MicroRNA-33b downregulates the differentiation and development of porcine preadipocytes.

Sterol regulatory element binding transcription factor (SREBF) is a key transcription regulator for lipid homeostasis. MicroRNA-33b (miR-33b) is embedded in intron 16 of porcine SREBF1 and is conserved among most mammals. Here, we investigated the effect of miR-33b on adipocyte differentiation and development in porcine subcutaneous pre-adipocytes (PSPA). PSPA were transiently transfected with miR-33b, and adipose differentiation was then induced. Delayed adipose differentiation and decreased lipid accumulation were observed in miR-33b-transfected PSPA. Computational predictions suggested that miR-33b may target early B cell factor 1 (EBF1), an adipocyte activator of lipogenesis regulators such as CCAAT-enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). Both gene and protein expression of EBF1 were downregulated in miR-33b-transfected PSPA, followed by considerable decreases in the expression of C/EBPα and PPARγ and their downstream lipogenic genes. However, miR-33b transfection did not markedly affect mRNA and protein expression of SREBF1. We also investigated differences in the expression of miR-33b and lipogenic genes in subcutaneous fat tissues between 5-month-old crossbred gilts derived from Landrace (lean-type) and Meishan (fatty-type) boars. Landrace-derived crossbred gilts expressed more miR-33b and less lipogenic genes than did gilts derived from Meishan. Our results suggest that miR-33b affected the differentiation and development of PSPA by attenuating the lipogenic gene expression cascade through EBF1 to C/EBPα and PPARγ. The differential expression of miR-33b observed in crossbred gilts may in part account for differences in lipogenic gene expression and the fat:lean ratio between pig breeds.

Effects of Cooking End-point Temperature and Muscle Part on Sensory 'Hardness' and 'Chewiness' Assessed Using Scales Presented in ISO11036:1994.

Texture and 'tenderness' in particular, is an important sensory characteristic for consumers' satisfaction of beef. Objective and detailed sensory measurements of beef texture have been needed for the evaluation and management of beef quality. This study aimed to apply the sensory scales defined in ISO11036:1994 to evaluate the texture of beef. Longissimus and Semitendinosus muscles of three Holstein steers cooked to end-point temperatures of 60°C and 72°C were subjected to sensory analyses by a sensory panel with expertise regarding the ISO11036 scales. For the sensory analysis, standard scales of 'chewiness' (9-points) and 'hardness' (7-points) were presented to the sensory panel with reference materials defined in ISO11036. As a result, both 'chewiness' and 'hardness' assessed according to the ISO11036 scales increased by increasing the cooking end-point temperature, and were different between Longissimus and Semitendinosus muscles. The sensory results were in good agreement with instrumental texture measurements. However, both texture ratings in this study were in a narrower range than the full ISO scales. For beef texture, ISO11036 scales for 'chewiness' and 'hardness' are useful for basic studies, but some alterations are needed for practical evaluation of muscle foods.

Radiocesium distribution in the tissues of Japanese Black beef heifers fed fallout-contaminated roughage due to the Fukushima Daiichi Nuclear Power Station accident.

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.

Comparative proteomic analysis of liver mitochondrial proteins derived from cloned adult pigs reconstructed with Meishan pig fibroblast cells and European pig enucleated oocytes.

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.

Comparison of liver mitochondrial proteins derived from newborn cloned calves and from cloned adult cattle by two-dimensional differential gel electrophoresis.

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.

Changes in thiamin contents in porcine muscles and liver during growth.

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.

A non-destructive method to monitor changes in a troponin T peptide in beef drip with a monoclonal antibody.

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.

Double phosphorylation of the myosin regulatory light chain during rigor mortis of bovine Longissimus muscle.

UNLABELLED: 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.

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.

Qualitative and Quantitative Comparisons of Texture Characteristics between Broiler and Jidori-niku, Japanese Indigenous Chicken Meat, Assessed by a Trained Panel.

The texture of jidori-niku (Japanese indigenous native chicken meat) was characterized and compared with those of Chunky broiler chicken meat. Experiment 1: A qualitative sensory test using jidori-niku and broiler breast (pectoralis major, PM), thigh (biceps femoris, BF) and sasami (deep pectoral) meat cooked to the end-point temperature 75°C by steam-heating was administered to a trained sensory panel (n=16-17) for the selection of descriptive texture items from ISO5492 texture words. By the correspondence analysis, the characteristics of 'chewiness,' 'hardness' and 'springiness' were found to be different between jidori-niku and broiler: they likely characterize jidori-niku texture. Experiment 2: Texture characteristics in the three above-mentioned muscles in jidori-niku and broiler were compared quantitatively using the three above-mentioned texture items by the trained sensory panel. Sensory chewiness and hardness were the highest in the broiler PM and second highest in the jidori-niku BF, whereas sensory springiness was the highest in the jidori-niku BF. These results suggest that jidori-niku-like texture was characterized as a springy texture as compared to broiler meat.

Difference in postmortem degradation pattern among troponin T isoforms expressed in bovine longissimus, diaphragm, and masseter muscles.

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.

Simultaneous imaging of fat crystallinity and crystal polymorphic types by Raman microspectroscopy.

The crystalline states of fats, i.e., the crystallinity and crystal polymorphic types, strongly influence their physical properties in fat-based foods. Imaging of fat crystalline states has thus been a subject of abiding interest, but conventional techniques cannot image crystallinity and polymorphic types all at once. This article demonstrates a new technique using Raman microspectroscopy for simultaneously imaging the crystallinity and polymorphic types of fats. The crystallinity and ß' crystal polymorph, which contribute to the hardness of fat-based food products, were quantitatively visualized in a model fat (porcine adipose tissue) by analyzing several key Raman bands. The emergence of the ß crystal polymorph, which generally results in food product deterioration, was successfully imaged by analyzing the whole fingerprint regions of Raman spectra using multivariate curve resolution alternating least squares analysis. The results demonstrate that the crystalline states of fats can be nondestructively visualized and analyzed at the molecular level, in situ, without laborious sample pretreatments.

Mechanism of production of troponin T fragments during postmortem aging of porcine muscle.

Troponin T (TnT) is one of the myofibrillar proteins that is easily degraded during postmortem aging of pork. In this study, we determined the N-terminal amino acid sequences of TnT degradation fragments produced during postmortem aging and by m-calpain hydrolysis. The N-terminal amino acid sequences of TnT fragments produced during postmortem aging were EVHEPEEKPRPKLTAP, EKPRPKLTAPKIPEG, and APKIPEGEKVDF. On the other hand, the N-terminal amino acid sequences of TnT fragments produced by the action of m-calpain were APPPPAEV, EVHEPEEK, and APK. These sequences of degradation fragments could be mapped on fast type TnT isoform 2. The peptide bonds of His37-Glu38 and Thr51-Ala52 in fTnT2 were cleaved during postmortem aging as well as by the calpain hydrolysis; therefore, calpain was concluded to have an important role in TnT degradation during postmortem aging. It was also found that the sourness-suppressing peptide APPPPAEVHEVHEEVH (Okumura et al. Biosci. Biotechnol. Biochem. 2004, 68, 1657-1662) derived from TnT degradation could be produced by the action of calpains on Glu21-Ala22 and His37-Glu38 sites.

The importance of subfragment 2 and C-terminus of myosin heavy chain for thick filament assembly in skeletal muscle cells.

In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 (S2) + light meromyosin (LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C-terminus on thick filament assembly. C-terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C-terminus of LMM.

Absence of the functional Myosin heavy chain 2b isoform in equine skeletal muscles.

Nucleotide sequences which included the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from equine skeletal muscles. The deduced amino acid sequences were 1937, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. No MyHC-2b isoform was amplified from the equine muscle cDNA except for one pseudogene fragment. One nucleotide was inserted in the coding region of the equine pseudogene product, a minute amount of which was expressed in the skeletal muscle. The 596 bp sequence of the equine MyHC pseudogene was categorized into the MyHC-2b genes on the phylogenetic tree of the mammalian MyHC genes. These results suggest that an ancestral MyHC-2b gene had lost its function and changed to a pseudogene during the course of horse history. The MyHC genes in some ungulates were analyzed through the PCR amplifications using the MyHC isoform-specific primers to confirm the presence of the MyHC-2b and -2x genes. The exon coding the 3' untranslated region of the MyHC-2x was successfully amplified from the all ungulates examined; however, that of the MyHC-2b gene was amplified only from horses, pigs and lesser mouse deer. The PCR analyses from rhinoceros, sika deer, moose, giraffes, water buffalo, bovine, Japanese serow and sheep genes implied the absence of the MyHC-2b-specific sequence in their genomes. These results suggest that the MyHC-2b gene independently lost its function in some ungulate species.

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

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.

A novel repeated sequence located on the bovine Y chromosome: its application to rapid and precise embryo sexing by PCR.

A novel repeated sequence specific to male cattle was identified and named S4. S4 is a highly repetitive sequence and is a 1.5 kb repeating unit that contains various internal repeated sequences. FISH analysis showed that S4 is localized on the whole long arm and the proximal region of the short arm of the Y chromosome. We found that a PCR primer set for S4 amplified a male-specific 178 bp product in addition to a 145 bp product common to both male and female cells. Although the origin of the 145 bp product is unknown, it acts as a positive internal control in practical embryo sexing. Due to the high copy number of S4, PCR required only 0.5 pg purified DNA for accurate amplification. This made it possible to reduce the amount of biopsy sample required for embryo sexing and thus result in less damage to embryos manipulated. These studies indicate that embryo sexing based on the S4 sequence is accurate and sensitive.

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).

CE-TOF MS-based metabolomic profiling revealed characteristic metabolic pathways in postmortem porcine fast and slow type muscles.

To determine key compounds and metabolic pathways associated with meat quality, we profiled metabolites in postmortem porcine longissimus lumborum (LL) and vastus intermedius (VI) muscles with different aging times by global metabolomics using capillary electrophoresis-time of flight mass spectrometry. Loading analyses of the principal component analysis showed that hydrophilic amino acids and ß-alanine-related compounds contributed to the muscle type positively and negatively, respectively, whereas glycolytic and ATP degradation products contributed to aging time. At 168h postmortem, LL samples were characterized by abundance of combinations of amino acids, dipeptides, and glycolytic products, whereas the VI samples were characterized by abundance of both sulfur-containing compounds and amino acids. The AMP and inosine contents in the VI were approx. 10 times higher than those in the LL at 4h postmortem, suggesting different rates of inosine 5'-monophosphate (IMP) accumulation by adenylate kinase 7 and 5'-nucleotidase, and subsequent different production levels of IMP and hypoxanthine between these two porcine muscles.

Characterization and classification of Japanese consumer perceptions for beef tenderness using descriptive texture characteristics assessed by a trained sensory panel.

Meat tenderness is an important characteristic in terms of consumer preference and satisfaction. However, each consumer may have his/her own criteria to judge meat tenderness, because consumers are neither selected nor trained like an expert sensory panel. This study aimed to characterize consumer tenderness using descriptive texture profiles such as chewiness and hardness assessed by a trained panel. Longissimus muscles cooked at four different end-point temperatures were subjected to a trained sensory panel (n=18) and consumer (n=107) tenderness tests. Multiple regression analysis showed that consumer tenderness was characterized as 'low-chewiness and low hardness texture.' Subsequently, consumers were divided into two groups by cluster analysis according to tenderness perceptions in each participant, and the two groups were characterized as 'tenderness is mainly low-chewiness' and 'tenderness is mainly low-hardness' for tenderness perception, respectively. These results demonstrate objective characteristics and variability of consumer meat tenderness, and provide new information regarding the evaluation and management of meat tenderness for meat manufacturers.