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.     

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.     

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.     

Characterization of the cells in the repair tissue of full-thickness articular cartilage defects

 It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue, cells of which are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair cells biochemically, full-thickness defects were created in rabbit knee joints and the repair tissues taken at 3, 6, and 12 weeks after surgery. The repair cells were cultured and examined biochemically to investigate the effects of four exogenous growth factors with regard to the metabolism of type II collagen and proteoglycans. A significant increase of carboxy-terminal type II procollagen peptide production was observed in the conditional medium of the repair cells, especially taken at 6 weeks after surgery, in the presence of each growth factor. Glycosaminoglycan content was also increased and proteoglycan synthesis stimulated. The repair cells taken at the early stage of the repair process could originally have more activity of type II collagen synthesis, and the growth factors used could enhance the differentiation of the repair cells in vitro. Accepted: 3 November 1997     

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     

Interaction of syntaxin with alpha-fodrin, a major component of the submembranous cytoskeleton

The soluble N-ethyl maleimide-sensitive factor attachment protein receptor machinery is involved in membrane docking and fusion. In this machinery, the syntaxin family is a central coordinator and participates in multiple protein-protein interactions. In this study we have shown that alpha-fodrin, nonerythroid spectrin, is a new binding partner of the syntaxin family. alpha-Fodrin bound to syntaxin-1a, -3, and -4, all of which are localized on the plasma membrane. Syntaxin-3 interacted with alpha-fodrin in dose-dependent and saturable manners but not with alpha-spectrin, erythroid spectrin. Syntaxin-3 interacted with alpha-fodrin through its C-terminal coiled-coil region. Binding of Munc18 or SNAP-25 to syntaxin-1a inhibited the interaction of alpha-fodrin with syntaxin-1a. Available evidence indicates that alpha-fodrin is implicated in exocytosis, but a precise mode of action of alpha-fodrin in exocytosis remains unclear. Our results suggest that alpha-fodrin regulates exocytosis through the interaction with members of the syntaxin family.