Single-molecule PCR using water-in-oil emulsion

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.     

New bulky chiral Lewis acid catalyst: 3,3'-di(2-mesitylethynyl)binaphthol-titanium(IV) complex

A new chiral Lewis acid catalyst 9 was prepared in situ from a 1:2 molar mixture of (R)-3,3'-di(2-mesitylethynyl)binaphthol (6) and titanium(IV) isopropoxide at ambient temperature. The 3- and 3'-substituents on 6 were effective for preventing undesired aggregation between Ti(IV) complexes and increasing the enantioselectivity (up to 82% ee) in the Diels-Alder reaction of methacrolein with cyclopentadiene.     

Stimulatory effect of a dietary casein phosphopeptide preparation on the mucosal IgA response of mice to orally ingested lipopolysaccharide from Salmonella typhimurium

The effect on immunoglobulin production of a commercially available casein phosphopeptide preparation (CPP-III) consisting mainly of bovine alpha s2-casein (1-32) and beta-casein (1-28) in mice that had orally ingested lipopolysaccharide (LPS) from Salmonella typhimurium was investigated. No significant difference in body weight gain was observed between the mice fed on the CPP-III-added diet and those fed on the control diet. The mice fed on the CPP-III-added diet exhibited similar serum and intestinal IgG, IgM, and IgE responses towards LPS to those fed on the control diet. In contrast, fecal and intestinal anti-LPS IgA and total IgA in mice fed on the CPP-III-added diet were significantly higher than in those fed on the control diet. Spleen cells from mice fed on the CPP-III-added diet produced larger amounts of IgA, IL-5, and IL-6 than cells from mice fed on the control diet. These results suggest that dietary casein phosphopeptide may protect a host from invasion of the intestinal mucosa by food-born pathogenic microorganisms.     

Expression of placental lactogen and cytokeratin in bovine placental binucleate cells in culture

Binucleate cells are present in ruminant placenta and play an endocrine role in the production of many hormones during pregnancy. We isolated and cultured binucleate cells from bovine placenta at middle to late gestation and characterized these cells using immunofluorescence techniques. Enriched preparations of binucleate cells were obtained using Percoll density gradient centrifugation following collagenase digestion. Binucleate cells in culture preferentially attached to collagen-coated dishes rather than to noncoated plastic dishes. The cells gradually extended their edges on collagen substrata, and finally assumed a flattened morphology. Antibodies to placental lactogen (PL) and pregnancy-associated glycoprotein-1 (PAG-1) specifically stained the majority of round binucleate cells, but not the flat cells. We found that PL-positive binucleate cells were consistently devoid of cytokeratin. In contrast, cytokeratin was expressed in PL-negative binucleate cells as well as mononuclear epithelial cells. Furthermore, the PL-negative flat binucleate cells also developed intense cytokeratin networks in the cytoplasm. These results indicate that cytokeratin expression is inversely proportionate to that of PL in cultured binucleate cells. We conclude that downregulation of cytokeratin in binucleate cells is a function of the state of cellular differentiation.     

Changes in cell volume of bacteria and heterotrophic nanoflagellates in a hypereutrophic pond

Changes in cell volume of planktonic bacteria and heterotrophic nanoflagellates (HNF) were examined in a hypereutrophic pond from April to October, 1997. There were marked changes in the abundance of bacteria, HNF and ciliates and in protistan bacterivory during this period. The cell volume of free-living bacteria (0.121 ± 0.031 m³, mean ± SD) was large relative to that reported in the literature. The cell volumes of HNF was 71.1 ± 24.8 m³. Both cell volumes did not follow a seasonal trend. The dominant size class of bacteria was seasonally variable, whereas density of filamentous bacteria was relatively high between August and September. Biomass of filamentous bacteria accounted for up to 33.6% of total bacterial biomass. A correlation analysis for cell volume of bacteria and HNF, density of filamentous bacteria and some microbial variates was performed. The positive correlations detected (p<0.05) were between density of bacteria and cell volume of HNF, and between density of filamentous bacteria and cell volume of HNF.     

The presence of a glycosyl phosphatidylinositol-anchored alpha-mannosidase in boar sperm

alpha-Mannosidase and beta-galactosidase were released from boar sperm into the medium by treatment with calcium ionophore A23187 or by 0.2% Brij-35/2% acetic acid. About half as much alpha-mannosidase activity as that in the acid extract was recovered by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the liberation rate of beta-galactosidase treated with PI-PLC was low. These results suggest that some alpha-mannosidase is anchored in the plasma membrane of the acrosomal region by attachment to the lipid phosphatidylinositol and that beta-galactosidase is localized mainly in the acrosome or integrated in the plasma membrane by a spanning stretch of hydrophobic peptides. beta-Galactosidase, which is present as an oligomers in the acid extract of sperm, dissociated into monomers under weakly alkaline conditions; under acidic conditions, the monomers associated again. No pH-sensitive association-dissociation of alpha-mannosidase was observed.     

Structure-activity relationships of trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenems. Part 2: J-111,225, J-114,870, J-114,871 and related compounds

Through further derivatization of J-111,347 (1a), a trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenem, undesired epileptogenicity in a rat intracerebroventricular assay (200 microg/rat) could be eliminated to afford J-111,225 (2a), J-114,870 (3a) and J-114,871 (3b) which preserved comparable broad antimicrobial activity.     

Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir

Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm(S)) to its mutant phenotype (Sm(R)). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.