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Biomedical subjects

M Koohmaraie

Publications and source records attributed to M Koohmaraie.

At least 55 records · Page 3Linked to original sources
PubMed2001

Effect of postmortem storage on mu-calpain and m-calpain in ovine skeletal muscle.

Casein zymography was used to determine the effect of postmortem storage on the proteolytic activity of mu-calpain and m-calpain in lamb longissimus. Casein zymography assays were conducted on crude muscle extracts (only one centrifugation). Six market weight crossbred lambs were slaughtered and a portion of the longissimus lumborum was removed at death (within 15 min of exsanguination) and after 3, 6, 9, 12, 24, 72, and 360 h postmortem. Muscle samples were snap-frozen in liquid nitrogen and stored at -70 degrees C. Soluble muscle proteins were extracted from muscle samples and analyzed by in-gel casein assay to measure calpain proteolytic activity. There was a gradual decline in mu-calpain activity (P < 0.05) such that after 24 and 72 h postmortem, mu-calpain had lost 42 and 95% of its activity, respectively. After 360 h postmortem, no mu-calpain activity could be detected (under the conditions used in this study). Autolysis of mu-calpain could be detected as early as 3 h postmortem. It was demonstrated that the detectable level of mu-calpain activity is a function of the amount of muscle protein electrophoresed. Hence, the activity data reported are in relative terms, rather than absolute values. Furthermore, it was demonstrated that the activity data also are a function of the assay methods used. Different methods have different lower detection limits. Of the three assays examined, 14C-labeled casein was the most sensitive, then the in-gel casein assay, and the least-sensitive method was the standard casein assay. Unlike mu-calpain, postmortem storage had no effect on m-calpain (P > 0.05). When the calcium concentration of a muscle extract was increased to the level that induces m-calpain autolysis, m-calpain was autolyzed and its autolysis was readily detected by the in-gel casein assay. Collectively, these results demonstrate that calcium concentration in postmortem muscle is only high enough to activate mu-calpain. These results support the widely believed conclusion that mu-calpain-mediated proteolysis of key myofibrillar and cytoskeletal proteins is responsible for postmortem tenderization. Hence, understanding the regulation of mu-calpain in postmortem muscle should be the focus of future studies.

Animals↗
PubMed2001

Properties of myofibril-bound calpain activity in longissimus muscle of callipyge and normal sheep.

Properties of the calpain bound to myofibrils in longissimus muscle from callipyge or noncallipyge sheep were examined after 0, 1, 3, and 10 d of postmortem storage at 4 degrees C. Western analysis has shown that most of this calpain is mu-calpain, although the sensitivity of the antibodies used in the earlier studies could not eliminate the possibility that up to 10% of the calpain was m-calpain. The calpain is bound tightly, and very little is removed by washing with the detergent Triton X-100; hence, it is not bound to phospholipids in the myofibril. Over 25% of total mu-calpain was bound to myofibrils from at-death muscle, and this increased to approximately 40% after 1 d postmortem. The amount of myofibril-bound mu-calpain increased only slightly between 1 and 10 d of postmortem storage. The percentage of autolyzed mu-calpain increases with time postmortem until after 10 d postmortem, when all myofibril-bound mu-calpain is autolyzed. The specific activity of the myofibril-bound calpain is very low and is only 6 to 13% as high as the specific activity of extractable mu-calpain from the same muscle. It is unclear whether this low specific activity is the result of unavailability of the active site of the myofibril-bound calpain to exogenous substrate. The myofibril-bound calpain degrades desmin, nebulin, titin, and troponin T in the myofibrils, and also releases undegraded alpha-actinin and undergoes additional autolysis when incubated with Ca2+; all these activities occurred slowly considering the amount of myofibril-bound calpain. Activity of the myofibril-bound calpain was partly (58 to 67%) inhibited by the calpain inhibitors, E-64 and iodoacetate; was more effectively inhibited by a broader-based protease inhibitor, leupeptin (84 to 89%); and was poorly inhibited (43 to 45%) by calpastatin. Release of undegraded alpha-actinin and autolysis are properties specific to the calpains, and it is unclear whether some of the myofibril-bound proteolytic activity originates from proteases other than the calpains or whether the active site of myofibril-bound calpain is shielded from the inhibitors. Activities and properties of the myofibril-bound calpain were identical in longissimus muscle from callipyge and normal sheep, although previous studies had indicated that the "normal" longissimus was much more tender than the callipyge longissimus. Hence, it seems unlikely that the myofibril-bound calpain has a significant role in postmortem tenderization of ovine longissimus.

Animals↗
PubMed2001

Development of a multiple-step process for the microbial decontamination of beef trim.

A multiple-hurdle antimicrobial process for beef trim was developed. The microbial profiles of inoculated lean beef trim tissue (BTL) and fat-covered lean beef trim (BTF) were monitored during prolonged refrigerated storage following the application of successive multiple antimicrobial treatments applied to inoculated beef trim on a processing conveyor belt set at a belt speed of 1 cm/s. Beef trim (meat size approximately 15 by 15 cm) was preinoculated with bovine feces before all treatments that included the following: control, no treatment; water wash at 65 psi for five passes; water plus lactic acid (2% [vol/vol] room temperature lactic acid wash at 30 psi for three passes); combination treatment 1 (water plus 65 degrees C hot water at 30 psi for one pass plus hot air at 510 degrees C for four passes plus lactic acid), combination treatment 2 (water plus hot water at 82 degrees C for one pass plus hot air at 510 degrees C for five passes plus lactic acid), and combination treatment 3 (water plus hot water at 82 degrees C for three passes plus hot air at 510 degrees C for six passes plus lactic acid). The effects of treatments on bacterial populations were monitored by enumerating mesophilic aerobic bacteria (APC), presumptive lactic acid bacteria (PLAB), psychrotrophic bacteria (PCT), coliforms, and Escherichia coli biotype 1 on product stored for up to 7 days at 4 degrees C. In the case of BTL, the numbers of APC, PCT, and PLAB increased during storage at 5 degrees C, whereas the numbers of coliform and E. coli decreased on average by 1.8 log CFU/cm2, then remained constant following the initial reduction. Negligible effects on color quality were observed from multihurdle treatment combination 1. In the case of the BTF, the microbial reductions by treatments were much greater than the reduction on BTL. The pH of treated BTF increased more slowly than the pH of treated BTL, resulting in further reduction of the microflora on BTF. Except for control and water treatments, all sample treatments involving lactic acid resulted in continuously decreasing microbial populations. Based on microbial reduction and quality aspects, it was concluded that successively applied combination antimicrobial treatments for meat trim could offer potential food safety benefits.

Animals↗
PubMed2001

Screening bovine carcass sponge samples for Escherichia coli O157 using a short enrichment coupled with immunomagnetic separation and a polymerase chain reaction-based (BAX) detection stept.

A bovine carcass sponge sample screening protocol for detecting Escherichia coli O157:H7 was composed of a short selective enrichment followed by an immunomagnetic separation (IMS) and target detection using the BAX E. coli O157 polymerase chain reaction assay. This screening protocol was compared to a culture-based method for detection of the organism in carcass sponge samples. Enriched samples were subjected to IMS; the bead suspension was divided and plated on selected media or stored at -20 degrees C, then subjected to BAX analysis. The results showed a high degree of agreement between the plating method and the BAX system. Fifty-two of the 59 culture-positive samples were also positive using the BAX system (88.1% sensitivity). Of the 76 samples that appeared negative for the presence of E. coli O157:H7 by the culture method, 66 were determined as negative using the BAX system (86.8% specificity). Four of the 10 samples found negative by the initial culture method and positive by the BAX method were subsequently found to be culture positive upon reanalysis. Based on these data, the BAX system combined with a short, selective enrichment and IMS may be a rapid, reliable, and simple method to screen for E. coli O157:H7 in carcass sponge samples. Our data indicate that optimization and subsequent testing of this protocol for use as a carcass screening tool are warranted.

Animals↗
PubMed2001

Microbial and quality attributes of ground pork prepared from commercial pork trim treated with combination intervention processes.

The effects of combination intervention treatments of commercial pork trim on microbial and quality attributes of the subsequent ground pork were examined. Fresh commercial pork trim was inoculated with swine feces and subjected to five different intervention treatments: (i) control (untreated), (ii) water (15 degrees C, 120 s), (iii) water followed by 2% lactic acid wash (15 degrees C, 75 s), (iv) Combination 1 (water plus lactic acid plus hot air [510 degrees C, 90 s]), and (v) Combination 2 (hot air plus water plus hot air). Following treatment, the pork trim was stored at 4 degrees C for 24 h, then ground, stuffed, vacuum packaged, and stored at 4 degrees C for 21 days. Populations of aerobic bacteria, coliforms, Escherichia coli, and lactic acid bacteria in the ground pork were monitored before treatment, after treatment (day 0), and at 2, 7, 14 and 21 days. In addition, uninoculated pork trim was treated as described above, and the color and emulsion stability of the ground product was evaluated. Ground pork prepared from trim treated with any of the treatment processes had lower initial microbial populations compared to the untreated samples. The applications of water plus lactic acid or Combination 1, which included a lactic acid wash, were more effective than water or Combination 2 at both reducing initial populations and suppressing the growth of aerobic bacteria, coliforms, and E. coli in ground pork during refrigerated storage. By day 21, populations of aerobic bacteria in ground pork prepared from control, water-treated, and Combination 2-treated trim were 8.22 to 8.32 log CFU/g, but in water plus lactic acid and Combination 1 ground pork, populations were 6.32 and 4.90 log CFU/g, respectively. Among the trim interventions examined, Combination 1 was most detrimental to the color and emulsion stability of the ground pork. The water plus lactic acid treatment provided the greatest microbial reduction and inhibition without large negative effects on quality attributes of the ground pork.

Animals↗
PubMed2001

Application of multiple antimicrobial interventions for microbial decontamination of commercial beef trim.

Commercially produced, irregularly sized (range, 100 to 400 cm2), uninoculated beef trim was treated by a previously optimized multihurdle antimicrobial process under spray system or hot air gun with set-up speed (1 cm/s): W (water wash at 65 psi for five passes) + HW (82 degrees C water at 30 psi for three passes) + HA (510 degrees C air for five passes) + L (2% [vol/vol] room temperature lactic acid wash at 30 psi for three passes). After treatment, the trim was finely ground, vacuum packaged, and stored at 4 degrees C for up to 20 days. At regular intervals (0, 5, 10, 15, and 20 days of storage at 4 degrees C), the ground beef was analyzed to measure mesophilic aerobic bacteria (APC), coliforms, psychrotrophic bacteria (PCT), and presumptive lactic acid bacteria (PLAB) and compared with the untreated control. The numbers of APC, coliforms, PCT, and PLAB were reduced to nearly nondetectable levels immediately after treatment, with significant differences compared with the control (P < 0.05), then started to increase after 5 to 10 days of storage at 4 degrees C. After 20 days, microbial populations of treated ground beef were significantly lower than those of nontreated ground beef for the numbers of APC, coliforms, PCT, and PLAB (P < 0.05), with differences of 1.2, 2.4, 1.6, and 1.6 log CFU/g, respectively. Based on microbial reduction and quality aspects, the multihurdle antimicrobial process was identified as an effective intervention to reduce coliforms on beef trim.

Animals↗
PubMed2001

Evaluation of combination treatment processes for the microbial decontamination of pork trim.

Combination treatment processes for the microbial decontamination of pork trim were developed and evaluated. Lean pork trim tissue (LPT) and fat-covered pork trim tissue (FPT) inoculated with swine feces were treated with intervention processes as follows: (i) control (untreated), (ii) water (15 degrees C, 120 s), (iii) water followed by lactic acid wash (15 degrees C, 75 s), (iv) combination 1 (water plus hot water [65.5 degrees C, 15 s] plus hot air [510 degrees C, 60 s] plus lactic acid), (v) combination 2 (water plus hot water [82.2 degrees C, 15 s] plus hot air [510 degrees C, 75 s] plus lactic acid), and (vi) combination 3 (water plus hot water [82.2 degrees C, 45 s] plus hot air [510 degrees C, 90 s] plus lactic acid). Populations of aerobic bacteria, psychrotrophic bacteria, coliforms, Escherichia coli, and lactic acid bacteria were determined before and after treatment and at days 2 and 7 of 4 degrees C storage. Regardless of the intervention treatment, lower microbial populations were observed on FPT than on LPT immediately after treatment and during the 7-day storage period. Both LPT and FPT treated with water plus lactic acid, combination 1, combination 2, and combination 3 had lower remaining populations of all microbial groups immediately after treatment than did water-treated samples. Populations of aerobic bacteria, coliforms, E. coli, and lactic acid bacteria on either LPT or FPT did not statistically increase during the 7-day storage period. On LPT, populations of psychrotrophic bacteria grew during 4 degrees C storage but remained lower at day 7 on LPT treated by combinations 2 and 3 (2.29 and 1.89 log10 CFU/cm2, respectively) than on LPT treated with water (4.07 log10 CFU/cm2) or water plus lactic acid (3.52 log10 CFU/cm2). Populations of psychrotrophic bacteria remained below detectable levels throughout the 7-day storage on FPT treated with water plus lactic acid or any of the three combination treatments. Treatment of pork trim with any of the combination treatments significantly (P < 0.05) affected the color and emulsion stability of the ground pork. Water and water plus lactic acid were the most favorable treatments in reducing microbial populations on pork trim without affecting the quality attributes of the ground pork.

Animals↗
PubMed2001

Effect of different levels of beef bacterial microflora on the growth and survival of Escherichia coli O157:H7 on beef carcass tissue.

The influence of various levels of endogenous beef bacterial microflora on the growth and survival of Escherichia coli O157:H7 on bovine carcass surface tissue was investigated. Bacterial beef microflora inoculum was prepared by enriching and harvesting bacteria from prerigor lean bovine carcass tissue (BCT) and was inoculated onto UV-irradiated prerigor BCT at initial levels of 10(5), 10(4), 10(3), and <10(3) CFU/cm2. Additional control BCT was inoculated with sterile H2O. E. coli O157:H7 was inoculated onto all tissues at an initial level of 10(2) CFU/cm2. Following a 48-h incubation at 4 degrees C, BCT was incubated up to 14 days at 4 or 12 degrees C, either aerobically or vacuum packaged. Regardless of the microflora level, there was no substantial growth of E. coli O157:H7 on BCT during storage at 4 degrees C under either aerobic or vacuum-packaged conditions. Instead, viable cell numbers at 4 degrees C remained constant, with no reduction in numbers associated with the different beef microflora levels. E. coli O157:H7 grew on all BCT stored at 12 degrees C, regardless of microflora inoculation treatment, reaching higher populations on aerobic samples than on vacuum-packaged samples in 10 days. However, the presence of the beef microflora did appear to delay the onset of growth or slow the growth of the pathogen, and E. coli O157:H7 counts on BCT without added microflora were generally higher following 7 to 10 days of 12 degrees C storage than those counts on BCT inoculated with beef microflora. These data demonstrate the importance of temperature control during meat handling and storage to prevent the outgrowth of this pathogen and indicate that proper sanitation and processing practices that prevent and reduce contamination of carcasses with E. coli O157:H7 are essential, regardless of background microflora levels.

Abattoirs↗
PubMed2000

Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing.

A survey was performed to estimate the frequency of enterohemorrhagic Escherichia coli O157:H7 or O157:nonmotile (EHEC O157) in feces and on hides within groups of fed cattle from single sources (lots) presented for slaughter at meat processing plants in the Midwestern United States, as well as frequency of carcass contamination during processing from cattle within the same lots. Of 29 lots sampled, 72% had at least one EHEC O157-positive fecal sample and 38% had positive hide samples. Overall, EHEC O157 prevalence in feces and on hides was 28% (91 of 327) and 11% (38 of 355), respectively. Carcass samples were taken at three points during processing: preevisceration, postevisceration before antimicrobial intervention, and postprocessing after carcasses entered the cooler. Of 30 lots sampled, 87% had at least one EHEC O157-positive preevisceration sample, 57% of lots were positive postevisceration, and 17% had positive postprocessing samples. Prevalence of EHEC O157 in the three postprocessing samples was 43% (148 of 341), 18% (59 of 332) and 2% (6 of 330), respectively. Reduction in carcass prevalence from preevisceration to postprocessing suggests that sanitary procedures were effective within the processing plants. Fecal and hide prevalence were significantly correlated with carcass contamination (P = 0.001), indicating a role for control of EHEC O157 in live cattle.

Animals↗
PubMed2000

Relationship of beef longissimus tenderness classes to tenderness of gluteus medius, semimembranosus, and biceps femoris.

The objective of this study was to determine the relationship of longissimus tenderness classes to tenderness of three other major muscles. Ninety-eight crossbred steers and heifers (14 to 17 mo of age) were humanely slaughtered over 9 wk and the carcasses were chilled 48 h at 0 degrees C. At 48 h postmortem, carcasses were assigned to one of three tenderness classes (tender < or = 26 kg, intermediate = 26 to 42 kg, tough > or = 42 kg) using slice shear force from the MARC Beef Classification System (n = 20, 67, and 11, respectively). The longissimus thoracis, gluteus medius, semimembranosus, and biceps femoris were removed, aged at 2 degrees C, and frozen at -30 degrees C at 14 d postmortem. Two 2.54-cm-thick steaks were obtained from each muscle, thawed to 5 degrees C, cooked with a belt grill at 163 degrees C for 5.5 min, and served warm to an eight-member trained descriptive attribute panel. Panelists evaluated each sample for tenderness, connective tissue amount, juiciness, and beef flavor intensity on 8-point scales. The mean 2-d longissimus slice shear force values were 20.7, 34.4, and 46.3 kg, respectively, for the "tender," "intermediate," and "tough" classes. Tenderness ratings were lowest (P < 0.05) for the "tough" class and highest (P < 0.05) for the "tender" class for all muscles except the gluteus medius, for which the "tender" and "intermediate" classes were not different (P > 0.05; longissimus, 7.7, 7.1, 6.3, and 7.1; semimembranosus, 6.4, 5.8, 5.1, and 5.8; biceps femoris, 5.9, 5.4, 4.8, and 5.4; gluteus medius, 6.8, 6.5, 5.8, and 6.5 for the "tender," "intermediate," "tough," and "unsorted" classes, respectively). The magnitude of the differences in tenderness ratings between the "tender" and "intermediate" classes and between the "intermediate" and "tough" classes was similar for all muscles. The percentages of tenderness ratings greater than 5.0 (slightly tender) for the "tender" and "unsorted" classes, respectively, were as follows: longissimus, 100 and 95%; semimembranosus, 95 and 85%; gluteus medius, 100 and 94%; and biceps femoris, 95 and 81%. The simple correlations between longissimus and the other muscles for tenderness ratings were as follows: semimembranosus, 0.58; biceps femoris, 0.43; and gluteus medius, 0.68. These data indicate that early-postmortem longissimus slice shear force could be used to classify top sirloin, top round, and bottom round cuts for tenderness.

Animals↗
PubMed2000

Protein kinetics in callipyge lambs.

The objectives for this experiment were to determine the effect of the callipyge phenotype on protein kinetics. We studied callipyge and normal lambs (n = 37) at 5, 8, and 11 wk of age (n = 4 to 7/ group) to determine how protein kinetics are altered by this trait. Total protein, DNA, and RNA and calpastatin activity were measured in five skeletal muscles and in the heart, kidneys, and liver, and protein accretion rates were calculated. At 8 wk, the fractional synthesis rates of proteins in these tissues were measured in vivo using a primed, continuous 8-h infusion of [2H5]phenylalanine. Fractional rates of protein degradation were estimated by differences. At 5 wk of age, muscle weights, protein mass, protein:DNA, RNA:DNA, and calpastatin activity were higher (P < .05) for callipyge, and protein mass differences continued to increase through 11 wk. At 8 wk, fractional rates of protein synthesis and degradation were lower (P < .05) in callipyge than in normal lambs. The organs of callipyge lambs exhibited reduced growth at 11 wk. Thus, enhanced muscle growth seems to be maintained in callipyge lambs by reduced protein degradation rather than increased protein synthesis. However, we cannot exclude the possibility that the initial onset of the callipyge condition may be caused by an increase in the fractional rate of protein synthesis.

Animals↗
PubMed2000

Quantitative trait loci affecting growth and carcass composition of cattle segregating alternate forms of myostatin.

The effects of the bovine myostatin gene on chromosome 2 on birth and carcass traits have been previously assessed. The objective of this study was to identify additional quantitative trait loci (QTL) for economically important traits in two families segregating an inactive copy of myostatin. Two half-sib families were developed from Belgian Blue x MARC III (n = 246) and Piedmontese x Angus (n = 209) sires. Traits analyzed were birth (kg) and yearling weight (kg); hot carcass weight (kg); fat depth (cm); marbling score; longissimus muscle area (cm2); estimated kidney, pelvic, and heart fat (%); USDA yield grade; retail product yield (%); fat yield (%); and wholesale rib-fat yield (%). Meat tenderness was measured as Warner-Bratzler shear force at 3 and 14 d postmortem. The effect of myostatin on these traits was removed by using phase information obtained from the previous study with six microsatellite markers flanking the locus. Selective genotyping was done on 92 animals from both families to identify genomic regions potentially associated with retail product yield and fat depth, using a total of 150 informative markers in each family. Regions in which selective genotyping indicated the presence of QTL were evaluated further by genotyping the entire population and additional markers. For the family with Belgian Blue inheritance (n = 246), a significant QTL for birth and yearling weight was identified on chromosome 6. Suggestive QTL were identified for longissimus muscle area and hot carcass weight on chromosome 6 and for marbling on chromosomes 17 and 27. For the family with Piedmontese inheritance (n = 209), suggestive QTL on chromosome 5 were identified for fat depth, retail product yield, and USDA yield grade and on chromosome 29 for Warner-Bratzler shear force at 3 and 14 d postmortem. Interactions suggesting the presence of QTL were observed between myostatin and chromosome 5 for Warner-Bratzler shear force at 14 d postmortem and between myostatin and chromosome 14 for fat depth. Thus, in families segregating an inactive copy of myostatin in cattle, other loci influencing quantitative traits can be detected. These results are the initial effort to identify and characterize QTL affecting carcass and growth traits in families segregating myostatin.

Alleles↗
PubMed2000

Variation in proteolysis, sarcomere length, collagen content, and tenderness among major pork muscles.

The objectives of this experiment were to determine the extent of variation in proteolysis, sarcomere length, and collagen content among pork muscles and the association of those factors with tenderness variation among muscles at 1 d postmortem. Twenty-three white composite barrows were slaughtered and carcasses (66 kg) were chilled at 0 degrees C for 24 h. At 1 d postmortem, the longissimus lumborum, biceps femoris, semimembranosus, semitendinosus, and triceps brachii, long head were dissected from one side of each carcass and frozen. Trained sensory panelists evaluated tenderness, amount of connective tissue, juiciness, and pork flavor intensity of grilled (70 degrees C) chops on 8-point scales. Raw chops were used for total collagen content, sarcomere length, and the extent of desmin proteolysis. Tenderness ratings were highest (P < .05) for semitendinosus (7.2) and triceps brachii (7.1), followed by longissimus lumborum (6.4) and semimembranosus (5.7) and were lowest (P < .05) for biceps femorus (4.0). The simple correlations between longissimus lumborum tenderness and the tenderness of other muscles were .54 (semimembranosus), .34 (semitendinosus), .36 (triceps branchii), and .17 (biceps femorus). Total collagen was highest (P < .05) for biceps femorus (7.1 mg/g muscle), followed by triceps branchii (6.0 mg/g) and semitendinosus (5.3 mg/g), and lowest for semimembranosus (4.5 mg/g) and longissimus lumborum (4.1 mg/g). Sarcomere length was longest (P < .05) for semitendinosus (2.5 microm) and triceps branchii (2.4 microm), followed by semimembranosus (1.8 microm), longissimus lumborum (1.8 microm), and biceps femorus (1.7 microm). Proteolysis of desmin was greatest (P < .05) in longissimus lumborum (39.3%), followed by semimembranosus (21.0%) and biceps femoris (18.5%), then semitendinosus (.2%) and triceps brachii (.2%). Multiple linear regression using total collagen, sarcomere length, and proteolysis accounted for 57% of the variation in tenderness rating among all samples. Piecewise linear regression was used to account for the interaction of sarcomere length with proteolysis and collagen. This analysis accounted for 72% of the variation in tenderness rating. Variation in collagen, proteolysis, and sarcomere length and the degree of their interaction with one another determine the tenderness of individual muscles.

Animals↗
PubMed2000

Ionic strength-induced inactivation of mu-calpain in postmortem muscle.

The present study was conducted to study the stability of autolyzed mu-calpain activity and determine whether measurement of mu-calpain activity after anion exchange chromatography accurately reflects its activity in postmortem muscle. Ionic strength and pH affected the stability of partially autolyzed mu-calpain. Complete loss of activity was observed as a result of binding of autolyzed mu-calpain to DEAE-Sephacel when the large subunit of mu-calpain was autolyzed from 80 to 76 kDa. Therefore, determination of mu-calpain by standard anion exchange chromatography may underestimate mu-calpain activity in postmortem muscle. The activity of autolyzed mu-calpain was stabilized by inclusion of glycerol in the buffers, and this permitted us to investigate whether the apparent loss of mu-calpain activity in postmortem muscle is an artifact of the methodology. Despite the inclusion of glycerol in the buffers, a decrease in mu-calpain activity was observed during postmortem storage of muscle, even though the autolyzed enzyme was readily detectable by Western blotting in muscle extracts and column eluates. This result indicates that instability of autolyzed mu-calpain is a major cause for the decline in mu-calpain activity in postmortem muscle.

Animals↗
PubMed1999

Molecular cloning and mapping of the bovine and ovine skeletal muscle-specific calpains.

The coding regions of the bovine and sheep skeletal muscle-specific calpains (CANP3 or p94) were cloned and sequenced by RT-PCR. Direct sequencing confirmed open reading frames of 2466 bp for both species, and bovine and sheep CANP3 shared 98.5% identity in their amino acid code. These sequences were greater than 88% identical to human, pig, rat and mouse CANP3 nucleotide sequences, and greater than 93% identical for the amino acid code. Single nucleotide polymorphisms were used to map the bovine and sheep CANP3 genes in two steps. The genes were placed into linkage groups based on two-point LOD scores (> or = 3.0) and the best order was determined with multipoint linkage analysis (CRI-MAP vs. 2.4). Bovine CANP3 mapped to bovine chromosome 10, relative position 33.9 CM with linkage to nine markers; LOD scores ranged from 4.89 to 8.61 (order, BMS2349-BL1035-RME25-CANP3-BM6305-BMS86 1-ILSTS053-BMS2742-CA090-BMS529). Ovine CANP3 mapped to chromosome 7, relative position 58 CM, with linkage to only one marker, BMS861 (a bovine microsatellite that has been used in sheep), with no recombination and a LOD score of 5.72. The observed heterozygosity was 50% for both CANP3 markers in bovine and sheep pedigrees.

Animals↗
PubMed1999

Effect of calpastatin on degradation of myofibrillar proteins by mu-calpain under postmortem conditions.

To improve our understanding of the regulation of mu-calpain activity in situ during postmortem storage of muscle, the effect of different calpastatin levels on proteolysis of myofibrillar proteins by mu-calpain in a system closely mimicking postmortem conditions was studied. Increasing the amount of calpastatin in the incubations limited both the rate and extent of proteolysis of myofibrillar proteins and autolysis of mu-calpain. Excess calpastatin (i.e., a mu-calpain:calpastatin ratio of 1:4) did not inhibit proteolysis completely. Western blot analysis revealed that proteolysis of myofibrillar proteins virtually ceased after 7 d of incubation, despite the presence of partly autolyzed, therefore seemingly active, mu-calpain. A series of incubations of autolyzed mu-calpain revealed that the autolyzed form of this enzyme is unstable at an ionic strength observed in postmortem muscle. The possible significance of these results in terms of the regulation of mu-calpain activity in postmortem muscle is discussed.

Animals↗
PubMed1999

Evaluation of slice shear force as an objective method of assessing beef longissimus tenderness.

Experiments were conducted to develop an optimal protocol for measurement of slice shear force (SSF) and to evaluate SSF as an objective method of assessing beef longissimus tenderness. Whereas six cylindrical, 1.27-cm-diameter cores are typically removed from each steak for Warner-Bratzler shear force (WBSF) determination, a single 1-cm-thick, 5-cm-long slice is removed from the lateral end of each longissimus steak for SSF. For either technique, samples are removed parallel to the muscle fiber orientation and sheared across the fibers. Whereas WBSF uses a V-shaped blade, SSF uses a flat blade with the same thickness (1.016 mm) and degree of bevel (half-round) on the shearing edge. In Exp. 1, longissimus steaks were acquired from 60 beef carcasses to determine the effects of belt grill cooking rate (very rapid vs. rapid) and conditions of SSF measurement (hot vs cold) on the relationship of SSF with trained sensory panel (TSP) tenderness rating. Slice shear force was more strongly correlated with TSP tenderness rating when SSF measurement was conducted immediately after cooking (r = -.74 to -.76) than when steaks were chilled (24 h, 4 degrees C) before SSF measurement (r = -.57 to -.72). When SSF measurement was conducted immediately after cooking, the relationship of SSF with TSP tenderness rating did not differ among the belt grill cooking protocols used to cook the SSF steak. In Exp. 2, longissimus steaks were acquired from 479 beef carcasses to compare the ability of SSF and WBSF of 1.27-cm-diameter cores to predict TSP tenderness ratings. Slice shear force was more strongly correlated with sensory panel tenderness rating than was WBSF (r = -.82 vs -.77). In Exp. 3, longissimus steaks were acquired from 110 beef carcasses to evaluate the repeatability (.91) of SSF over a broad range of tenderness. Slice shear force is a more rapid, more accurate, and technically less difficult technique than WBSF. Use of the SSF technique could facilitate the collection of more accurate data and should allow the detection of treatment differences with reduced numbers of observations and reduced time requirements, thereby reducing research costs.

Animals↗