Myofibrillar Protein Breakdown Research Articles

Myofibrillar proteolysis in response to voluntary or electrically stimulated muscle contractions in humans

Knowledge about the effects of exercise on myofibrillar protein breakdown in human subjects is limited. Our purpose was to measure the changes in the degradation of myofibrillar proteins in response to different ways of eliciting muscle contractions using the local interstitial 3-methyl-histidine (3-MH) concentration as a marker for myofibrillar protein breakdown. Untrained males (n=8, 22-27 years, range) performed 210 maximal isokinetic eccentric contractions with each leg on an isokinetic dynamometer. One leg performed voluntary (VOL) and the other leg performed electrically induced contractions (ES). Microdialysis probes were placed in m. vastus lateralis in both the legs immediately after, and 1 and 3 days post-exercise. Interstitial 3-MH was higher in ES vs VOL immediately after exercise (P<0.05). One and 3 days post-exercise no difference between the two exercise types was observed. Only after ES did the histochemical stainings show significant disruption of cytoskeletal proteins. Furthermore, intracellular disruption and destroyed Z-lines were markedly more pronounced in ES vs VOL. In conclusion, the local level of interstitial 3-MH in the skeletal muscle was significantly enhanced after ES compared with VOL immediately after exercise, while the level of 3-MH did not change in the post-exercise period after VOL. These results indicate that the local myofibrillar breakdown is accelerated after ES associated with severe myofiber damage.

Reliability of results and interpretation of measures of 3-methylhistidine in muscle interstitium as marker of muscle proteolysis

TO THE EDITOR: We were interested in the contribution by Tesch and colleagues to the evidence base concerning the events subsequent to acute immobilization in human muscle. Tesch and colleagues (14) used a microdialysis technique applied to human muscle in an attempt to measure an index of myofibrillar proteolysis 72 h after immobilization using the unilateral leg suspension technique. The authors discuss their results as though they had good evidence that the technique they used produces results that are sufficiently firm to be the basis of conjecture about mechanisms of protein balance in muscle after immobilization. We demur and propose that they are on very shaky ground. First, we wonder about the size of the effect reported. The authors claim it to be 44%, but calculation of the change shows it to be actually 29% with the SEs sufficiently large that they almost span the difference; SDs must overlap. Significance at the 5% level can only barely have been reached. Are the authors convinced that they have not ignored a type I error? The authors (14) begin their explanation of the advantages of the dialysis technique using 3-methylhistidine (3MeHis) by describing the gold standard method, i.e., the stable isotopelabeled amino acid tracer dilution method, as cumbersome and possibly unjustified in study of otherwise healthy subjects. We find this notion strange since Ferrando and colleagues (6, 12) have used the technique in many studies of how immobilization affects muscle protein turnover subsequent to disuse. We have also used the techniques in a wide variety of studies of proteolysis in healthy and infirm subjects (1, 2, 10, 11, 15) with no problems and, we would propose, producing results with a greater degree of certainty and biological appropriateness than those obtained by the 3MeHis dialysis technique.

Protein breakdown on whole-body and organ level in non-cachectic tumour-bearing mice undergoing surgery

Since both cancer and surgery are known to alter protein turnover, we investigated how the presence of tumour affects post-operative protein breakdown. 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. 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. 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.

Growth, carcass quality, and protein and energy metabolism in beef cattle with different growth potentials and residual feed intakes1,2

Twenty-four beef steers (predominantly Angus x Hereford, 14 to 18 mo of age, 403 +/- 3 kg of BW), were housed and fed in individual pens for about 122 d. Twelve steers came from a herd that had been selected for growth (high growth; HG) and the other 12 from a herd with no selection program (low growth; LG). Another 6 steers (3 from each group) were slaughtered at the beginning to obtain the initial composition. All steers were fed the same corn-based diet (3.06 Mcal of ME/kg of DM, 13.6% CP) on an ad libitum basis. Two weeks before slaughter, total urine was collected for 5 d for estimation of 3-methylhistidine excretion and myofibrillar protein breakdown rates. Compared with LG steers, HG steers had less initial BW but greater final BW, DMI (7.52 vs. 6.37 kg/d), ADG (1.33 vs. 0.853 kg/d), G:F (0.176 vs. 0.133 kg/kg), ME intake (0.233 vs. 0.201 Mcal x kg of BW(0.75) x d(-1)), and retained energy (RE; 0.0711 vs. 0.0558 Mcal x kg of BW(0.75) x d(-1)); gained more fat (676 vs. 475 g/d); and tended to gain more whole body protein (100 vs. 72 g/d), with no difference in residual feed intake (RFI). Estimated net energetic efficiency of gain (k(g)) and ME for maintenance (ME(m)) did not differ between the 2 groups, averaging 0.62 and 0.114, respectively. The HG steers had greater HCW (350 vs. 329 kg), backfat (16.1 vs. 11.6 mm), and yield grades (3.53 vs. 2.80), with a similar dressing percent, KPH fat, LM area, and marbling score. Skeletal muscle protein gain (70.2 vs. 57.6 g/d) and fractional protein accretion rate (0.242 vs. 0.197%/d) tended to be greater in HG than in LG steers. Steers were classified into low (-0.367 kg/d) and high (0.380 kg/d) RFI classes. Compared with the high RFI steers, low RFI steers consumed less DM (6.61 vs. 7.52 kg/d) and ME (0.206 vs. 0.234 Mcal x kg of BW(0.75) x d(-1)) and tended to gain less fat (494 vs. 719 g/d), but were similar for initial and final BW, ADG, G:F, protein gain, HCW, dressing percent, backfat, KPH fat, LM area, marbling score, and yield grade, as well as for all observations related to myofibrillar protein metabolism. Residual feed intake may be positively [corrected] correlated with ME for maintenance. The maintenance energy requirement increased by 0.0166 Mcal x kg(-0.75) x d(-1) for each percentage increase in fractional protein degradation rate, confirming the importance of this process in the energy economy of the animal.

Greater whole-body myofibrillar protein breakdown in cachectic patients with chronic obstructive pulmonary disease

Experimental studies indicate that greater skeletal muscle protein breakdown is a trigger for the cachexia that often is prevalent in chronic obstructive pulmonary disease (COPD). We compared myofibrillar protein breakdown (MPB) with whole-body (WB) protein breakdown (PB) in 9 cachectic COPD patients [x +/- SEM forced expiratory volume in 1 s (FEV(1)): 48 +/- 4% of predicted], 7 noncachectic COPD patients (FEV(1): 53 +/- 5% of predicted), and 7 age-matched healthy control subjects, who were matched by body mass index with the noncachectic patients. After the subjects fasted overnight (10 h) and discontinued the maintenance medication, a primed constant and continuous infusion protocol was used to infuse L-[ring-(2)H(5)]-phenylalanine and L-[ring-(2)H(2)]-tyrosine to measure WB protein turnover and L-[(2)H(3)]-3-methylhistidine to measure WB MPB. Three arterialized venous blood samples were taken between 80 and 90 min of infusion to measure amino acid concentrations and tracer enrichments. Body composition, WB protein turnover, and WB MPB did not differ significantly between the noncachectic COPD and control subjects. Cachectic COPD patients had lower fat mass and fat-free mass values (both: P < 0.01) than did the noncachectic COPD patients. WB MPB was significantly (P < 0.05) higher in the cachectic COPD group (18 +/- 3 nmol . kg(-1) . min(-1)) than in the combined control and noncachectic COPD groups (10 +/- 1 nmol . kg(-1) . min(-1)), but WB protein turnover did not differ significantly between the groups. Correlations with fat-free mass were significant (P < 0.05) for plasma glutamate and branched-chain amino acids, and that for WB MPB trended toward significance (P = 0.07). Cachexia in clinically stable patients with moderate COPD is characterized by increased WB MPB, which indicates that myofibrillar protein wasting is an important target for nutritional and pharmacologic modulation.

A reexamination of the effect of exercise on rate of muscle protein degradation.

The purpose of this study was to examine the effect of exercise on the rate of protein degradation in rat skeletal muscle. The rates of total and myofibrillar protein degradation were determined by the measurement of the rates of release of tyrosine and 3-methylhistidine, respectively, from the perfused single rat leg. This method measures the rate of protein degradation in the entire lower leg and does not suffer from the limitations inherent in methods that rely on urinary excretion. The rate of total protein degradation was increased by exercise and involved increased flux through the lysosomal pathway, while the breakdown of myofibrillar protein was unchanged. The changes in the rates of protein degradation during the recovery period were greatly influenced by energy intake. Again the rate of myofibrillar protein degradation was unchanged or slightly increased during the recovery period, after either level or downgrade running. Exercise did prevent the increase in the rate of total protein degradation caused by food restriction, which may have important implications in weight reduction diets.

Mechanisms of Statin-Induced Myopathy

The advent of the HMG-CoA reductase inhibitors, or statins, in the 1980’s as highly efficacious agents for the lowering of low-density lipoprotein-cholesterol (LDL-C) revolutionized treatment of hypercholesterolemia, a long established risk factor for premature coronary heart disease. Indeed, a recent prospective meta-analysis of data from more than 90 000 participants in 14 randomized clinical trials revealed that a statin-mediated reduction of 1 mmol/L (40 mg/dL) in LDL-C that is sustained for 5 years may produce a proportional reduction in major vascular events of some 23%.1 Greater reductions in LDL-C, which may be attained with intensive statin therapy as exemplified in the recent Pravastatin or Atorvastatin Evaluation and Infection Therapy–Thrombolysis in Myocardial Infarction 22 (PROVE IT-TIMI 22), Treating to New Targets (TNT), and Reversal of Atherosclerosis with Aggressive Lipid Lowering (REVERSAL) trials involving use of atorvastatin (80 mg/d), produce larger reductions in vascular disease risk.2–4 Importantly, risk reductions are proportional to the absolute reduction in LDL-C1, and moreover clinical benefit may be apparent with intensive statin treatment as early as 30 days after initiation in acute coronary syndrome patients, with significant decrement in cardiovascular morbi-mortality.5 See page 2560 Statins not only exhibit a remarkably high benefit to risk ratio, but are equally characterized by a safety profile with excellent tolerance.6,7 Nonetheless, statins may exert toxic effects on skeletal muscle which are generally referred to as myopathy, and whose overall incidence is typically <0.1% of patients receiving statin monotherapy.6 Although myopathy can refer to any muscular disease, the recent clinical advisory on the use and safety of statins differentiated myalgia as muscle ache or weakness in the absence of elevation in creatine kinase (CK), and myositis as adverse muscular symptoms associated with increased CK levels.7 Rhabdomyolysis is a severe form of myositis involving myoglobulinuria, …

Overexpression of UCP3 in both murine and human myotubes is linked with the activation of proteolytic systems: A role in muscle wasting?

Overexpression of the UCP3 gene in both murine and human myotube cell cultures leads to a significant activation of the different proteolytic systems involved in muscle myofibrillar protein breakdown. Thus, lysosomal (cathepsin B) and non-lysosomal (m-calpain and ubiquitin-proteasome) mRNA content was significantly increased in the different cell culture systems used. Interestingly, the overexpression of the UCP3 gene was not associated with any changes in apoptosis. Although the function of the UCP3 protein is not completely understood (uncoupling, oxidative stress), these results suggest a possible relation between these main mechanisms involved in muscle wasting during cancer.

The Effect of Preventive Use of Alanyl-Glutamine on Diaphragm Muscle Function in Cecal Ligation and Puncture-Induced Sepsis Model

Low muscle glutamine levels during sepsis are associated with reduced protein synthesis and elevated protein breakdown, in particular myofibrillar protein breakdown. Thus, in a cecal ligation and puncture (CLP)-induced sepsis model in the rat, we hypothesized that glutamine pretreatment would protect the diaphragm muscle function. Eighty-four male Wistar rats weighing between 180 g and 200 g received standard amino acid solution 1.2 g kg(-1) per day intraperitoneally (IP) or standard amino acid solution 1.2 g kg(-1) per day plus alanyl-glutamine (GLN) 0.25 g kg(-1) per day (IP) during the first 6 days of the experiment. On the seventh day, CLP or sham procedures were applied. The sham and CLP groups were equally divided into 3 subgroups according to the termination of the experiment, which took place at either the 24th hour, 48th hour, or 72nd hour. After the compound muscle action potentials (CMAP) were recorded from the diaphragms of the rats at these selected times, they were decapitated under ketamine/xylazine anesthesia, and diaphragms were harvested for biochemical and histopathological examination. The mean area and amplitude of CMAP were significantly larger in sham+GLN groups when compared with CLP and CLP+GLN groups at all times (p < .05). Diaphragm Ca+2 -ATPase levels were found to be significantly decreased in CLP group at all times compared to sham groups (p < .05). Diaphragm reduced glutathione levels were significantly higher in sham+GLN groups when compared with CLP and CLP+GLN groups at all times (p < .05). In histopathologic assessment, moderate neutrophil infiltration, which was observed in CLP48, was significantly reduced with alanyl-glutamine supplementation in CLP+GLN48 group (p < .05). This study showed that glutamine pretreatment did not improve diaphragm muscle function, but prevented the biochemical and histopathological changes in diaphragmatic muscle in CLP-induced sepsis. However, further studies are needed to clarify whether a higher dose of glutamine supplementation might protect the diaphragmatic muscle functions.

The Effect of Preventive Use of Alanyl‐Glutamine on Diaphragm Muscle Function in Cecal Ligation and Puncture–Induced Sepsis Model

Background: Low muscle glutamine levels during sepsis are associated with reduced protein synthesis and elevated protein breakdown, in particular myofibrillar protein breakdown. Thus, in a cecal ligation and puncture (CLP)‐induced sepsis model in the rat, we hypothesized that glutamine pretreatment would protect the diaphragm muscle function. Methods: Eighty‐four male Wistar rats weighing between 180 g and 200 g received standard amino acid solution 1.2 g kg−1 per day intraperitoneally (IP) or standard amino acid solution 1.2 g kg−1 per day plus alanyl‐glutamine (GLN) 0.25 g kg−1 per day (IP) during the first 6 days of the experiment. On the seventh day, CLP or sham procedures were applied. The sham and CLP groups were equally divided into 3 subgroups according to the termination of the experiment, which took place at either the 24th hour, 48th hour, or 72nd hour. After the compound muscle action potentials (CMAP) were recorded from the diaphragms of the rats at these selected times, they were decapitated under ketamine/xylazine anesthesia, and diaphragms were harvested for biochemical and histopathological examination. Results: The mean area and amplitude of CMAP were significantly larger in sham+GLN groups when compared with CLP and CLP+GLN groups at all times (p &lt; .05). Diaphragm Ca+2‐ATPase levels were found to be significantly decreased in CLP group at all times compared to sham groups (p &lt; .05). Diaphragm reduced glutathione levels were significantly higher in sham+GLN groups when compared with CLP and CLP+GLN groups at all times (p &lt; .05). In histopathologic assessment, moderate neutrophil infiltration, which was observed in CLP48, was significantly reduced with alanyl‐glutamine supplementation in CLP+GLN48 group (p &lt; .05). Conclusions: This study showed that glutamine pretreatment did not improve diaphragm muscle function, but prevented the biochemical and histopathological changes in diaphragmatic muscle in CLP‐induced sepsis. However, further studies are needed to clarify whether a higher dose of glutamine supplementation might protect the diaphragmatic muscle functions.

Protein utilisation and turnover in lines of chickens selected for different aspects of body composition

1. Protein utilisation and turnover were measured in male chickens sampled from a line selected for high breast yield and a randombred control line (lines QL and CL, experiment 1) and in male chickens sampled from lines selected for either high or low abdominal fatness (lines FL and LL, experiment 2). In each experiment, 18 birds per line were given iso-energetic (12·9 MJ ME/kg) diets containing either 120 or 220 g CP/kg from 21 to 29 d (experiment 1) and 33 to 43 d (experiment 2). 2. Measurements were made of growth rate, food intake, body composition, excreta production and Nτ-methylhistidine excretion as a measure of myofibrillar protein breakdown, and fractional rates (%/d) of protein deposition, breakdown and synthesis were calculated. 3. In experiment 1, there were no significant differences between the line means for the fractional measures of protein turnover, but there was marked differential response in the two lines in the fractional rates of protein deposition, breakdown and synthesis, to increase in protein intake. The positive slope of the regressions of fractional (%/d) protein deposition and synthesis rates on protein intake (g/d/kg BW) were approximately 1·4- and 2·0-fold higher respectively in the QL than the CL line birds, and the negative slope of the regression of fractional breakdown rate on protein intake was approximately threefold greater in the CL than the QL line birds. 4. In experiment 2, fractional deposition rate was 6·2% lower, but fractional breakdown rate 9·4% higher in the LL than the FL birds, whilst there was essentially no difference in response of the FL and LL birds in the components of protein turnover to increase in protein intake. Line differences in deposition and breakdown rates were thus a reflection of the considerably higher (20%) food and hence protein intake in the FL than the LL birds. 5. The differential line responses in protein turnover in the two experiments suggest that selection for increased breast muscle yield and for reduced body fatness manipulate different physiological pathways in relation to protein turnover, but neither selection strategy results in an improvement in net protein utilisation at typical levels of protein intake by birds on commercial broiler diets, through a reduction in protein breakdown rate.

Creatine monohydrate enhances strength and body composition in Duchenne muscular dystrophy

To determine whether creatine monohydrate (CrM) supplementation increases strength and fat-free mass (FFM) in boys with Duchenne muscular dystrophy (DD). Thirty boys with DD (50% were taking corticosteroids) completed a double-blind, randomized, cross-over trial with 4 months of CrM (about 0.10 g/kg/day), 6-week wash-out, and 4 months of placebo. Measurements were completed of pulmonary function, compound manual muscle and handgrip strength, functional tasks, activity of daily living, body composition, serum creatine kinase and gamma-glutamyl transferase activity and creatinine, urinary markers of myofibrillar protein breakdown (3-methylhistidine), DNA oxidative stress (8-hydroxy-2-deoxyguanosine [8-OH-2-dG]), and bone degradation (N-telopeptides). During the CrM treatment phase, there was an increase in handgrip strength in the dominant hand and FFM (p < 0.05), with a trend toward a loss of global muscle strength (p = 0.056) only for the placebo phase, with no improvements in functional tasks or activities of daily living. Corticosteroid use, but not CrM treatment, was associated with a lower 8-OH-2-dG/creatinine (p < 0.05), and CrM treatment was associated with a reduction in N-telopeptides (p < 0.05). Four months of CrM supplementation led to increases in FFM and handgrip strength in the dominant hand and a reduction in a marker of bone breakdown and was well tolerated in children with DD.

The role of ubiquitin-proteasome-dependent proteolysis in the remodelling of skeletal muscle.

In skeletal muscle, as in any mammalian tissue, protein levels are dictated by relative rates of protein synthesis and breakdown. Recent studies have shown that the ubiquitin-proteasome-dependent proteolytic pathway is mainly responsible for the breakdown of myofibrillar proteins. In this pathway proteins that are to be degraded are first tagged with a polyubiquitin degradation signal. Ubiquitination is performed by the ubiquitin-activating enzyme, ubiquitin-conjugating enzymes and ubiquitin-protein ligases, which are responsible for the recognition of specific substrates. Polyubiquitinated protein substrates are then specifically recognised and degraded by the 26S proteasome. The present review focuses on: (1) the mechanisms of ubiquitination-deubiquitination that make the system highly selective; (2) the mechanisms of proteolysis in skeletal muscle. In particular, the role of the system in the remodelling of skeletal muscle during exercise and disuse and in recovery or regeneration that prevails during post-atrophic conditions is reviewed.

Proteasome production in human muscle during nutritional inhibition of myofibrillar protein degradation

Protein undernutrition inhibits adenosine triphosphate (ATP)-dependent muscle protein degradation—a hallmark of the proteasome system. Here we report decreased myofibrillar protein degradation during dietary protein restriction without a concomitant decrease in proteasome gene expression, proteasome protein abundance, or proteasome in vivo fractional synthesis rate. Healthy human subjects consuming the average minimum adult protein requirement (0.71 g · kg −1 fat-free mass · d −1) exhibited substantially lower (68%) excretion of 3-methylhistidine, an indicator of myofibrillar protein breakdown, when compared with subjects consuming an ample, American-style protein intake (1.67 g · kg −1 fat-free mass · d −1). However, they displayed no difference in the expression of mRNA for proteasome subunits C2 or C3, in the content of C2 protein, or in the rate of incorporation of stable isotopically labeled l-[1- 13 C]-leucine into proteasome proteins. The results demonstrate that nutritional inhibition of myofibrillar protein degradation does not involve suppression in vivo of proteasome production in man. This suggests that other elements of the ubiquitin-proteasome system, such as ubiquitination pathways, are more important than proteasome abundance in the nutritional regulation of skeletal muscle mass.

Sepsis induces the transcription of the glucocorticoid receptor in skeletal muscle cells.

Evidence from a recent study indicates that glucocorticoids (GCs) mediate skeletal muscle proteolysis during sepsis via the GC receptor (GR) pathway. Attempts to identify the mechanisms regulating GR gene expression in skeletal muscle during sepsis have been hampered by the lack of an appropriate in vitro model system that can mimic in vivo septic conditions. In the present study, we report that GR gene transcription in L6 myocytes in vitro is up-regulated by treatment with sera from septic rats in a manner similar to that measured in septic rats in vivo. Sera from septic rats were collected from animals in which sepsis was induced by caecal ligation and puncture and from control rats that were sham-operated. Finally, by treating L6 myotubes with the GR antagonist RU 38486, thereby preventing sepsis-induced GR transcription, we confirmed that the possible septic effect on the GR was due to increased GCs. L6 myocytes treated with sera from septic rats might therefore be useful as an experimental model for identifying the molecular mechanisms by which the GR regulates muscle cachexia during sepsis. Furthermore, RU 38486 inhibited the sepsis-induced increase in total and myofibrillar energy-dependent protein breakdown rates in incubated extensor digitorum longus muscles from septic and sham-operated rats, as measured by release of tyrosine and 3-methylhistidine respectively. Our results demonstrate for the first time that sepsis induces GR transcription in skeletal muscle, and supports the hypothesis that the GC-induced proteolysis under sepsis is partially a consequence of GR activation.

Triiodothyronine but Not Thyroxine Accelerates Myofibrillar ProteolysisviaATP Production in Cultured Muscle Cells

These experiments were done to clarify that the differential effects of thyroxine (T(4)) and triiodothyronine (T(3)) on skeletal muscle protein turnover are caused by their roles on ATP production. Primary cultured chick muscle cells were treated with a physiological level of T(4) (60 ng/ml), T(3) (12 ng/ml), or ATP (0.5 mM) for 6 days and the protein content, ATP production, proteasome activity, and myofibrillar protein breakdown were measured. The protein content measured as an index of cell growth was not affected by T(4), T(3), or ATP. The cellular ATP level was increased by T(3) and ATP, but not by T(4). Proteasome activity and N(tau)-methylhistidine (MeHis) release measured as an index of myofiblillar protein breakdown was also increased by T(3) and ATP, but not by T(4). These results indicate that T(3) but not T(4) increases ATP production followed by an increase in proteasome activity, and thus stimulates myofibrillar proteolysis.

Effects of corticosterone on Ca2+ uptake and myofibrillar disassembly in primary muscle cell culture.

This study was done to examine the effects of corticosterone, a glucocorticoid, on Ca2+ uptake, proteolysis, and Ca2+ channels in primary cultures of chick muscle cells, to clarify the mechanism of glucocorticoid action on muscle proteolysis. Chick muscle cells were incubated for 24 h in a medium containing corticosterone (30 ng/ml) when the cells were confluent (6 days). To examine the contribution of Ca2+ channels, nifedipine, a Ca2+ channels antagonist, was used. Ca2+ uptake measured with 45CaCl2 was increased three-fold by corticosterone, with a peak at 12 h after the treatment started. The growth of the cells estimated from the protein content and creatine kinase activity was not affected by corticosterone. Proteolysis, evaluated with [3H]tyrosine as a label of the protein and Ntau-methylhistidine release, was unchanged by corticosterone. However, the amount of easily releasable myofilament as a measure of myofibrillar disassembly in the muscle cells was increased by corticosterone, and prevented by nifedipine. These results show that corticosterone increases Ca2+ uptake and starts myofibrillar protein breakdown.

Effects of Papain and a Microbial Enzyme on Meat Proteins and Beef Tenderness

ABSTRACT: The relative effects of an aspartic proteinase (AP) and papain on meat proteins and beef tenderness were evaluated by measuring release of hydroxyproline in collagen, and breakdown of myofibrillar proteins. Tenderness was objectively measured by Warner‐Bratzler shear. AP showed self‐limiting hydrolysis of myofibrillar proteins resulting in 25 to 30% improvement in meat tenderness and was not adversely affected by pH, salt, phosphate, and ascorbate concentrations often encountered in meat processing. Like papain, its tenderizing effect was expressed primarily during cooking and caused no ignificant changes (p &gt; 0.05) in tenderness during frozen or refrigerated storage. It was also inactivated at cooking temperatures in excess of 60 °C, therefore eliminating any undesirable side effects that may be associated with residual protease activity.

Effect of curing conditions and Lactobacillus casei CRL705 on the hydrolysis of meat proteins.

The effect of the common curing conditions used during the manufacture of dry fermented sausage on the proteolytic activity of Lactobacillus casei CRL705 against meat proteins was investigated. Hydrolysis of pork muscle sarcoplasmic and myofibrillar proteins was evaluated by SDS-PAGE and reverse phase-HPLC analysis. Ascorbic acid exerted a stimulatory effect on both sarcoplasmic and myofibrillar protein breakdown by Lact. casei CRL705 with the release of hydrophilic peptides and free amino acids, while NaCl and NaNO2 mainly stimulated myofibrillar degradation. Even when processing temperature (25 degrees C) did not positively affect bacterial protein hydrolysis, the presence of curing salts accounted for a remarkable increase in the non-volatile components that constitute taste-active compounds that strongly influence the final flavour of the product. To predict the suitability of Lact. casei CRL705 and its proteolytic enzymes as a starter culture for the dry processing of dry fermented sausages.

The effect of glutamine on protein balance and amino acid flux across arm and leg tissues in healthy volunteers.

Glutamine is important in nitrogen transportation and the physiological control of acid-base regulation. In addition, it has been assumed that glutamine regulates protein balance in skeletal muscles based on findings in both experimental and clinical studies. However, little information on glutamine and its effect on protein dynamics in normal individuals is available. Therefore, the aim of this study was to evaluate whether glutamine improves protein balance and uptake of various indispensable amino acids across peripheral tissue in healthy individuals. Standard primed constant infusions of L-[ring-2H5]phenylalanine and [ring 3,3-2H2]tyrosine (2 micromol kg(-1) h(-1)) were performed after overnight fast in five healthy male volunteers before and during infusions of a standard and a glutamine/tyrosine enriched amino acid solution. Flux measurements of amino acids (AA) including 3-methylhistidine, glucose, lactate and free fatty acids (FFA) were performed across arm and leg tissues. Infusion of the standard AA solution (0.2 g N kg(-1) day(-1)) increased the net uptake of individual amino acids, but provision of the enriched solution (0.4 g N kg(-1) day(-1)) with increased amounts of glutamine and tyrosine seemed to compete unfavourably with the net uptake of other key amino acids as methionine and phenylalanine, which are indispensable in muscles for protein synthesis. Increased flux of amino acids across peripheral tissues did not influence on flux of glucose, free fatty acid and lactate. Glutamine provision did neither stimulate protein synthesis nor attenuate breakdown of either globular or myofibrillar proteins in skeletal muscles of healthy volunteers.