Isolation and properties of fecal proteins and fecal alkaline phosphatase from germfree and conventional rats.

Fecal proteins from germfree and conventional rats were isolated. The proteins from the two kinds of feces differed in molecular weight, judging from Sephadex gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The conventional feces contained a greater amount of high-molecular-weight and a lesser amount of low-molecular-weight proteins than did the germfree feces. The fecal proteins of both kinds contained carbohydrates. Both feces contained considerable enzyme activity. The germfree feces contained extremely high activity in alkaline phosphatase and leucine aminopeptidase. Both feces showed the same level of trehalase activity. The conventional feces contained higher levels of activity of protease and acid phosphatase than did the germfree feces. Lactase activity was observed only in the conventional feces. The fecal alkaline phosphatase resembled the intestinal enzyme in response to L-phenylalanine inhibition and urea denaturation. From these results it was inferred that the germfree feces contained some of the intestinal proteins and that the conventional feces contained bacterial proteins in addition to intestinal proteins.     

An endo-(1→6-β-D-glucanase from Mucor hiemalis

An endo-(1→6)-β-D-glucanase (EC 3.2.1), isolated from the culture filtrate of Mucor hiemalis, was purified by ammonium sulphate fractionation and gel filtration. The homogeneity of the enzyme was confirmed by disc electrophoresis. The enzyme had a wide range of temperature and pH stability, high substrate specificity, and an action pattern of the endo-type.     

Studies on the interaction between coxsackievirus A9 and HeLa cells. II. Mode of growth of coxsackievirus A9 in HeLa cell cultures and the effect of sulfated polysaccharide on plaque formation.

For the purpose of clarifying the mechanism of plaque formation in HeLa cell cultures by coxsackievirus A9, which does not show definite CPE in fluid cultures, we investigated the growth pattern of the virus in HeLa cells, comparing plaque (HeLA)-forming and non-plaque (HeLa)-forming viruses. It was revealed that the yield of both viruses per cell was nearly the same, but non- plaque (HeLa)-forming virus was far less efficient in infecting HeLa cells. Dextran sulfate was effective in releasing more virus from cells, when HeLa cell cultures were infected with plaque (HeLa)-forming virus, but not in cultures infected with non-plaque (HeLa)-forming virus. From these experimental results, the mechanism by which plaques are formed in HeLa cell cultures by coxsackievirus A9 was discussed.     

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using γ-³²P ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.     

Mass mortality of the Japanese pearl oyster Pinctada fucata martensii in relation to water temperature, chlorophyll a and phytoplankton composition

Mass mortalities of the Japanese pearl oyster Pinctada fucata martensii have widely occurred in western Japan since 1994. The causes of these mass mortalities are at present not thoroughly understood. In this study, we investigated oyster survival in relation to some environmental factors such as water temperature, concentration of chlorophyll a and density or composition of phytoplankton. The examined mass mortality occurred from September to December 1998, and the color on the adductor muscle of the oysters was red-brown, suggesting an infectious disease. Oysters that became moribund during the experiment lost weight, while the weight of unaffected oysters increased. The cell density of Nitzschia spp., an inedible algae for the oyster, in Uchiumi Bay increased before and during the mass mortality event. From the results of our study, we hypothesize that P. fucata martensii was weakened by starvation because of the dominance of inedible food and then contracted an infectious disease that resulted in mortality.     

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.     

Protoplast culture and plant regeneration of several species in the genus Dianthus

Summary Seventeen cultivars belonging to the genus Dianthus were examined for protoplast isolation, culture and shoot regeneration under the same conditions. These included D. caryophyllus, D. chinensis, D. barbatus, D. plumarius, D. superbus and D. japonicus as well as interspecific hybrid cultivars (D. caryophyllus x D. chinensis and D. chinensis x D. barbatus). In all cultivars, viable protoplasts were isolated at high yields from leaves of axenic shoot cultures and some of these protoplasts divided and formed colonies. However, shoot regeneration frequencies were markedly different among the species. High frequency shoot regeneration was obtained from D. chinensis and interspecific hybrid cultivars, while only low frequency or no shoot regeneration was obtained from other species.Abbreviations MS Murashige and Skoog (1962) - FW fresh weight - MES 2-N-morpholinoethane sulfonic acid - FDA fluoroscein diacetate - NAA 1-naphthaleneacetic acid     

Ancient Mitochondrial DNA Reveals the Origin of Sus scrofa from Rebun Island, Japan

The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north–northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences. Received: 18 April 2000 / Accepted: 24 November 2000