Genotypic analysis of the FAS2-F1279Y (3836T>A) polymorphism conferring high ethyl caprylate productivity in industrial sake yeast strains.

In Saccharomyces cerevisiae, ethyl caprylate is produced by the esterification of caprylic acid, which is synthesized through the action of fatty acid synthase. A recent study reported a yeast mutant with a single nucleotide substitution in the alpha subunit of fatty acid synthase (FAS2) gene (F1279Y; 3836T>A) that produced large amounts of ethyl caprylate. Here, we designed two primer sets (P1/P2 and P3/P4) with mismatches that incorporate restriction sites for the enzymes NdeI and SspI, respectively and developed an easy and rapid polymerase chain reaction-restriction fragment length polymorphism assay to identify yeasts harboring the FAS2-F1279Y mutation associated with high ethyl caprylate productivity.

Genomic and Metabolomic Analyses of a Piezosensitive Mutant of Saccharomyces cerevisiae and Application for Generation of Piezosensitive Niigata-Sake Yeast Strains.

A sparkling-type draft cloudy sake (Japanese rice wine), AWANAMA, was recently developed using high hydrostatic pressure (HHP) treatment as a non-thermal pasteurization method. This prototype sake has a high potential market value, since it retains the fresh taste and flavor similar to draft sake while avoiding over-fermentation. From an economic point of view, a lower pressure level for HHP pasteurization is still required. In this study, we carried out a genome analysis of a pressure-sensitive (piezosensitive) mutant strain, a924E1, which was generated by UV mutagenesis from a laboratory haploid Saccharomyces cerevisiae strain, KA31a. This mutant strain had a deletion of the COX1 gene region in the mitochondrial DNA and had deficient aerobic respiration and mitochondrial functions. A metabolomic analysis revealed restricted flux in the TCA cycle of the strain. The results enabled us to use aerobic respiration deficiency as an indicator for screening a piezosensitive mutant. Thus, we generated piezosensitive mutants from a Niigata-sake yeast strain, S9arg, which produces high levels of ethyl caproate but does not produce urea and is consequently suitable for brewing a high-quality sake. The resultant piezosensitive mutants showed brewing characteristics similar to the S9arg strain. This study provides a screening method for generating a piezosensitive yeast mutant as well as insight on a new way of applying HHP pasteurization.

Seroprevalence of Immunoglobulin G Antibodies Against Mycobacterium avium subsp. paratuberculosis in Dogs Bred in Japan.

In this study, the seroprevalence of immunoglobulin G (IgG) antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) in dogs bred in Japan was evaluated. Ninety-two non-clinical samples were obtained from three institutes and fifty-seven clinical samples were obtained from a veterinary hospital in Japan. Serum titers of total IgG, IgG1 and IgG2 isotype antibodies against MAP were measured using an indirect enzyme-linked immunosorbent assay (ELISA). The IgG antibodies against MAP in non-clinical serum obtained from three institutes was observed to be 2.4%, 20% and 9.0%. Similarly, the IgG1 antibodies titers against MAP were observed to be 7%, 20% and 0%. Lastly, the IgG2 antibodies against MAP were observed to be 7%, 20% and 4.4%. No significance differences in these titers were observed among the three institutes. The IgG, IgG1 and IgG2 antibodies in serum obtained from a veterinary hospital were observed to be 55.3%, 42% and 42%, respectively. Significant differences were found between the non-clinical and clinical samples. The titers in the clinical samples showed a high degree of variance, whereas low variance was found in the non-clinical samples. The IgG antibody levels were thought to be induced following exposure to MAP-contaminated feed. The difference in titers between the clinical and non-clinical samples is likely to be related to the amount of MAP antigen contamination in dog foods.

Correlation between synthesis of α2-macroglobulin as acute phase protein and degree of hepatopathy in rats.

The degree of hepatopathy affecting the synthesis of α2-macroglobulin (α2M) as an acute phase protein in rats was investigated. Hepatopathy was induced in Sprague-Dawley rats by intravenous administration of galactosamine at a dose of 30 mg/kg for 7 days. Inflammation was induced by intramuscular injection of turpentine oil at a dose of 2 mL/kg. Blood was collected before turpentine oil injection and at 24, 48, 72 and 96 h after injection. Serum concentrations of α2M were measured by enzyme-linked immunosorbent assay. Mean values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in rats administered galactosamine were significantly higher than in controls. Mean values of body weight and total protein were significantly lower than in controls. Serum concentrations of α2M in the galactosamine group were significantly lower than in controls. Kinetic parameters, area under the concentration-time curve (AUC0-96) and maximum serum concentration (Cmax), were significantly lower than in controls. The cut-off value for detecting the effects on synthesis of α2M in liver was 46.9 mgˑh/mL. Seven rats (77.8%) were assessed for decreases in the synthesis of α2M due to hepatopathy. Two rats showed no influence on the synthesis of α2M, despite administration of galactosamine. AST and ALT in these two rats were ≤ 285 and ≤ 174 U/L, respectively. In conclusion, synthesis of α2M in rats is evidently suppressed in the severe stages of hepatopathy.

Erratum: Kuribayashi, T. et al. Seroprevalence of Immunoglobulin E Antibodies against Japanese Cedar Pollen Allergens Cry j1 and Cry j2 in Dogs Bred in Japan. Vet. Sci. 2018, 5, 79.

Due to an error during production, the column title of Figure 2 and Figure 3 are misaligned in the Results section of the published paper [...].

Seroprevalence of Immunoglobulin E Antibodies against Japanese Cedar Pollen Allergens Cry j 1 and Cry j 2 in Dogs Bred in Japan.

Levels of Japanese cedar pollen (Cryptomeria japonica) have increased in Japan and cedar pollinosis caused by Japanese cedar pollen has been reported in dogs. Serum levels of immunoglobulin E (IgE) against Cry j 1 and Cry j 2 in dogs raised in institutes and treated at veterinary hospitals in Japan were thus investigated. A total of 71 sera obtained from two institutes and 87 sera obtained from veterinary hospitals in the Hyogo and Kanagawa Prefectures were analyzed in this study. Serum levels of IgE were measured using the enzyme-linked immunosorbent assay with commercial purified Cry j 1 and Cry j 2. IgE against Cry j 1 and Cry j 2 in sera obtained from the two institutes were detected, despite the dogs being bred in enclosed areas. Moreover, significant differences were noted in the serum levels of IgE against Cry j 1 and Cry j 2 between the two institutes. The number of samples showing Cry j 1 or Cry j 2 levels above the cut-off values was greater in the Kanagawa Prefecture than in the Hyogo Prefecture. In total, 14 dogs showed Cry j 1 and Cry j 2 levels greater than the cut-off values in the Hyogo Prefecture, and only three such dogs were seen in the Kanagawa Prefecture. A significant correlation between serum levels against both allergens was observed (r² = 0.6931, p < 0.0001).

Elimination half-lives of interleukin-6 and cytokine-induced neutrophil chemoattractant-1 synthesized in response to inflammatory stimulation in rats.

The elimination half-lives of in Interleukin-6 (IL-6) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) in rats after inflammatory stimulation were investigated. Five male Sprague-Dawley rats were used (age, 9 weeks; body weight, 235-375 g). Turpentine oil was intramuscularly injected at a dose of 2 mL/kg body weight to induce acute inflammation. Blood was collected pre-injection and 6, 12, 24, 36, 48, 60, 72, 84, and 96 h after the turpentine oil injection. Serum concentrations of IL-6, CINC-1, and α2-macroglobulin (α2M) were measured by enzyme-linked immunosorbent assay. Half-lives were calculated as 0.693/elimination rate constant. The serum concentration of α2M peaked at 48 h after turpentine oil injection. Serum concentrations of IL-6 and CINC-1 increased and peaked at 12 and 24 h, respectively. The terminal elimination half-lives of IL-6 and CINC-1 were 15.5 and 29.9 h, respectively. The half-life of CINC-1 was significantly longer than that of IL-6 (P=0.006). These results suggested that these cytokines synthesized in response to inflammatory stimulation were rapidly eliminated in rats. The serum concentrations of these cytokines should be measured at an early stage if these cytokines will be used as surrogate inflammatory markers instead of acute-phase proteins.

Stable Isotope Analysis of Hydrogen and Oxygen in a Traditional Japanese Alcoholic Beverage, Sake, from Niigata Prefecture in Japan and Other Countries.

Stable hydrogen and oxygen isotopic compositions (δD and δ18O values, respectively) were analyzed for "sake," a traditional Japanese alcohol beverage, to assess these values for the identification of its geographic origin. We collected sake (Junmai-shu; made of only rice, water, and koji) and its source water (i.e., brewing water) from breweries in Niigata Prefecture, Japan, and measured the isotopic compositions of water in these samples. The δD and δ18O values for the sake are well correlated with their respective values for the corresponding brewing water (δD; r = 0.92, δ18O; r = 0.80). Furthermore, based on the δD-δ18O cross plot, sake brewed in Niigata Prefecture is distinguishable from that brewed in countries other than Japan. These results imply that this dual isotope (δD and δ18O) analysis is potentially useful in identifying the geographic origin of sake.

Simple differentiation of sake (Japanese alcoholic beverage) based on trace inorganic components using colorimetric methods.

Several colorimetric methods were combined and used for the discrimination of commercial sake samples, based on their constituent inorganic components. The method was very rapid, simple, and did not require expensive equipment. Further, we showed that this method has potential application in immediate differentiation of sake by the visual inspection.

Seroprevalence of IgG1 and IgG4 class antibodies against Mycobacterium avium subsp. paratuberculosis in Japanese population.

Mycobacterium avium subsp. paratuberculosis (MAP) is the established causative agent of Johne's disease in cattle and other ruminants, and it has also been speculated to be a putative etiological agent of several human autoimmune diseases. It is acknowledged that dairy products deriving from infected animals play a role (could be vehicles) in exposing humans to MAP. MAP could stimulate the human immune system by means of their complex antigen (in the case of lipids, multivalent antigens) and may modulate it, acting as adjuvant molecules such as Freund's complete adjuvant. The immune system might be abnormally stimulated by the constant presence of MAP antigens (for example, in the dairy products), and this might be particularly relevant in genetically predisposed individuals. However, there is limited understanding about the current human exposure to MAP. The present study analyzed the antibody recognition profile of MAP lipophilic antigens in a cohort of 126 healthy Japanese. We measured the serum levels of total immunoglobulin G (IgG) and subclasses targeting MAP surface antigens through ethanol vortex indirect enzyme-linked immunosorbent assay (EVELISA) by using serum absorbed with Mycobacterium phlei. Elevated IgG (especially IgG1 and IgG4) responses were observed in 14% of the sera. To assess the specificity of EVELISA, the same samples were analyzed by means of a commercially available Johnelisa II kit. It was noteworthy that a high degree of correlation was observed when comparing the two methodologies (rs=0.7, p<0.0001). Moreover, in order to investigate the specificity of the binding, inhibition assay experiments were carried out also searching for antibodies against Bacillus Calmette-Guérin antigens, but no cross-reaction was observed. The result obtained represents the first evidence implying that the Japanese population is exposed to MAP, and additionally the existence of a foodborne chain of exposure that transmits MAP antigens to humans.

Elimination Half-Lives of Acute Phase Proteins in Rats and Beagle Dogs During Acute Inflammation.

The half-lives of typical acute phase proteins in rats and beagle dogs during acute inflammation were investigated. Acute inflammation was induced by injection of turpentine oil in rats and administration of indomethacin in beagle dogs. Serum concentrations of α2-macroglobulin (α2M) and C-reactive protein (CRP) were measured by enzyme-linked immunosorbent assay and α1-acid glycoprotein (AAG) was measured by single radial immunodiffusion. Half-life was calculated as 0.693/elimination rate constant (K). The mean half-lives in the terminal elimination phase of α2M and AAG were 68.1 and 164.8 h, respectively. The half-life of AAG was significantly longer than that of α2M. Mean half-lives in the terminal elimination phase of CRP and AAG were 161.9 and 304.4 h, respectively. The half-life of AAG was significantly longer than that of CRP in beagle dogs. No significant differences in the half-life of AAG were observed between rats and beagle dogs. Furthermore, serum concentrations in the terminal elimination phase could be simulated with the K data acquired in this study.

Inhibition of verotoxin (VT) 2 absorption into systemic blood from intestine by repeated administration of bovine immune colostral antibody against VT2 in mice.

BACKGROUND/PURPOSE: Whether absorption of verotoxin (VT) 2 from the intestine in mice is inhibited by administration bovine immune colostral antibody against VT2 was investigated. METHODS: Three-week-old mice were administered VT2 solution at 477.8 ng/mL or 955.6 ng/mL, and bovine immune colostral antibody against VT2 was then administered three times. Whey without antibody against VT2 was administered to control mice. Serum levels of VT2 were measured by fluorescence enzyme immunoassay. RESULTS: Serum levels of VT2 in mice administered VT2 solution at 477.8 ng/mL and bovine immune colostral antibody against VT2 scarcely changed. By contrast, serum levels of VT2 in control mice increased and peaked 12 hours after administration. Peak values were 15.4 ± 5.04 ng/mL. Furthermore, serum levels of VT2 at 12 hours and 16 hours in control mice were significantly higher than in mice administered bovine colostral antibody against VT2. Serum levels of VT2 in mice administered antibody at 955.6 ng/mL showed no significant differences between repeated administration of bovine immune colostral antibody and controls. CONCLUSION: These results suggest that absorption of VT2 from the intestine was inhibited by repeated administration of bovine immune colostral antibody against VT2 at early stages of Escherichia coli O157:H7 infection, whereas VT2 in the intestine remained at low levels.

Differentiation of industrial sake yeast strains by a loop-mediated isothermal amplification method that targets the PHO3 gene.

We developed a loop-mediated isothermal amplification method that targets the PHO3 gene for discriminating sake yeast strains. Our data indicate that this assay is simple, rapid, and useful to use for differentiation of specific yeasts in sake mash.

New application of indirect fluorescent antibody (IFA) technique using latex particles coupled with verotoxin 2 from Escherichia coli O157:H7 in order to determine colostral antibody titers in immunized dairy cows.

A simple and novel assay method for determining colostral and serum against soluble verotxin 2 (VT2) titers by indirect fluorescent antibody (IFA) assay using latex sensitized with VT2 was devised. The latex particles did not auto-fluoresce, and non specific reactions disappeared after washing with phosphate buffered saline containing 3 M Nacl. The highest titer measured by neutralizing test was observed at 1 day after delivery. The highest titer for each immunoglobulin class measured by enzyme-linked immunosorbent assay (ELISA) or IFA using latex sensitized with VT2 was also observed at 1 day after delivery. The changes in titer measured by each method showed similar patterns. Furthermore, the titers for IgG antibody were higher than those for IgM or IgA antibodies. Thus, the titers of bovine immune colostral antibody and each immunoglobulin class could be measured by IFA using latex sensitized with VT2.

Isolation of a non-urea-producing sake yeast strain carrying a discriminable molecular marker.

In the fermentation industry, the traceability of microorganisms during the process is important to ensure safety and efficacy. Ethyl carbamate, a group-2A carcinogen, is produced from ethanol and urea during the storage of food/alcoholic beverages. We isolated non-urea-producing sake yeast car1 mutants carrying a discriminable molecular marker, and demonstrated, by the use of PCR assays, that these mutants are useful for traceability analysis and identification during the sake brewing process.

Comparison of α2-macroglobulin synthesis by juvenile vs. mature rats after identical inflammatory stimulation.

Synthesis of α2-macoglobulin (α2M) by 3-week-old juvenile rats was compared to that of mature 7- and 11-week-old rats. Serum concentrations of α2M, interleukin (IL)-6- and cytokine-induced neutrophil chemoattractant (CINC)-1 were measured by enzyme-linked immunosorbent assay. The area under the concentration vs. time curve (AUC) for α2M was significantly different among the three groups. The synthesis of α2M increased in an age-dependent manner. No significant difference was observed for the AUC of IL-6, but that of CINC-1 in 3-week-old rats was significantly lower than that in 7- or 11-week-old rats. These results suggest that synthesis of α2M was increased in mature compared to juvenile rats, possibly due to differences in liver function. The maximum concentration of CINC-1 in 3-week-old rats was observed 6 h after turpentine oil injection. The serum concentrations of IL-6 and CINC-1 increased more quickly in juvenile rats than in mature rats after inflammatory stimulation.

Molecular characterization of a lipoxygenase from the basidiomycete mushroom Pleurotus ostreatus.

The full-length cDNA of the gene PoLOX1 encoding a lipoxygenase (LOX) and its corresponding genomic DNA were isolated from the basidiomycete mushroom Pleurotus ostreatus strain H1. The deduced amino acid sequence of PoLOX1 showed similarity to a valencene dioxygenase of Pleurotus sapidus, putative LOX-like proteins from ascomycete, basidiomycete, and deuteromycete fungi, and known LOXs from plants, animals, and bacteria. PoLOX1 also contained the LOX iron-binding catalytic domain in the C-terminal region, but not the polycystin-1, lipoxygenase, alpha-toxin (PLAT) domain, which is usually found in the N-terminal region of eukaryotic LOXs. Genomic sequence analysis revealed that PoLOX1 was interrupted by one intron, and that the promoter region included TATA and CAAT boxes. Southern blot analysis indicated that PoLOX1 is a member of a small gene family comprising highly similar genes. Northern blot analysis revealed that it is transcribed more abundantly in the stipes of the fruit bodies than in the caps.

Determination of antibody titers against soluble antigen by indirect fluorescent antibody (IFA) assay using latex particles coupled with soluble antigen: a preliminary study.

A method for indirect fluorescent antibody (IFA) assay for soluble antigen has been developed using latex particles (latex) as a carrier for soluble antigen. Two types of latex having grain sizes of 1.0 and 6.0 µm were used for IFA assay and were evaluated in this study. Bovine IgG and rabbit anti-bovine IgG antibody were used as soluble antigen and as primary antibody, respectively. Bovine IgG-sensitized latex on slide glass was not washed off after immersion for 45 min. IFA using latex appears to have high specificity, as no reactions between fluorescein isothiocyanate (FITC)-conjugated antibody and protein-sensitized latex were observed, and the latex did not show auto-fluorescence or non-specific reactions. Latex with a grain size of 6.0 µm was appropriate as a carrier of soluble antigen, as the amount of soluble antigen necessary for sensitization was approximately one-eighth that necessary for the small-diameter latex (1.0 µm), and large-diameter latex is easier to distinguish under the fluorescence microscope. IFA assay using soluble antigen-sensitized latex is suitable for more widespread use in the future.

Comparison of efficacies of bovine immune colostral antibody and each immunoglobulin class against verotoxin 2, flagellum and somatic cells of Escherichia coli O157:H7 in mice.

PURPOSE: The efficacy of bovine immune colostral (colostral) antibodies against verotoxin (VT) 2, flagellum and somatic cells of Escherichia coli (E. coli) O157:H7 in mice was determined. METHODS: Three major immunoglobulin (Ig) classes were isolated from the colostral antibody against VT2 by affinity chromatography and were used for estimation. Mice inoculated with VT2 were administered each Ig class from the colostral antibody, colostral antibody (colostral whey containing antibody) or serum antibody against VT2 at 1 hour after VT2 inoculation. RESULTS: All control mice (20/20) died after administration of sterilized saline instead of the colostral antibody. The survival rate was 93.3% (14/15) after administration of S-IgA or IgM antibody, or colostral antibody. Survival rates for IgG antibody and serum antibody administration were 80% (12/15) and 60% (9/15), respectively. Serum concentrations of VT2, which was absorbed from the small intestine in mice after administration of VT2 and colostral antibody, were measured by fluorescence enzyme immunoassay (FEIA). Serum concentrations of VT2 after administration of colostral antibody were lower than those after administration of sterilized saline. Mice inoculated with VT2-producing E. coli 157:H7 were administered anti-flagellum or anti-somatic colostral antibodies. Survival rates for E. coli O157:H7-infected mice administered the anti-flagellum and anti-somatic colostral antibodies were 52.4% (11/21) and 22.2% (4/18), respectively. Furthermore, survival rates increased to 89.5% (17/19) with combined administration of anti-flagellum and anti-VT2 colostral antibodies. CONCLUSION: These results suggest that colostral antibodies against VT2, flagellum and somatic cells are effective against E. coli O157:H7 infection.