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Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue N δ -methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRS MIFAF ) for encoding MeHis into proteins, by transplanting five known active site mutations from Methanomethylophilus alvus ( Ma PylRS) into the single domain PylRS from Methanogenic archaeon ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the Ma system, G1PylRS MIFAF can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRS MIFAF is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.

Background: Chronic kidney disease is an important contributor to morbidity and mortality. 3-methylhistidine (3-MH) is the by-product of actin and myosin degradation reflecting skeletal muscle turnover. Markedly elevated 3-MH levels have been documented in uraemic patients, but the interpretation of high 3-MH concentration in maintenance haemodialysis (MHD) patients remains unclear. Indeed, it is not known whether elevated serum 3-MH levels are a marker of excessive muscle catabolism or a better lean tissue mass. Here, we evaluated the association between serum 3-MH levels and clinical outcomes in these patients., Methods: Serum 3-MH concentration was measured by reverse-phase liquid chromatography/tandem mass spectrometry in a cohort of MHD patients. We analysed the relationships between various clinical/laboratory indices, lean tissue mass measured by bioimpedance spectroscopy, mortality and cardiovascular (CV) events., Results: Serum 3-MH concentration was positively correlated with serum albumin, normalized protein catabolic rate (nPCR), simplified creatinine index (SCI) and lean tissue mass. Of 291 MHD patients, during a mean follow-up of 847 days, 91 patients died and 101 patients experienced a CV event. Survival was significantly better in patients with high 3-MH concentrations (P = .002). A higher level of 3-MH was also associated with a lower CV mortality and lower incidence of CV events (P = .015 and P < .001, respectively). Low serum 3-MH levels remained significantly associated with CV events but not with mortality after adjustment for demographic, metabolic and CV risk factors., Conclusion: Elevated serum 3-MH concentration appears to be a marker of better lean tissue mass and nutritional status. Low serum 3-MH is a robust and independent predictor of CV events in the MHD population., (© The Author(s) 2022. Published by Oxford University Press on behalf of the ERA.)

Ovothiols are π-methyl-5-thiohistidines produced in great amounts in sea urchin eggs, where they can act as protective agents against the oxidative burst at fertilization and environmental stressors during development. Here we examined the biological relevance of ovothiol during the embryogenesis of the sea urchin Paracentrotus lividus by assessing the localization of the key biosynthetic enzyme OvoA, both at transcript and protein level, and perturbing its protein translation by morpholino antisense oligonucleotide-mediated knockdown experiments. In addition, we explored the possible involvement of ovothiol in the inflammatory response by assessing ovoA gene expression and protein localization following exposure to bacterial lipopolysaccharide. The results of the present study suggest that ovothiol may be a key regulator of cell proliferation in early developing embryos. Moreover, the localization of OvoA in key larval cells and tissues, in control and inflammatory conditions, suggests that ovothiol may ensure larval skeleton formation and mediate inflammatory processes triggered by bacterial infection. This work significantly contributes to the understanding of the biological function of ovothiols in marine organisms, and may provide new inspiration for the identification of the biological activities of ovothiols in humans, considering the pharmacological potential of these molecules.

Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-β-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC-tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021. Published by Elsevier Inc.)

Ovothiols are sulfur-containing amino acids synthesized by marine invertebrates, protozoans, and bacteria. They act as pleiotropic molecules in signaling and protection against oxidative stress. The discovery of ovothiol biosynthetic enzymes, sulfoxide synthase OvoA and β-lyase OvoB, paves the way for a systematic investigation of ovothiol distribution and molecular diversification in nature. In this work, we conducted genomic and metagenomics data mining to investigate the distribution and diversification of ovothiol biosynthetic enzymes in Bacteria. We identified the bacteria endowed with this secondary metabolic pathway, described their taxonomy, habitat and biotic interactions in order to provide insight into their adaptation to specific environments. We report that OvoA and OvoB are mostly encountered in marine aerobic Proteobacteria, some of them establishing symbiotic or parasitic relationships with other organisms. We identified a horizontal gene transfer event of OvoB from Bacteroidetes living in symbiosis with Hydrozoa. Our search within the Ocean Gene Atlas revealed the occurrence of ovothiol biosynthetic genes in Proteobacteria living in a wide range of pelagic and highly oxygenated environments. Finally, we tracked the evolutionary history of ovothiol biosynthesis from marine bacteria to unicellular eukaryotes and metazoans. Our analysis provides new conceptual elements to unravel the evolutionary and ecological significance of ovothiol biosynthesis., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.

Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where "x" is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.

Sulfoxide synthases are non-heme iron enzymes that catalyze oxidative carbonsulfur bond formation in the biosynthesis of thiohistidines such as ergothioneine and ovothiol. The catalytic mechanism of these enzymes has been studied by protein crystallography, steady-state kinetics, non-natural amino acid incorporation and computational modeling. This review discusses the current status of this research and also highlights similarities between the CS bond forming activity of sulfoxide synthases with that of synthetic coordination compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Reactive oxygen species present a challenge for marine organisms releasing gametes into the water. Thiol-containing molecules protect cells against oxidative stress, and ovothiol (OSH), an antioxidant-reducing mercaptohistidine, has been described as especially relevant in the oocytes of marine invertebrates. Ovothiol synthase ( ovoA ), in charge of the first step in OSH synthesis, was sequenced in mussels, Mytilus galloprovincialis . Transcription levels of ovoA in mantle did not significantly change along the reproductive cycle. No alterations of ovoA transcription were observed after a laboratory copper (10 µg/L) exposure or in mussels captured in a highly polluted site. Conversely, the metabolomic analysis of the hydrophilic metabolite content in mantle clearly classified mussels according to their site of origin, especially at the most advanced stages of oogenesis. Quantification of OSH-A and -B and glutathione (GSH), revealed stable levels in mantle at early gametogenesis in the unpolluted sampling site, but a strong increase in female mantle previous to spawning in the polluted site. These increased concentrations under pollution suggest that OSH-A accumulates along oogenesis, independent of gene transcription regulation. The concerted accumulation of OSH-A and GSH suggests the building of a balanced cellular redox-system to scavenge ROS produced in the oocyte before and during fertilization., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Protein histidine methylation is a rarely studied posttranslational modification in eukaryotes. Although the presence of N-methylhistidine was demonstrated in actin in the early 1960s, so far, only a limited number of proteins containing N-methylhistidine have been reported, including S100A9, myosin, skeletal muscle myosin light chain kinase (MLCK 2), and ribosomal protein Rpl3. Furthermore, the role of histidine methylation in the functioning of the protein and in cell physiology remains unclear due to a shortage of studies focusing on this topic. However, the molecular identification of the first two distinct histidine-specific protein methyltransferases has been established in yeast (Hpm1) and in metazoan species (actin-histidine N-methyltransferase), giving new insights into the phenomenon of protein methylation at histidine sites. As a result, we are now beginning to recognize protein histidine methylation as an important regulatory mechanism of protein functioning whose loss may have deleterious consequences in both cells and in organisms. In this review, we aim to summarize the recent advances in the understanding of the chemical, enzymological, and physiological aspects of protein histidine methylation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Sepsis is a systemic inflammatory response syndrome resulting from infection. This study aimed at exploring the role of microRNA-140 (miR-140) in septic mice. Wnt family member 11 (WNT11) was verified to be a target gene of miR-140 after bioinformatic prediction and dual luciferase reporter gene assay. Importantly, miR-140 negatively regulated WNT11. We initially induced the model of sepsis by endotoxin, and then ectopic expression and knockdown experiments were performed to explore the functional role of miR-140 in sepsis. Additionally, cross-sectional areas of muscle fiber, lactic acid production, 3-methylhistidine (3-MH) and tyrosine (Tyr) production in extensor digitorium longus (EDL) muscles, and serum levels of inflammatory factors were examined. The effect of miR-140 on the expression of WNT signaling pathway-related and apoptosis-related factors in skeletal muscle tissue was determined. The experimental results indicated that upregulated miR-140 or silenced WNT11 increased cross-sectional areas of muscle fiber while decreasing lactic acid production, skeletal muscle cell apoptosis [corresponding to downregulated B cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and caspase-3 and upregulated Bcl-2], and the proteolytic rate of Tyr and 3-MH. Also, overexpressed miR-140 or silenced WNT11 reduced inflammation as reflected by decreased serum levels of IL-6, IL-10, and TNF-α. Furthermore, overexpression of miR-140 was shown to suppress the activation of the WNT signaling pathway, accompanied by decreased expression of WNT11, β-catenin, and GSK-3β. Taken together, upregulation of miR-140 could potentially inhibit skeletal muscle lactate release, an indirect measure of glycolysis, and atrophy in septic mice through suppressing the WNT signaling pathway via inhibiting WNT11 expression.

The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions 1-4 . However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis 5 , here we report the generation of a hydrolytic enzyme that uses N δ -methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design 6-10 . Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free N δ -methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. N δ -methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine 11 , and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

Vitamin B 6 deficiency is associated with cardiovascular disease (CVD). Although plasma biomarkers have been proposed, no studies have yet directly profiled heart tissue, and the mechanisms have to be fully defined. Thus, in order to provide better insight into vitamin B 6 -deficient effects on cardiac functions, we sought to identify the metabolic profile in heart tissue consequent to change in dietary vitamin B 6 levels by applying metabolomics. Heart tissues of rats fed a basal diet containing a marginal vitamin B 6 -deficient, vitamin B 6 -recommended or vitamin B 6 -supplemented level were analyzed by metabolomics analysis. Among over 500 detected metabolites, imidazole metabolites including carnosine, anserine, homocarnosine and histamine exhibited the highest decrease upon vitamin B 6 deficiency (>-45%, P<.01), along with their precursors β-alanine, γ-aminobutyric acid (GABA) and 1-methylhistidine. Ornithine was the only metabolite exhibiting an increased level in the vitamin B 6 -deficient group. Vitamin B 6 deficiency significantly attenuated the activity of heart tissue glutamate decarboxylase (GAD), although there was undetectable activity of aspartate decarboxylase (ADC), suggesting that the involvement of vitamin B 6 in imidazole metabolite synthesis occurs partly through GABA production by regulating GAD rather than through a straightforward β-alanine production pathway via ADC in the heart. Notably, vitamin B 6 deficiency significantly attenuated citric acid cycle metabolite levels, suggesting cardiac energy metabolism impairment. This study provides a new link between vitamin B 6 and cardiac functions, in which marginal vitamin B 6 deficiency impairs imidazole and energy metabolism in heart. This newly revealed cardiac metabolic profile may reveal novel molecular targets or foodstuffs for CVD prevention., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Covering: up to 2018 Ovothiols are sulfur-containing natural products biosynthesized by marine invertebrates, microalgae, and bacteria. These compounds are characterized by unique chemical properties suggestive of numerous cellular functions. For example, ovothiols may be cytoprotectants against oxidative stress, serve as building blocks of more complex structures and may act as molecular messengers for inter- and intracellular signaling. Detailed understanding of ovothiol physiological role in marine organisms may unearth novel concepts in cellular redox biochemistry and highlight the therapeutic potential of this antioxidant. The recent discovery of ovothiol biosynthetic genes has paved the way for a systematic investigation of ovothiol-modulated cellular processes. In this highlight we review the early research on ovothiol and we discuss key questions that may now be addressed using genome-based approaches. This highlight article provides an overview of recent progress towards elucidating the biosynthesis, function and potential application of ovothiols.

The aim was to determine the effects of enhanced availability of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on ammonia detoxification to glutamine (GLN) and protein metabolism in two types of skeletal muscle under hyperammonemic conditions. Isolated soleus (SOL, slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscles from the left leg of white rats were incubated in a medium with 1 mM ammonia (NH3 group), BCAAs at four times the concentration of the controls (BCAA group) or high levels of both ammonia and BCAA (NH3 + BCAA group). The muscles from the right leg were incubated in basal medium and served as paired controls. L-[1- 14 C]leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. We observed decreased protein synthesis and glutamate and α-ketoglutarate (α-KG) levels and increased leucine oxidation, GLN levels, and GLN release into medium in muscles in NH3 group. Increased leucine oxidation, release of branched-chain keto acids and GLN into incubation medium, and protein synthesis in EDL were observed in muscles in the BCAA group. The addition of BCAAs to medium eliminated the adverse effects of ammonia on protein synthesis and adjusted the decrease in α-KG found in the NH3 group. We conclude that (i) high levels of ammonia impair protein synthesis, activate BCAA catabolism, enhance GLN synthesis, and decrease glutamate and α-KG levels and (ii) increased BCAA availability enhances GLN release from muscles and attenuates the adverse effects of ammonia on protein synthesis and decrease in α-KG.

Ovothiols are thiolhistidine derivatives. The first step of ovothiol biosynthesis is OvoA-catalyzed oxidative coupling between histidine and cysteine. In this report, the remaining steps of ovothiol A biosynthesis were reconstituted in vitro. ETA&#95;14770 (OvoB) was reported as a PLP-dependent sulfoxide lyase, responsible for mercaptohistidine production. OvoA was found to be a bifunctional enzyme, which mediates both oxidative C-S bond formation and methylation of mercaptohistidine to afford ovothiol A. Besides reconstituting the whole biosynthetic pathway, two unique features proposed in the literature were also examined: a potential cysteine-recycling mechanism of the C-S lyase (OvoB) and the selectivity of the π- N methyltransferase.

As one of the most abundant elements on earth, sulfur is part of many small molecular metabolites and is key to their biological activities. Over the past few decades, some general strategies have been discovered for the incorporation of sulfur into natural products. In this review, we summarize recent efforts in elucidating the biosynthetic details for two sulfur-containing metabolites, ergothioneine and ovothiol. Their biosyntheses involve an unprecedented trans-sulfur strategy, a combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation reaction and a PLP enzyme-mediated C-S lyase reaction.

Branched-chain amino acids (BCAA) have been clearly demonstrated to have anabolic effects on muscle protein synthesis. However, little is known about their roles in the regulation of net AA fluxes across skeletal muscle in vivo. This study was aimed to investigate the effect and related mechanisms of dietary supplementation of BCAA on muscle net amino acid (AA) fluxes using the hindlimb flux model. In all fourteen 4-week-old barrows were fed reduced-protein diets with or without supplemental BCAA for 28 d. Pigs were implanted with carotid arterial, femoral arterial and venous catheters, and fed once hourly with intraarterial infusion of p-amino hippurate. Arterial and venous plasma and muscle samples were obtained for the measurement of AA, branched-chain α-keto acids (BCKA) and 3-methylhistidine (3-MH). Metabolomes of venous plasma were determined by HPLC-quadrupole time-of-flight-MS. BCAA-supplemented group showed elevated muscle net fluxes of total essential AA, non-essential AA and AA. As for individual AA, muscle net fluxes of each BCAA and their metabolites (alanine, glutamate and glutamine), along with those of histidine, methionine and several functional non-essential AA (glycine, proline and serine), were increased by BCAA supplementation. The elevated muscle net AA fluxes were associated with the increase in arterial and intramuscular concentrations of BCAA and venous metabolites including BCKA and free fatty acids, and were also related to the decrease in the intramuscular concentration of 3-MH. Correlation analysis indicated that muscle net AA fluxes are highly and positively correlated with arterial BCAA concentrations and muscle net BCKA production. In conclusion, supplementing BCAA to reduced-protein diet increases the arterial concentrations and intramuscular catabolism of BCAA, both of which would contribute to an increase of muscle net AA fluxes in young pigs.

Dexmedetomidine is generally used for sedaton in critically ill, it could shorten duration of mechanical ventilation, ICU stay and lower basic metabolism. However, the exact mechanism of these positive effects remains unkown. Here we investigated the hypothesis that dexmedetomidine could ameliorate muscle wasting in endotoxemic rats and whether it was related to hypothalamic neuropeptides alteration and inflammation. Fourty-eight adult male Sprague-Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS) (5 mg/kg) or saline, followed by 50 μg/kg dexmedetomidine or saline administration via the femoral vein catheter (infusion at 5 μg·kg-1·hr-1). Twenty-four hours after injection, hypothalamus tissues and skeletal muscle were obtained. Muscle wasting was measured by the mRNA expression of two E3 ubiquitin ligases, muscle atrophy F-box (MAFbx) and muscle ring finger 1 (MuRF-1) as well as 3-methylhistidine (3-MH) and tyrosine release. Hypothalamic inflammatory markers and neuropeptides expression were also detected in all four groups. Results showed that LPS administration led to significant increase in hypothalamic inflammation together with muscle wasting. Increased hypothalamic neuropeptides, proopiomelanocortin (POMC), cocaine and amphetamine-related transcript (CART) and neuropeptides Y (NPY) and decreased agouti-related protein (AgRP) were also observed. Meanwhile dexmedetomidine administration ameliorated muscle wasting, hypothalamic inflammation and modulated the alteration of neuropeptides, POMC, CART and AgRP, in endotoxemic rats. In conclusion, dexmedetomidine could alleviate muscle wasting in endotoxemic rats, and it could also attenuate the alteration of hypothalamic neuropeptides and reduce hypothalamic inflammation.

Ovothiol, isolated from marine invertebrate eggs, is considered one of the most powerful antioxidant with potential for drug development. However, its biological functions in marine organisms still represent a matter of debate. In sea urchins, the most accepted view is that ovothiol protects the eggs by the high oxidative burst at fertilization. In this work we address the role of ovothiol during sea urchin development to give new insights on ovothiol biosynthesis in metazoans. The gene involved in ovothiol biosynthesis OvoA was identified in Paracentrotus lividus genome (PlOvoA). PlOvoA embryo expression significantly increased at the pluteus stage and was up-regulated by metals at concentrations mimicking polluted sea-water and by cyclic toxic algal blooms, leading to ovothiol biosynthesis. In silico analyses of the PlOvoA upstream region revealed metal and stress responsive elements. Structural protein models highlighted conserved active site residues likely responsible for ovothiol biosynthesis. Phylogenetic analyses indicated that OvoA evolved in most marine metazoans and was lost in bony vertebrates during the transition from the aquatic to terrestrial environment. These results highlight the crucial role of OvoA in protecting embryos released in seawater from environmental cues, thus allowing the survival under different conditions.

Muscle wasting impairs physical function and leads people to a bedridden state. We previously demonstrated that lysine (Lys) suppresses autophagic-proteolysis through the Akt pathway. However, the effect of metabolites of Lys on proteolysis is unclear. In this study, we investigated the effect of saccharopine (Sac), a metabolite of Lys, on proteolysis in C2C12 cells. When C2C12 myotubes were incubated in serum-free medium containing Sac, the rate of proteolysis, which was evaluated by 3-methylhistidine released from C2C12 myotubes, and autophagy activity, which was assessed by amount of light chain 3-II, were suppressed. Sac stimulated Akt and mammalian target of rapamycin signaling, which was evaluated from eIF4E-binding protein 1 phosphorylation. The suppressive effects of Sac on proteolysis and autophagy were completely abolished by an Akt inhibitor. Therefore, we concluded that Sac suppresses autophagic-proteolysis through Akt as with Lys.

Background: Enhanced glutamine (GLN) intake may affect the catabolism of branched-chain amino acids (BCAAs; valine, leucine, and isoleucine), which play a regulatory role in protein turnover. We examined the effects of enhanced GLN availability on leucine oxidation, amino acid concentrations, and protein metabolism in muscles from healthy and septic rats., Methods: Cecal ligation and puncture were used as a model of sepsis. Twenty-four hours after surgery, the soleus (SOL, red muscle) and the extensor digitorum longus (EDL, white muscle) were incubated in medium containing 0.5 or 2.0 mM GLN. Protein breakdown, protein synthesis, and leucine oxidation were determined via 3-methylhistidine release, muscle L-[1-(14)C]leucine radioactivity, and the radioactivity of released (14)CO2, respectively., Results: In muscles from septic animals, increased proteolysis and leucine oxidation and decreased protein synthesis were detected. These effects were more pronounced in the EDL. In septic muscles, the addition of GLN decreased leucine oxidation in both muscles and increased protein synthesis in the EDL. In muscles from untreated animals, decreased leucine oxidation after the addition of GLN to the medium was associated with decreased protein synthesis in the SOL and decreased concentrations of serine, glycine, histidine, alanine, arginine, proline, and lysine in both muscles., Conclusions: White muscle fibers are more sensitive to septic stimuli than red fibers are. In sepsis, enhanced GLN intake may ameliorate GLN deficiency, inhibit BCAA catabolism, and stimulate protein synthesis. In the healthy state, surplus of GLN may lead to severe alterations in the intramuscular concentration of several amino acids and impair protein synthesis., (© 2014 American Society for Parenteral and Enteral Nutrition.)

Background: The neurobiological changes underlying depression resistant to treatments remain poorly understood, and failure to respond to selective serotonin reuptake inhibitors may result from abnormalities of neurotransmitter systems that excite serotonergic neurons, such as histamine., Methods: Using behavioral (tail suspension test) and neurochemical (in vivo microdialysis, Western-blot analysis) approaches, here we report that antidepressant responses to selective serotonin reuptake inhibitors (citalopram or paroxetine) are abolished in mice unable to synthesize histamine due to either targeted disruption of histidine decarboxylase gene (HDC(-/-)) or injection of alpha-fluoromethylhistidine, a suicide inhibitor of this enzyme., Results: In the tail suspension test, all classes of antidepressants tested reduced the immobility time of controls. Systemic reboxetine or imipramine reduced the immobility time of histamine-deprived mice as well, whereas selective serotonin reuptake inhibitors did not even though their serotonergic system is functional. In in vivo microdialysis experiments, citalopram significantly increased histamine extraneuronal levels in the cortex of freely moving mice, and methysergide, a serotonin 5-HT1/5-HT2 receptor antagonist, abolished this effect, thus suggesting the involvement of endogenous serotonin. CREB phosphorylation, which is implicated in the molecular mechanisms of antidepressant treatment, was abolished in histamine-deficient mice treated with citalopram. The CREB pathway is not impaired in HDC(-/-) mice, as administration of 8-bromoadenosine 3', 5'-cyclic monophosphate increased CREB phosphorylation, and in the tail suspension test it significantly reduced the time spent immobile by mice of both genotypes., Conclusions: Our results demonstrate that selective serotonin reuptake inhibitors selectively require the integrity of the brain histamine system to exert their preclinical responses., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

OvoA in ovothiol biosynthesis is a mononuclear non-heme iron enzyme catalyzing the oxidative coupling between histidine and cysteine. It can also catalyze the oxidative coupling between hercynine and cysteine, yet with a different regio-selectivity. Due to the potential application of this reaction for industrial ergothioneine production, in this study, we systematically characterized OvoA by a combination of three different assays. Our studies revealed that OvoA can also catalyze the oxidation of cysteine to either cysteine sulfinic acid or cystine. Remarkably, these OvoA-catalyzed reactions can be systematically modulated by a slight modification of one of its substrates, histidine.

Objectives: Muscle release of the amino acid 3-methyl-histidine (3MH) is a sensitive index of myofibrillar protein overdegradation (MPO). We hypothesized that patients with chronic heart failure (CHF) could have increased muscle release of 3MH, which in turn reflects MPO, and that serum electrolyte sodium (Na(+)) and potassium (K(+)) levels may be associated with this 3MH muscle release., Methods: Thirty-one overweight outpatients (body mass index, 27 ± 4.4 kg/m(2); 22 men and 9 women; age, 56 ± 8.7 y) with clinically stable CHF were studied. After a 24-hour meat-free diet and overnight fasting, patients underwent blood sampling from a cannulated arm vein (V) and concomitantly from the arterial artery (A) to determine plasma 3MH levels and to calculate the A-V difference. Serum levels of Na(+) and K(+) in the venous blood were determined, and the Na(+)/K(+) ratio was calculated. Ten healthy subjects who were matched for gender, age, and body mass index served as controls and underwent the same protocol as the patients with CHF., Results: The patient group had higher arterial (P = 0.02) and venous (P = 0.005) 3MH levels but a similar A-V 3MH difference (P = 0.28) as compared with the controls. Within the CHF group, 67.7% of patients released 3MH, which resulted in a negative A-V value (P < 0.02 as compared with controls). In patients with CHF, the A-V 3MH difference correlated positively with the serum K(+) level (r = 0.62; P = 0.0002) and negatively with Na(+)/K(+) ratio (r = -0.55; P = 0.002). No association was found between the A-V 3MH difference and the Na(+) level., Conclusions: The study demonstrated the existence of MPO in resting overweight patients with CHF, thereby suggesting that low serum levels of K(+) may contribute to MPO., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ergothioneine (5) and ovothiol (8) are two novel thiol-containing natural products. Their C-S bonds are formed by oxidative coupling reactions catalyzed by EgtB and OvoA enzymes, respectively. In this work, it was discovered that in addition to catalyzing the oxidative coupling between histidine and cysteine (1 → 6 conversion), OvoA can also catalyze a direct oxidative coupling between hercynine (2) and cysteine (2 → 4 conversion), which can shorten the ergothioneine biosynthetic pathway by two steps.

OvoA is an iron(II) dependent sulfoxide synthase which catalyzes the first step in ovothiol A biosynthesis. This enzyme sulphurizes the C5 position of the imidazole side chain of L-histidine. We report the substrate specificity profile of this catalyst and present data which indicate that OvoA catalysis follows an thiol-ene type mechanism.

Ergothioneine and ovothiol A are sulfur-containing histidine derivatives produced by microorganisms including Mycobacterium tuberculosis, Trypanosoma cruzi or Erwinia amylovora and may also play important roles in human physiology. Based on our recent identification of thiohistidine biosynthetic enzymes from Mycobacterium smegmatis and Erwinia tasmaniensis we investigate several aspects of sulfur-based redox biochemistry. For example, we are characterizing the catalytic mechanism of two thiohistidine biosynthetic enzymes which afford O2-dependent sulfur insertion into the C(5)-H and C(2)-H bonds of the imidazolyl side chain of histidine.

Combined amylin+leptin (AMN+LEP) can reduce diet induced obesity and is very effective in combating LEP resistance. The purpose of this study was to evaluate the effect of AMN+LEP on central histaminergic signaling in lean and obese rats. Male rats were administered LEP (300 μg/kg/d), AMN (100 μg/kg/d), AMN+LEP or vehicle (SAL, 0.9% normal saline), via a subcutaneous mini-osmotic pump or single injection (LEP, 300 μg/kg and AMN, 100 μg/kg) for acute studies. AMN+LEP administration increased expression of histamine H1 receptor (HIR) and histidine decarboxylase (HDC) mRNA in the hypothalamus. Increased levels of H1R were seen in arcuate (Arc) and ventromedial hypothalamus (VMH) as well as the area postrema (APOS) and nucleus of solitary tract (NTS) following AMN+LEP administration. APOS and NTS also showed expression of immediate early gene c-FOS in the hindbrain in AMN+LEP-treated rats. We confirmed previous evidence indicating that AMN+LEP increased STAT-3 protein phosphorylation in Arc and VMH. Finally, by in vivo microdialysis, we observed an increase in methyl HIS levels in the VMH of AMN, LEP and AMN+LEP-treated rats. Taken together, these observations are consistent with an important role that neuronal HIS may play in mediating the potent effects of AMN+LEP on food intake and body weight., (Copyright © 2011 Elsevier B.V. All rights reserved.)

It is unclear whether the muscle hypertrophy induced by loss of myostatin signaling in mature muscles is maintained only by increased protein synthesis or whether reduced proteolysis contributes. To address this issue, we depleted myostatin by activating Cre recombinase for 2 wk in mature mice in which Mstn exon 3 was flanked by loxP sequences. The rate of phenylalanine tracer incorporation into myofibrillar proteins was determined 2, 5, and 24 wk after Cre activation ended. At all of these time points, myostatin-deficient mice had increased gastrocnemius and quadriceps muscle mass (≥27%) and increased myofibrillar synthesis rate per gastrocnemius muscle (≥19%) but normal myofibrillar synthesis rates per myofibrillar mass or RNA mass. Mean fractional myofibrillar degradation rates (estimated from the difference between rate of synthesis and rate of change in myofibrillar mass) and muscle concentrations of free 3-methylhistidine (from actin and myosin degradation) were unaffected by myostatin knockout. Overnight food deprivation reduced myofibrillar synthesis and ribosomal protein S6 phosphorylation and increased concentrations of 3-methylhistidine, muscle RING finger-1 mRNA, and atrogin-1 mRNA. Myostatin depletion did not affect these responses to food deprivation. These data indicate that maintenance of the muscle hypertrophy caused by loss of myostatin is mediated by increased protein synthesis per muscle fiber rather than suppression of proteolysis.

Nuclear magnetic resonance (NMR)-based metabolomic profiling identified urinary 1- and 3-methylhistidine (1- and 3-MH) as potential biomarkers of skeletal muscle toxicity in Sprague-Dawley rats following 7 and 14 daily doses of 0.5 or 1mg/kg cerivastatin. These metabolites were highly correlated to sex-, dose- and time-dependent development of cerivastatin-induced myotoxicity. Subsequently, the distribution and concentration of 1- and 3-MH were quantified in 18 tissues by gas chromatography-mass spectrometry. The methylhistidine isomers were most abundant in skeletal muscle with no fiber or sex differences observed; however, 3-MH was also present in cardiac and smooth muscle. In a second study, rats receiving 14 daily doses of 1mg/kg cerivastatin (a myotoxic dose) had 6- and 2-fold elevations in 1- and 3-MH in urine and had 11- and 3-fold increases in 1- and 3-MH in serum, respectively. Selectivity of these potential biomarkers was tested by dosing rats with the cardiotoxicant isoproterenol (0.5mg/kg), and a 2-fold decrease in urinary 1- and 3-MH was observed and attributed to the anabolic effect on skeletal muscle. These findings indicate that 1- and 3-MH may be useful urine and serum biomarkers of drug-induced skeletal muscle toxicity and hypertrophy in the rat, and further investigation into their use and limitations is warranted., (2010 Elsevier Inc. All rights reserved.)

Ovothiols are histidine-derived thiols that were first isolated from marine invertebrates. We have identified a 5-histidylcysteine sulfoxide synthase (OvoA) as the first ovothiol biosynthetic enzyme and characterized OvoAs from Erwinia tasmaniensis and Trypanosoma cruzi . Homologous enzymes are encoded in more than 80 genomes ranging from proteobacteria to animalia.

Oral contraceptive (OC) treatment has an inhibiting effect on protein synthesis in tendon and muscle connective tissue. We aimed to investigate whether OC influence myofibrillar protein turnover in young women. OC-users (24±2 years; Lindynette® n=7, Cilest® n=4) and non-OC-users (controls, 24±4 years n=12) performed one-legged kicking exercise. The next day, the myofibrillar protein fractional synthesis rate (FSR) was measured using stable isotopic tracers ((13)C-proline) while the subjects were fed standardized nutrient drinks. Simultaneously, a marker for myofibrillar protein breakdown, 3-methyl-histidine (3-MH), was measured in the interstitial fluid of the vastus lateralis. Measurements were performed in both legs. In general, myofibrillar protein FSR was lower in OC-users (two-way analysis of variance, P<0.05), although the difference seemed to depend on the OC type. Interstitial 3-MH in the skeletal muscle was not different between groups and did not vary by OC type. Exercise did not change myofibrillar protein FSR or 3-MH concentrations. Serum androstenedione and bioavailability of testosterone were lower in OC-users. In conclusion, the results indicate that the use of OC has an inhibiting effect on myofibrillar protein synthesis and the magnitude of the effect may depend on the type of OC. In contrast, there was no effect of OC on myofibrillar protein breakdown in the fed state., (© 2009 John Wiley & Sons A/S.)

We have shown that Rpl3, a protein of the large ribosomal subunit from baker's yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-(3)H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven β-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.

The physiologic response to trauma results in the efflux of large amounts of amino acids from skeletal muscle. This is extreme in large burn injuries. Protein kinetic studies, although useful in determining the rates of protein synthesis and breakdown, do not provide information about muscle loss. This study determined the contribution of muscle protein to whole-body protein breakdown in children throughout their course of burn injury. Children aged 0 to 18 years with initial burn size ≥30% TBSA underwent ¹⁵N glycine and 3 methylhistidine (3MH) analysis during three phases of care: A, early acute; B, wound closure; and C, convalescence. Muscle protein breakdown was estimated using a factor of 4.2 μmol 3MH per 1 g of mixed protein. Twenty-two patients with a mean of 54.5 ± 20.1% TBSA burn were studied. Protein balance did not change remarkably and remained positive by 2 g/kg during hospitalization. However, muscle protein breakdown dropped from 1.1 to 0.6 g/kg with wound closure (P < .0001), representing a decrease in the contribution of muscle protein to whole-body protein breakdown from 20 to 7%. Ten patients returned for a third measurement after discharge. Although protein turnover was high, muscle breakdown was consistent with 3MH values reported in healthy children. Serial determination of 3MH excretion is a simple way to track muscle catabolism throughout burn injury. Our data suggest that despite accelerated protein turnover, muscle catabolism significantly decreases with wound closure and begins to normalize around discharge. In convalescence, 3MH excretion is comparable with normal children.

Despite several decades of active research, the success of large-scale clinical trials involving antioxidants remains equivocal given the complex biological interactions of reactive oxygen/nitrogen species in human health. Herein, we outline a differential metabolomics strategy by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) to assess the efficacy of nutritional intervention to attenuate oxidative stress induced by strenuous exercise. A healthy volunteer was recruited to perform a submaximal prolonged ergometer cycling trial until volitional exhaustion with frequent blood collection over a 6 h time interval, which included pre-, during, and postexercise periods while at rest. A follow-up study was subsequently performed by the same subject after high-dose oral intake of N-acetyl-L-cysteine (NAC) prior to performing the same exercise protocol under standardized conditions. Time-dependent changes in global metabolism of filtered red blood cell lysates by CE-ESI-MS were measured to reveal a significant attenuation of cellular oxidation associated with high-dose oral NAC intake relative to a control. Untargeted metabolite profiling allowed for the identification and quantification of several putative early- and late-stage biomarkers that reflected oxidative stress inhibition due to nutritional intervention, including oxidized glutathione (GSSG), reduced glutathione (GSH), 3-methylhistidine (3-MeHis), L-carnitine (C0), O-acetyl-L-carnitine (C2), and creatine (Cre). Our work demonstrates the proof-of-principle that NAC pretreatment is effective at dampening acute episodes of oxidative stress by reversible perturbations in global metabolism that can provide deeper insight into the mechanisms of thiol-specific protein inhibition relevant to its successful translation as a prophylaxis in clinical medicine.

Chronic vitamin A deficiency induces a substantial delay in the rates of weight and height gain in both humans and experimental animals. This effect has been associated with an impaired nutrient metabolism and loss of body protein. Therefore, we analyzed the effect of vitamin A deficiency on endogenous proteolysis and nitrogen metabolism and its reversibility with all-trans retinoic acid (RA). Male weanling rats, housed in pairs, were pair-fed a vitamin A-deficient (VAD) or control diet until they were 60 d old. A group of deficient rats were further treated with daily intraperitoneal injections of all-trans RA for 10 d. Final body and tissue (i.e. liver and heart) weights were significantly lower and tissue:body weight ratios were similar in VAD rats and in controls. Conversely, the epididymal white fat:body weight ratio and the plasma concentrations of alanine aminotransferase and adiponectin were significantly higher in VAD rats, which also had hepatic macrovesicular lipid accumulations. Plasma and gastrocnemius muscle 3-methylhistidine, urine nitrogen, and plasma and urine urea concentrations were all significantly higher in the VAD group. The expression of the genes encoding urea cycle enzymes and their activities increased in VAD livers. These changes were partially reverted by all-trans RA. We propose that fuel partitioning in vitamin A deficiency may shift from fatty acids to protein catabolism as an energy source. Our results emphasize the importance of vitamin A on the energy balance control system and they provide an explanation for the role of vitamin A in protein turnover, development, and growth.

We fed common brushtail possums artificial diets containing a buffer and the plant secondary metabolite (PSM), orcinol, to test the hypothesis that organic acids, common products of PSM metabolism, limit feeding by common brushtail possums (Trichosurus vulpecula). We introduced several diets containing orcinol and a buffer (urinary alkalising agent) over a course of three experiments. A diet containing 2% orcinol (wet matter) caused possums to reduce their food intake immediately, but feeding returned to normal 1-2 days later. Even though possums excreted strongly acidic urine (pH 5.1) and had perturbed nitrogen metabolism, they maintained their food intake and body mass until the experiment terminated 9 days after the introduction of orcinol. Possums ate 52% less when the basal diet contained 4% orcinol. As expected, the acid loads caused a change in the composition of urinary nitrogen with possums excreting more ammonium than urea and a large amount of unidentified nitrogenous material. Supplementing the diet containing orcinol with buffer neutralised the metabolic acid load and partly restored normal nitrogen metabolism, but did not restore feeding. Also, animals eating orcinol excreted normal amounts of 3-methylhistidine, indicating no increase in muscle protein catabolism. This suggests that a limitation to the rate of detoxification or toxicosis, rather than acid loads, limits the ingestion of acid-inducing PSMs.

The human endotoxin model has been used to study the early phase of sepsis. The aim of the present study was to assess leg muscle protein kinetics after an endotoxin challenge given to healthy human volunteers. Six healthy male subjects were studied in the post-absorptive state before and during 4 h following an intravenous endotoxin bolus (4 ng/kg of body weight). Primed continuous infusion of [(2)H(5)]phenylalanine and [(2)H(3)]3-methylhistidine in combination with sampling from the radial artery, femoral vein and muscle tissue were used to assess leg muscle protein kinetics. Both two- and three-compartment models were used to calculate protein kinetics. In addition 26S proteasome activity and protein ubiquitination were assessed. An increase in the net release of phenylalanine from the leg following the endotoxin challenge was observed; however, this phenylalanine originates from the free intracellular pool and not from protein. Net protein balance was unchanged, whereas both protein synthesis and breakdown were decreased. Degradation rates of contractile proteins were not affected by endotoxin, as indicated by an unchanged rate of appearance of 3-methylhistidine from leg muscle. In addition, proteasome activity and protein ubiquitination were unaffected by endotoxaemia. In conclusion, intravenous endotoxin administration to healthy volunteers resulted in an increased release of free phenylalanine from skeletal muscle, whereas protein balance was unaffected. Both protein synthesis and breakdown were decreased to a similar extent.

Background: Oral glutamine decreases whole body protein breakdown in Duchenne muscular dystrophy (DMD). We evaluated the functional benefit of 4 months oral glutamine in DMD., Methodology/principal Findings: 30 ambulant DMD boys were included in this double-blind, randomized crossover trial with 2 intervention periods: glutamine (0.5 g/kg/d) and placebo, 4 months each, separated by a 1-month wash-out, at 3 outpatient clinical investigation centers in France. Functional benefit was tested by comparing glutamine versus placebo on change in walking speed at 4 months. Secondary outcome measures were: 2-minute walk test, work, power, muscle mass (urinary creatinine), markers of myofibrillar protein breakdown (urinary 3-methyl-histidine/creatinine), serum creatine phospho-kinase, body composition (fat free mass, fat mass percentage), safety and oral nutrient intake. There was no improvement in the primary end point (walking speed) or in secondary measures of muscle function (2-minute walk test, work, power) in the glutamine group compared with placebo. However, subjects receiving glutamine or placebo showed no deterioration in functional measures over the course of the 9-month trial. No differences in muscle mass, markers of protein breakdown or serum creatine phosho-kinase were observed, except for a blunted increase in fat free mass in the glutamine group which led to a greater increase in fat mass percentage. Glutamine was safe and well-tolerated., Conclusions: This trial did not identify additional benefit of 4 months oral glutamine over placebo on muscle mass or function in ambulatory DMD boys. Although apparently safe, current data cannot support routine supplementation in this population as a whole, until further research proves otherwise., Trial Registration: (ClinicalTrials.gov) NCT00296621.

Our previous results show that leptin, as well as nitric oxide (NO) and some antioxidants (histidine dipeptides - HDP) change the secretion of gonadotrophins from ovine adenohypophysis cells in vitro. NO and HDP are produced by pituitary and can modulate gonadotropin secretion by autocrine action. It is possible that these compounds mediate leptin influence on gonadotropin secretion. Therefore, the objective of the present study was to analyse leptin effect on NO and HDP (3-metyl-L-histidine, carnosine and anserine) release from ovine pituitary in vitro. Adenohypophysis cells were cultured in McCoy 5A medium with GnRH (4 x 10(-9) M) and 10(-10)-10(-5) M/l of leptin, respectively. Next, the media for analysis of NO (Griess method) and HDP (HPLC) were collected. Leptin in concentration of 10(-8)-10(-6) M/l caused a significant augmentation in NO in the culture medium, whereas in the dose of 10(-5) M/l reduced (P< or =0.05) NO release. The level of 3-metyl-L-histidine and anserine, but not carnosine, was significantly lower in the culture with 10(-8)-10(-7) M/l of leptin. Taking into account that 10(-8)-10(-7) M/l leptin stimulates LH and FSH secretion, as show in our previous study, it is possible that this effect in ewes is mediated by augmented release of NO and reduction of HDP level.

Skeletal muscle atrophy is evident after muscle disuse, unloading, or spaceflight and results from decreased protein content as a consequence of decreased protein synthesis, increased protein breakdown or both. At this time, there are essentially no human data describing proteolysis in skeletal muscle undergoing atrophy on Earth or in space, primarily due to lack of valid and accurate methodology. This particular study aimed at assessing the effects of short-term unloading on the muscle contractile proteolysis rate. Eight men were subjected to 72-h unilateral lower limb suspension (ULLS) and intramuscular interstitial levels of the naturally occurring proteolytic tracer 3-methylhistidine (3MH) were measured by means of microdialysis before and on completion of this intervention. The 3MH concentration following 72-h ULLS (2.01 +/- 0.22 nmol/ml) was 44% higher (P < 0.05) than before ULLS (1.56 +/- 0.20 nmol/ml). The present experimental model and the employed method determining 3MH in microdialysates present a promising tool for monitoring skeletal muscle proteolysis or metabolism of specific muscles during conditions resulting in atrophy caused by, e.g., disuse and real or simulated microgravity. This study provides evidence that the atrophic processes are evoked rapidly and within 72 h of unloading and suggests that countermeasures should be employed in the early stages of space missions to offset or prevent muscle loss during the period when the rate of muscle atrophy is the highest.

Background & Aims: Since both cancer and surgery are known to alter protein turnover, we investigated how the presence of tumour affects post-operative protein breakdown., Methods: Controls and tumour-bearing non-cachectic mice were studied, both with and without laparotomy (n=8 per group). One day after laparotomy, stable isotopes of phenylalanine and 3-methylhistidine were used in a steady-state protocol to assess total protein breakdown on whole-body level and in muscle, intestines, liver and kidney, in addition to whole-body myofibrillar protein breakdown and body composition. Proteasomal chymotrypsin-like activity was measured to determine activity of the ATP-dependent ubiquitin pathway., Results: On whole-body level, the presence of tumour increased total protein breakdown from 47+/-6 to 58+/-4 nmol/10 g/min (p<0.05) and myofibrillar protein breakdown from 0.70+/-0.04 to 1.22+/-0.14 nmol/10 g/min (p<0.05) without affecting protein breakdown in organs, body composition or proteasomal activity. Laparotomy increased myofibrillar protein breakdown in controls (from 0.70+/-0.04 to 0.98+/-0.12 nmol/10 g/min, p<0.05) and tumour-bearing mice (from 1.22+/-0.14 to 1.54+/-0.22 nmol/10 g/min, p=0.15) to a similar extent., Conclusions: Whole-body total protein breakdown, total protein breakdown across organs, body composition or proteasomal activity were not affected by laparotomy. Tumour-bearing mice had increased total and myofibrillar protein breakdown on whole-body level even before weight loss was obvious. However, this did not affect the post-operative response in protein breakdown or body composition.

Background: Negative nitrogen balance and skeletal muscle loss are common in critically injured patients and may contribute to morbidity, mortality and resource utilization. Juven, an enteral supplement which is a combination of beta-hydroxy-beta-methylbutyrate (HMB), arginine (ARG), and glutamine (GLN) has been shown to restore muscle in cachetic acquired immunodeficiency syndrome (AIDS) and cancer patients. More recently HMB has been shown to attenuate cancer-induced muscle loss by decreasing muscle proteolysis. The purpose of this study was to analyze whether HMB alone or in combination with ARG and GLN would have a similar effect on critically injured trauma patients. We hypothesized that nitrogen balance would be improved and muscle proteolysis decreased with HMB and HMB/ARG/GLN supplementation., Methods: There were 100 adult trauma patients with Injury Severity Score (ISS) >18 were enrolled in this prospective, randomized, blinded study. All patients received standard tube feeds and one of three iso-nitrogenous supplements; HMB, HMB/ARG/ GLN, or placebo (PLAC) for 28 days. Urine, serum, and clinical data were collected for 72 patients receiving at least 7 days of supplementation during the first 14 days of treatment. Urinary 3-methylhistidine (3-MH) was used as a proxy for muscle proteolysis., Results: The three groups were similar in age, gender, mechanism, and severity of injury, with the average ISS being 31.9. Utilizing covariant (ISS) repeated measure (days 1-14) mixed model (SAS) analysis, there was a significant treatment effect (p = 0.05) on nitrogen balance (g/d). Change in nitrogen balance from the first 7 days to the last 7 days was -4.3 for the HMB and -5.6 g/d HMB/ARG/GLN groups compared with -8.9 g/d for the PLAC group. 3-MH to creatinine ratios were not different in the PLAC group as compared with the HMB/ARG/GLN and HMB groups (Treatment Effect, p = 0.80)., Conclusions: These data suggest that supplementation with HMB alone may improve nitrogen balance in critically injured adult patients and that this effect is not a result of lowered muscle protein turnover as originally hypothesized.

The present study deals with the application of high-performance-liquid-chromatography (HPLC) method for a quantitative detection of carnosine, anserine, L-histidine and 3-methyl-L-histidine in biological material with o-phthaldialdehyde (OPA) post column derivatisation at the constant temperature of 50 degrees C. For this purpose, some mobile-phases were prepared with scalar acetonitrile concentrations. A complete separation of all molecules, particularly for carnosine and 3-methyl-L-histidine, was obtained with a solution of acetonitrile and 6mM hydrochloric acid with 0.48 M sodium chloride (5%:95% v/v). Post column derivatisation reaction at temperature of 50'C permitted to obtain an increase in sensibility of all molecules. This method has been utilised for detection of histidine dipeptides in boar spermatozoa and in sheep milk. Concentrations (mean +/- S.E. nmol/10(9) spermatozoa) of carnosine (0.96 +/- 0.14) and anserine (0.83 +/- 0.18) in boar spermatozoa were significantly lower than those of L-histidine (52.85 +/- 4.86) and 3-methyl-L-histidine (83.07 +/- 7.1). Positive correlation was found between carnosine and anserine contents (r = 0.740; p < 0.01) and between L-histidine and 3-methyl-L-histidine (r = 0.657; p < 0.01). All histidine dipeptides studied were also present in 40 samples of sheep milk. In a case of samples without unit-forming colonies (UFC) of Staphylococcus coagulase-positive, carnosine concentrations (9.17 +/- 0.89 nmol/ml) were higher than anserine (0.51 +/- 0.02 nmol/ml) and both were significantly lower in respect to L-histidine (49.51 +/- 6.48 nmol/ml) and 3-metyl-L-histidine (81.21 +/- 6.82 nmol/ml). A negative correlation was observed between carnosine milk levels (r = -0.773; p < 0.01) and UFC/ml of Staphylococcus coagulase-positive. In conclusion this very simple and fast method can be used to detect histidine dipeptides in biological compartments where their concentrations are very low.

Numerous proteins are known to be lost following myocardial ischemia/reperfusion yet little is known about the mediating proteinases. This study examines the hypothesis that proteasome plays a significant role in the removal of proteins oxidized during myocardial ischemia. Proteasome was inhibited by perfusing isolated rat hearts with buffer containing lactacystin, 2 micromol/L, for 10 min, which resulted in 51 and 42% decreases in 20S and 26S proteasome activities that persisted for a minimum of 90 min. Lactacystin pretreatment had minor effects on postischemic recovery of isolated hearts exposed to 30 min global ischemia and 60 min reperfusion. Protein carbonyl content of lactacystin-pretreated ischemic hearts was significantly (P < 0.05) increased. One band with approximate molecular mass of 50 kDa is known to contain oxidized actin. Actin degradation was quantitated by analysis of 3-methylhistidine which was significantly (P < 0.05) decreased by 15% following 30 min ischemia and 60 min reperfusion. Pretreatment of ischemic hearts with lactacystin prevented much of the loss (-6.5%) of 3-methylhistidine. Probing immunoprecipitated actin with an antibody specific for ubiquitin revealed no bands containing ubiquitinated homologues of this protein. These observations support the conclusion that proteasome mediates removal of some of the proteins oxidized during myocardial ischemia/reperfusion, and that at least oxidized actin is removed by the 20S proteasome.

Objective: To study the changes of skeletal muscle proteolysis in severely burned patients with sepsis and analyze its possible mechanism., Methods: Blood samples were collected from 20 burned patients with sepsis, aged 42 +/- 10 (burn sepsis group), and 12 patients undergoing plastic operation after burn, aged 43 +/- 8 (control group) to determine the plasma concentrations of cortisol and tumor necrosis factor (TNF-alpha) by radioimmunoassay. 24 h urine was retained to determine the level of 3-methylhistidine (3-MH) by high performance liquid chromatography. An appropriate amount of quadriceps femoris muscle sample was collected during operation to determine the concentration of 3-methylhistidine with high performance liquid chromatography, the expression of ubiquitin mRNA, E(2)-14k mRNA and C(2) subunit mRNA by Northern blot analysis, and the protein expression of C(2) subunit by Western blot analysis., Results: The plasma concentrations of cortisol and TNF-alpha in the burn sepsis group were 478 +/- 56 microg/L and 22.4 +/- 3.8 microg/L respectively, both significantly higher than the those in the normal control group, 72 +/- 12 microg/L and 0.5 +/- 0.1 microg/L respectively (both P < 0.01). The amount of 3-MH in the quadriceps femoris muscle of the burn sepsis group was 4.3 +/- 0.6 micromol/g, significantly higher than that of the control group (2.5 +/- 0.4 micromol/g, P < 0.01). The 24 h urinary output of 3-MH of the burn sepsis group was 456 +/- 25 micromol, significantly higher than that of the control group (202 +/- 29 micromol, P < 0.091). The expressions of ubiquitin mRNA 2.4 Kilobase pair (kb) and 1.2 kb, E(2)-14k mRNA 1.2 kb and C(2) subunit mRNA in quadriceps femoris muscles of the burn sepsis group were increased with 51%, 32%, 75%, and 39% respectively, as compared with those of the control group (all P < 0.01). The protein expression of C(2) subunit in quadriceps femoris muscles of the burn sepsis group was increased with 130% than that of the control group (P < 0.01)., Conclusion: The proteolytic rate in skeletal muscle is increased during sepsis after severe burn and the mechanism is related to the enhanced function of the ubiquitin system at both gene and protein levels with the hypersecretion of glucocorticoid and TNF-alpha.

Purpose of Review: To describe the latest innovations in measuring protein breakdown in vivo, particularly in muscle., Recent Findings: The traditional method of using 3-methylhistidine excretion to measure muscle protein breakdown has been updated to include arteriovenous or microdialysis measurements, which address the concern that there are alternative sources of 3-methylhistidine in the body other than muscle. Several variations of a precursor-product method to measure fractional breakdown rate of tissues have been developed that are analogous to fractional synthesis rate of tissues. These methods are more generally applicable than the 3-methylhistidine methods and are less invasive than arteriovenous methods. The various precursor-product methods are distinguished by whether they require an isotopic steady state or multiple tracers and by how many biopsies are required., Summary: The new precursor-product methods have enabled assessment in clinical trials of protein breakdown for proteins other than myofibrillar proteins and in circumstances in which arteriovenous sampling is not feasible.

Objective: To study the gene expression of C2 subunit of proteasome and proteolysis in cardiac muscle in burned animals with sepsis., Methods: Male Wistar rats were randomly divided into burn group, burn sepsis group and control group. The extent of burn injury was 30% total body surface area (TBSA) III degree burn, and the model of burn sepsis was replicated by administration of endotoxin (6 mg/kg) into the peritoneal cavity immediately after burn injury. The contents of 3-methylhistidine (3-MH) in cardiac muscle were determined with high performance liquid chromatography. The expression of C2 subunit mRNA in the cardiac muscle was assessed with Northern blot analysis., Results: The contents of 3-MH in cardiac muscle of burned rats with sepsis increased significantly at 2 hours and 6 hours, as compared with burned rats and normal control (all P<0.01). There was no marked difference of contents of 3-MH in cardiac muscle between burned rats at 2 hours and control group, however, the contents of 3-MH in cardiac muscle of burned rats at 6 hours increased significantly compared with control group. The gene expression of C2 subunit in cardiac muscle of burned rats with sepsis increased significantly at 2 hours and 6 hours compared with burned rats and normal control (all P<0.01). There were significant differences in the intensity of gene expression of C2 subunit in cardiac muscle between burned rats at 2 hours and 6 hours and normal control (all P<0.01)., Conclusion: The data suggest that the activity of ubiquitin system, one of important protein degradation pathways, and the proteolysis in cardiac muscle is continuously enhanced after burn injury, especially when sepsis set in, probably contributes to abnormal protein metabolism with malfunction of the heart during burn sepsis.