Muscle RNA Research Articles

Targeting nuclear RNA for in vivo correction of myotonic dystrophy

Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat1–3 and are retained in the nucleus4, 5. The mutant RNA exerts a toxic gain-of-function6, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target mRNAs7, 8. Here we show that nuclear-retained transcripts containing expanded CUG (CUGexp) repeats are extraordinarily sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUGexp RNA in skeletal muscle, correcting the physiological, histopathologic, and transcriptomic features of the disease. The effect was sustained for up to one year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat-1, a long noncoding RNA (lncRNA) that is retained in the nucleus9. These results provide a general strategy to correct RNA gain-of-function and modulate the expression of expanded repeats, lncRNAs, and other transcripts with prolonged nuclear residence.

Functional muscle impairment in facioscapulohumeral muscular dystrophy is correlated with oxidative stress and mitochondrial dysfunction

Facioscapulohumeral muscular dystrophy (FSHD), the most frequent muscular dystrophy, is an autosomal dominant disease. In most individuals with FSHD, symptoms are restricted to muscles of the face, arms, legs, and trunk. FSHD is genetically linked to contractions of the D4Z4 repeat array causing activation of several genes. One of these maps in the repeat itself and expresses the DUX4 (the double homeobox 4) transcription factor causing a gene deregulation cascade. In addition, analyses of the RNA or protein expression profiles in muscle have indicated deregulations in the oxidative stress response. Since oxidative stress affects peripheral muscle function, we investigated mitochondrial function and oxidative stress in skeletal muscle biopsies and blood samples from patients with FSHD and age-matched healthy controls, and evaluated their association with physical performances. We show that specifically, oxidative stress (lipid peroxidation and protein carbonylation), oxidative damage (lipofuscin accumulation), and antioxidant enzymes (catalase, copper–zinc-dependent superoxide dismutase, and glutathione reductase) were higher in FSHD than in control muscles. FSHD muscles also presented abnormal mitochondrial function (decreased cytochrome c oxidase activity and reduced ATP synthesis). In addition, the ratio between reduced (GSH) and oxidized glutathione (GSSG) was strongly decreased in all FSHD blood samples as a consequence of GSSG accumulation. Patients with FSHD also had reduced systemic antioxidative response molecules, such as low levels of zinc (a SOD cofactor), selenium (a GPx cofactor involved in the elimination of lipid peroxides), and vitamin C. Half of them had a low ratio of gamma/alpha tocopherol and higher ferritin concentrations. Both systemic oxidative stress and mitochondrial dysfunction were correlated with functional muscle impairment. Mitochondrial ATP production was significantly correlated with both quadriceps endurance (TLimQ) and maximal voluntary contraction (MVCQ) values (rho=0.79, P=0.003; rho=0.62, P=0.05, respectively). The plasma concentration of oxidized glutathione was negatively correlated with the TLimQ, MVCQ values, and the 2-min walk distance (MWT) values (rho=−0.60, P=0.03; rho=−0.56, P=0.04; rho=−0.93, P<0.0001, respectively). Our data characterized oxidative stress in patients with FSHD and demonstrated a correlation with their peripheral skeletal muscle dysfunction. They suggest that antioxidants that might modulate or delay oxidative insult may be useful in maintaining FSHD muscle functions.

Temporal dynamics in growth and white skeletal muscle composition of the mummichog Fundulus heteroclitus during chronic hypoxia and hyperoxia

Specific growth rate (G(S) ) and white skeletal muscle composition were measured in the mummichog Fundulus heteroclitus over a period of 28 days at four levels of dissolved oxygen (DO): severe hypoxia (c. 1.2 mg O(2) l(-1) ), moderate hypoxia (3.0 mg O(2) l(-1) ), normoxia (7.1 mg O(2) l(-1) ) and hyperoxia (10.6 mg O(2) l(-1) ). The G(S) was calculated over 0-8, 0-14, 0-28 and 14-28 days, and muscle protein, lactate dehydrogenase (LDH), DNA, RNA and water were measured at 0, 8, 14 and 28 days. Exposure of fish to severe hypoxia was associated with significantly reduced G(S) , lower muscle protein content and lower RNA:DNA compared with other DO treatments. When calculated over the first and second half of the 28 day exposure, however, G(S) of fish in severe hypoxia increased significantly during the second two-week interval, to the same rate as that of normoxic fish. Muscle LDH activity and water content were not significantly affected by DO level. Neither moderate hypoxia nor hyperoxia significantly affected G(S) or any biochemical variable. The results demonstrate that F. heteroclitus can tolerate wide variation in ambient oxygen concentration and, during prolonged exposure to severe hypoxia, shows significant compensation for the initial negative effects on growth. The capacity of F. heteroclitus to grow over a wide range of DO probably contributes to its ability to exploit habitats characterized by marked variation in oxygen availability.

Ecological Effects on Food Utilization, Trypsin Isozymes, and Performance Qualities of Growth and Maturation in Northeast Arctic Cod (Gadus morhua L.)

The work demonstrates, for the first time, natural biological changes in fish during maturing processes using a unique combination of different biochemical techniques. Northeast Arctic cod caught by demersal trawls were studied in three Barents Sea areas during February-March 2008. Older larger cod from area A (between North Kanin Bank and Eastern Basin) had higher food utilization efficiency (with females showing highest white muscle protein synthesis capacity) than those from areas B (Kanin Bank) and C (Central Bank). Populations in areas A and C living in separate environments showed parallel regressions of pyloric caecal slope T/C ratio, with different elevations of trypsin specific activities and trypsin isozymes expressions. Approximately 30 trypsin isozymes were observed, with 13 isozymes of possibly exogenous trypsin isozyme fragments from prey items found at higher percentage of cod in areas A and B with higher food varieties. Larger maturing cod required higher energy from carbohydrate (probably phytoplankton), as amylase specific activity correlated with body weight. White muscle RNA levels were varied among females from different areas, but not among males. RNA and RNA/protein ratio levels were higher in oocytes than white muscle, and these levels decreased in higher developed oocytes. Oocyte trypsin-like specific activity in areas B A > C) illustrated that females from area B (youngest with lowest oocyte protein turnover) had highest maturation rate, and would reach their spawning area(s) before those from areas A and C. Younger females (probably also males) living closer to coastal area with higher temperature and food varieties would reach sexual maturity faster than older cod and those living far from coastal area.

Unique microRNA expression patterns associated with macrophage infiltration following skeletal muscle injury

Skeletal muscle regeneration following injury requires macrophages. MicroRNAs (miRNAs) are vital to macrophage biology and may be key regulators of muscle regeneration. We compared temporal expression of miRNA in cardiotoxin‐injured muscle derived from wild type (WT, C57Bl/6J) and CCR2−/− mice to identify macrophage‐specific miRNA patterns. MiRNA expression was determined on muscle RNA by qRT‐PCR in TaqMan arrays. Compared to uninjured muscle, log‐fold changes in miRNA expression at 1, 3, or 7 days following injury (n=4 mice/group/timepoint) were determined. Flow cytometry confirmed impaired macrophage recruitment in CCR2−/− mice. The temporal expression patterns of 60 miRNAs were significantly different between strains, including miRNAs known to be highly expressed in macrophages (e.g., miR‐146a, miR‐106b, and miR‐221). Interestingly, 15 miRNAs with no known relationship to macrophages were matched with the temporal expression patterns of macrophage‐related miRNAs. These 15 miRNAs may be unique, macrophage‐related miRNA that regulate the muscle regenerative response. This study furthers our knowledge of miRNA regulation of the regenerative response in injured skeletal muscle and identifies potential miRNA targets for advancing tissue regeneration following traumatic injury. Funding: Veterans Administration and NIH (HL110743 and HL074236).

Investigations on Transgenerational Epigenetic Response Down the Male Line in F2 Pigs

We investigated the nutritional effects on carcass traits, gene expression and DNA methylation in a three generation Large White pig feeding experiment. A group of experimental (E) F0 boars were fed a standard diet supplemented with high amounts of methylating micronutrients whereas a control group (C) of F0 boars received a standard diet. These differentially fed F0 boars sired F1 boars which then sired 60 F2 pigs. Carcass traits were compared between 36 F2 descendants of E F0 boars and 24 F2 descendants of C F0 boars. The two F2 offspring groups differed with respect to backfat percentage (P = 0.03) and tended to differ with respect to adipose tissue (P = 0.09), fat thickness at the 10th rib (P = 0.08) and at the croup (P = 0.09) as well as percentages of shoulder (P = 0.07). Offspring from the experimental F0 boars had a higher percentage of shoulder and were leaner compared to the control group. Gene expression profiles showed significant twofold differences in mRNA level between 8 C F2 offspring and 8 E F2 offspring for 79, 64 and 53 genes for muscle, liver and kidney RNA, respectively. We found that in liver and muscle respective pathways of lipid metabolism and metabolic pathway were over-represented for the differentially expressed genes between these groups. A DNA methylation analysis in promoters of differentially expressed genes indicated a significant difference in DNA methylation at the IYD gene. If these responses on carcass traits, gene expression and DNA methylation withstand verification and can indeed be attributed to transgenerational epigenetic inheritance, it would open up pioneering application in pork production and would have implications for human health.

Generation of a reference transcriptome for evaluating rainbow trout responses to various stressors

BackgroundFish under intensive culture conditions are exposed to a variety of acute and chronic stressors, including high rearing densities, sub-optimal water quality, and severe thermal fluctuations. Such stressors are inherent in aquaculture production and can induce physiological responses with adverse effects on traits important to producers and consumers, including those associated with growth, nutrition, reproduction, immune response, and fillet quality. Understanding and monitoring the biological mechanisms underlying stress responses will facilitate alleviating their negative effects through selective breeding and changes in management practices, resulting in improved animal welfare and production efficiency.ResultsPhysiological responses to five treatments associated with stress were characterized by measuring plasma lysozyme activity, glucose, lactate, chloride, and cortisol concentrations, in addition to stress-associated transcripts by quantitative PCR. Results indicate that the fish had significant stressor-specific changes in their physiological conditions. Sequencing of a pooled normalized transcriptome library created from gill, brain, liver, spleen, kidney and muscle RNA of control and stressed fish produced 3,160,306 expressed sequence tags which were assembled and annotated. SNP discovery resulted in identification of ~58,000 putative single nucleotide polymorphisms including 24,479 which were predicted to fall within exons. Of these, 4907 were predicted to occupy the first position of a codon and 4110 the second, increasing the probability to impact amino acid sequence variation and potentially gene function.ConclusionWe have generated and characterized a reference transcriptome for rainbow trout that represents multiple tissues responding to multiple stressors common to aquaculture production environments. This resource compliments existing public transcriptome data and will facilitate approaches aiming to evaluate gene expression associated with stress in this species.

Genetic variability of transcript abundance in pig skeletal muscle at slaughter: Relationships with meat quality traits1

A family structured population of 325 pigs (females and barrows) was produced as an intercross between 2 commercial sire lines and was subjected to a systematic transcriptome analysis of LM samples obtained shortly after slaughter. Additionally, measurements of meat quality traits of fresh and cooked loin were gathered from the same animals. The transcriptome analysis was achieved by microarray hybridization, using a custom repertoire of 15,000 6mer DNA probes targeting transcripts expressed in growing pig skeletal muscle. These data allowed us to estimate the heritability of expression abundance for each of the quantified RNA species. The abundance of 9,765 RNA was estimated as heritable with a false discovery rate of 5%, from which 1,174 were deemed as highly heritable (h(2) > 0.50). We also observed a large number of transcripts whose LM expression abundance is genetically correlated with 4 meat quality traits: the loin pH measured at 45 min postmortem (pH45), 253 transcripts; the loin cooking loss (CL), 134 transcripts; the cooked loin shear force (SFc), 184 transcripts; and the loin color redness (a*) value, 190 transcripts. Heritable and meat quality genetically correlated transcripts showed an over-representation of biological processes involved in the induction of apoptosis (genetically correlated with CL), complement activation (genetically correlated with SFc), glucose metabolism (genetically correlated with a*), and cation channel activity (genetically correlated with pH45). Overall, the biological functions highlighted in the highly heritable transcripts and the lack of transcript that would be genetically correlated with LM glycolytic potential suggest that the genetic variability of the LM postmortem transcriptome is focused on muscle tissue response to postmortem ischemia and reflects more distantly the antemortem muscle physiology. Because of the contrasting distributions of the genetic correlations between LM RNA concentrations and the different meat quality traits studied, indirect selection strategies of meat quality traits based on measurements of selected LM RNA species could be only proposed for a subset of the analyzed meat characteristics (pH45, SFc, a*, CL). A substantial improvement in the efficiency of selection for these meat quality traits could result from measuring muscle RNA concentrations on selection candidates, if the same genetic parameters can be verified using in vivo-sampled muscles.

Effects of growth rate on carcass and meat quality traits and their association with metabolism‐related gene expression in finishing pigs

The objective of this study was to evaluate the effect of different growth rates on carcass characteristics and meat quality and their relationship with myogenesis and lipogenesis in finishing pigs. Pigs were slaughtered at the same age and were assigned to high (HGR) or low (LGR) growth rates with 120 kg or 110 kg final body weights at slaughter. The results indicated that pigs with HGR had heavier (P < 0.05) final body weight, carcass weight, dressing percentage, backfat thickness, higher (P < 0.05) concentrations of fat in the muscle and higher (P < 0.05) total RNA concentration in muscle than pigs with LGR. The messenger RNA (mRNA) expression of lipoprotein lipase, fatty acid synthase, malic enzyme, peroxisome proliferator-activated receptor γ, and sterol regulatory element binding protein-1 in fat were higher (P < 0.05) in pigs with HGR. Additionally, the mRNA expression of glycogen synthase in muscle was lower (P < 0.05) in pigs with HGR. These results indicate differences in postmortem myogenesis and lipogenesis traits of pigs with different growth rates; these differences in turn might have affected carcass characteristics and meat quality.

Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia–hyperaminoacidaemia in healthy young and middle-aged men and women

Increased dietary LCn-3PUFA (long-chain n-3 polyunsaturated fatty acid) intake stimulates muscle protein anabolism in individuals who experience muscle loss due to aging or cancer cachexia. However, it is not known whether LCn-3PUFAs elicit similar anabolic effects in healthy individuals. To answer this question, we evaluated the effect of 8weeks of LCn-3PUFA supplementation (4g of Lovaza®/day) in nine 25-45-year-old healthy subjects on the rate of muscle protein synthesis (by using stable isotope-labelled tracer techniques) and the activation (phosphorylation) of elements of the mTOR (mammalian target of rapamycin)/p70S6K (p70 S6 kinase) signalling pathway during basal post-absorptive conditions and during a hyperinsulinaemic-hyperaminoacidaemic clamp. We also measured the concentrations of protein, RNA and DNA in muscle to obtain indices of the protein synthetic capacity, translational efficiency and cell size. Neither the basal muscle protein fractional synthesis rate nor basal signalling element phosphorylation changed in response to LCn-3PUFA supplementation, but the anabolic response to insulin and amino acid infusion was greater after LCn-3PUFA [i.e. the muscle protein fractional synthesis rate during insulin and amino acid infusion increased from 0.062±0.004 to 0.083±0.007%/h and the phospho-mTOR (Ser2448) and phospho-p70S6K (Thr389) levels increased by ∼50%; all P<0.05]. In addition, the muscle protein concentration and the protein/DNA ratio (i.e. muscle cell size) were both greater (P<0.05) after LCn-3PUFA supplementation. We conclude that LCn-3PUFAs have anabolic properties in healthy young and middle-aged adults.

Advances in Exercise, Fitness, and Performance Genomics in 2010

This review of the exercise genomics literature emphasizes the strongest articles published in 2010 as defined by sample size, quality of phenotype measurements, quality of the exercise program or physical activity exposure, study design, adjustment for multiple testing, quality of genotyping, and other related study characteristics. One study on voluntary running wheel behavior was performed in 448 mice from 41 inbred strains. Several quantitative trait loci for running distance, speed, and duration were identified. Several studies on the alpha-3 actinin (ACTN3) R577X nonsense polymorphism and the angiotensin-converting enzyme (ACE) I/D polymorphism were reported with no clear evidence for a joint effect, but the studies were generally underpowered. Skeletal muscle RNA abundance at baseline for 29 transcripts and 11 single nucleotide polymorphisms (SNPs) were both found to be predictive of the V˙O2max response to exercise training in one report from multiple laboratories. None of the 50 loci associated with adiposity traits are known to influence physical activity behavior. However, physical activity seems to reduce the obesity-promoting effects of at least 12 of these loci. Evidence continues to be strong for a role of gene-exercise interaction effects on the improvement in insulin sensitivity after exposure to regular exercise. SNPs in the cAMP-responsive element binding position 1 (CREB1) gene were associated with training-induced HR response, in the C-reactive protein (CRP) gene with training-induced changes in left ventricular mass, and in the methylenetetrahydrofolate reductase (MTHFR) gene with carotid stiffness in low-fit individuals. We conclude that progress is being made but that high-quality research designs and replication studies with large sample sizes are urgently needed.

Regulation of organic nucleic acids and serum biochemistry parameters by dietary chromium picolinate supplementation in swine model

The relationships between chromium and metabolism are sophisticated. Organic nucleic acids and serum biochemistry parameters are affected by dietary chromium levels. The objective of this work was to study the effect of chromium picolinate (CrPic) supplementation on total DNA and RNA contents, the ratio of RNA/DNA in muscle and in pancreatic tissue, the level of insulin receptor (IR) mRNA and some serum biochemistry parameters in a porcine model. Young animals (48) were assigned randomly into three groups of 16 piglets, fed with three different dietary levels of Cr (common basal feedstuff alone or supplemented with CrPic at a dose of 1.61 μg/g or 3.22 μg/g, which corresponds to 0.2 μg/g and 0.4 μg/g Cr). After 80 days, the animals were sacrificed and skeletal muscle and pancreatic tissues were analyzed to detect differences caused by different levels of dietary Cr. The total content of RNA in muscle was increased significantly ( P < 0.05) in the CrPic supplemented groups. There was no significant difference between groups in the concentrations of total RNA in the pancreas or DNA in the muscle and pancreatic tissues. The RNA/DNA ratio in pancreas showed no significant change but the ratio was increased significantly ( P < 0.05) in muscle. There was a slight increase of the mRNA level of IR but there was no significant difference between groups. The content of serum cholesterol and insulin were reduced significantly ( P < 0.05) in the CrPic-supplemented groups and the content of high-density lipoprotein cholesterol (HDLC) was increased significantly ( P < 0.05) as the CrPic dose increased. There was a slight (non-significant) reduction of the concentrations of serum triglyceride and low-density lipoprotein cholesterol (LDLC) in the CrPic supplementation groups. Supplementary CrPic caused no significant change of muscular mRNA level of IR in healthy animals. An increased content of RNA in muscle, improved cholesterol metabolism and improved insulin sensitivity were found in these CrPic-treated groups in the porcine model.

Evaluation of early biomarkers of muscle anabolic response to testosterone

BackgroundEarly biomarkers of skeletal muscle anabolism will facilitate the development of therapies for sarcopenia and frailty.Methods and resultsWe examined plasma type III collagen N-terminal propeptide (P3NP), skeletal muscle protein fractional synthesis rate, and gene and protein expression profiles to identify testosterone-induced changes in muscle anabolism. Two placebo-controlled studies enrolled community-dwelling men (study 1, 60–75 years; study 2, 18–40 years) with low to normal testosterone levels. Men were randomized to lower dose (study 1, 100 mg; study 2, 200 mg) or higher dose (study 1, 300 mg; study 2, 600 mg) single intramuscular testosterone or saline injection. After 1 week, testosterone acutely increased plasma P3NP levels in a dose-dependent manner and altered the expression of several skeletal muscle transcripts and proteins. Though not statistically significant, mixed muscle protein fractional synthesis rate tended to increase (1.08-fold with 100 mg testosterone, 1.12-fold with 300 mg testosterone). Testosterone exposure also increased skeletal muscle expression of the collagen type III gene that encodes P3NP.ConclusionP3NP is a potentially useful early biomarker for muscle anabolic therapy. Skeletal muscle protein and RNA profiling are useful tools for the discovery of novel muscle anabolic biomarkers.

Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

Histidine-rich glycoprotein (HRG) is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD). We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD) cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

Effect of branched-chain amino acid supplementation during unloading on regulatory components of protein synthesis in atrophied soleus muscles

Maintenance of skeletal muscle mass depends on the equilibrium between protein synthesis and protein breakdown; diminished functional demand during unloading breaks this balance and leads to muscle atrophy. The current study analyzed time-course alterations in regulatory genes and proteins in the unloaded soleus muscle and the effects of branched-chain amino acid (BCAA) supplementation on muscle atrophy and abundance of molecules that regulate protein turnover. Short-term (6days) hindlimb suspension of rats resulted in significant losses of myofibrillar proteins, total RNA, and rRNAs and pronounced atrophy of the soleus muscle. Muscle disuse induced upregulation and increases in the abundance of the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), increases in gene and protein amounts of two ubiquitin ligases (muscle RING-finger protein 1 and muscle atrophy F-box protein), and decreases in the expression of cyclin D1, the ribosomal protein S6 kinase 1, the mammalian target of rapamycin (mTOR), and ERK1/2. BCAA addition to the diet did not prevent muscle atrophy and had no apparent effect on regulators of proteasomal protein degradation. However, BCAA supplementation reduced the loss of myofibrillar proteins and RNA, attenuated the increases in 4E-BP1, and partially preserved cyclin D1, mTOR and ERK1 proteins. These results indicate that BCAA supplementation alone does not oppose protein degradation but partly preserves specific signal transduction proteins that act as regulators of protein synthesis and cell growth in the non-weight-bearing soleus muscle.

Effect of Concomitant Administration of a-tocopherol and ACEI on Cardiovascular Performance and Nucleic Acid Changes in DOCA Hypertensive Rats

Background: Evidence for cardiovascular oxidative stress in animals with hypertension has been accumulated. Antioxidant agents have been used to treat endothelial dysfunction and arterial hypertension. Aim: The aim of present study is to elucidate the effect of concomitant administration of α-tocopherol (antioxidant) and angiotensin converting enzyme inhibitor (ACEI; anti-hyptertensive) on cardiac smooth muscle DNA and RNA changes in DOCA induced hypertension in rats. Methods: 48 male Sprague-Dawley rats were used in this study and were divided into 6 groups (n=8): C control rats; CE control rats received α-tocopherol (60 mg/kg) intragastrically (IG); DOCA rats were made hypertensive by SC injection of DOCA (50 mg/kg once weekly for 4 weeks); DOCA+E; DOCA hypertensive rats received α-tocopherol (60 mg/kg) IG; DOCA+ACEI; DOCA hypertensive rats received ACEI (captopril; 15 mg/kg) IG; and DOCA+E+ACEI; DOCA hypertensive rats received both α-tocopherol and captopril in the same doses as DOCA+E and DOCA+ACEI groups respectively. All IG treatments were given to the animals 6 days weekly for 6 weeks. At the end of experimental protocol, systolic blood pressure (MSP), renal vascular flow velocity (RFV), renal vascular resistance (RVR), myocardial contractility (MC) and heart rate (HR) were measured in rats of all groups. Also, serum sodium (Na + ), potassium (K + ) and total plasma antioxidant capacity (TOC) were measured. DNA and RNA analysis were done for the myocardial tissue homogenate. Results: While DOCA administration in rats produced significant increase in serum Na + , MSP, RVR, and RNA levels; it reduced significantly serum K + , TOC, MC and DNA levels compared with the control group. Concomitant administration of both α-tocopherol and ACEI resulted in significant reduction of serum Na + ,

Effects of various processed soybeans meal on growth and protein metabolism of &lt;I&gt;Cyprinus carpio&lt;/I&gt; juveniles

The effect of replacing fish meal with raw and various processed soybeans meal on growth and protein metabolism of common carp Cyprinus carpio was evaluated in an 8-week trial. The common carp fingerlings[initial body weight(5.89±0.36) g] were fed five isonitrogenous and isocaloric feeds(30% digestible protein and 15 MJ/kg digestible energy) containing SBM replacing 50% of the fish meal protein,such as raw soybean(RSB),processed soybean by chemical inactivator,processed soybean by heating,processed soybean by heat alcohol,respectively. Results showed that weight gain of control were significantly different(P0.05),but those fed RSB were significantly lower than control(P0.05),those fed processed soybeans by chemical inactivator and by heating and by heat alcohol had no significant difference,but all significantly higher than control. Protease specific activity of control in the intestine and hepatopancreas was significantly higher than those fed RSB and processed soybeans by heat alcohol,but both had no significant difference with those fed processed soybeans by chemical inactivator and by heating(P0.05). RNA of 100% fish meal and 50% processed soybean by heating in white muscle were no significant difference(P0.05),but both were significantly higher than those fed RSB and processed soybeans by heat alcohol and by chemical inactivator and by heating,RNA/DNA of 100% fish meal and 50% processed soybean by heating and by chemical inactivator in white muscle had no significant difference(P0.05),but all were higher than those fed raw soybean(RSB) and processed soybean by heat alcohol(P0.05). RNA in white muscle and serum IGF-I had a positive correlation with growth(P0.05).The degree of negative effect of soybean anti-nutritional factors on fish is anti-trypsin inhibitoragglutininsoybean antigen protein under the experimental conditions,and proper control could reduce the negative impact of soybean anti-nutritional factors on fish.

Beneficial Effects of a Q-ter® Based Nutritional Mixture on Functional Performance, Mitochondrial Function, and Oxidative Stress in Rats

BackgroundMitochondrial dysfunction and oxidative stress are central mechanisms underlying the aging process and the pathogenesis of many age-related diseases. Selected antioxidants and specific combinations of nutritional compounds could target many biochemical pathways that affect both oxidative stress and mitochondrial function and, thereby, preserve or enhance physical performance.Methodology/Principal FindingsIn this study, we evaluated the potential anti-aging benefits of a Q-ter® based nutritional mixture (commercially known as Eufortyn®) mainly containing the following compounds: terclatrated coenzyme Q10 (Q-ter®), creatine and a standardized ginseng extract. We found that Eufortyn® supplementation significantly ameliorated the age-associated decreases in grip strength and gastrocnemius subsarcolemmal mitochondria Ca2+ retention capacity when initiated in male Fischer344 x Brown Norway rats at 21 months, but not 29 months, of age. Moreover, the increases in muscle RNA oxidation and subsarcolemmal mitochondrial protein carbonyl levels, as well as the decline of total urine antioxidant power, which develop late in life, were mitigated by Eufortyn® supplementation in rats at 29 months of age.Conclusions/SignificanceThese data imply that Eufortyn® is efficacious in reducing oxidative damage, improving the age-related mitochondrial functional decline, and preserving physical performance when initiated in animals at early midlife (21 months). The efficacy varied, however, according to the age at which the supplementation was provided, as initiation in late middle age (29 months) was incapable of restoring grip strength and mitochondrial function. Therefore, the Eufortyn® supplementation may be particularly beneficial when initiated prior to major biological and functional declines that appear to occur with advancing age.

Molecular Identification of Carnosine Synthase as ATP-grasp Domain-containing Protein 1 (ATPGD1)

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyryl-L-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the "ATP-grasp family" of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15-25-fold higher catalytic efficiencies on beta-alanine than on gamma-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown.

Histochemical pattern of gastrocnemius muscle in fluoride toxicity syndrome

Objective To evaluate the effect of sodium fluoride (NaF) on cytochemical distribution of proteins, DNA, and RNA in the gastrocnemius muscle of rat in experimental fluorosis. Methods Young Sprague Dawley albino rats were administered with NaF at 30, 45, and 75 mg/kg bw/day subcutaneously for 15 and 30 days, respectively. The control animals were given the vehicle (1cc deionized double distilled water/kg bw/day). Results In the first phase of 15 days experimentation, the gastrocnemius muscle of rats intoxicated with NaF at 30, 45, and 75 mg/kg bw/day showed decline in proteins including α amino acids as compared to control. In the second phase of 30 days experimentation, the muscle fibers of rat showed elevation in sarcolemmal and sarcoplasmic proteins in 30 mg NaF dose group, and angulated fibers exhibited increase in sarcolemmal proteins in 45 mg NaF group. The marginal regions of angular and rim fibers showed deeply stained rings of sarcoplasmic proteins whereas the split fibers were faintly stained in rats treated with NaF at 75 mg/kg bw/day for 30 days. In rats treated with NaF at 30 mg/kg bw/day for 15 days, the hypertrophied peripheral muscle fibers contained more DNA, however the atrophied fibers had more RNA. In 45 mg NaF group, RNA was located in sarcolemmal regions, while DNA content decreased as compared to control. In 75 mg NaF group, the muscle fibers had dark and light staining regions of DNA and RNA. In the 30 days of experimentation, the DNA level decreased whereas RNA content increased in the gastrocnemius muscle fibers of the rat treated with NaF at 30 mg/kg bw/day. In treatment group with NaF at 45 mg/kg bw/day, the RNA content slightly declined in comparison to control, in all treatments for 15 days as well as in treatment group with NaF at 30 mg/kg bw/day for 30 days whereas the amount of DNA slightly increased as compared to treatment group with NaF at 75 mg/kg bw/day for 15 days. The highest dose group revealed elevated amount of RNA whereas DNA content remained stable. Conclusions The findings of present study demonstrate that certain concentrations of fluoride can induce muscle lesions and damage DNA, RNA, and protein in muscle cells and excessive intake and accumulation of fluoride is therefore a serious risk factor for muscular abnormalities in fluorosis.