Apoptotic cell death in the fission yeast Schizosaccharomyces pombe induced by valproic acid and its extreme susceptibility to pH change.

Schizosaccharomyces pombe treated with valproic acid died with apoptotic markers such as DNA fragmentation, loss of a mitochondrial electrochemical gradient and chromatin condensation, independently of metacaspase, a yeast homolog of metazoan caspase. Sensitivity to valproic acid was strongly dependent on growth phase. Cells in a later growth phase were much more sensitive to valproic acid than those in an earlier one. Altering the pH of the medium with HCl and with NaOH also caused remarkable changes in sensitivity. Cells in an acidic medium were more sensitive to valproic acid. This pH-dependent change in sensitivity did not require de novo protein synthesis, and a change in pH 60 min after the administration of valproic acid affected sensitivity. These results suggest that the intracellular cell death process was susceptible to extracellular pH. Although a sir2 mutant of Saccharomyces cerevisiae has been reported to be resistant to valproic acid, mutations in sir2 did not affect the sensitivity to valproic acid of S. pombe.

Polyphasic characterization of a PCP-to-phenol dechlorinating microbial community enriched from paddy soil.

Dechlorination of PCP has been observed previously under anaerobic condition in paddy soil. However, there is poor information about the dechlorination pathway of PCP and the microbial community associated with the PCP dechlorination in paddy soil. In this study, an anaerobic microbial community dechlorinating PCP was enriched by serial transfers from a paddy soil using a medium containing PCP, lactate and the steam-sterilized paddy soil. The enriched microbial community dechlorinated PCP completely to phenol under the anaerobic condition by a dechlorinating pathway as follows; PCP-->2,3,4,5-tetrachlorophenol-->3,4,5-trichlorophenol-->3,5-dichlorophenol-->3-chlorophenol-->phenol. Intermediate products such as 3-chlorophenol were not accumulated, which were immediately dechlorinated to phenol. The enriched microbial community was characterized physiologically by testing the effects of electron donors and electron acceptors on the dechlorinating activity. The dechlorinating activity was promoted with lactate, pyruvate, and hydrogen as electron donors but not with acetate. Electron acceptors, nitrate and sulphate, inhibited the dechlorinating activity competitively but not iron (III). The microbial group associated with the anaerobic dechlorination was characterized by the effect of specific inhibitors on the PCP dechlorination. Effects of specific metabolic inhibitors and antibiotics indicated the involvement of Gram-positive spore-forming bacteria with the PCP dechlorinating activity, which was represented as bacteria of phylum Firmicutes. The structure of the microbial community was characterized by fluorescence in situ hybridization, quinone profiling, and PCR-DGGE (denaturing gel gradient electrophoresis). The combined results indicated the predominance of Clostridium species of phylum Firmicutes in the microbial community. Desulfitobacterium spp. known as anaerobic Gram-positive spore-forming bacteria dechlorinating PCP were not detected by PCR using a specific primer set. These indicated a probable presence of novel anaerobic Gram-positive spore-forming bacteria dechlorinating PCP in the microbial community.

Syndecan-dependent binding of Drosophila hemocytes to laminin alpha3/5 chain LG4-5 modules: potential role in sessile hemocyte islets formation.

Heparin-column chromatography and elastase-digestion of medium from hemocyte Kc167 gave Drosophila laminin alpha3/5betagamma trimer, alpha3/5LG2-3 and alpha3/5LG4-5 modules with eluting NaCl concentrations of 450, 280 and 450 mM, respectively. Kc167 cells bound dish surface with alpha3/5betagamma trimer or alpha3/5LG4-5, but not with alpha3/5LG2-3 modules. Cell binding was counteracted by treating with heparin or heparan sulfate. RNA interference of syndecan in Kc167 cells impaired the binding, but that of dally or dally-like did not. Green fluorescent protein-expressing hemocytes also bound surface with alpha3/5betagamma trimer or alpha3/5LG4-5 module. Thus, syndecan-dependent binding of hemocytes to laminin may have a potential role in sessile hemocytes islets formation in T2-A8 segments of Drosophila.

A rice semi-dwarf gene, Tan-Ginbozu (D35), encodes the gibberellin biosynthesis enzyme, ent-kaurene oxidase.

A rice (Oryza sativa L.) semi-dwarf cultivar, Tan-Ginbozu (d35Tan-Ginbozu), contributed to the increase in crop productivity in Japan in the 1950s. Previous studies suggested that the semi-dwarf stature of d35Tan-Ginbozu is caused by a defective early step of gibberellin biosynthesis, which is catalyzed by ent-kaurene oxidase (KO). To study the molecular characteristics of d35Tan-Ginbozu, we isolated 5 KO-like (KOL) genes from the rice genome, which encoded proteins highly homologous to Arabidopsis and pumpkin KOs. The genes (OsKOL1 to 5) were arranged as tandem repeats in the same direction within a 120 kb sequence. Expression analysis revealed that OsKOL2 and OsKOL4 were actively transcribed in various organs, while OsKOL1 and OsKOL5 were expressed only at low levels; OsKOL3 may be a pseudogene. Sequence analysis and complementation experiments demonstrated that OsKOL2 corresponds to D35. Homozygote with null alleles of D35 showed a severe dwarf phenotype; therefore, d35Tan-Ginbozu is a weak allele of D35. Introduction of OsKOL4 into d35Tan-Ginbozu did not rescue its dwarf phenotype, indicating that OsKOL4 is not involved in GA biosynthesis. OsKOL4 and OsKOL5 are likely to take part in phytoalexin biosynthesis, because their expression was promoted by UV irradiation and/or elicitor treatment. Comparing d35Tan-Ginbozu with other high yielding cultivars, we discuss strategies to produce culm architectures suitable for high crop yield by decreasing GA levels.

The evolution of the phenylacetic acid degradation pathway in bacteria.

The phenylacetic acid (PhAc) degradation pathway becomes an interesting model for the catabolism of aromatic compounds. To determine the molecular basis for this environmentally important process, we did a phylogenic analysis based on the PhAc CoA ligase gene. It suggests that the PhAc CoA ligase genes are distributing widely and subject to frequent lateral gene transfer within and across bacterial phylum.

Effects of various culture environments on expression of major outer membrane proteins from Porphyromonas gingivalis.

We examined the effects of various culture environments on major outer membrane proteins from Porphyromonas gingivalis ATCC 33277. Major outer membrane protein patterns on gel electrophoresis showed little difference over the culturable range of osmolarity and pH. With elevated temperature or prolonged culture, the intensities of the gingipain bands decreased; however, bands of RagA, RagB and the putative porins were relatively stable. Similar results were observed with several different culture media. Although the precise functions of RagA, RagB and the putative porins are unknown, these factors may be strongly related to the initiation and progression of adult periodontitis.

Flagellum-independent trail formation of escherichia coli on semi-solid agar.

Escherichia coli can form linear trails and move in a flagellum-independent manner on semisolid agar containing carbon sources. Trail formation seemed to correlate with the growth speed and/or carbon metabolism. Cell morphology in linear trails changed into larger cell sizes. We speculate that the flagellum-independent trail formation is a new mechanism for migration of E. coli cells.

Spatial distribution of mDLG6 mRNA in embryonic and adult mouse brain.

We have isolated mouse DLG6 (mDLG6) cDNA clones by RT-PCR and then by using the RT-PCR products to screen a mouse brain cDNA library. The deduced amino acid sequence of mDLG6 shows 79.2% and 82.7% overall identity to human (hDLG6) and rat DLG6 (rDLG6), respectively. In situ hybridization revealed that mDLG6 mRNA is predominantly expressed in embryonic and adult brain.

Reduction of protein degradation by use of protease-deficient mutants in cell-free protein synthesis system of Escherichia coli.

In an Escherichia coli in vitro transcription/translation system, the degradation of produced proteins is often caused by endogenous proteases from E. coli extracts. To reduce the extent of this degradation, several extracts were prepared from E. coli mutants that genetically lacked DegP, OmpT, or Lon proteases. Then, these extracts were used with 14C-leucine in a system for synthesizing single-chain Fv against gp120 (anti-gp120), and phospholipase D (PLD) of Streptomyces antibioticus. The proteins synthesized in vitro were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and autoradiography. The use of extracts from mutants that were deficient in the structural genes encoding OmpT and Lon markedly repressed the degradation of anti-gp120. Similarly, extracts from degP- and ompT-deleted mutants were able to significantly stabilize in vitro-synthesized PLD, which otherwise disappeared within 30 min. Such protease-deficient mutants were suggested to be useful for preventing the degradation of heterologous proteins in in vitro systems.