The effect of calcium activated protease (CAF) and cathepsin D on bovine muscle myofibrils under varying conditions of pH and temperature

This study examined effects of purified CAF and cathepsin D on bovine skeletal myofibrillar proteins and structure when incubated under postmortem-like conditions of pH and temperature;When CAF (purified from bovine cardiac muscle) was incubated with isolated myofibrils under near optimal conditions (pH 7.5 and 25(DEGREES)C), a rapid and significant release of soluble protein, including intact alpha-actinin, from the myofibrils was observed. SDS-PAGE analysis of treated sedimented myofibrils showed CAF digestion had degraded titin and, at least partly, tropomyosin, and troponins T and I. A trio of bands in the 30,000-dalton region appeared in the gels of the sedimented myofibrils. CAF treatment caused a significant increase in myofibril fragmentation;Lowered incubation pH or temperature reduced the effect of CAF on myofibrils. However, CAF effectively caused alterations in myofibrillar proteins and structure at pH 7.5 and 5(DEGREES)C, pH 6.5 and 15(DEGREES)C, or pH 5.5 and 25(DEGREES)C, suggesting that it may play a significant role in the tenderization process occurring in muscle postmortem;When cathepsin D (purified from bovine cardiac muscle) was incubated with myofibrils under conditions resembling those found in postmortem muscle, several alterations occurred. Cathepsin D at pH 5.5 and 37(DEGREES)C released a significant amount of soluble protein after 30 to 60 min of incubation. SDS-PAGE analysis of sedimented myofibrils showed degradation of myosin heavy chains and titin. A small amount of actin, tropomyosin, troponins T and I, and myosin light chains also were degraded. Myofibrils incubated with cathepsin D under these conditions (pH 5.5 and 37(DEGREES)C) and then subjected to a brief homogenization step showed no change in the degree of myofibril fragmentation, even though a significant narrowing of the A bands occurred. Raising the pH and/or lowering the temperature greatly reduced the effect of cathepsin D on myofibrillar proteins and structure. Cathepsin D had only a small effect at pH 6.5 and 37(DEGREES)C or at pH 5.5 and 25(DEGREES)C, suggesting that cathepsin D does not play a principal role in the tenderization process occurring in muscle postmortem

Microarray profiling of skeletal muscle sarcoplasmic reticulum proteins

Microarrays were developed to profile the level of proteins associated with calcium regulation in sarcoplasmic reticulum (SR) isolated from porcine Longissimus muscle. The microarrays consisted of SR preparations printed onto to glass slides and probed with monoclonal antibodies to 7 target proteins. Proteins investigated included: ryanodine receptor, (RyR), dihydropyridine receptor, (DHPR), triadin (TRI), calsequestrin (CSQ), 90 kDa junctional protein (JSR90), and fast-twitch and slow-twitch SR calcium ATPases (SERCA1 and SERCA2). Signal from a fluorescentlylabeled detection antibody was measured and quantitated using a slide reader. The microarray developed was also employed to profile Longissimus muscle SR proteins from halothane genotyped animals. Significant (P\u3c0.05) reductions in levels of several proteins were found including: RyR, CSQ, TRI, DHPR and SERCA2 in SR samples from halothane positive animals. The results illustrate the potential of microarrays as a tool for profiling SR proteins and aiding investigations of calcium regulation

Microarray profiling of skeletal muscle sarcoplasmic reticulum proteins

Microarrays were developed to profile the level of proteins associated with calcium regulation in sarcoplasmic reticulum (SR) isolated from porcine Longissimus muscle. The microarrays consisted of SR preparations printed onto to glass slides and probed with monoclonal antibodies to 7 target proteins. Proteins investigated included: ryanodine receptor, (RyR), dihydropyridine receptor, (DHPR), triadin (TRI), calsequestrin (CSQ), 90 kDa junctional protein (JSR90), and fast-twitch and slow-twitch SR calcium ATPases (SERCA1 and SERCA2). Signal from a fluorescentlylabeled detection antibody was measured and quantitated using a slide reader. The microarray developed was also employed to profile Longissimus muscle SR proteins from halothane genotyped animals. Significant (P\u3c0.05) reductions in levels of several proteins were found including: RyR, CSQ, TRI, DHPR and SERCA2 in SR samples from halothane positive animals. The results illustrate the potential of microarrays as a tool for profiling SR proteins and aiding investigations of calcium regulation

α-Actinin and Filamin Cooperatively Enhance the Stiffness of Actin Filament Networks

BACKGROUND: The close subcellular proximity of different actin filament crosslinking proteins suggests that these proteins may cooperate to organize F-actin structures to drive complex cellular functions during cell adhesion, motility and division. Here we hypothesize that alpha-actinin and filamin, two major F-actin crosslinking proteins that are both present in the lamella of adherent cells, display synergistic mechanical functions. METHODOLOGY/PRINCIPAL FINDINGS: Using quantitative rheology, we find that combining alpha-actinin and filamin is much more effective at producing elastic, solid-like actin filament networks than alpha-actinin and filamin separately. Moreover, F-actin networks assembled in the presence of alpha-actinin and filamin strain-harden more readily than networks in the presence of either alpha-actinin or filamin. SIGNIFICANCE: These results suggest that cells combine auxiliary proteins with similar ability to crosslink filaments to generate stiff cytoskeletal structures, which are required for the production of internal propulsive forces for cell migration, and that these proteins do not have redundant mechanical functions

Synthetic receptors for the recognition and discrimination of post-translationally methylated lysines

Post-translational modifications (PTMs) describe the chemical alteration of proteins after their biosynthesis in ribosomes. PTMs play important roles in cell biology including the regulation of gene expression, cell-cell interactions and the development of different diseases. A prominent class of PTMs is the side chain methylation of lysine. For the analysis and discrimination of differently methylated lysines antibodies are widely used, though, methylated peptide and protein targets are known to be particularly difficult to be differentiated by antibody-based affinity reagents; an additional challenge can be batch-to-batch reproducibility. The application of mass spectrometry techniques for methyllysine discrimination requires a complex sample preparation and is not suited for working in cells. The desire to overcome above-named challenges promoted the development of synthetic receptor molecules that recognize and bind methyllysines. Such ‘artificial antibodies’ are of interest for a number of applications, e.g. as reagents in biochemical assays, for the isolation and purification of post-translationally methylated proteins and for the tracking of signalling pathways. Moreover, they offer new approaches in diagnostics and therapy. This review delivers an overview of the broad field of methyllysine binding and covers a wide range of synthetic receptors used for the recognition of methylated lysines including calixarenes, resorcinarenes, pillararenes, disulfide cyclophanes, cucurbituriles and acyclic receptors

Early childhood professionals' perception of their occupational worth

Sociological trends indicate that as more parents enter the work force, increasing numbers of children and families become involved with early childhood professionals. Little is known about the perceived occupational worth of these workers. Early childhood professionals' perception of their own and their peers' occupational worth, as measured by responses on the Occupational Worth Inventory (OWI) and career-pattern profiled were investigated;Data were collected from 104 family day care providers, 26 group day care providers, 101 day care center teachers, 26 Head Start Teachers, and 95 nursery school teachers. Results of Friedman's rank-order analyses of variance on job pay, job status, and job value were supportive of two major conclusions. First, subjects varied in statistically significant ways in their views about which occupation within the early childhood profession should command the most pay or derive the greatest social status. Second, for job value a positional trend indicated nursery school teaching was most valued followed by group day care home providing, day care center teaching, family day care providing, and Head Start teaching;Analyses of variance on the ratings by group of occupations predominantly held by male workers, female workers, and teachers yielded statistically significant group differences. Nearly one-half (46.8%) of survey respondents reported career pattern profiles which ended in non-career jobs;Results indicated that early childhood professionals varied in perceptions of their own and peers' occupational worth as measured by responses to items on the OWI. Lack of consensus on job worth, professionalism, and support systems for occupational choice suggested that the early childhood profession is fragmented and multi-focused.</p

The effect of calcium activated protease (CAF) and cathepsin D on bovine muscle myofibrils under varying conditions of pH and temperature

This study examined effects of purified CAF and cathepsin D on bovine skeletal myofibrillar proteins and structure when incubated under postmortem-like conditions of pH and temperature;When CAF (purified from bovine cardiac muscle) was incubated with isolated myofibrils under near optimal conditions (pH 7.5 and 25(DEGREES)C), a rapid and significant release of soluble protein, including intact alpha-actinin, from the myofibrils was observed. SDS-PAGE analysis of treated sedimented myofibrils showed CAF digestion had degraded titin and, at least partly, tropomyosin, and troponins T and I. A trio of bands in the 30,000-dalton region appeared in the gels of the sedimented myofibrils. CAF treatment caused a significant increase in myofibril fragmentation;Lowered incubation pH or temperature reduced the effect of CAF on myofibrils. However, CAF effectively caused alterations in myofibrillar proteins and structure at pH 7.5 and 5(DEGREES)C, pH 6.5 and 15(DEGREES)C, or pH 5.5 and 25(DEGREES)C, suggesting that it may play a significant role in the tenderization process occurring in muscle postmortem;When cathepsin D (purified from bovine cardiac muscle) was incubated with myofibrils under conditions resembling those found in postmortem muscle, several alterations occurred. Cathepsin D at pH 5.5 and 37(DEGREES)C released a significant amount of soluble protein after 30 to 60 min of incubation. SDS-PAGE analysis of sedimented myofibrils showed degradation of myosin heavy chains and titin. A small amount of actin, tropomyosin, troponins T and I, and myosin light chains also were degraded. Myofibrils incubated with cathepsin D under these conditions (pH 5.5 and 37(DEGREES)C) and then subjected to a brief homogenization step showed no change in the degree of myofibril fragmentation, even though a significant narrowing of the A bands occurred. Raising the pH and/or lowering the temperature greatly reduced the effect of cathepsin D on myofibrillar proteins and structure. Cathepsin D had only a small effect at pH 6.5 and 37(DEGREES)C or at pH 5.5 and 25(DEGREES)C, suggesting that cathepsin D does not play a principal role in the tenderization process occurring in muscle postmortem.</p