Minoru Yamanoue

Effect of ascorbic acid and alpha-tocopherol on storage stability of beef steaks

Fresh beef loin steaks were dipped in solutions of (1) 70% ethyl alcohol (EtOH). (2) 70% EtOH containing 3% l-ascorbic acid (AsA), (3) 70% EtOH containing 0·08% dl-α-tocopherol (Toc) and (4) 70% EtOH containing 3% AsA plus 0·08% Toc for 20 s, then drained for 10 s. Control samples received no dip treatment. After 3, 6, 9 and 13 days of storage at 4°C, the samples were evaluated for MetMb formation, TBA number and viable bacterial count. The beef steaks given AsA treatment maintained an acceptable meat colour for 13 days of storage. The TBA numbers of samples dipped in EtOH containing AsA stored for 13 days were significantly lower than those of controls, and in samples dipped in solutions without AsA. At 9 and 13 days of storage, the AsA plus Toc-treated samples gave lower TBA numbers than the samples given AsA treatment only. Viable bacterial counts were not affected by AsA, Toc or AsA plus Toc treatment. Beef loin steaks dipped in EtOH solution maintained a viable bacterial count of 10(6) after 13 days of storage.

Effect of cooking temperature on the percentage colour formation, nitrite decomposition and sarcoplasmic protein denaturation in processed meat products.

The effect of cooking temperature and time on the percentage colour formation, nitrite decomposition and denaturation of sarcoplasmic proteins in processed meat products was investigated in detail. The colour forming percentage increased with a rise in temperature of heating, especially at 50-60°C (P < 0·05). The percentage nitrite decomposition was promoted by the retention time of cooking rather than by the cooking temperature (P < 0·05). The percentage of sarcoplasmic proteins denatured was enhanced by heating temperature in the range 50-80°C (especially at 50-60°C) (P < 0·05). The relationship between the percentage colour formation and the percentage of sarcoplasmic proteins denatured is discussed. The SDS-PAGE patterns of the heat-treated samples revealed the components of the sarcoplasmic proteins which had been denatured.

The expression of diacylglycerol kinase theta during the organogenesis of mouse embryos.

BackgroundDiacylglycerol kinase (DGK) is a key enzyme that regulates diacylglycerol (DG) turnover and is involved in a variety of physiological functions. The isoform DGKθ has a unique domain structure and is the sole member of type V DGK. To reveal the spatial and temporal expression of DGKθ we performed immunohistochemical staining on paraffin sections of mouse embryos.ResultsAt an early stage of development (E10.5 and 11.5), the expression of DGKθ was prominently detected in the brain, spinal cord, dorsal root ganglion, and limb bud, and was also moderately detected in the bulbus cordis and the primordium of the liver and gut. At later stages (E12.5 and 14.5), DGKθ expression persisted or increased in the neocortex, epithalamus, hypothalamus, medulla oblongata, and pons. DGKθ was also evident in the epidermis, and nearly all epithelia of the oropharyngeal membrane, digestive tract, and bronchea. At prenatal developmental stages (E16.5 and E18.5), the expression pattern of DGKθ was maintained in the central nervous system, intestine, and kidney, but was attenuated in the differentiated epidermis.ConclusionThese results suggest that DGKθ may play important physiological roles not only in the brain, but also in diverse organs and tissues during the embryonic stages.

Both the C1 domain and a basic amino acid cluster at the C-terminus are important for the neurite and branch induction ability of DGKβ.

We previously reported that diacylglycerol kinase β (DGKβ) induces neurites and branches, contributing to higher brain function including emotion and memories. However, the detailed molecular mechanism of DGKβ function remains unknown. Therefore, we constructed various mutants of DGKβ and compared their enzyme activity, intracellular localization, and ability to induce neurites and branching in SH-SY5Y cells. Even when RVH-domain and EF-hand motif were deleted, the mutant showed similar plasma membrane localization and neurite induction compared to wild type (WT), although the kinase activity of the mutant was three times higher than that of WT. In contrast, further deletion of C1 domain reduced the activity to 50% and abolished plasma membrane localization and neurite induction ability. When 34 amino acids were deleted from C-terminus, the mutants completely lost enzyme activity, plasma membrane localization, and the ability to induce neurites. A kinase-negative mutant of DGKβ retained plasma membrane localization and induced significant neurites and branches; however, the rate of induction was weaker than that of WT. Furthermore, C1A and C1B mutants, which have a mutation in a cysteine residue in the C1A or C1B domain, and the RK/E mutant, which has substitutions of arginine and lysine to glutamic acid in a cluster of basic amino acids at the C-terminus, lost their plasma membrane localization and neurite induction ability. These results indicate that in addition to kinase activity, plasma membrane localization via the C1 domain and basic amino acids at the C-terminus were indispensable for neurite induction by DGKβ.

The N-Terminal Sequence of Paratropomyosin Binding Fragments from β-Connectin

In order to clarify the position where paratropomyosin binds to connectin in the A-I junction region of a sarcomere, chicken β-connectin was digested by Staphylococcus aureus V8 protease under denaturing conditions and the digested peptides were electrophoretically separated. Five peptides, 150-kDa, 100-kDa, 70-kDa, and 43-kDa fragments, were simultaneously detected by biotinylated paratropomyosin and an anti-connectin monoclonal antibody. The N-terminal sequence of the 43-kDa fragment was found to be YQFRVYAVNK, similar to the sequence of 7556-7565 amino acids in the I51 fibronectin type 3 domain that was located at the A-I junction region of human cardiac titin/connectin. Therefore, we propose that paratropomyosin binds to the 43-kDa fragment from β-connectin at the A-I junction region in both living muscle and in muscle immediately postmortem, and the N-terminus of the 43-kDa fragment is localized in the I51 domain.

Diacylglycerol Kinase alpha is Involved in the Vitamin E-Induced Amelioration of Diabetic Nephropathy in Mice

Diabetic nephropathy (DN) is one of vascular complications of diabetes and is caused by abnormal protein kinase C activation as a result of increased diacylglycerol (DG) production in diabetic hyperglycaemia. Diacylglycerol kinase (DGK) converts DG into phosphatidic acid. Therefore, it is expected that the activation of DGK would ameliorate DN. Indeed, it has been reported that vitamin E (VtE) ameliorates DN in rat by activating DGK, and we recently reported that VtE specifically activates DGKα isoform in vitro. However, whether DGKα is involved in the VtE-induced amelioration of DN in vivo remains unknown. Therefore, we investigated the VtE-induced amelioration of DN in wild-type (DGKα+/+) and DGKα–deficient (DGKα−/−) mice in which diabetes was induced by streptozocin. Several symptoms of DN were ameliorated by VtE treatment in the DGKα+/+ mice but not in the DGKα−/− mice. Moreover, transmission electron microscopy of glomeruli and immunofluorescent staining of glomerular epithelial cells (podocytes) indicated that VtE ameliorates podocyte pathology and prevents podocyte loss in the DGKα+/+ mice but not in the DGKα−/− mice. We showed that VtE can ameliorate DN in mice and that DGKα is involved in the VtE-induced amelioration of DN in vivo, suggesting that DGKα is an attractive therapeutic target for DN.

Chicken heat shock protein HSPB1 increases and interacts with αB-crystallin in aged skeletal muscle

International trading markets of meat require the animal’s age information to prevent cross-contamination of ineligible meat products. Individual livestock age is either evaluated from physiological features or verified by breeding history. However, it remains impossible to perform age verification on meat when a suspicion of error occurred in the importing country. To investigate an age-related protein in skeletal muscle of livestock, we compared protein expression among chicken pectoralis major of different ages. Results indicated that the level of expression of chicken HSPB1, one of the small heat shock proteins, was increased in aged muscles. On the other hand, other heat shock proteins, heat shock factors, and myosin heavy chain isoform did not change the expression levels in aged chicken muscle. In addition, we identified that αB-crystallin interacted with HSPB1 in aged chicken muscle. These results suggest that HSPB1 protein forms complexes with αB-crystallin in aged chicken muscle and suppose to become the candidate of age-related bio-marker for verifying the age of chicken meat. Graphical abstract Immunohistochemical analysis of HSPB1 in chicken muscle (50 days and 500 days of age). Longitudinal sections show HSPB1 expression depending on the age in the muscle.

Amelioration of diabetic nephropathy by oral administration of d-α-tocopherol and its mechanisms

Abstract Diabetic nephropathy (DN) is a diabetic vascular complication, and abnormal protein kinase C (PKC) activation from increased diacylglycerol (DG) production in diabetic hyperglycemia is one of the causes of DN. Diacylglycerol kinase (DGK) converts DG into phosphatidic acid. In other words, DGK can attenuate PKC activity by reducing the amount of DG. Recently, we reported that intraperitoneally administered d-α-tocopherol (vitamin E, αToc) induces an amelioration of DN in vivo through the activation of DGKα and the prevention of podocyte loss. However, the effect of the oral administration of αToc on DN in mice remains unknown. Here, we evaluated the effect of oral administration of αToc on DN and its molecular mechanism using streptozocin-induced diabetic mice. Consequently, the oral administration of αToc significantly ameliorated the symptoms of DN by preventing the loss of podocytes, and it was revealed that the inhibition of PKCactivity was involved in this amelioration. The assumed mechanisms of the protective effect of d-α-tocopherol on diabetic nephropathy.

Comparative metabolomics of Japanese Black cattle beef and other meats using gas chromatography–mass spectrometry

ABSTRACT Progress in metabolomic analysis now allows the evaluation of food quality. This study aims to identify the metabolites in meat from livestock using a metabolomic approach. Using gas chromatography–mass spectrometry (GC/MS), many metabolites were reproducibly detected in meats, and distinct differences between livestock species (cattle, pigs, and chickens) were indicated. A comparison of metabolites between tissues types (muscle, intramuscular fat, and intermuscular fat) in marbled beef of Japanese Black cattle revealed that most metabolites are abundant in the muscle tissue. Several metabolites (medium-chain fatty acids, etc.) involved in triacylglycerol synthesis were uniquely detected in fat tissue. Additionally, the results of multivariate analysis suggest that GC/MS analysis of metabolites can distinguish between cattle breeds. These results provide useful information for the analysis of meat quality using GC/MS-based metabolomic analysis. ABBREVIATIONS: GC/MS: gas chromatography-mass spectrometry; NMR: nuclear magnetic resonance; MS: mass spectrometry; IS: 2-isopropylmalic acid; MSTFA: N-Methyl-N-trimethylsilyltrifluoroacetamide; CV: coefficient of variation; TBS: Tris-buffered saline; MHC: myosin fast type; PCA: principal component analysis; OPLS-DA: orthogonal partial least-squares discriminant analysis; O2PLS: two-way orthogonal partial least-squares Graphical Abstract Comparative metabolomics of Japanese Black cattle beef and other meats using gas chromatography–mass spectrometry.

Structural differences between myofibrillar protein, paratropomyosin, and tropomyosin as revealed by high‐performance liquid chromatography

Paratropomyosin (PTM) composes myofibril functions to weaken the rigor linkages formed between actin and myosin during postmortem aging of muscles. PTM has the similar physico-chemical properties as tropomyosin (TM) that is a regulatory protein of myofibrils. So far, it is unclear whether PTM is definitely different from TM, because the primary structure of PTM has not been determined yet. The aim of this study was to clarify structural difference of PTM from TM. PTM was prepared by column chromatography immediately after slaughter from broiler breast muscle, and purified by high-performance liquid chromatography (HPLC). Purified PTM was successfully separated from TM, and the recovered PTM molecule was reduced with dithiothreitol to separate again by HPLC. Two subunits were obtained and peptides from each digested subunit by V8 protease were recovered by HPLC, and then amino acid sequences of the peptides were analyzed by protein sequencing. As a result, some amino acid residues were replaced from that of TMα1 isoform which is the major isoform of TM, and also was different between the two subunits. Therefore, it is concluded that PTM clearly differs from TM and it is suggested that functional difference in PTM from TM is attributed to amino acid replacements in subunits composing PTM.