of the proteins present in the membrane EGTA extract.

Cytosol prepared in the absence of EGTA showed an even lower specific activity

in the triggering assay compared with regular cytosol (Fig. 2A).

Identification of Calmodulin as an Active Triggering Factor in the EGTA Extract

Biochemical fractionation of the bovine brain membrane EGTA extract was carried out

to identify the active components capable of reconstituting NE release.

Activity was assayed in a composite reaction mixture containing

cracked cells,
ATP,
Ca2+, and
the test protein(s).

Except for the presence of bovine serum albumin in the basal buffer,

no other proteins were added to the cell ghosts except for the test protein(s).

Initial tests indicated that at least

part of the activity in the membrane EGTA extract binds to and
can be efficiently eluted from an anion exchanger and hydroxyapatite resin,
but does not bind to Amicon color resins.

The starting material was, therefore, sequentially purified using

DEAE, Affi-Gel Blue (or Matrex Green-A), and gel filtration chromotography.

Gel filtration fractionation indicated the presence of two peaks of activity with

predicted molecular masses of 25 and 85 kDa, respectively (Fig. 3).

FIG. 3. Gel filtration chromatography reveals two stimulatory factors in the membrane EGTA extract.

In order to purify the active component(s) in the membrane EGTA extract, the crude extract from one bovine brain was fractionated chromatographically (see Experimental Procedures” for details). Fractions from a Sephacryl S-200 gel filtration column were tested for their activity in stimulating NE release in the composite assay. The two activity peaks have predicted molecular masses of 85 and 25 kDa, respectively. The arrows indicate the retention volume of standard proteins run on the same column.

The low molecular weight active factor was purified to homogeneity, as judged by a

Coomassie-stained SDS-PAGE gel, after a subsequent Mono-Q fractionation (Fig. 4).

FIG. 4. The low molecular wen.ight active factor is calmodulin

A, the membrane EGTA extract from one bovine brain (Start) was subjected to sequential fractionation on DEAE, Blue A, and
Sephacryl S-200 columns. The pooled material containing the activity after each chromotographic step was analyzed by SDS-
PAGE and Coomassie staining. The arrowheads indicate the presence of calmodulin in all the lanes. Calmodulin shows a
mobility shift depending on whether or not Ca2+ is present during electrophoresis (see panel C).
B, the active material pooled from Sephacryl S-200 was fractionated on a Mono-Q FPLC column and the fractions
(5 ml/fraction) were tested for activity in a composite assay. The activity peak is shown.
C, the active Mono-Q fractions (5 ml/fraction) were subjected to SDS-PAGE in the presence of 1 mM EGTA or 0.1 mM Ca2+,
and the gels stained with Coomassie Blue.
D, fraction 47 (1 ml) was probed by Western blotting with a monoclonal anti-calmodulin antibody. No Ca2+ or EGTA was
added during SDS-PAGE.

We reasoned that the protein might be calmodulin (43) based on the following:

1) It is a relatively small protein (14–18 kDa) that is abundant in the
starting extract (Fig. 4A).
2) It elutes at a very high salt concentration (0.41 M KCl) on the
Mono-Q column.
3) It stains negatively in silver stain (data not shown).
4) Its electrophoretic mobility shifts depending on the presence or
absence of Ca21 (Fig. 4C).

A Western blot with an anti-calmodulin monoclonal antibody gave a
positive signal (Fig. 4D), confirming our prediction.

Properties of Calmodulin-stimulated Exocytosis

We used commercial calmodulin or bacterially expressed recombinant calmodulin to confirm our purification result; both sources of authentic calmodulin stimulated NE release as expected. Moreover, we found that calmodulin stimulates secretion in a triggering assay as well as in a composite assay (Fig. 5A).

The half-maximal increase was at 75 nM (250 ng/200 ml) final calmodulin concentration. This is within the broad
range of affinities between calmodulin and its various targets and suggests that the interaction between
calmodulin and its target molecule in exocytosis is in the physiological range. When the triggering reaction was
performed at different Ca2+ concentrations, calmodulin increased NE release only at high [Ca2+] (0.4 – 2 mM)
similar to the crude EGTA extract (Fig. 5B),

suggesting that calmodulin contributes to the triggering activity of the membrane EGTA extract. Calmodulin’s affinity for Ca2+ has
been reported to be around 1 mM (25),

consistent with the Ca2+ requirement for
calmodulin-stimulated secretion that we observed.

FIG. 5. Calmodulin stimulates NE release in the triggering stage.
A, calmodulin (obtained from Sigma) increased NE release in the
triggering assay in a dose-dependent fashion, in the absence of ATP
or any other cytosolic proteins. In this particular experiment, the
maximal release achieved by addition of rat brain cytosol was 46.5%.

B, the triggering assay was performed with different concentrations
of free Ca2+. Calmodulin (3 mg bacterially expressed recombinant
protein; closed squares) increased NE release with a similar Ca2+
sensitivity to rat brain membrane EGTA extract (10 mg; closed
triangles), as compared with conditions in which no protein was
added (open squares).

Western analysis with commercial protein as standards indicated that calmodulin

constitutes about 5% of total proteins in the rat brain membrane EGTA extract
and about 2% of total proteins in the rat brain cytosol (data not shown).

In addition, a significant amount of calmodulin appears to be left

in the washed cell ghosts (data not shown).

Based on the activity of saturating levels of

pure calmodulin (releasing 6–10% of total [3H]NE)
and crude EGTA extract (releasing ;45% of total [3H]NE),

we estimated that

calmodulin accounts for 13–22% of total activity of the extract.

Consistent with this,

a high affinity calmodulin-binding peptide
(CaMKIIa(291–312) (44), used at 5 mM) and
an anti-calmodulin antibody (2 mg/200 ml)
inhibited about 20% of the membrane EGTA extract-stimulated release
(6.7 mg of extract added; data not shown).

We showed that calmodulin increased NE release

in the triggering stage.

Since regular triggering reactions were performed

in the absence of any added ATP,

this suggests that

calmodulin enhanced secretion in an ATP-independent fashion.

Furthermore, residual ATP in the cell ghosts did not play a role, since

addition of a hexokinase ATP depletion system that
can deplete millimolar concentrations of ATP
- within a few minutes (11) had little effect, as did
- addition of 5 mM AMPPNP,
which blocks ATP-dependent enzymatic activity (Fig.8A).

Therefore, we ruled out the possibility that a kinase mediates calmodulin’s effect.

FIG. 8. PKC and calmodulin stimulate the late triggering reaction in
an ATP-dependent and ATP-independent manner respectively.
A, triggering assays were performed to test the activity of calmodulin
(recombinant; black bars) and PKC (purified rat brain PKC from
Calbiochem; shaded bars) in the absence of ATP. A regular triggering
assay is done in the absence of ATP (2ATP). To deplete residual ATP
in the cells, hexokinase-based ATP depletion was employed (1Hexo).
Alternatively, 5 mM AMP-PNP (1AMP-PNP) was added in the triggering
reaction. Under all three conditions, calmodulin increased release
as compared with the background (buffer only; white bars), whereas
PKC did not.
B, NE release in a composite assay was measured with varying
concentrations of free Ca2+ in the presence of 10 mg of calmodulin
(recombinant; closed triangles), 70 ng of PKC (purified rat brain PKC
from Calbiochem; closed squares), or buffer only (open squares).

A series of calmodulin mutants from Paramecium and chicken were tested

for their ability to enhance Ca2+-stimulated secretion, and
none of the mutations abolished the calmodulin effect (data not shown).

These mutations include

S101F, M145V, E54K, G40E/D50N, V35I/D50N within Paramecium
calmodulin (45), and M124Q, M51A/V55A, and M51A/V55A/L32A
within chicken calmodulin (46, 47).

The Paramecium calmodulin mutants are the result of

naturally occurring mutations that result in aberrations in their behavior.

These mutants can be grouped into two categories according to their
behavior, reflecting their loss of either

a Ca2+-dependent Na1 current
(calmodulin N-terminal lobe mutants: E54K, G40E/D50N, and
V35I/D50N) or
a Ca21-dependent K1 current
(calmodulin C-terminal lobe mutants: S101F and M145V) (45).

The chicken calmodulin mutants have been shown to

differentially activate myosin light chain kinase
(M124Q, M51A/V55A, and M51A/V55A/L32A),
CaMKII (M124Q), and CaMKIV (M124Q),

and the mutated residues are thought to be important in

defining calmodulin’s binding specificity (46, 47).

Our finding that these mutant calmodulins can stimulate exocytosis suggests that

calmodulin-binding domains similar to those of Paramecium Ca2+/calmodulin-dependent
ion channels, myosin light chain kinase, CaMKII, and CaMKIV,
are unlikely to mediate release utilizing the conserved SNARE fusion machinery, as they
could be completely abolished by addition of exogenous syntaxin H3 domains (data not shown).
the same molecular pathway was not activated, since their effects were additive (data not shown).

Acknowledgments—

We thank Diana Bautista and Dr. Richard S.Lewis for generous help

with [Ca21]free determination; Dr. Ching Kung for providing the Paramecium calmodulin

mutants, and Dr. Anthony R. Means for providing the chicken calmodulin mutants. We also

thank Dr. Jesse C. Hay for the initial setup of the cracked cell assay, and Dr. Suzie J.

Scales for helpful comments on the manuscript.

REFERENCES

1. Calakos, N., and Scheller, R. H. (1996) Physiol. Rev. 76, 1–29

2. Su¨ dhof, T. C. (1995) Nature 375, 645–653

3. Zucker, R. S. (1996) Neuron 17, 1049–1055

4. Hanson, P. I., Heuser, J. E., and Jahn, R. (1997) Curr. Opin. Neurobiol. 7, 310–315

5. Chen, Y. A., Scales, S. J., Patel, S. M., Doung, Y.-C., and Scheller, R. H. (1999) Cell 97, 165–174

6. Neher, E., and Zucker, R. S. (1993) Neuron 10, 21–30

7. Kamiya, H., and Zucker, R. S. (1994) Nature 371, 603–606

SOURCE

J Biol Chem 1999; 274(37): 25469-26476.

ION CHANNEL and Cardiovascular Diseases

http://pharmaceuticalintelligence.com/?s=Ion+Channel

Calcium Role in Cardiovascular Diseases

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-
that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and 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/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease
Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-
exchange-mechanism-in-health-and-disease/

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-
involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-
post-ischemic-arrhythmia-similarities-and-differen/

Part V: Ca2+-Stimulated Exocytosis: The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP and 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-exocytosis/

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary

Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-
for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure –
Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and 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/

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells:
The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-
muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction
(Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-
and-calcium-release-related-contractile-dysfunction-ryanopathy/

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of
vesicles with cell membranes during Neurotransmission
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-
regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Part XI: Sensors and Signaling in Oxidative Stress
Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/