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How Protein Networks Stabilize Muscle

Fibers: Same Mechanism Known for DNA

Now Found for Muscle Proteins The same mechanism that stabilises the DNA in the cell nucleus is also important for the

structure and function of vertebrate muscle cells. This has been established by RUB-researchersled by Prof. Dr. Wolfgang Linke (Institute of Physiology) in cooperation with American and

German colleagues. An enzyme attaches a methyl group to the protein Hsp90, which then forms

a complex with the muscle protein titin. When the researchers disrupted this protein network through genetic manipulation in zebrafish the muscle structure partly disintegrated. The scientists

have thus shown that methylation also plays a significant role outside the nucleus.

They published their results in Genes and Development.

<<< Protein complexes in a muscle cell:

 Myofibrils are the building blocks of muscle

cells which show a regular cross-striated 

 pattern (blue). These cells are elastic owing to

the presence of the giant protein titin (red) in the

myofibrils. Methylated heat shock protein Hsp90

binds together with the methyltransferase Smyd2

(green) to the elastic titin springs and stabilises

them. The bottom right panel shows the co-

localisation of Smyd2 and the elastic titin

region. (Credit: Illustration: Prof. Wolfgang A.

 Linke)

Methylation in the nucleus

Enzymes, called methyltransferases, transfer methyl (CH3) groups to specific sections of theDNA in the nucleus. In this way, they mark active and inactive regions of the genes. However,

not only DNA but also nuclear proteins incur methylation, mostly at the amino acid lysine.

Methylated lysines on nuclear proteins promote the formation of protein complexes that control,for example, DNA repair and replication. However, methyltransferases are not only found in the

nucleus, but also in the cellular fluid (cytoplasm). Yet, it is not well established which proteins

they methylate in the cytoplasm and how this methylation may affect function.

Shown for the first time: methylation in the cytoplasm promotes protein complex

formation

The researchers first identified an enzyme which is mainly present in the cytoplasm and whichmethylates the amino acid lysine (Smyd2). Then they searched for interaction partners of the

enzyme Smyd2 and found the heat shock protein Hsp90. The scientists went on to show that

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Smyd2 and methylated Hsp90 form a complex with the muscle protein titin. "Titin is the largest

protein in the human body and known primarily for its role as an elastic spring in muscle cells"explains Linke. "Precisely this elastic region of titin is protected by the association with

methylated Hsp90."

Titin requires protection by methylated proteins

In skeletal muscle cells of the zebrafish, Linke's team explored what happens when the

protection by the methylated heat shock protein is repressed. By genetic manipulation theyaltered the organism in such a way that it no longer produced the enzyme Smyd2, which blocked

the methylation of Hsp90. Without methylated Hsp90, the elastic titin region was unstable and

muscle function strongly impaired; the regular muscle structure was partially disrupted.

Strong Communication Between Brain and

Muscle Requires Both Having the ProteinLRP4 

Communication between the brain and muscle must be strong for us to eat, breathe or walk. Now

scientists have found that a protein known to be on the surface of muscle cells must be present in both

tissues to ensure the conversation is robust.

Scientists at the Medical College of Georgia at Georgia Health Sciences University have shown

that without LRP4 in muscle cells and neurons, communication between the two cells types is

inefficient and short-lived.

Problems with the protein appear to contribute to disabling disorders such as myasthenia gravis

and other forms of muscular dystrophy. The MCG scientists reported finding antibodies to LRP4in the blood of about 2 percent of patients with muscle-degenerating myasthenia gravis in

 Archives of Neurology earlier this year.

Scientists know that LRP4 plays an important role in the muscle cell, where it receives cues fromthe brain cell that it's time to form the receptors that will be enable ongoing communication

between the two, said Dr. Lin Mei, Director of the GHSU Institute of Molecular Medicine and

Genetics and corresponding author of the study in the journal  Neuron.

However when Dr. Haitao Wu deleted LRP4 just from muscle cells, a connection -- albeit a

weak one -- still formed between muscle and brain cells. The mice survived several days duringwhich they experienced some of the same muscle weakness as patients with myasthenia gravis."That's against the dogma," Mei said. "If LRP4 is essential only in the muscle cells, how could

the mice survive?" When they totally eliminated LRP4, neuromuscular junctions never formed

and the mice didn't survive.

Additional evidence suggests that LRP4 in the neurons is vital, said Wu, postdoctoral fellow and

the study's first author. "When we knocked out the LRP4 gene in the muscles, there was some

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redundant function coming from the motor neuron, like a rescue attempt," he said. They

documented the neuron reaching out to share LRP4 with the muscle cell. Unfortunately, thegesture was not sufficient.

"The nerve does not get the stop signal," Mei said, referencing images of too-long neurons that

never got the message from the muscle that they have gone far enough. When they cut theelongated nerves, they found they didn't contain enough vesicles, little packages of chemical

messengers that are the hallmark of brain cell communication. On the receiving end, muscle cells

developed receptors that were too small and too few -- hence, the tenuous communicationnetwork. "When LRP4 in the muscle is taken out, not surprisingly, the muscle has some kind of a

problem," Mei said. "What was very surprising was that the motor neurons also have problems."

"The talk between motor neurons and muscle cells is very critical to the synapse formation

and the very precise action between the two," Wu said. Mei's lab earlier established that the

conversation goes both ways.

The scientists believe about 60 percent of the LRP4 comes from muscle cells, about 20 percentfrom brain cells -- which helps explain why the brain's effort to share is insufficient -- and the

remainder from cells in spaces between the two. In addition to better explaining nerve-musclecommunication, the scientists hope their findings will eventually enable gene therapy that

delivers LRP4 to bolster insufficient levels in patients.

Other early and key players in establishing nerve-muscle conversation include agrin, a proteinthat motor neurons release to direct construction of the synapse, a sort of telephone line between

the nerve and muscle. MuSK on the muscle cell surface initiates critical internal cell talk so

synapses can form and receptors that enable specific commands will cluster at just the right spot.Mei's lab reported in Neuron in 2008 that agrin starts talking with LRP4 on the muscle cell

surface, then recruits the enzyme MuSK to join the conversation. LRP4 and MuSK become

major components of the receptor needed for the muscle cell to receive the message agrin is

sending.

The agrin-MuSK signaling pathway has been implicated in muscular dystrophy, a group of 

genetic diseases that lead to loss of muscle control because of problems with neurons, musclecells and/or their communication. Some reports have implicated a mutant MuSK as a cause of 

muscular dystrophy and autoantibodies (antibodies the body makes against itself) to MuSK have

been found in the blood of some patients.

New Clue To Muscular Dystrophy

Uncovered: Mediator In Communication

Between Neurons And Muscle Cells Found 

A missing piece of the puzzle of how neurons and muscle cells establish lifelong communication has

been found by researchers who suspect this piece may be mutated and/or attacked in muscular

dystrophy.

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Agrin is a protein that motor neurons release to direct construction of the nerve-muscle contact

or synapse. MuSK on the muscle cell surface initiates critical internal cell talk so synapses canform and receptors that enable specific commands will cluster at just the right spot.

The conundrum was agrin and MuSK don't directly communicate, explains Dr. Lin Mei, chief of 

developmental neurobiology at the Medical College of Georgia and Georgia Research AllianceEminent Scholar in Neuroscience.

Much as a friend gets two other friends together, MCG researchers have found that agrin startstalking with LRP4, a protein also on the muscle cell surface, then recruits MuSK to join the

conversation. LRP4 and MuSK become major components of the receptor needed for the muscle

cell to receive the message agrin is sending, says Dr. Bin Zhang, postdoctoral fellow in Dr. Mei'slab and first author on the paper published in the Oct. 23 issue of the journal Neuron.

"We know agrin is important and we know MuSK is important. The puzzle or greatest gap in this

field for a long time is there was no evidence these two would shake hands but they must have

each other," says Dr. Mei, corresponding author. "This paper shows that this protein, LRP4,forms a nice bridge between the two and solves this long puzzle."

The agrin-MuSK signaling pathway has been implicated in muscular dystrophy, a group of 

genetic diseases that lead to loss of muscle control because of problems with neurons, muscle

cells and/or their communication. Some reports have implicated a mutant MuSK as a cause of 

muscular dystrophy and autoantibodies (antibodies the body makes against itself) to MuSK havebeen found in the blood of some patients.

Now that researchers know LRP4's critical role, they will look in patient samples where theysuspect they also will find mutant forms and autoantibodies to it as well, Dr. Zhang says. They'll

also look to see if LRP4 has a role in later development and maintenance of the neuromuscular

 junction.

In research published in the Oct. 9 issue of Neuron, the same research team used the

neuromuscular junction to identify Hsp90β as a stabilizing protein that helps receptors on muscle

cells get and stay where needed to catch a neuron's signal. This process showed the need forteamwork as well since rapsyn, a protein responsible for anchoring the receptor, is oddly

unstable. They showed Hsp90β gives rapsyn needed stability. 

Unusual Alliances Enable Movement 

Some unusual alliances are necessary for you to wiggle your fingers, researchers report. Understanding

those relationships should enable better treatment of neuromuscular diseases, such as myasthenia

gravis, which prevent muscles from taking orders from your brain, said Dr. Lin Mei, Director of the

Institute of Molecular Medicine and Genetics at Georgia Health Sciences University.

During development, neurons in the spinal cord reach out to muscle fibers to form a direct line of communication called the neuromuscular junction. Once complete, motor neurons send chemical

messengers, called acetylcholine, via that junction so you can text, walk or breathe.

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As a first step in laying down the junction, motor neurons release the protein agrin, which

reaches out to LRP4, a protein on the muscle cell surface. This activates MuSK, an enzyme thatsupports the clustering of receptors on the muscle cell surface that will enable communication

between the brain and muscle. The precise alignment between the neuron and muscle cell that

occurs during development ensures there is no confusion about what the brain is telling the

muscle to do.

A missing piece was how agrin and LRP4 get together.

A study published in the journal Genes & Development shows that in the space between the

neuron and its muscle cell, agrin and LRP4 first form two diverse work teams: each team has one

agrin and one LRP4. The two teams then merge to form a four-molecule complex essential toMuSK activation and to the clustering of receptors that will receive the chemical messenger

acetylcholine on the muscle cell.

It was expected that the two agrins would get together first then prompt the LRP4s to merge.

"This is very novel," said Mei, and an important finding in efforts to intervene in diseases thatattack the neuromuscular junction.

Mei and Dr. Rongsheng Jin, neuroscientist and structural biologist in the Del E. Webb

Neuroscience, Aging and Stem Cell Research Center at Sanford-Burnham Medical Research

Institute in La Jolla, Calif., are co-corresponding authors of the study.

Myasthenia gravis, which paralyzes previously healthy individuals, targets these protein workers.

The condition, which can run in families, likely results from a process called mimicry in which

the immune system starts making antibodies to the workers, which it confuses with a previousviral or bacterial infection. The majority of patients have antibodies to acetylcholine receptors

and a smaller percentage have antibodies to MuSK. Most recently, GHSU researchers also

helped identify LRP4 as an antibody target.

The scientists already are looking at the impact of the antibodies on the LRP4 complex.

Understanding its unique structure is essential to designing drugs that could one day block such

attacks. "Prior to this we had no idea how they interacted," Mei said.

In addition to providing new information on muscle diseases, this study might also have a far-

reaching ripple effect in the field of neuroscience.

"This is just the beginning," says Jin. "Now that we know more about how signals are transferred

during the formation of neuromuscular junctions, we can start looking at how a similar system

might work in brain synapses and how it malfunctions in neurodegenerative conditions likeAlzheimer's and Parkinson's diseases. If we can figure out how to trigger the formation of new

brain synapses, maintain old synapses, or simply slow their disappearance, we'd be much better

equipped to prevent or treat these diseases."

To reveal the novel mechanism, researchers used a technique known as X-ray crystallography,

which produces 3-D "pictures" of protein at the atomic level using powerful X-ray beams.

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Boosting Supply of Key Brain Chemical

Reduces Fatigue in Mice 

Researchers at Vanderbilt University have "engineered" a mouse that can run on a treadmill twice aslong as a normal mouse by increasing its supply of acetylcholine, the neurotransmitter essential for

muscle contraction.

The finding, reported this month in the journal Neuroscience, could lead to new treatments for

neuromuscular disorders such as myasthenia gravis, which occurs when cholinergic nerve signalsfail to reach the muscles, said Randy Blakely, Ph.D., director of the Vanderbilt Center for

Molecular Neuroscience.

Blakely and his colleagues inserted a gene into mice that increased the production of a proteincalled the choline transporter at the neuromuscular junction.

The choline transporter is vital to the capacity for muscle contraction -- including the ability to

breathe -- because it regulates the supply of choline, the precursor to acetylcholine. "Wereasoned that giving more of this protein might enhance muscle function and reduce nerve-

dependent fatigue," Blakely said.

Other researchers have manipulated the gene for the muscle tissue growth factor myostatin to

produce animals with greater strength and endurance, but Blakely said this may be the first time

"neural endurance" was enhanced by manipulating the nerves that innervate muscle.

Drugs that increase choline transporter activity "could represent a novel therapeutic strategy" for

myasthenia gravis and a wide range of other disorders that involve cholinergic signaling deficits,

Blakely said.

These disorders include muscular dystrophy, congestive heart failure, depression, schizophrenia,

Alzheimer's disease and attention-deficit hyperactivity disorder (ADHD). "The brain usesacetylcholine for a wide variety of functions, including the ability to sustain attention," Blakely

noted.

Last year, Blakely and his colleagues reported that a variation in the choline transporter gene isassociated with the "combined" type of ADHD, which is characterized by both inattention and

hyperactivity/impulsivity.

With funding from the National Institutes of Health (NIH), the researchers are developingcholine transporter-targeted agents that could lead to new medications for these conditions.

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Mechanism For Regulation Of Growth And

Differentiation Of Adult Muscle Stem Cells Is

Revealed During muscle regeneration, which is a natural response to injury and disease, environmental cues cause

adult muscle stem cells (satellite cells) to shift from dormancy to actively building new muscle tissue.

Although the signaling pathways controlling muscle regeneration are fairly well known, how

these signals lead to altered chromatin structure remains undiscovered.

A group of scientists at the Burnham Institute for Medical Research in La Jolla, CA, analyzed themechanism by which certain cellular signaling cues cause epigenetic modifications when

released within the regenerative microenvironment, thus controlling the expression of genes that

regulate growth and differentiation of muscle stem cells that repair injured muscle.

In a recent publication in Molecular Cell, the scientific group, led by Pier Lorenzo Puri, MD,

Ph.D., shows how two signaling pathways, PI3K/AKT and p38, work together to assemble

components of the protein complexes responsible for muscle-specific transcription, and howeach pathway is responsible for a distinct step in the transcription process. Additionally, the team

was able to pharmacologically separate these two steps, showing that selective interference with

either cascade leads to incomplete assembly of protein complexes, thus preventing muscle-specific gene expression. The results point to possible pharmacological avenues for selective

control of gene expression in adult muscle stem cells that may have therapeutic potential in

regenerative medicine.

Peak Muscle Performance Requires Two

Forms of nNOS Protein 

The protein nNOS-mu, which is just one form of the nNOS protein, is essential for skeletal muscle health,

and signaling via nNOS-mu is commonly reduced in neuromuscular disease. Now, Justin Percival and

colleagues, at the University of Washington, Seattle, have identified a crucial role for the nNOS-beta

form of nNOS in mouse skeletal muscle, where it enables skeletal muscle to maintain force production

during and after exercise.

In the study, mouse muscles lacking both nNOS-beta and nNOS-mu were found to be smaller inmass, to be intrinsically weaker, to be more susceptible to fatigue, and to exhibit more markedpostexercise weakness than mouse muscles lacking only nNOS-mu.

The specific function of nNOS-beta was determined to be regulation of skeletal muscle structural

and functional integrity. By contrast, previous data have indicated that nNOS-mu helps matchblood supply with the metabolic demands of active muscle. These distinct roles for nNOS-beta

and nNOS-mu are likely a result of the fact that the authors found the two proteins at distinct

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locations within skeletal muscle cells; the former was localized to the Golgi apparatus and the

latter to the sarcolemma. These data indicate that nNOS proteins are critical regulators of skeletalmuscle exercise performance.

The research appears in the Journal of Clinical Investigation.

Nitric oxide synthases (EC 1.14.13.39) (NOSs) are a family of  enzymes that catalyze theproduction of  nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule,

having a vital role in many biological processes. It is the intercellular signal that controls

vascular tone (hence blood pressure), insulin secretion, airway tone, and peristalsis, and isinvolved in angiogenesis (growth of new blood vessels) and in the development of nervous

system. It is believed to function as a retrograde neurotransmitter and hence is likely to be

important in learning. Nitric oxide signalling is mediated in mammals by the calcium / calmodulin 

controlled isoenzymes eNOS (endothelial NOS) and nNOS (neuronal NOS); the inducibleisoform iNOS is involved in immune response, binds calmodulin at all physiologically relevant

concentrations, and produces large amounts of NO as a defense mechanism. It is the proximate

cause of  septic shock  and may play a role in many diseases with an autoimmune etiology.

The canonical reaction catalyzed by NOS is:

  L-arginine + 3/2 NADPH + H+ + 2 O2 = citrulline + nitric oxide + 3/2 NADP+ 

NOS isoforms catalyze many other leak and side reactions such as superoxide production at the

expense of NADPH, so this stoichiometry is not generally observed. The unusual stoichiometry

reflects the three electrons supplied per NO by NADPH; NO is a free radical with an unpaired

electron.

NOSs are unusual in that they require five cofactors. Eukaryotic NOS isozymes are catalyticallyself-sufficient. The electron flow in the NO synthase reaction is: NADPH --> FAD --> FMN -->

heme --> O2. Tetrahydrobiopterin provides an additional electron during the catalytic cycle

which is replaced during turnover. ). NOS is the only known enzyme that binds flavin adenine

dinucleotide (FAD), flavin mononucleotide (FMN), heme, tetrahydrobiopterin (BH4) andcalmodulin. 

Species distribution

Arginine-derived NO synthesis has been identified in mammals, fish, birds, invertebrates, and

bacteria.[2]

 Best studied are mammals, where three distinct genes encode NOS isozymes: 

neuronal (nNOS or NOS-1), cytokine-inducible (iNOS or NOS-2) and endothelial (eNOS orNOS-3).[3] iNOS and nNOS are soluble and found predominantly in the cytosol, while eNOS is

membrane associated. Evidence has been found for NO signaling in plants, but plant genomes

are devoid of homologs to the superfamily which generates NO in other kingdoms.

Function

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In mammals, the endothelial isoform is the primary signal generator in the control of vascular

tone, insulin secretion, and airway tone, is involved in regulation of cardiac function andangiogenesis (growth of new blood vessels). NO produced by eNOS has been shown to be a

vasodilator identical to the endothelium-derived relaxing factor ( see nitric oxide (NO) ),

produced in response to shear from increased blood flow in arteries. This dilates blood vessels by

relaxing smooth muscle in their linings. ENOS is the primary controller of smooth muscle tone.NO activates guanylate cyclase, which induces smooth muscle relaxation by:

  Increased intracellular cGMP, which inhibits calcium entry into the cell, and decreases

intracellular calcium concentrations

  Activation of K+ channels, which leads to hyperpolarization and relaxation

  Stimulates a cGMP-dependent protein kinase that activates myosin light chain phosphatase, the

enzyme that dephosphorylates myosin light chains, which leads to smooth muscle relaxation.

The neuronal isoform is involved in the development of nervous system. It functions as a

retrograde neurotransmitter important in long term potentiation and hence is likely to be

important in memory and learning. NNOS has many other physiological functions, includingregulation of cardiac function and peristalsis and sexual arousal in males and females. An

alternatively spliced from of nNOS is a major muscle protein that produces signals in response to

calcium release from the SR. The primary receiver for NO produced by eNOS and nNOS is

soluble guanylate cyclase, but many secondary targets have been identified. S-nitrosylationappears to be an important mode of action. The inducible isoform iNOS produces large amounts

of NO as a defense mechanism. It is synthesized by many cell types in response to cytokines and

is an important factor in the response of the body to attack by parasites, bacterial infection, andtumor growth. It is also the cause of septic shock and may play a role in many diseases with an

autoimmune etiology. NOS signaling is involved in development and in fertilization in

vertebrates. It has been implicated in transitions between vegetative and reproductive states in

invertebrates, and in differentiation leading to spore formation in slime molds. NO produced bybacterial NOS is protective against oxidative damage.

Classification

Different members of the NOS family are encoded by separate genes.[4]

 NOS is one of the mostregulated enzymes in biology. There are three known isoforms, two are constitutive (cNOS) andthe third is inducible (iNOS).[5] Cloning of NOS enzymes indicates that, cNOS include both

brain constitutive (NOS1) and endothelial constitutive (NOS3), the third is the inducible (NOS2) 

gene.[5]

 Recently, NOS activity has been demonstrated in several bacterial species, including

such notorious pathogens as Bacillus anthracis and Staphylococcus aureus.[6]

 

The different forms of NO synthase have been classified as follows:

Name  Gene(s)  Location  Function 

Neuronal NOS 

(nNOS or NOS1) 

NOS1,

Chromosome

12

  nervous tissue 

  skeletal muscle type

  cell communication 

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II 

Inducible NOS 

(iNOS or NOS2) 

NOS2,

Chromosome

17

  immune system 

  cardiovascular

system 

  immune defense against

pathogens 

Endothelial NOS 

(eNOS or NOS3 or

cNOS) 

NOS3,

Chromosome 7 

  endothelium    vasodilation 

Bacterial NOS 

(bNOS) 

multiple   various Gram-

positive bacteria 

  defense against oxidative

stress, antibiotics, 

immune attack 

nNOS

Neuronal NOS (nNOS) produces NO in nervous tissue in both the central and peripheral nervoussystem. The gene coding for nNOS is located on Chromosome 12.[7] Neuronal NOS also

performs a role in cell communication and is associated with plasma membranes. nNOS action

can be inhibited by NPA (N-propyl-L-arginine). This form of the enzyme is specifically inhibited

by 7-nitroindazole.[8]

 

The subcellular localisation of nNOS in skeletal muscle is mediated by anchoring of nNOS to

dystrophin. nNOS contains an additional N-terminal domain, the PDZ domain.[9]

 

iNOS

As opposed to the critical calcium-dependent regulation of constitutive NOS enzymes (nNOSand eNOS), iNOS has been described as calcium-insensitive, likely due to its tight non-covalentinteraction with calmodulin (CaM) and Ca2+. The gene coding for iNOS is located on

Chromosome 17.[7] While evidence for ‘baseline’ iNOS expression has been elusive, IRF1 and

NF-κB-dependent activation of the inducible NOS promoter supports an inflammation mediated

stimulation of this transcript.

Induction of the high-output iNOS usually occurs in an oxidative environment, and thus high

levels of NO have the opportunity to react with superoxide leading to peroxynitrite formationand cell toxicity. These properties may define the roles of iNOS in host immunity, enabling its

participation in anti-microbial and anti-tumor activities as part of the oxidative burst of 

macrophages.[10]

 

It has been suggested that pathologic generation of  nitric oxide through increased iNOS 

production may decrease tubal ciliary beats and smooth muscle contractions and thus affectembryo transport, which may consequently result in ectopic pregnancy.[11] 

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eNOS

Main article: Endothelial NOS 

Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3), generates NO in bloodvessels and is involved with regulating vascular function. Gene coding for eNOS is located on

Chromosome 7.[7]

 A constitutive Ca2+

dependent NOS provides a basal release of NO. eNOS isassociated with plasma membranes surrounding cells and the membranes of Golgi bodies within

cells. eNOS localisation to endothelial membranes is mediated by cotranslational N-terminal

myristoylation and post-translational palmitoylation.[12]

 

bNOS

Bacterial NOS (bNOS) has been shown to protect bacteria against oxidative stress, diverse

antibiotics, and host immune response. bNOS plays a key role in the transcription of  superoxide

dismutase (SodA). Bacteria late in the log phase who do not possess bNOS fail to upregulateSodA, which disables the defenses against harmful oxidative stress. Initially, bNOS may have

been present to prepare the cell for stressful conditions but now seems to help shield the bacteriaagainst conventional antimicrobials. As a clinical application, a bNOS inhibitor could be

produced to decrease the load of Gram positive bacteria.[13][14]

 

Chemical reaction

Nitric oxide synthases produce NO by catalysing a five-electron oxidation of a guanidino

nitrogen of L-arginine (L-Arg). Oxidation of L-Arg to L-citrulline occurs via two successive

monooxygenation reactions producing N ω-hydroxy-L-arginine (NOHLA) as an intermediate.

2 mol of O2 and 1.5 mol of NADPH are consumed per mole of NO formed.[2] 

Structure

The enzymes exist as homodimers. In eukaryotes, each monomer consisting of two major

regions: an N-terminal oxygenase domain, which belongs to the class of heme-thiolate proteins,

and a multi-domain C-terminal reductase, which is homologous to NADPH:cytochrome P450reductase (EC 1.6.2.4) and other flavoproteins. The FMN binding domain ins homologous to

flavodoxins, and the two domain fragment containing the FAD and NADPH binding sites is

homologous to flavodoxin-NADPH reductases. The interdomain linker between the oxygenase

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See also  Biological functions of nitric oxide 

References

1. 

^ PDB 3N5P; Delker SL, Xue F, Li H, Jamal J, Silverman RB, Poulos TL (December 2010). "Role of zinc in isoform-selective inhibitor binding to neuronal nitric oxide synthase". Biochemistry  49 

(51): 10803 –10. DOI:10.1021/bi1013479. PMID 21138269. 

2.  ^ a b Liu Q, Gross SS (1996). "Binding sites of nitric oxide synthases". Meth. Enzymol.. Methods in

Enzymology 268: 311 –324. DOI:10.1016/S0076-6879(96)68033-1. ISBN 978-0-12-182169-2. 

PMID 8782597. 

3.  ^ Knowles RG, Moncada S (1994). "Nitric oxide synthases in mammals". Biochem. J. 298 (Pt 2):

249 –258. PMC 1137932. PMID 7510950. 

4.  ^ Taylor BS, Kim YM, Wang Q, Shapiro RA, Billiar TR, Geller DA (November 1997). "Nitric oxide

down-regulates hepatocyte-inducible nitric oxide synthase gene expression". Arch Surg 132 (11):

1177 –83. PMID 9366709. 

5.  ^ a b Stuehr DJ (May 1999). "Mammalian nitric oxide synthases". Biochim. Biophys. Acta 1411 (2 –

3): 217 –

30. DOI:10.1016/S0005-2728(99)00016-X. PMID 10320659. 

6.  ^ Gusarov I, Starodubtseva M, Wang ZQ, McQuade L, Lippard SJ, Stuehr DJ, Nudler E (May 2008).

"Bacterial Nitric-oxide Synthases Operate without a Dedicated Redox Partner".  J. Biol. Chem. 283 

(19): 13140 –7. DOI:10.1074/jbc.M710178200. PMC 2442334. PMID 18316370. 

7.  ^ a b c  [1] 

8.  ^ Southan GJ, Szabó C (February 1996). "Selective pharmacological inhibition of distinct nitric

oxide synthase isoforms". Biochem. Pharmacol. 51 (4): 383 –94. DOI:10.1016/0006-

2952(95)02099-3. PMID 8619882. 

9.  ^ Ponting CP, Phillips C (March 1995). "DHR domains in syntrophins, neuronal NO synthases and

other intracellular proteins". Trends Biochem. Sci. 20 (3): 102 –3. DOI:10.1016/S0968-

0004(00)88973-2. PMID 7535955. 

10. ^ Mungrue IN, Husain M, Stewart DJ (October 2002). "The role of NOS in heart failure: lessonsfrom murine genetic models". Heart Fail Rev  7 (4): 407 –22. DOI:10.1023/A:1020762401408. 

PMID 12379825. 

11. ^ Al-Azemi M, Refaat B, Amer S, Ola B, Chapman N, Ledger W (August 2010). "The expression of 

inducible nitric oxide synthase in the human fallopian tube during the menstrual cycle and in

ectopic pregnancy". Fertil. Steril. 94 (3): 833 –40. DOI:10.1016/j.fertnstert.2009.04.020. 

PMID 19482272. 

12. ^ Liu J, Hughes TE, Sessa WC (June 1997). "The First 35 Amino Acids and Fatty Acylation Sites

Determine the Molecular Targeting of Endothelial Nitric Oxide Synthase into the Golgi Region of 

Cells: A Green Fluorescent Protein Study".  J. Cell Biol. 137 (7): 1525 –35.

DOI:10.1083/jcb.137.7.1525. PMC 2137822. PMID 9199168. 

13. ^ Gusarov I, Nudler E (September 2005). "NO-mediated cytoprotection: Instant adaptation to

oxidative stress in bacteria". Proc. Natl. Acad. Sci. U.S.A. 102 (39): 13855 –60.

DOI:10.1073/pnas.0504307102. PMC 1236549. PMID 16172391. 

14. ^ Gusarov I, Shatalin K, Starodubtseva M, Nudler E (September 2009). "Endogenous Nitric Oxide

Protects Bacteria Against a Wide Spectrum of Antibiotics". Science 325 (5946): 1380 –4.

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  "RCSB Protein Data Bank - Structure Summary for 3N5P - Structure of endothelial nitric oxide

synthase heme domain complexed with 4-(2-(6-(2-(6-amino-4-methylpyridin-2-yl)ethyl)pyridin-

2-yl)ethyl)-6-methylpyridin-2-amine". 

Stem Cell Surprise For Tissue Regeneration Scientists working at the Carnegie Institution's Department of Embryology, with colleagues, have

overturned previous research that identified critical genes for making muscle stem cells. It turns out that

the genes that make muscle stem cells in the embryo are surprisingly not needed in adult muscle stem

cells to regenerate muscles after injury. The finding challenges the current course of research into

muscular dystrophy, muscle injury, and regenerative medicine, which uses stem cells for healing tissues,

and it favours using age-matched stem cells for therapy.

The study is published in the June 25 advance online edition of  Nature. 

Previous studies have shown that two genes Pax3 and Pax7, are essential for making the

embryonic and neonatal muscle stem cells in the mouse. Lead researcher Christoph Lepper, a

predoctoral fellow in Carnegie's Chen-Ming Fan's lab and a Johns Hopkins student, for the firsttime looked at these two genes in promoting stem cells at varying stages of muscle growth in live

mice after birth.

As Christoph explained: "The paired-box genes, Pax3 and Pax7 are involved in the development

of the skeletal muscles. It is well established that both genes are needed to produce muscle stem

cells in the embryo. A previous student, Alice Chen, studied how these genes are turned on in

embryonic muscle stem cells (also published in Nature). I thought that if they are so important inthe embryo, they must be important for adult muscle stem cells. Using genetic tricks, I was able

to suppress both genes in the adult muscle stem cells. I was totally surprised to find that themuscle stem cells are normal without them."

The researchers then looked at whether the same was true upon injury, after which the repair

process requires muscle stem cells to make new muscles. For this, they injured the leg musclesbetween the knee and ankle. They were again surprised that these muscle stem cells, without the

two key embryonic muscle stem cell genes, could generate muscles as well as normal muscle

stem cells. They even performed a second round of injury and found that the stem cells were stillactive.

The scientists then wondered when these genes become unnecessary for muscle stem cells to

regenerate muscles. It turned out that these embryonic genes are important to muscle stem cellcreation up to the first three weeks after birth. What makes the muscle stem cells different after

three weeks? The scientist believe that these two embryonic muscle stem cell genes also tell the

stem cells to become quiet as the organism matures. After that time is reached, they "hand over"their jobs to a different set of genes. The researchers suggest that since the adult muscle stem

cells are only activated when injury occurs (by trauma or exercise), they use a new set of genes

from those used during embryonic development, which proceeds without injury. The scientistsare eager to find these adult muscle stem cell genes.

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"We are just beginning to learn the basics of stem cell biology, and there are many surprises,"

remarked Allan Spradling, director of Carnegie's Department of Embryology. "This work illustrates the importance of carrying out basic research using animal models before rushing into

the clinic with half-baked therapies."

New Way To Enhance Stem Cells ToStimulate Muscle Regeneration 

Scientists at the Ottawa Hospital Research Institute (OHRI) and the University of Ottawa have discovered

a powerful new way to stimulate muscle regeneration, paving the way for new treatments for

debilitating conditions such as muscular dystrophy.

The research, to be published in the June 5 issue of Cell Stem Cell, shows for the first time that aprotein called Wnt7a increases the number of stem cells in muscle tissue, leading to accelerated

growth and repair of skeletal muscle.

"This discovery shows us that by targeting stem cells to boost their numbers, we can improve the

body's ability to repair muscle tissue," said senior author Dr. Michael Rudnicki. Dr. Rudnicki is

the Scientific Director of Canada's Stem Cell Network and a Senior Scientist at OHRI andDirector of OHRI's Sprott Centre for Stem Cell Research, as well as a Professor of Medicine at

the University of Ottawa.

Stem cells give rise to every tissue and organ in the body. Satellite stem cells are specialized

muscle stem cells that live in adult skeletal muscle tissue and have the ability to both replicate

and differentiate into various types of muscle cells. Dr. Rudnicki's team found that the Wnt7aprotein, when introduced into mouse muscle tissue, significantly increased the population of 

these satellite stem cells and fueled the regeneration process, creating bigger and stronger

muscles. Muscle tissue mass was increased by nearly 20 per cent in the study.

"Our findings point the way to the development of new therapeutic treatment for muscular

diseases such as muscular dystrophy, sarcopenia and muscle wasting conditions resulting fromextended hospital stays and surgeries," said Dr. Rudnicki.

Building Muscle Requires Foxo1 

The mechanisms by which Foxo proteins regulate metabolism are relatively well characterized.

However, little was known about the mechanisms by which these same proteins regulate cellular

differentiation.

New data generated by Domenico Accili and colleagues at Columbia University, New York,now indicates that Foxo1 cooperates with Notch to control muscle cell differentiation in vitro.

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In addition to examining the cells in vivo, the researchers studied the cells' response to strain on

different substrates. They found that MSC response is very sensitive to the mechanicalenvironment, indicating that conditions of muscle strain affect the cells' activity.

"These findings are important because we've identified an adult stem cell in muscle that may

provide the basis for muscle health with exercise and enhanced muscle healing withrehabilitation/movement therapy," Boppart said. "The fact that MSCs in muscle have the

potential to release high concentrations of growth factor into the circulatory system duringexercise also makes us wonder if they provide a critical link between enhanced whole-body

health and participation in routine physical activity."

Next, the group hopes to determine whether these cells contribute to the decline in muscle mass

over a person's lifetime. Preliminary data suggest MSCs become deficient in muscle with age.

The team hopes to develop a combinatorial therapy that utilizes molecular and stem-cell-based

strategies to prevent age-related muscle loss.

"Although exercise is the best strategy for preserving muscle as we age, some individuals are justnot able to effectively engage in physical activity," Boppart said. "Disabilities can limit

opportunities for muscle growth. We're working hard to understand how we can best utilize these

cells effectively to preserve muscle mass in the face of atrophy."

The team published its findings in the journal PLoS One.

Types of tissue

Main article: Muscle tissue 

Types of muscle (shown at different magnifications)

Muscle tissue is a soft tissue, and is one of the four fundamental types of  tissue present in

animals. There are three types of muscle tissue recognized in vertebrates: 

  Skeletal muscle or "voluntary muscle" is anchored by tendons (or by aponeuroses at a few

places) to bone and is used to effect skeletal movement such as locomotion and in maintaining

posture. Though this postural control is generally maintained as an unconscious reflex, the

muscles responsible react to conscious control like non-postural muscles. An average adult male

is made up of 42% of skeletal muscle and an average adult female is made up of 36% (as a

percentage of body mass).[2]

 

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  Smooth muscle or "involuntary muscle" is found within the walls of organs and structures such

as the esophagus, stomach, intestines, bronchi, uterus, urethra, bladder, blood vessels, and the

arrector pili in the skin (in which it controls erection of body hair). Unlike skeletal muscle,

smooth muscle is not under conscious control.

  Cardiac muscle is also an "involuntary muscle" but is more akin in structure to skeletal muscle,

and is found only in the heart.

Cardiac and skeletal muscles are "striated" in that they contain sarcomeres and are packed into

highly regular arrangements of bundles; smooth muscle has neither. While skeletal muscles arearranged in regular, parallel bundles, cardiac muscle connects at branching, irregular angles

(called intercalated discs). Striated muscle contracts and relaxes in short, intense bursts, whereas

smooth muscle sustains longer or even near-permanent contractions.

Skeletal (voluntary) muscle is further divided into two broad types: slow twitch and fast twitch:

  Type I, slow twitch, or "red" muscle, is dense with capillaries and is rich in mitochondria and

myoglobin, giving the muscle tissue its characteristic red color. It can carry more oxygen and

sustain aerobic activity using fats or carbohydrates as fuel.[3] Slow twitch fibers contract for long

periods of time but with little force.

  Type II, fast twitch muscle, has three major subtypes (IIa, IIx, and IIb) that vary in both

contractile speed[4] and force generated.[3] Fast twitch fibers contract quickly and powerfully but

fatigue very rapidly, sustaining only short, anaerobic bursts of activity before muscle contraction

becomes painful. They contribute most to muscle strength and have greater potential for

increase in mass. Type IIb is anaerobic, glycolytic, "white" muscle that is least dense in

mitochondria and myoglobin. In small animals (e.g., rodents) this is the major fast muscle type,

explaining the pale color of their flesh.

The density of mammalian skeletal muscle tissue is about 1.06 kg/liter.[5] This can be contrasted

with the density of  adipose tissue (fat), which is 0.9196 kg/liter.[6] This makes muscle tissueapproximately 15% denser than fat tissue.

Histogenesis

  Main articles: Histogenesis and Mesoderm 

All muscles derive from paraxial mesoderm.[7]

 The paraxial mesoderm is divided along theembryo's length into somites, corresponding to the segmentation of the body (most obviously

seen in the vertebral column.[7] Each somite has 3 divisions, sclerotome (which forms vertebrae),

dermatome (which forms skin), and myotome (which forms muscle).[7]

 The myotome is dividedinto two sections, the epimere and hypomere, which form epaxial and hypaxial muscles,

respectively.[7] Epaxial muscles in humans are only the erector spinae and small intervertebralmuscles, and are innervated by the dorsal rami of the spinal nerves.[7] All other muscles,

including limb muscles, are hypaxial muscles, formed from the hypomere, and inervated by the

ventral rami of the spinal nerves.[7]

 

During development, myoblasts (muscle progenitor cells) either remain in the somite to formmuscles associated with the vertebral column or migrate out into the body to form all other

muscles.[7] Myoblast migration is preceded by the formation of  connective tissue frameworks,

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usually formed from the somatic lateral plate mesoderm.[7]

 Myoblasts follow chemical signals to

the appropriate locations, where they fuse into elongate skeletal muscle cells.[7]

 

http://en.wikipedia.org/wiki/Muscle 

http://en.wikipedia.org/wiki/Skeletal_muscle