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ABSTRACT: From the adipose tissues of pork carcasses stored in a refrigerator, Raman spectra were observed in situ by a portable Raman spectrometer. The observed Raman spectra, which were almost completely due to fat, showed clear dependence on the refrigeration time and the carcass temperature. This dependence reflected an increase in the crystallinity of the fat and a change in the fraction of the β´ polymorph. Evidence of changes in the packing order of the aliphatic chains of acylglycerol molecules was obtained, and the changes lasted for a long time after the temperature reached the lowest (4.3 °C). Possibilities of using Raman spectrometry as a tool for routine monitoring of the conditions of carcasses as well as for research on the improvement of the mechanical strength of the adipose tissue are discussed.
Journal of Agricultural and Food Chemistry 12/2012; · 2.82 Impact Factor
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Keisuke Sasaki,
Masayuki Hayashi,
Takumi Narita,
Michiyo Motoyama,
Mika Oe,
Koichi Ojima,
Ikuyo Nakajima,
Susumu Muroya, Koichi Chikuni,
Katsuhiro Aikawa,
Yasuyuki Ide,
Naoto Nakanishi,
Nobuaki Suzuki,
Shigeru Shioya,
Akio Takenaka
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ABSTRACT: This study examined the accumulation and tissue distribution of radioactive cesium nuclides in Japanese Black beef heifers raised on roughage contaminated with radioactive fallout due to the accident at the Fukushima Daiichi Nuclear Power Station on March 2011. Radiocesium feeding increased both (134)Cs and (137)Cs levels in all tissues tested. The kidney had the highest level and subcutaneous adipose had the lowest of radioactive cesium in the tissues. Different radioactive cesium levels were not found among parts of the muscles. These results indicate that radiocesium accumulated highly in the kidney and homogenously in the skeletal muscles in the heifers.
Bioscience Biotechnology and Biochemistry 08/2012; 76(8):1596-9. · 1.28 Impact Factor
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ABSTRACT: Messenger RNA (mRNA) expression of calpain-1 (µ-calpain), -2 (m-calpain), -3 (p94), small subunit (calpain-4; 28 kDa), and three types of calpastatin (CSTN) isoform were investigated for 10 skeletal muscles of Holstein cattle by real-time and/or semi-quantitative reverse transcription polymerase chain reaction. Noticeably, effect of muscle type was observed on 28 kDa expression (P < 0.001) with a tendency of higher 28 kDa expression in myosin heavy chain (MyHC)-2x-rich muscles compared to MyHC-slow-rich muscles. The CSTN-I and -III expression in Longissimus thoracis (LT) showed the lowest value among the muscles tested. Moreover, 28 kDa/CSTN-I ratio was higher in the diaphragm (DP), psoas major (PM), and LT than those in the lingual muscles (TN), masseter (MS) and pectoralis (PP) (P < 0.05). Calpain-1/CSTN I, calpain-2/CSTN I in LT and PM were higher than that in TN (P < 0.05). Calpain-3/CSTN-I and -III in LT and/or PM showed higher values than that in TN (P < 0.05). These results indicated that the calpain and CSTN expressions are regulated by muscle type, suggesting especially by muscle fiber type. Calpains/CSTN-I ratios, especially 28 kDa/CSTN-I, may account for higher extent of post mortem proteolysis previously observed in LT and PM muscles.
Animal Science Journal 03/2012; 83(3):252-9. · 0.86 Impact Factor
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Kumiko Takeda,
Mariko Tasai,
Masaki Iwamoto,
Mika Oe, Koichi Chikuni,
Yoshiaki Nakamura,
Takahiro Tagami,
Keijiro Nirasawa,
Hirofumi Hanada,
Carl A Pinkert,
Akira Onishi
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ABSTRACT: Somatic cell nuclear transfer (SCNT) has been exploited in efforts to clone and propagate valuable animal lineages. However, in many instances, recipient oocytes are obtained from sources independent of donor cell populations. As such, influences of potential nuclear-cytoplasmic incompatibility, post SCNT, are largely unknown. In the present study, alterations in mitochondrial protein levels were investigated in adult SCNT pigs produced by microinjection of Meishan pig fetus fibroblast cells into enucleated matured oocytes (maternal Landrace genetic background). Mitochondrial fractions were prepared from liver samples by mechanical homogenization and differential centrifugation. Liver mitochondria were then subjected to two-dimensional difference gel electrophoresis (2-D DIGE). Protein expression changes were confirmed with a volume ratio greater than 2 fold (P<0.05). 2-D DIGE analysis further revealed differential expression of three proteins between the Meishan (n=3) and Landrace (n=3) breeds. Differential expression patterns of 16 proteins were detected in SCNT pig liver tissue (n=3) when compared with Meishan control samples. However, none of the 16 proteins correlated with the three differentially expressed Meishan and Landrace liver mitochondrial proteins. In summary, alteration of mitochondrial protein expression levels was observed in adult SCNT pigs that did not reflect the breed difference of the recipient oocytes. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.
Journal of Reproduction and Development 12/2011; 58(2):248-53. · 1.46 Impact Factor
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ABSTRACT: This study investigated the growth performance and gene expression for muscle development between grass hay-fed (GH) and concentrate-fed (CT) steers. Daily gain and energy intake during the fattening period of the GH group were lower than those of the CT group. Analysis of C/EBPα, PPARγ2, myosin heavy chain (MHC), and myostatin gene expressions was performed by real-time PCR. Expressions of C/EBPα and myostatin in semitendinosus and longissimus lumborum (LL) muscles were higher in the CT group than in the GH group at the end of fattening. In LL muscle, MHC expression at the end of fattening was greater in the GH group than in the CT group. These results suggest that regulation of adipogenesis and myogenesis by the expression of genes involved in muscle development might have occurred in the skeletal muscle of the GH group by the feeding of grass hay and/or because of the low energy intakes.
Meat Science 05/2011; 89(4):451-6. · 2.28 Impact Factor
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ABSTRACT: Experiments were designed to compare the adipocyte cellularity of subcutaneous adipose tissue between growing Landrace (low backfat) and Meishan (high backfat) pigs at 1 week, 3 weeks, 6 weeks, 3 months and 5 months of age. As pigs aged, body weight and backfat thickness of both breeds significantly increased. When compared at equal ages, backfat thickness adjusted to equal body weight was greater for Meishan pigs. The mean diameter of fat cell size also increased with age, and by 6 weeks adipocytes from both outer and inner layers of subcutaneous adipose tissue were larger in Meishan pigs. At 5 months, approximately 80% of the adipose tissue mass in Meishan pigs was attributable to adipocytes measuring 95-165 µm in diameter, whereas adipocytes of 75-145 µm comprised most of the tissue mass in the Landrace. Although the contribution of smaller adipocytes (25-45 µm) to the tissue volume was negligible, both breeds showed a biphasic diameter distribution at all ages, suggesting that adipocyte hyperplasia is still active. Our results demonstrate that cellularity differences exist between the subcutaneous adipose tissues of Landrace and Meishan pigs, and adipocyte hypertrophy is the most overwhelming contributor to the greater backfat deposition for Meishan pigs.
Animal Science Journal 02/2011; 82(1):144-9. · 0.86 Impact Factor
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Kumiko Takeda,
Mariko Tasai,
Satoshi Akagi,
Shinya Watanabe,
Mika Oe, Koichi Chikuni,
Mayumi Ohnishi-Kameyama,
Hirofumi Hanada,
Yoshiaki Nakamura,
Takahiro Tagami,
Keijiro Nirasawa
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ABSTRACT: Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for such a developmental deficiency. However, detailed knowledge of protein expression during somatic cell nuclear transfer (SCNT) in cattle is lacking. In the present study, variations in mitochondrial protein levels between SCNT-derived and control cattle, and from calves derived by artificial insemination were investigated. Mitochondrial fractions were prepared from frozen liver samples and subjected to two-dimensional (2-D) fluorescence differential gel electrophoresis (DIGE) using CyDye™ dyes. Protein expression changes were confirmed with a volume ratio greater than 2.0 (P < 0.05). 2D-DIGE analysis revealed differential expression of three proteins for SCNT cattle (n = 4) and seven proteins for SCNT calves (n = 6) compared to controls (P < 0.05). Different protein patterning was observed among SCNT animals even if animals were generated from the same donor cell source. No differences were detected in two of the SCNT cattle. Moreover, there was no novel protein identified in any of the SCNT cattle or calves. In conclusion, variation in mitochondrial protein expression concentrations was observed in non-viable, neonatal SCNT calves and among SCNT individuals. This result implicates mitochondrial-related gene expression in early developmental loss of SCNT embryos. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.
Molecular Reproduction and Development 02/2011; 78(4):263-73. · 2.53 Impact Factor
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ABSTRACT: To assess both quantitative and qualitative differences between the slow- and fast-type muscles, masseter (slow) and semitendinosus (fast) from four Holstein cows were analyzed by two-dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry. The proteome analysis identified 27 spots as 20 proteins in the whole protein fraction extracted with 8 mol/L urea solution, and 16 spots were identified as 11 proteins in the water-soluble protein fraction. Two slow-type myofibrillar proteins (myosin light chain-1 slow-b and myosin light chain-2 slow), and aconitase-2 mitochondria were present at higher levels in the masseter muscle (P < 0.05). Four fast-type myofibrillar proteins (myosin light chain-1 fast, myosin light chain-2 fast, myosin light chain-3 fast and tropomyosin-1), and three enzymes of glycolytic pathway (enolase-3, aldolase-A and triosephosphate isomerase), were present at higher levels in the semitendinosus muscle (P < 0.05). Our proteome analysis showed that the composition of sarcoplasmic proteins as well as myofibrillar proteins was clearly different between slow- and fast-type muscles.
Animal Science Journal 02/2011; 82(1):181-6. · 0.86 Impact Factor
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ABSTRACT: To assess the role of muscle fiber type in beef taste-traits, we analyzed cooked meats from bovine masseter, diaphragm, psoas major, longissimus thoracis, and semitendinosus muscles with an electric taste sensing system (INSENT SA402B). The system is composed of five taste sensors of polymer membranes fixing different lipids. The sensors, CT0, CA0, AAE, C00 and AE1 are designed to respond to the individual tastes of salty, sour, umami, bitter and astringent, respectively. The system found significant differences in the converted outputs of CA0 (cvCA0), C00 (cvC00) and AE1 (cvAE1) among the bovine muscles. The slow-type muscles (masseter and diaphragm) showed lower cvCA0, higher cvC00, and higher cvAE1 than did the fast-type muscles (psoas major, longissimus thoracis, and semitendinosus). Lactic acid content was different among muscle types and was highly related to the cvCA0 output and pH. carbonyl compounds and free fatty acids were higher in the slow-type muscles. Free fatty acids were major components causing the difference in the C00 output among the muscle types. Iron content was also different among the muscle types and related to the cvC00 and cvAE1 outputs. These results suggested that the muscle fiber type affects the beef taste characteristics.
Animal Science Journal 10/2010; 81(5):600-5. · 0.86 Impact Factor
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ABSTRACT: In this study, we examined the contribution of the cathepsins (cathepsin D and crude cathepsins containing cathepsins B and L) to troponin T degradation during postmortem aging. The action of cathepsin D on troponin T was examined at various pHs (pH 4.0-6.5). The degradation of intact troponin T was observed at pH 4.0, but not observed at pH 5.5 and 6.5. As a result of the degradation of troponin T, the 30 kDa fragment was not generated in any pH condition. The action of the crude cathepsins on troponin T was also examined at various pHs (pH 4.0-6.5). The intact troponin T was degraded at pH 4.0 and the 30 kDa fragments were observed. These 30 kDa fragments disappeared during further incubation. On the other hand, at pH 5.5 and 6.5, the intact troponin T was degraded and the 30 kDa fragment was accumulated. These results suggested that the cathepsin D scarcely contributed to the degradation of troponin T during postmortem aging, but crude cathepsins containing cathepsins B and L were partially involved in the degradation of troponin T and the generation of 30 kDa fragments.
Animal Science Journal 08/2010; 81(4):501-5. · 0.86 Impact Factor
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Susumu Muroya,
Kouichi Watanabe,
Shinichiro Hayashi,
Masato Miyake,
Shigeru Konashi,
Youichi Sato,
Manabu Takahashi,
Shigeki Kawahata,
Yoshisato Yoshikawa,
Hisashi Aso, Koichi Chikuni,
Takahiro Yamaguchi
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ABSTRACT: To clarify muscle type-specific effect of myostatin on myogenic regulatory factors (MRFs), we examined mRNA expression of MRFs in five skeletal muscles of normal (NM) and myostatin-deficient double-muscled (DM) adult Japanese Shorthorn cattle by quantitative reverse-transcribed PCR. Among the four MRFs, namely, Myf5, MyoD, myogenin, and MRF4, MyoD expression was different among the muscles of the DM cattle (P < 0.01) but not of the NM cattle. Meanwhile, MyoD expression was significantly elevated only in masseter (MS) muscle in the DM cattle due to the myostatin deficiency (P < 0.05). Myf5 and MRF4 expression in semitendinosus (ST) was higher in the DM than in the NM cattle (P < 0.05). According to analysis of myosin heavy chain (MyHC) isoform expression, more MyHC-2x and -2a and less -slow isoforms were expressed in the longissimus and ST muscles compared to the MS muscle in both cattle (P < 0.05), but no significant difference in MyHC expression was observed between the NM and DM cattle. Taken together, myostatin has influences on Myf5 and MRF4 expression in faster-type muscles and on MyoD expression in slower-type muscles, suggesting a possible muscle type-specific effect of myostatin in skeletal muscle growth and maintenance.
Animal Science Journal 12/2009; 80(6):678-85. · 0.86 Impact Factor
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ABSTRACT: Vascular cell adhesion molecule-1 (VCAM-1), which has several alternatively splicing variants, plays a role in myotube formation. To investigate which form functions in myogenesis, we analyzed VCAM-1 mRNA expression in bovine skeletal muscle cells. We detected the expression of two VCAM-1 splice forms in the muscle tissue and in the primary satellite cell culture. The longer form was predominantly expressed at the muscle and during myotube formation of the cells. The nucleotide sequences of the two forms were determined by cDNA direct sequencing. The sequence data showed that the predominant form in skeletal muscle was a full-length VCAM-1 (VCAM-7D) that consists of seven immunoglobulin-like (Ig) domains, and the minor form was a novel six-domain form that lacks the seventh Ig domain. Compared to this, bovine pulmonary artery endothelial cells also express a variant which lacks domain 7, but VCAM-7D was not detected by RT-PCR in the culture. No VCAM-1 expression was detected in bovine kidney epithelial cell, lymph node epithelial cell, or leukemic B-lymphocyte culture even under stimulation by tumor necrosis factor-α. These data suggest that the splicing of the VCAM-1 gene alternatively varies depending on the cell type where it is expressed, and that VCAM-7D plays a predominant role in myotube formation.
ZOOLOGICAL SCIENCE 08/2009; · 0.95 Impact Factor
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ABSTRACT: To simplify the monitoring of postmortem beef aging, we established a system to detect a troponin T (TnT) peptide fragment in bovine muscle drip (natural exudates) with an original monoclonal antibody. The antibody was raised against a synthetic peptide APPPPAEVPEVHEEVH corresponding to the N-terminal region of bovine fast-type TnT. In a competitive enzyme-linked immunosorbent assay (ELISA), our antibody detected the standard peptide dose-dependently. According to the monitoring examination with a competitive ELISA during 22days postmortem, the concentration of the peptide in both the drip and trichloroacetic acid extracts from the longissimus muscle (n=4) significantly increased in parallel, up to 10nmol/ml and 16.4nmol/g at day 14 postmortem, respectively. These events were accompanied by an increase in the conventional 30kDa fragment in western blot analysis and a decrease in the Warner-Bratzler shear force value of the beef from 5.0 to 2.4N/cm(2). The peptide detection system using drips with the antibody has advantages applicable to a non-destructive, simple, quick, and on-site monitoring method, such as immunochromatography.
Meat Science 05/2009; 83(1):155-60. · 2.28 Impact Factor
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ABSTRACT: Changes in the thiamin contents in three types of porcine muscle and porcine liver during growth were investigated. The muscular thiamin content was lower at the newborn stage than at fetal stage, and increased after the weaning period. The liver thiamin content, however, remained unchanged from the fetal stage to 5 months old. The changes in thiamin contents were different between Landrace and Meishan pigs.
Bioscience Biotechnology and Biochemistry 02/2009; 73(1):177-9. · 1.28 Impact Factor
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ABSTRACT: The full amino acid coding sequences of adrenergic receptor genes beta1, beta2, and beta3 (ADRB1, ADRB2, and ADRB3)were determined for Jinhua, Meishan, Duroc and Landrace pigs. Non-synonymous substitution of Arg458Pro was found in the porcine ADRB1 gene, resulting in a 469 amino acid sequence. Continuous substitutions of Asn29Asp and Glu30Gln were found in the porcine ADRB2 gene, resulting in a 418 amino acid sequence. Additionally, a Lys30 polymorphism of the ADRB2 gene was found in the Jinhua pigs. There were three non-synonymous substitutions of Asn24Thr, Arg264Gln and Asn398Asp on the porcine ADRB3 gene. A thymine insertion in the ADRB3 gene, resulting in a protein with two fewer amino acids, was found in the Jinhua and Meishan pigs. To assess the effect of ADRB polymorphisms on porcine subcutaneous fat layer thickness, we calculated the genetic frequency of the variants in fatty and lean groups, each consisting of 24 pigs that were crossbreds of Duroc and Jinhua pigs. The effect of the ADRB3 gene polymorphism was not evaluated, because there was insufficient variation on the ADRB3 gene in the examined groups. Although Fisher's exact test showed no significant difference in the frequency of ADRB1 and ADBR2 variants between the two groups, the Arg458 variant of ADRB1 was higher (P = 0.11) in the lean group, and pigs in that group had a thinner fat layer than did those with the Pro458 variant. These results imply a possibility of ADRB1 polymorphism as a minor factor in porcine fat layer thickness. The Asp29 variant of ADRB2 was higher in the lean group (P = 0.11), and the Glu30 variant was higher in the fatty group (P = 0.15), but the Asp29 variant was found only in the Chinese pigs. Thus, the effect of ADRB2 polymorphisms was not clear in this study.
Animal Science Journal 08/2008; 79(6):665 - 672. · 0.86 Impact Factor
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ABSTRACT: To investigate changes in myosin light chains (MyLCs) during postmortem aging of the bovine longissimus muscle, we performed two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results of fluorescent differential gel electrophoresis showed that two spots of the myosin regulatory light chain (MyLC2) at pI values of 4.6 and 4.7 shifted toward those at pI values of 4.5 and 4.6, respectively, by 24 h postmortem when rigor mortis was completed. Meanwhile, the MyLC1 and MyLC3 spots did not change during the 14 days postmortem. Phosphoprotein-specific staining of the gels demonstrated that the MyLC2 proteins at pI values of 4.5 and 4.6 were phosphorylated. Furthermore, possible N-terminal region peptides containing one and two phosphoserine residues were detected in each mass spectrum of the MyLC2 spots at pI values of 4.5 and 4.6, respectively. These results demonstrated that MyLC2 became doubly phosphorylated during rigor formation of the bovine longissimus, suggesting involvement of the MyLC2 phosphorylation in the progress of beef rigor mortis. Keywords: Bovine; myosin regulatory light chain (RLC, MyLC2); phosphorylation; rigor mortis; skeletal muscle.
Journal of Agricultural and Food Chemistry 06/2007; 55(10):3998-4004. · 2.82 Impact Factor
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ABSTRACT: 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.
Meat Science 03/2007; 75(3):506-14. · 2.28 Impact Factor
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ABSTRACT: In this study, 10 troponin T isoforms from adult porcine skeletal muscle messenger RNA were clarified. These were eight fast- and two slow-type isoforms. Fast-type isoforms had three and two variable exons in the N-terminal and the C-terminal region respectively. Slow-type isoforms had one variable exon in the N-terminal region.
Bioscience Biotechnology and Biochemistry 04/2006; 70(3):726-8. · 1.28 Impact Factor
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ABSTRACT: The postmortem degradation of troponin T (TnT) in bovine longissimus (LT), diaphragm (DP), and masseter (MS) was analyzed. A 28.3kDa (conventional 30kDa) fragment of fast-type TnT isoforms showed the highest content in both LT and DP, where a 35.4kDa isoform had the highest expression among the other fast isoforms. Meanwhile, a 26.0kDa fragment was found to be the most highly produced among the fast TnT fragments in MS, where the expression of 36.5 and 32.8kDa isoforms was higher than that of 35.4 and 34.8kDa isoforms. Thus, the compositions of both the intact TnT isoform proteins and the postmortem fragments differed among the muscles examined, indicating that each TnT isoform degrades into a specific fragment in each muscle. Among the muscles, the LT muscle showed a high extent of TnT degradation and the highest expression of fast TnT isoforms containing a taste-related peptide sequence.
Meat Science 02/2006; 72(2):245-51. · 2.28 Impact Factor
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ABSTRACT: To investigate the roles played by MyoD in the terminal differentiation of satellite cell-derived myoblasts, the effect of antisense inhibition of MyoD expression was examined in bovine adult myoblast culture, in which inhibition treatment was limited to the terminal differentiation phase. MyoD antisense oligonucleotide DNA (AS-mD) suppressed the formation of multinucleated myotubes in the cell culture. Myotube formation was suppressed even when AS-mD treatment was limited to the period preceding the onset of myotube formation. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that treatment with AS-mD suppressed the expression of myosin heavy chain embryonic isoform and troponin T isoforms at 4 days after the induction of differentiation. AS-mD also suppressed the expression of MRF4, but did not alter the expression of either Myf5 or myogenin, in contrast to previous results using mouse cells possessing MyoD(-/-) genetic background. These findings suggest that MyoD controls myogenesis but not Myf5 or myogenin mRNA expression during the terminal differentiation phase. Furthermore, among the alpha4, alpha5, alpha6, and alpha7 integrins, alpha4, alpha5, and alpha7 integrin expression was suppressed by AS-mD treatment, in parallel with the suppression of myotube formation, which suggests that MyoD controls myotube formation by regulating the expression of alpha4, alpha5, and alpha7 integrins.
Embryologia 10/2005; 47(7):483-92. · 2.21 Impact Factor
