Susumu Muroya

A quick and simple method for the identification of meat species and meat products by PCR assay

The polymerase chain reaction (PCR) was applied to identify six meats (cattle, pig, chicken, sheep, goat and horse) as raw materials for products. By mixing seven primers in appropriate ratios, species-specific DNA fragments could be identified by only one multiplex PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial cytochrome b gene, and reverse primers on species-specific DNA sequences for each species. PCR primers were designed to give different length fragments from the six meats. The products showed species-specific DNA fragments of 157, 227, 274, 331, 398 and 439 bp from goat, chicken, cattle, sheep, pig and horse meats, respectively. Identification is possible by electrophoresis of PCR products. Cattle, pig, chicken, sheep and goat fragments were amplified from cooked meat heated at 100 or 120°C for 30 min, but horse DNA fragments could not be detected from the 120°C sample. Detection limits of the DNA samples were 0.25 ng for all meats.

Extracellular matrix development during differentiation into adipocytes with a unique increase in type V and VI collagen

In order to study how adipose conversion affects the extracellular environment, levels of extracellular matrix (ECM) proteins during differentiation were analyzed by 125I‐labeled antibody binding to each specific primary antibody. When confluent bovine intramuscular preadipocytes (BIP) were stimulated with adipogenic medium, there was a significant accretion on the cell surface of type I—VI collagens, laminin and fibronectin, compared with undifferentiated cells. The deposition amount of ECM proteins had reached near maximal levels at an early stage of differentiation and lasted throughout the culture. However, the increasing manners were not all the same in these eight proteins. Type V and type VI collagen tended to show a transient decline after the rapid rise at the beginning of stimulation, and fibronectin instead, subsequently decreased. Further analysis by immunocytochemical staining showed that remodeling occurred in type V and VI collagen matrices during this period; extensive fibrillar networks seen at 10 d after stimulation were quite unlike that formed earlier. These specific increases and development of matrix during adipocyte differentiation imply some significance for organizing fat lobules in each ECM proteins, especially type V and VI collagens.

Difference in tenderness and pH decline between water buffalo meat and beef during postmortem aging.

The objective of this research was to determine the difference in tenderness and some characteristics of water buffalo meat and beef during postmortem aging. Five female crossbred water-buffalo (Philippine Carabao×Bulgarian Murrah) and five female crossbred cattle (Brahman×Philippine Native), were finished on the same diet for 6 months and slaughtered at 30 months of age. The muscle pH was measured at 40min, 3h, 7h, 24h, and 48h postmortem. Longissimus thoracis (LT) and semimembranosus (SM) muscles were excised at 2d postmortem, and shear force was measured at 2, 4, 7, and 14d postmortem. Glycogen and lactate concentrations were determined from 0, 2, and 4d LT samples, and myosin heavy chain type of buffalo and cattle LT was determined by ELISA methods. Myofibrillar protein degradation was also observed by SDS-PAGE and Western blotting of fast-type troponin T. Results showed that the buffalo meat had significantly lower shear force values compared to beef for LT and SM muscles, which was supported by a difference in troponin T degradation. Postmortem pH decline of buffalo meat was significantly slower than that of beef, which was confirmed by lactic acid concentrations, but was not explained by glycogen content. In addition, there was no significant difference in the ratio of slow to fast type muscle fibers in buffalo and cattle, indicating that myosin heavy chain type was not responsible for the difference in pH decline and tenderness between the buffalo meat and beef. This study demonstrated that the tenderness of water buffalo meat was superior to that of Brahman beef, which may have been due to the difference in pH decline and the subsequent effect on muscle protease activity.

Differential expression of the skeletal muscle proteome in grazed cattle.

The objective of this study was to investigate the differences in the muscle proteome of grass-fed and grain-fed cattle. Eight Japanese Black Cattle 10 mo of age were separated randomly into 2 groups: 1) grazing (grass-fed) and 2) concentrate (grain-fed) groups. All cattle were first housed individually in a stall barn and fed a combination of concentrate ad libitum and Italian ryegrass hay until 21 mo of age. After this control period, the 4 grass-fed cattle were placed on outdoor pasture, whereas the other 4 grain-fed cattle continued on the concentrate diet. The cattle were slaughtered at 27 mo of age, and tissues from the semitendinosus muscle were obtained for use in proteome analysis. Differential expression of muscle proteins in the 2 groups was carried out using 2-dimensional gel electrophoresis (2DE) and Western blot analyses, with subsequent mass spectrometry. Approximately 200 individual protein spots were detected and compared in each group using 2DE, of which 20 and 9 spots, respectively, showed differences in the spot intensity for the sarcoplasmic fraction and myofibrillar fraction. In the grazing group, the relative intensity of spots was significantly greater for adenylate kinase 1 and myoglobin in the sarcoplasmic fraction, and for slow-twitch myosin light chain 2 in the myofibrillar fraction (P < 0.05), than the concentrate group. The relative spot intensity of several glycolytic enzymes was significantly greater in the grazing group, such as beta-enolase 3, fructose-1,6-bisphosphate aldolase A, triosephosphate isomerase, and heat shock 27 kDa protein (P < 0.05). Moreover, significantly greater slow twitch of troponin T, troponin I, and myosin heavy chain of semitendinosus muscle was detected in the grazing group than in the concentrate group using Western blot analysis (P < 0.05). Several previous reports have described that the slow-twitch muscle contents affect elements of nutrition, flavor, and food texture of meat. This study revealed muscle fiber type conversion to slow-twitch tissues from fast-twitch tissues occurring with change in the energy metabolic enzyme when cattle were grazed in the latter fattening period. Although analyses of the influence on elements of nutrition, flavor, and food texture were not done for this study, these results show that slow-twitch converted muscle resulting from the grazing of cattle might modify several meat characteristics.

SEQUENCING OF THE 2A, 2X, AND SLOW ISOFORMS OF THE BOVINE MYOSIN HEAVY CHAIN AND THE DIFFERENT EXPRESSION AMONG MUSCLES

Myosin is a major structural protein of the thick filament of the sarcomere. It constitutes 40% of myofibrillar protein. Each myosin molecule consists of two identical heavy chains (MyHC, 220 kDa each) and two pairs of non-identical myosin light chains (20 kDa each; Warrick and Spudich 1987). There are four major sarcomeric MyHC isoforms in adult mammalian skeletal muscle, MyHC-2a, -2b, -2x (also referred to as -2d), and ‘‘-slow’’. Each MyHC isoform is encoded by a distinct gene (Schiaffino and Reggiani 1996). Individual muscle fibers are characterized according to their MyHC content: type II A fiber (containing MyHC-2a), type II B fiber (containing MyHC-2b), type II X fiber (containing MyHC2x), and type I fiber (containing MyHC-slow; Pette and Staron 1990). Skeletal muscle fibers can alter their MyHC isoform composition in response to electrical stimuli and mechanical factors such as stretch (Ausoni et al. 1990; Loughna et al. 1990; Termin et al. 1989). However, the molecular mechanisms determining genespecific activation of these MyHC genes are not well defined (Schiaffino and Reggiani 1996). Expression of the four major MyHC isoform genes was reported in rat muscles (Schiaffino and Reggiani 1996). Expressions of these four MyHC isoforms in the porcine skeletal muscles were also detected by in situ hybridization (Chang and Fernandes 1997) and RT-PCR (Tanabe et al. 1999). Expression pattern of a fetal bovine MyHC gene was reported (Young et al. 1994); however, expression pattern MyHC isoform genes in bovine skeletal muscles has not been reported. Cattle are large ruminant mammals, whose physiology is different from that of non-ruminant mammals; therefore, bovine MyHC isoform composition would differ from that of other mammals. Moreover, MyHC isoform composition would also affect the quality of the meat originating from bovine muscles (Xiong 1994). In this report, we determined the sequences containing 58 non-coding region of bovine MyHC-2a, -2x and -slow isoforms, which differed among the isoforms. Based on these sequences, we developed a procedure to detect the expression of these three MyHC isoforms in bovine muscles by the RT-PCR method with the use of the multiplex PCR technique. Published sequences of porcine MyHCs (Chang et al. 1993, 1995; Tanabe et al. 1999) were used to amplify the bovine cDNA fragments by PCR. From the reported sequences, three sense primers (MYO1, MYO103, MYO401) and four antisense primers (MYO2, MYO6, MYO102, MYO404) were synthesized. The sequences of these primers are described in Table 1. We obtained first-strand cDNAs from M. biceps femoris of 7-month-old cattle and diaphragm of 3-month-old cattle by using CapFinderTM PCR cDNA Library Construction Kit (CLONTECH, Calif.). This kit contained a primer named 58PCR (the sequence is described in Table 1) for cDNA amplification. Two PCR fragments were obtained from the first-strand cDNAs of M. biceps femoris by using the primer pairs MYO1/MYO2 and 58PCR/MYO6 and then were directly sequenced. The resulting sequence was highly different from the porcine MyHC-2a sequence, especially on the non-coding region; therefore, two sense primers (MYO201, MYO203) and one antisense primer (MYO202) on the bovine non-coding region were synthesized to confirm the bovine sequence. The sequences of these primers are described in Table 1. With MYO201, MYO202, MYO203, MYO2, and 58PCR, a cDNA fragment of the M. biceps femoris was amplified again. Consequently, this bovine cDNA fragment was confirmed to be homologous to the porcine MyHC2x isoform reported by Chang and Fernandes (1997). We amplified and sequenced a cDNA fragment of the bovine MyHC-2a isoform from the cDNAs of M. biceps femoris with the use of MYO102, MYO103, MYO2, and 58PCR. We amplified and seCorrespondence to: R. Tanabe

Myosin heavy chain isoforms expressed in bovine skeletal muscles.

Nucleotide sequences including the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from bovine adult skeletal muscles. The deduced amino acid sequences were 1940, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. Like other mammalian MyHC isoforms, the bovine MyHC isoforms had homologous sequences except for substitutions concentrated on the loop 1, loop 2, and light chain binding regions. RT-PCR amplifications showed that the adult bovine skeletal muscles expressed the MyHC-2a, -2x, and -slow isoforms but no -2b isoform. The absence of the MyHC-2b isoform and substitutions on the loop2 region could explain some differences in meat quality between beef and pork.

Related expression of MyoD and Myf5 with myosin heavy chain isoform types in bovine adult skeletal muscles.

Abstract Skeletal muscles are characterized as fast and slow muscles, according to the expression pattern of myosin heavy chain (MyHC) isoforms in the muscle fibers. To investigate the relationships between MyHC isoforms and myogenic regulatory factors (MRFs) including MyoD, Myf5, myogenin, and MRF4 in adult skeletal muscles, expressions of these MRFs in the ten muscles of three cows were analyzed by a semi-quantitative RT-PCR. The results showed that MyoD expression was significantly lower in the lingual muscles (TN), masseter (MS) and diaphragm (DP), which lack MyHC-2x (fast glycolytic) expression and abound with MyHC-slow (slow oxidative) and/or MyHC-2a (fast oxidative), than it was in the pectoralis (PP), psoas major (PM), longissimus thoracis (LT), spinnalis (SP), semitendinosus (ST), semimembranosus (SM), and biceps femoris (BF). In contrast, the Myf5 expression in TN, MS, and DP was significantly higher than in PM, LT, ST, SM, and BF. No significant difference was observed in myogenin and MRF4 expression among the muscles tested. The results suggest that MyoD and Myf5 influence the MyHC isoform expression, although the effects are not decisive in specifying the phenotypes of adult muscles.

N-terminal amino acid sequences of troponin T fragments, including 30 kDa one, produced during postmortem aging of bovine longissimus muscle

We have determined the amino (N)-terminal amino acid (AA) sequences of five troponin T (TnT) fragments produced during postmortem aging of bovine longissimus muscle. Western blot analysis showed that 32.1, 28.8, 27, and 25.8 kDa anti-fast-type TnT (fTnT)-positive fragments and a 31 kDa anti-slow-type TnT (sTnT)-positive fragment were present at 14 d postmortem. The N-terminal AA sequences of the 32.1, 28.8 (conventional 30 kDa), 27, and 25.8 kDa fragments were APPPPAEV, EVHEPEEK, EKPRPRLT, and APKIPEGE, respectively, and they were mapped to the N-terminal region of bovine fTnT isoforms. The N-terminal sequences of the 31 kDa fragment, EAPEEPEP, were mapped to the sTnT isoforms. These findings indicate that the two isoform types of fTnT predominantly expressed in the longissimus muscle are cleaved specifically at Glu(21)-Ala(22) and Glu(15)-Ala(16), His(37)-Glu(38) and His(31)-Glu(32), Glu(43)-Glu(44) and Glu(37)-Glu(38), and/or Thr(51)-Ala(52) and Thr(45)-Ala(46), respectively, and that a sTnT isoform is cleaved specifically at Glu(23)-Glu(24).

Profiling of differentially expressed microRNA and the bioinformatic target gene analyses in bovine fast- and slow-type muscles by massively parallel sequencing.

MicroRNA (miRNA) are highly conserved, noncoding small RNA involved in post-transcriptional gene regulation in a variety of biological processes. To elucidate roles of miRNA in bovine muscle type specification and maintenance, we sought to determine differentially expressed miRNA between semitendinosus (STD) and masseter (MS) muscles from 3 Japanese black cattle by massively parallel sequencing. Differential gene expression of myosin heavy chain (MyHC) isoforms confirmed that STD and MS were MyHC-2x- and MyHC-1-abundant muscles, respectively. In total, 192 known miRNA and 20 potential new bovine miRNA were obtained from the sequencing. The differentially expressed miRNA with more than 2-fold difference in each muscle were identified. In particular, miR-196a and miR-885 were exclusively expressed in STD muscle, which was validated by quantitative reverse transcription-PCR (P=0.045 and P<0.001, respectively), whereas a slow type-directing miR-208b was highly expressed in MS compared with STD (false discovery rate<0.05). In addition, 16 potential novel miRNA were mapped and confirmed for their precursor structures by computational analyses. The results of functional annotation combined with in silico target analysis showed that the predicted target genes of miR-196a/b and miR-885 enriched gene ontology (GO) terms related to skeletal system development and regulation of transcription, respectively. Moreover, GO terms enriched from predicted targets miRNA suggested that STD-abundant- and MS-abundant-miRNA were associated with embryonic body planning and organ/tissue pattern formation, respectively. The present results revealed that the differentially expressed miRNA between the STD and MS muscles may play key roles to determine muscle type-specific tissue formation and maintenance in cattle thorough attenuating putative target genes involved in different developmental events.

Postmortem changes in bovine troponin T isoforms on two-dimensional electrophoretic gel analyzed using mass spectrometry and western blotting: The limited fragmentation into basic polypeptides

To comprehend postmortem changes in troponin T (TnT), whole beef proteins were developed on a two-dimensional electrophoretic gel. Multiple TnT-related spots were identified by both western blotting and MALDI-TOF MS utilizing bovine TnT isoform mRNA sequences. More than 10 TnT fast-type isoform spots (pI 5.7-9.6<) and the two slow-type isoform spots (pI 5.6-5.7) were observed at slaughter. All the isoforms were degraded exclusively into basic spots (pI 9.6<) at day 14 postmortem. Some TnT-related phosphorylated spots present at slaughter had disappeared by day 14, suggesting that the phosphorylated N-terminal region was cut off during beef aging. The intact isoforms and the fragments were identified by the MS with sequence coverage of 20.8-62.7%, and two of the fragments included the cutting site peptide of a conventional 30kDa or of a slow TnT-derived fragment. These results revealed that all of the TnT isoforms are cut exclusively in the glutamic acid-rich amino-terminal region during postmortem aging.