Ultra-rapid real-time polymerase chain reaction assay for genotyping of the NT5E gene in Japanese black beef using a mobile PCR device, PCR 1100.

The concentration of inosine 5'-monophosphate (IMP) in beef is an important factor contributing to beef palatability. A previous study suggested that single nucleotide polymorphisms (SNPs) in the ecto-5'-nucleotidase (NT5E) gene strongly affect the concentration of IMP under postmortem conditions by regulating NT5E enzymatic activity in beef. Genotyping of the NT5E gene is performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or real-time PCR assay. However, these conventional laboratory assays require large installed instruments. They also involve complicated procedures and are time-consuming. Here, the PCR primers and probes for the NT5E gene (rs42508588 SNP) were designed and synthesized, and we examined the rapid genotyping of the NT5E gene using a PicoGene PCR 1100 mobile PCR device. The results showed that this system enabled rapid amplification of each allele at approximately 19.4 s per cycle, with a total run time of 13 min 36 s. This device is portable and does not require a power supply, which facilitates its use not only in specific laboratories but also in meat production farms and distribution stages of beef.

Estimates of genetic parameters for adenosine triphosphate-related compounds at different aging periods and NT5E genotypes in Japanese Black beef.

Previous studies on Japanese Black beef showed that single nucleotide polymorphisms in the ecto-5'-nucleotidase (NT5E) gene affected the degradation rate of inosine 5'-monophosphate (IMP), which has contributed to the umami taste, especially between Postmortem Days 4 and 7. Therefore, this study estimated the genetic parameters of IMP and its degradation products on Postmortem Days 4 and 7 using the model with or without the NT5E genotype. The heritability estimates of IMP on Postmortem Days 4 and 7 were moderate by the model without the NT5E genotype (0.44 and 0.32, respectively). When the NT5E genotype was included in the model, the heritability of IMP on Postmortem Day 4 did not change, whereas that on Day 7 decreased from 0.32 to 0.08. The genetic correlation of IMP between Postmortem Days 4 and 7 was highly positive using the model with the NT5E genotype. Regarding the estimated breeding values (EBVs) of IMP, the ranking of EBVs among NT5E genotypes was not changed between Postmortem Days 4 and 7, when the model with the NT5E genotype was used. The study suggested that the model including NT5E genotype would allow for appropriate genetic parameter estimation and breeding value evaluation in adenosine triphosphate-related compounds (ATPRCs) under different aging periods.

MEATabolomics: Muscle and Meat Metabolomics in Domestic Animals.

In the past decades, metabolomics has been used to comprehensively understand a variety of food materials for improvement and assessment of food quality. Farm animal skeletal muscles and meat are one of the major targets of metabolomics for the characterization of meat and the exploration of biomarkers in the production system. For identification of potential biomarkers to control meat quality, studies of animal muscles and meat with metabolomics (MEATabolomics) has been conducted in combination with analyses of meat quality traits, focusing on specific factors associated with animal genetic background and sensory scores, or conditions in feeding system and treatments of meat in the processes such as postmortem storage, processing, and hygiene control. Currently, most of MEATabolomics approaches combine separation techniques (gas or liquid chromatography, and capillary electrophoresis)-mass spectrometry (MS) or nuclear magnetic resonance (NMR) approaches with the downstream multivariate analyses, depending on the polarity and/or hydrophobicity of the targeted metabolites. Studies employing these approaches provide useful information to monitor meat quality traits efficiently and to understand the genetic background and production system of animals behind the meat quality. MEATabolomics is expected to improve the knowledge and methodologies in animal breeding and feeding, meat storage and processing, and prediction of meat quality.

The NT5E gene variant strongly affects the degradation rate of inosine 5'-monophosphate under postmortem conditions in Japanese Black beef.

Inosine 5'-monophosphate (IMP) contributes to the umami taste in Japanese Black beef. In a previous study, it was suggested that single nucleotide polymorphisms (SNPs) in the ecto-5'-nucleotidase (NT5E) gene affect the concentration of IMP under postmortem conditions by regulating NT5E enzymatic activity in Japanese Black beef. The present study showed that the degradation rate of IMP in Japanese Black beef was significantly different among NT5E genotypes in the middle stage of postmortem, indicating a significant difference in the concentrations of IMP and its degradation products among NT5E genotypes. In addition, no significant difference was observed among NT5E genotypes in the concentrations of IMP precursors or other taste-active compounds. These results indicate the significant effect of the interaction between the NT5E genotype and the aging period on the degradation rate of IMP in beef.

NMR-based metabolomics for simultaneously evaluating multiple determinants of primary beef quality in Japanese Black cattle.

Analytical methodologies to comprehensively evaluate beef quality are increasingly needed to accelerate improvement in both breeding and post-mortem processing. Consumer palatability towards beef is generally attributed to tenderness, flavor, and/or juiciness. These primary qualities are modified by post-mortem aging and the crude content and fatty acid composition of intramuscular fat. In this study, we report a nuclear magnetic resonance (NMR)-based metabolic profiles of Japanese Black cattle to evaluate the compositional attributes of intramuscular fat and the long-term post-mortem aging. The unsaturation degree of triacylglycerol was estimated by the 1H NMR spectra and was correlated with the content ratio of unsaturated fatty acids (R 2 = 0.944) and the melting point of intramuscular fat (R 2 = 0.871). NMR-detected profiles of water-soluble metabolites revealed overall metabolic change (R 2 = 0.951) and several metabolites (R 2 > 0.818) linearly correlated with long-term aging duration, which can be used to evaluate the aging rate and aging duration of beef. This approach also provided the pH profile during aging, which is related to the water-holding capacity of beef. Thus, NMR-based metabolomics has the potential to evaluate multiple parameters related to the beef qualities of Japanese Black cattle.

Search for an index for the taste of Japanese Black cattle beef by panel testing and chemical composition analysis.

To search for an index for chemical composition related to superior taste in Japanese Black beef, we conducted panel tests and analyzed the chemical composition of seven beef brands. Thirty-five sirloin beefs from five heifers were used in this study, sold under seven beef brands graded as more than A4 on the Japanese Meat Grade scale. The chemical composition analyses assessed both raw and roasted meat, the latter of which was roasted under the same conditions as those used for the panel test. Results of the panel test and chemical composition analyses revealed that fatty acid composition, sugar content, adenosine triphosphage (ATP)-related compounds, amino acid composition and odor composition in the sirloin meat differed among beef brands. Furthermore, the correlations of chemical compositions between roasted and raw meat were significantly high. Sugar content and ATP-related compounds in roasted meat were significantly correlated with the item 'overall evaluation' of the panel test. ATP-related compounds, such as inosinic acid, carnosine and taurine, in roasted and raw meat were correlated significantly with the item 'umami intensity' of the panel test. These results suggest that the composition of these components is important for an index related to the overall evaluation of beef.

Allelic frequencies and association with carcass traits of six genes in local subpopulations of Japanese Black cattle.

Marker-assisted selection (MAS) is expected to accelerate the genetic improvement of Japanese Black cattle. However, verification of the effects of the genes for MAS in different subpopulations is required prior to the application of MAS. In this study, we investigated the allelic frequencies and genotypic effects for carcass traits of six genes, which can be used in MAS, in eight local subpopulations. These genes are SCD, FASN and SREBP1, which are associated with the fatty acid composition of meat, and NCAPG, MC1R and F11, which are associated with carcass weight, coat color and blood coagulation abnormality, respectively. The frequencies of desirable alleles of SCD and FASN were relatively high and that of NCAPG was relatively low, and NCAPG was significantly associated with several carcass traits, including carcass weight. The proportions of genotypic variance explained by NCAPG to phenotypic variance were 4.83 for carcass weight. We thus confirmed that NCAPG is a useful marker for selection of carcass traits in these subpopulations. In addition, we found that the desirable alleles of six genes showed no negative effects on carcass traits. Therefore, selection using these genes to improve target traits should not have negative impacts on carcass traits.

Effects of genetic and environmental factors on muscle glycogen content in Japanese Black cattle.

Monosaccharides such as glucose contribute to the development of meat flavor upon heating via the Maillard reaction; therefore, monosaccharide content is related to beef palatability. Here, we analyzed the effects of genetic and environmental factors on the content of glycogen, one of the precursors of monosaccharides, in the muscles of 958 fattened Japanese Black cattle from Yamagata Prefecture. Analysis of variance showed that muscle glycogen content was affected by the farm and postmortem periods, but not by sex, slaughter age, slaughter month or number of days detained at the slaughter yard. Additionally, consumption of digestible brown rice feed elevated muscle glycogen levels. Glycogen heritability was estimated to be 0.34, and genetic correlations between glycogen and carcass weight (CW) or beef marbling standard (BMS) were weak. The predicted breeding values varied among paternal lines. These results demonstrated that genetic factors might improve muscle glycogen content and therefore beef palatability, but do not influence CW or BMS.

The assessment of genetic diversity within and among the eight subpopulations of Japanese Black cattle using 52 microsatellite markers.

Japanese Black cattle are at risk for genetic homogeneity due to intensive use of a few sires. Therefore, assessment of the actual genetic diversity of this breed is important for future breeding plans. In the present study, we investigated the genetic diversity within and among eight subpopulations of Japanese Black cattle using 52 microsatellite markers. The parameters for genetic diversity of Japanese Black cattle were comparable to those of other cattle breeds, suggesting that the relatively high genetic diversity of the breed. However, upon comparison among the eight subpopulations, the Hyogo subpopulation showed markedly low genetic diversity. The results of the pairwise FST values, phylogenetic network and structure analysis indicated that the Hyogo population has remarkably high level of genetic differentiation from other populations, while Yamagata, Niigata, Hiroshima and Kagawa populations have low levels of genetic differentiation. Furthermore, multidimensional scaling plots indicated that individuals in some subpopulations were separated from individuals in the other subpopulations. We conclude that while the overall genetic diversity of Japanese Black cattle is still maintained at a relatively high level, that of a particular subpopulation is significantly reduced, and therefore the effective population size of the breed needs to be controlled by correct mating strategies.

Extrusion of Na,K-ATPase and transferrin receptor with lipid raft-associated proteins in different populations of exosomes during reticulocyte maturation in dogs.

The present study characterizes canine reticulocyte exosomes. Exosomes are small membrane vesicles involved in membrane remodeling that are released from reticulocytes during the final maturation step of red blood cells. The vesicles collected from reticulocyte culture supernatants by differential centrifugation contained major exosomal proteins including heat shock protein cognate 70 (Hsc70) and transferrin receptors (TfR), consistent with the definition of the exosome. In addition, the Na,K-ATPase alpha-subunit and stomatin, a lipid raft-associated protein, were extruded by the exosome pathway, possibly leading to the absence of these proteins in erythrocytes, while the major protein constituents of erythrocyte membranes, spectrin and band 3 were retained in reticulocytes and not expelled into exosomes. The Na,K-ATPase alpha-subunit, as well as TfR and about half of the stomatin contained in exosomes, was obtained in a detergent-soluble fraction that was distinct from the lipid raft microdomain. Moreover, Na,K-ATPase and a portion of stomatin were distributed differently to Hsc70, TfR, stomatin, and ganglioside GM1 in vesicles separated with sucrose density gradient centrifugation. These results demonstrate that a heterogeneous group of exosomes participates in the loss of Na,K-ATPase and membrane remodeling during reticulocyte maturation in dogs.

Parallel reductions in stomatin and Na,K-ATPase through the exosomal pathway during reticulocyte maturation in dogs: stomatin as a genotypic and phenotypic marker of high K(+) and low K(+) red cells.

Dogs can be divided into two genetic groups (a minor HK phenotype and a major LK phenotype) based on erythrocyte monovalent cation concentrations, which are controlled by the putative hk and lk allelic genes. HK dogs retain Na,K-ATPase in their erythrocytes due to the high activity of the enzyme in their precursor cells, whereas total loss of reticulocyte Na,K-ATPase occurs in LK dogs. Here, we report that the levels of the lipid raft-associated membrane protein stomatin decrease in parallel with those of Na,K-ATPase during reticulocyte maturation due to its extrusion in exosomes. The stomatin content of HK reticulocytes is higher than that of LK reticulocytes, and remains in the erythrocytes at levels compatible with that in human erythrocytes. However, it is almost absent from LK erythrocytes with the lk/lk genotype; similar to the deficiency seen in human red cells with overhydrated stomatocytosis. LK erythrocytes from hk/lk genotype dogs show reduced, but not negligible, levels of stomatin. These results indicate that the erythrocyte stomatin level is a suitable genotypic marker for the HK/LK red cell phenotype, and suggests a functional association between stomatin and Na,K-ATPase. The absence of morphological abnormalities in the erythrocytes of stomatin-deficient LK dogs also confirms that stomatin deficiency and stomatocytic shape change are independent from each other.

The covalent modification of spectrin in red cell membranes by the lipid peroxidation product 4-hydroxy-2-nonenal.

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein-protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that alpha- and beta-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly alpha-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential beta-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE-spectrin adduct formation with a similar preponderance of HNE-beta-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE-spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.

Expression of alpha-hemoglobin stabilizing protein and cellular prion protein in a subclone of murine erythroleukemia cell line MEL.

Alpha-Hemoglobin stabilizing protein (AHSP) functions as the erythroid-specific molecular chaperon for alpha-globin. AHSP gene expression has been reported to be downregulated in hematopoietic tissues of animals suffering from prion diseases though the mechanism remains to be clarified. Herein, we demonstrate that MELhipod8 cells, a subclone of murine erythroleukemia (MEL) cells, have prion protein (PrPc) on the cell surface and have highly inducible expression of the AHSP and alpha- and beta-globin genes, resembling the expression pattern of the PrP and AHSP genes in bipotential erythroid- and megakaryocyte-lineage cells followed by erythroid differentiation in normal erythropoiesis. Moreover, MELhipod8 cells exhibit greater effective erythroid differentiation with a population of hemoglobinized normoblast-like cells than that observed for the parental MEL cells. These findings suggest that MELhipod8 cells could provide a mechanism for downregulation of the AHSP gene in prion diseases.

Identification of genes for two major sialoglycoproteins, glycophorin A and glycophorin C in canine red cell membranes.

Glycophorins are the major sialoglycoproteins in red blood cell membranes, possessing various physiological and pathological roles. We examined membrane glycoproteins in canine red cells and cloned cDNAs for two major glycophorins, glycophorins A (GPA) and C (GPC) from bone marrow cells. Periodic acid-Schiff staining and immunoblotting analyses showed that canine red cell membranes contained several glycoproteins immunoreactive to an anti-bovine GPC antibody, whereas the most abundant sialoglycoproteins, the candidates for GPA, did not react with an anti-human GPA antibody. The amino acid sequences of the extracellular domains of GPA and GPC had no significant homology to those from other mammalian species, including humans, and had O-linked and/or N-linked glycosylation sites. On the other hand, the C-terminal cytoplasmic domain and/or the transmembrane helices of GPA and GPC were conserved among species, indicating some functional significance of those regions in red cell membranes that include dimerization of GPA in the membrane-spanning region, and association of GPC with membrane skeletal proteins through binding with protein 4.1 and p55 in the cytoplasmic domain. These findings provide insights for clinical studies to evaluate the involvement of GPA and GPC in the pathogenesis of red cell diseases.

Identification of heat shock protein 70 in canine reticulocytes and mature erythrocytes.

In the present study, we demonstrated that heat shock protein 70 (Hsp70) was present in both canine reticulocytes and mature erythrocytes, and that the canine Hsp70 in reticulocytes was decreased along with the maturation of the cells into erythrocytes. These results suggest that the Hsp70 in canine reticulocytes might act as a chaperone to remove unnecessary proteins during reticulocyte maturation. We also demonstrated that Hsp70 was present in exosomes from reticulocytes during their maturation in in vitro culture. Furthermore, the concentration of Hsp70 in reticulocyte membranes was increased in proportion to an increase of the protein in exosomes until 48 hours after the incubation of reticulocytes in vitro. At 96 hours of the incubation, however, only a trace amount of Hsp70 was detected in the membrane, while a large amount of the protein was present in the exosomes. These results suggest that Hsp70 in canine reticulocytes might play an important role for exosome formation in reticulocytes, resulting in the maturation of the cells.