Postmortem Metabolic Rate and Calpain System Activities on Beef Longissimus Tenderness Classifications

In this study, we adopted a model of tenderness classification in order to determine the factors affecting the tenderness and tenderization characteristics of beef longissimus, using cluster analysis on the basis of Warner-Bratzler shear force and myofibril fragmentation index, at 1, 7, and 14 d. The rate of tenderization was effectively differentiated by pH, R-values, μ-calpain activity, and calpastatin activity. Differences among tenderness classes were generally detected at 3 and 9 h postmortem for metabolic rate, and at 9 and 24 h for the activities of μ-calpain and calpastatin. Early postmortem metabolic rate and calpain system activities were verified as important factors with regard to longissimus tenderization.     

The effect of calcium chloride injection on shear force and caspase activities in bovine longissimus muscles during postmortem conditioning

Tenderness is considered as the most important quality determinant of meat. Calcium chloride application has been shown to improve tenderness by regulating endogenous proteinases. This study was designed to determine the effect of 300 mM calcium chloride injection on myofibrillar structures, caspase activities and shear force in longissimus muscles of bulls during postmortem storage of 7 days. Myofibrillar fragmentation index was determined as an index of proteolysis occurring in muscle fibers and associated proteins. Maximum tenderness was observed at days 4 and 7 in both treated and control samples. The injection of calcium chloride significantly increased myofibrillar proteolysis and improved tenderness at postmortem days 4 and 7. The treatment reduced caspase-9 activity at 4 h and day 4, whereas those of caspase-8 and -3 activities at days 1 and 4 with respect to control. The improved tenderness and increased myofibril fragmentation with decreased caspase activities suggested that the proteolytic systems activated with calcium chloride injection possibly behave independent of the caspase system.     

Correlation between a novel calpastatin biosensor and traditional calpastatin assay techniques

An optical fiber biosensor to detect calpastatin has been investigated as a preliminary step in developing tenderness detection instrumentation. Longissimus dorsi samples were taken from beef carcasses (n=21) at 0, 24, 36 and 48h postmortem. Muscle homogenates were assayed for calpastatin activity using traditional methods and an optical fiber biosensor. Warner-Bratzler shear force was also performed on a steak from each carcass at 14d postmortem. Results demonstrated that the measurements with highest correlation between traditional calpastatin assays and optical biosensor readings were taken at 48h postmortem (r=0.597, P< or =0.01), suggesting that this is the best time for use of this biosensor in an on-line grading system. This research further advances the development of a calpastatin biosensor and would be useful in laboratory determination of the presence of biologically active calpastatin concentrations.     

Relationships among calpastatin single nucleotide polymorphisms,calpastatin expression and tenderness in pork longissimus1

Genome scans in the pig have identified a region on chromosome 2 (SSC2) associated with tenderness. Calpastatin is a likely positional candidate gene in this region because of its inhibitory role in the calpain system that is involved in postmortem tenderization. Novel single nucleotide polymorphisms (SNP) in calpastatin were identified and used to genotype a population (n = 1042) of Duroc–Landrace–Yorkshire swine for association with longissimus lumborum slice shear force (SSF) measured at days 7 and 14 postmortem. Three genetic markers residing in the calpastatin gene were significantly associated with SSF (P < 0.0005). Haplotypes constructed from markers in the calpastatin gene were significantly associated with SSF (F‐ratio = 3.93; P‐value = 0.002). The levels of normalized mRNA expression of calpastatin in the longissimus lumborum of 162 animals also were evaluated by real‐time RT‐PCR and were associated with the genotype of the most significant marker for SSF (P < 0.02). This evidence suggests that the causative variation alters expression of calpastatin, thus affecting tenderness. In summary, these data provide evidence of several significant, publicly available SNP markers associated with SSF that may be useful to the swine industry for marker assisted selection of animals that have more tender meat.     

Effect of Ca2+ on binding of the calpains to calpastatin

Autolyzed mu-calpain, unautolyzed mu-calpain, autolyzed m-calpain, and unautolyzed m-calpain (mu-calpain is the micromolar Ca2+-requiring proteinase, m-calpain is the millimolar Ca2+-requiring proteinase) were passed through a calpastatin-affinity column at different free Ca2+ concentrations, and binding of the calpains to calpastatin was compared with proteolytic activity of that calpain at each Ca2+ concentration. Unautolyzed m-calpain, autolyzed m-calpain, and autolyzed mu-calpain required less Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Unautolyzed mu-calpain, however, required slightly more Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Half-maximal binding of oxidatively inactivated mu- or m-calpain to calpastatin required approximately the same Ca2+ concentrations as half-maximal binding of unautolyzed mu- or m-calpain, respectively, to calpastatin. Binding of unautolyzed m-calpain and autolyzed mu-calpain to calpastatin occurred over a wide range of Ca2+ concentrations, and it seems likely that two or more Ca2+-binding sites with different Ca2+-binding constants are involved in binding of the calpains to calpastatin. Proteolytic activity occurs at different Ca2+ concentrations than calpastatin binding, suggesting a second set of Ca2+-binding sites associated with proteolytic activity. Third and fourth sets of Ca2+-binding sites may be involved in autolysis and in binding to phosphatidylinositol or cell membranes; these four Ca2+-dependent properties of the calpains may require the eight potential Ca2+-binding sites that amino acid sequences predict are present in the calpain molecules.     

Changes in proteolytic enzymes mRNAs and proteins relevant for meat quality during myogenesis and hypoxia of primary bovine satellite cells

The current study was conducted to evaluate the functions of μ-calpain (CAPN1), calpastatin, HSPs (heat shock proteins), and caspases during myogenesis and cell death induced by sodium azide (NaN(3)) hypoxia. The cell samples were divided into three groups: satellite cells formed at confluent monolayer (stage 1), stage 1 cells fusion into myotubes on d eight post-differentiation (stage 2), and stage 2 cells treated with 1 mM NaN(3) for 24 h (stage 3). Real-time RT-PCR showed that stage 2 cells had increased CAPN1, calpastatin, caspase 7, and CARD9 (Caspase activation and recruitment domain 9) mRNA expressions compared to stage 1 cells (*p < 0.05). By Western blotting caspase 3, caspase 7, caspase 8, and caspase 9 protein levels increased in cells at stage 2 compared to cells at stage 1 (*p < 0.05). Real-time RT-PCR showed that stage 3 cells had increased CAPN1, calpastatin, caspase 7, HSP70 (70 kDA heat shock proteins), and HSP90 (90 kDA heat shock proteins-alpha) and decreased CARD9 mRNA expression compared to stage 2 cells (*p < 0.05). Stage 3 samples had increase caspase 7 and caspase 12 activities compared to stage 2 samples, and by Western blotting protein levels of both HSP70 and HSP90 expressions, increased significantly under hypoxia condition (*p < 0.05). Here, we conclude that CAPN1, calpastatin, caspase 3, caspase 7, caspase 8, and CARD9 have important roles for satellite cell myogenesis; and that caspase 7, 12, HSP70, and HSP90 are involved in the process of apoptotic cell death under hypoxia conditions and we speculate that these proteins may be involved in early postmortem proteolysis and meat tenderization.     

The role of ultimate pH in proteolysis and calpain/calpastatin activity in bovine muscle.

Of a total of three Friesian cows, two of which had been treated with adrenalin before slaughter, Mm longissimus (LO), supraspinatus (SS), triceps brachii (TB) and rectus abdominis (RA) were sampled at different times post mortem (pm). pH, calpain/calpastatin activities and degradation of myofibrillar proteins, as evidenced by SDS-PAGE, were assessed. Contraction characteristics were measured by determining myofibrillar ATPase activities. Adrenalin treatment resulted in a high ultimate pH (6.48 +/- 0.40) and a faster decline pm of calpain I activity. The effect was similar in all four investigated muscles (72.4 +/- 5.4% decline at 24 h pm). The decline in calpain I activity in the control muscles was muscle-dependent and ranged from 22.8-74.3% at 24 h pm. Differences in ultimate pH did not lead to distinct rates of breakdown of proteins with molecular weights lower than that of myosin heavy chain. Calpastatin levels were muscle-dependent and correlated with myofibrillar ATPase activity (r = -0.99). In a second experiment Mm rectus abdominis (RA) and psoas major (PM) of adrenalin-treated (n = 6) and control (n = 6) Friesian-Holstein calves were sampled at 1 and 29 h pm for assessment of calpain activities. At seven days pm the M longissimus (LO) was sampled for tenderness evaluation. pH values were measured at 30 min, 4 h and 29 h pm. Adrenalin treatment resulted in a higher ultimate pH in the three muscles. Higher ultimate pH resulted in lower calpain activities in the RA at 29 h pm (P less than or equal to 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)     

The calpain system in human placenta

The calpain system is involved in a number of human pathologies ranging from the muscular dystrophies to Alzheimer's disease. It is important, therefore, to be able to obtain and to characterize both mu-calpain and m-calpain from human tissue. Although human mu-calpain can be conveniently obtained from either erythrocytes or platelets, no readily available source of human m-calpain has been described. Human placenta extracts contain both mu-calpain and m-calpain in nearly equal proportions and in significant quantities (3-4 mg mu-calpain and 4-5 mg m-calpain/1000 g placenta tissue). Placenta also contains calpastatin that elutes off ion-exchange columns over a wide range of KCl concentrations completely masking the mu-calpain activity eluting off these columns and even partly overlapping m-calpain elution. Placenta mu-calpain requires 50-70 microM Ca2+ and placenta m-calpain requires 450-460 microM Ca2+ for half-maximal proteolytic activity. Western analysis of washed placenta tissue shows that placenta contains both mu- and m-calpain, although some of the mu-calpain in whole placenta extracts likely originates from the erythrocytes that are abundant in the highly vascularized placenta. Placenta calpastatin could not be purified with conventional methods. The most prominent form of calpastatin in Western analyses of placenta obtained as soon as possible after birth was approximately 48-51 kDa; partly purified preparations of placenta calpastatin also contained 48-51 and 70 kDa polypeptides. Human placenta extracts likely contain two different calpastatin isoforms, a 48-51 kDa "placenta calpastatin" and a 70 kDa erythrocyte calpastatin.     

Identification of calpastatin and mu-calpain and studies of their association in pulmonary smooth muscle mitochondria

Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of micro-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of mu-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as mu-calpain and calpastatins. Immunoprecipitation study revealed molecular association between mu-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of mu-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis.     

Viability of a FRET dual binding technique to detect calpastatin

We have been investigating a fluorescence dual binding biosensor to detect calpastatin. Calpastatin is a protein found in meat and it is a regulator of meat tenderness. The ability to accurately predict the calpastatin concentration of beef with a biological sensor at the time of grading would lead to a more accurate assessment of the overall palatability of beef when it reaches the consumer. Meat can then be labeled as tender or tough, which would greatly enhance meat processors' ability to grade meat, allowing them to recover lost revenue. The biosensor technique utilized the chemical transduction principle of fluorescence resonance energy transfer (FRET). FRET requires the use of two fluorophores, termed a donor and acceptor. In this study, the donor fluorophore was conjugated to the protein, mu-calpain, while the acceptor fluorophore was conjugated to a monoclonal antibody. The results showed that in the presence of calpastatin, the labeled mu-calpain and antibody would bind to calpastatin, reducing the distance between the two proteins and eliciting a measurable change in fluorescence. The FRET dual binding technique was tested in heated and unheated meat extract, and a limit of detection for calpastatin was 120 ng/ml in diluted heated meat extract with no significant response in the unheated meat extract. Stable response times were achieved within 5 min. The proof-of-principle of utilizing a FRET dual binding technique to detect calpastatin in heated meat extract has been established.     

Single nucleotide polymorphisms in an intron of the ovine calpastatin gene

Calpastatin is the specific inhibitor of the ubiquitous calcium-dependent proteases mu-calpain and m-calpain. Enzyme assay data from sheep and cattle inversely correlates post-mortem muscle calpastatin levels with ultimate meat tenderness. Genetic markers of meat quality may therefore be found linked to the calpastatin gene (CAST). A three-allele system detected by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) has been observed in the ovine CAST. The three allele amplimers have been fully nucleotide sequenced and their differences in terms of single nucleotide polymorphism (SNPs) in the intron region of the amplimer are reported and compared to a consensus sequence of the orthologous region of the cattle CAST. A PCR-RFLP for more rapid CAST genotyping of all three ovine alleles was also developed.     

A comparison of the intracellular distribution of mu-calpain, m-calpain, and calpastatin in proliferating human A431 cells.

Little is known about the relative intracellular localizations of the calcium-dependent proteases, calpains, and their naturally occurring inhibitor, calpastatin. In the present study, the intracellular localization of mu-calpain, m-calpain, and calpastatin was studied at the light microscopic level in proliferating A431 cells. Highly specific antibodies against the three antigens revealed distinct staining patterns in interphase and mitotic cells. Most notably, calpastatin in interphase cells was localized near the nucleus in tube-like, or large granular structures, while the calpains were more uniformly distributed through the cytoplasm in either a fibrillar form (mu-calpain) or a diffuse or fine granular form (m-calpain). The distribution patterns of the two calpain isozymes were distinctly different during mitosis. m-Calpain was concentrated at the mitotic spindle poles and midbody, while mu-calpain appeared to accumulate at the cell membrane and the spindles. Four other human cell lines as well as normal human monocytes were examined to determine if the calpains-calpastatin segregation patterns are common to other cells or are unique to the A431 line. With the exception of abundant nuclear mu-calpain in the C-33A cervical carcinoma, the segregation of the proteins was similar to that of A431. These studies indicate that calpains may be localized at regions which are relatively poor in calpastatin content. Proteins at these sites may be susceptible to calpain-catalyzed cleavage.     

Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

mu-Calpain regulates receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis via NF-kappaB activation in RAW 264.7 cells

To clarify the role of calpain in the receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on alpha-spectrinolysis and that mu-calpain, rather than m-calpain, was activated during RANKL-supported osteoclastogenesis in RAW 264.7 cells. Overexpression of mu-calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-kappaB activation via inhibition of the cleavage of inhibitor of NF-kappaB(IkappaBalpha)in RAW 264.7 cells. Taken together, our findings suggest that mu-calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-kappaB activation.     

The purification and characterization of mu-calpain and calpastatin from ostrich brain

Calcium-activated neutral proteinases (CANPs) and their endogenous specific inhibitor calpastatin are found in a wide variety of vertebrate and invertebrate tissues. The CANPs are cysteine proteinases that have an absolute requirement for Ca(2+) for activity. mu-Calpain and calpastatin were purified by successive chromatographic steps on Toyopearl-Super Q 650S and Pharmacia Mono Q HR 5/5 columns. The enzyme has a M(r) of 84KDa using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), a M(min) of 79KDa from amino acid analysis and an pI of 5.2. Calpastatin has a M(r) of 323KDa using denaturing gradient PAGE and a pI of 4.7. The amino acid composition of mu-calpain revealed 689 residues and the pH and temperature optima were found to be 7.5 and 37 degrees C, respectively. mu-Calpain underwent a Ca(2+)-dependent autoproteolysis producing a fragment of 82KDa. The N-terminal sequence of mu-calpain showed 24 and 18% sequence identity with human and bovine mu-calpain.     

Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells

Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14 - 18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CTheta, Src, Bax and mu-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, mu-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells.     

Single nucleotide polymorphisms in caprine calpastatin gene

The calpains and calpastatin (CAST) make up a major cytosolic proteolytic system, the calpain-calpastatin system, found in mammalian tissues. The relative levels of the components of the calpain-calpastatin system determine the extent of meat tenderization during postmortem storage. Calpastatin (CAST) is a protein inhibitor of the ubiquitous calcium-dependent proteases, μ-calpain, and m-calpain. Polymorphisms in the bovine, ovine and pig CAST gene have been associated with meat tenderness but little is known about how caprine CAST gene may affect goat meat quality traits. In this study we selected different parts of the CAST gene: (1) that have been previously reported to be polymorphic, intron 5 and 12 and 3’UTR; (2) first time explored (exon 3, 7 and 8 and part of intron 7 and 8) to investigate polymorphic status of caprine CAST gene. Using comparative sequencing ten novel SNPs located in exon 3 and intron 5, 7 and 8 were identified. Previously reported SNPs in intron 5, 3’UTR and intron 12 were absent. Sequence analysis revealed a non synonymous amino acid variation in exon 3, which would result in Lys/Arg substitution in the corresponding protein sequence. Considerable variation was detected in intronic regions. Twenty-four InDel were also recognized in intronic regions (15) and 3’UTR (9). All the sequences shared high homology with published bovine and ovine sequences. Three PCR-RFLP loci have been established for further analyzing genetic polymorphism in indigenous goats.     

The calpastatin/calpain system in trout Salmo trutta trutta muscle

Many recent reports suggest that the calpastatin/calpain system plays a role in cellular growth and differentiation. Defects of the calpastatin/calpain system have been linked to cellular dysfunctions, apoptosis, myocardial infarct, and dystrophies. The calpastatin/calpain system has also been implicated in post‐mortem tenderization of skeletal muscle through degradation of key myofibrillar and associated proteins, a process of key importance to meat quality. In the present study we investigate the presence and activity of the calpastatin/calpain system in trout muscle samples, collected at 0, 3, 18 and 28 h post‐mortem, by immunohistochemistry method. Calpastatin is a specific endogenous enzyme of cytosol, modulating the ubiquitous calpains. Calpastatin was found in samples obtained in vivo and immediately post‐mortem, but its concentration declined rapidly in samples obtained 3, 18 and 28 h post‐mortem. The ubiquitous m e m‐calpains, which are localized on Z line proteins and activated by intracellular Ca²⁺ increase, showed a rapid decline within 3 h post‐mortem. By contrast p94 calpain, which is specific to skeletal muscle, showed a slow decrease post‐mortem which was independent of intracellular Ca²⁺ increase. Our results suggest that the mechanism of activation and activity of the calpastatin/calpain system in trout is similar to that described in mammals.     

Meat quality traits and the expression of tenderness-related genes in the loins of young goats at different ages

Goat meat is considered healthy because of its low fat content, but it is often rather tough. Tenderness is the most important attribute of quality during meat consumption and there is scarce information about the expression of genes involved in the meat tenderization process in goats. The aim of this trial was to assess certain meat quality traits and the expression, at the messenger RNA (mRNA) and protein levels, of specific genes involved in the tenderization process of the longissimus lumborum (LL) in young male goats (Capra hircus) at different ages. Samples of LL were collected at slaughter from 32 Alpine goats that were divided into three categories: 9 suckling kids (Sk) at 5.4±0.15 weeks of age, 16 chevons (Ch) at 17.1±0.55 weeks of age and 7 post-puberal goats (Pu) at 34.3±2.5 weeks of age. Animal and carcass variables (live weight gain, live weight, carcass weight and fat deposits) and quality traits of meat (lipid content, ultimate pH, color parameters, cooking loss and shear force) were determined. The mRNA abundances of calpain-1 (Capn1), calpain-2 (Capn2), calpastatin (Cast), caspase 3 (Casp3), caspase 9 (Casp9), αB-crystallin (Cryab), heat shock protein 27 (Hsp27), heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) were detected by quantitative PCR. Capn1, Cast, Cryab and Hsp27 protein expression was investigated by ELISA. The Sk group had the leanest carcasses. The meat of the Pu group was the darkest (P<0.05) and the toughest (P<0.05). The redness of meat increased with the age of the goats. The Sk group showed lower mRNA abundances for the Capn2/Cast ratio, Casp3, Cryab, Hsp27, Hsp40 and Hsp70 than the Pu group (P<0.05). Intermediate values were found for the Ch group. Similar results were highlighted for the protein expression of Cryab and Hsp27. The experiment acknowledged a differentiation of the experimental groups based on performance, carcass and meat characteristics, and the genes considered. Moreover, Sk and Pu groups, characterized by a different tenderness of their meat, were clearly discriminated by a different expression of the Hsp.