Host-specific plant signal and G-protein activator, mastoparan, trigger differentiation of zoospores of the phytopathogenic oomycete Aphanomyces cochlioides

We found that the gradient of a host-specific attractant, cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone) isolated from the roots of spinach triggered encystment followed by germination of zoospores of Aphanomyces cochlioidesat a concentration less than micromolar order. This compound did not affect the growth and reproduction of this phytopathogen up to 10^–6 M concentration in the culture medium. We also observed that mastoparan, an activator of heterotrimeric G-protein could inhibit the motility of zoospores and then strikingly effect encystment followed by 60–80% germination of cysts. Concomitant application of cochliophilin A and mastoparan showed stronger encystment followed by 100% germination of cysts. In addition, we have observed that chemicals interfering with phospholipase C activity (neomycin) and Ca²⁺ influx/release (EGTA and loperamide) suppress cochliophilin A or mastoparan induced encystment and germination. These results suggest that G-protein mediated signal transduction mechanism may be involved in the differentiation of the A. cochlioides zoospores. This is the first report on the differentiation of oomycete zoospores initiated by a host-specific plant signal or a G-protein activator.     

Planning continuity and the actual conditions of shopping malls

The main purpose of this study is to investigate the continuity of the planning of shopping malls in downtown areas of Japan and to look into the tendencies of the current existing malls until today. This paper is a summary of a survey conducted on the actual conditions of current shopping malls and a questionnaire administered to local governments in the survey areas. The results of this study allow us to summarize the reasons for and changes caused by renewal efforts directed toward the streets, public spaces, and urban elements (pavement, bench, streetlight, arcade, sculpture, etc.) in shopping malls. Furthermore, these results also help us to understand the scale of the renewal efforts as well as their timing in relation to when the shopping mall was originally constructed.     

Isolation and structure elucidation of Peronosporomycetes hyphal branching-inducing factors produced by Pseudomonas jessenii EC-S101

Pseudomonas jessenii EC-S101 produced hyphal branching-inducing and mitosis-accelerating factors active towards Peronosporomycetes, Aphanomyces cochlioides hyphae. In searching for the active substances, EtOAc-solubles extracted from EC-S101-cultured solid medium were fractionated under the guidance of a paper disc assay using an A. cochlioides mycelium. Two active substances were subsequently isolated and the structure was elucidated by spectroscopic analysis to be (+)-4,5-didehydroacaterin (1) and 3-[(1R)-hydroxyhexyl]-5-methylene-2(5H)-furanone (2), both of which accelerated the mitotic process of A. cochlioides hyphae along with excessive branching at 1.0 microg per disc. These compounds are likely to affect the morphophysiological development of certain eukaryotic organisms in the terrestrial ecosystem.     

DETECTION AND ENUMERATION OF H2S+ BACTERIA: APPLICATION TO SHEWANELLA PUTREFACIENS (CIP)

H₂S⁺ bacteria responsible for the degradation of sulfur-containing amino acids of fish muscle are currently little used to evaluate the microbiological pal quality of fish. Shewanella putrefaciens greatly predominates in this flora, and was therefore used to define a suitable culture method and medium. Inoculations by the Spiral surface method at 25C, with an incubation of 72h, gave the best counts on a medium containing two sources of sulfur (organic and inorganic) for H₂S⁺ bacteria. The culture medium and the NaCl concentration were determinant in the evaluation of this flora. At present there is no standard medium which meets these requirements.     

Antifungal nitro compounds from skunk cabbage (Lysichitum americanum) leaves treated with cupric chloride

Two nitro compounds, 2-(4-methoxyphenyl)-1-nitroethane named as lysichitalexin and 2-(4-hydroxyphenyl)-1-nitroethane were isolated as stress metabolites from the leaves of Lysichitum americanum Hultén and St. John treated with cupric chloride. Their structures were determined by spectroscopic methods and chemical reactions. The former compound showed antifungal activities against Fusarium oxysporum and Cladosporium herbarum. Both compounds were isolated for the first time from this species and the former was isolated from natural sources for the first time. This is the first report on stress metabolites from a member of the Araceae.     

Accumulation of oxidative DNA damage, 8-oxo-2'-deoxyguanosine, and change of repair systems during in vitro cellular aging of cultured human skin fibroblasts

Effects of in vitro cellular aging on the content of 8-oxo-2'-deoxyguanosine, a typical oxidation product of DNA bases, were examined in cultured human skin fibroblasts. The 8-oxo-2'-deoxyguanosine content in the DNA of TIG-3S cells established from skin tissues of a fetal donor increased immediately before the cessation of proliferation. TIG-114 and TIG-104 cells established from skin tissues of adult and aged donors, respectively, showed similar changes in 8-oxo-2'-deoxyguanosine content during in vitro cellular aging. The accumulation of 8-oxo-2'-deoxyguanosine in late-passage cells was dependent on the number of cell divisions, and not on the cultivation time. Increases in the activities of superoxide dismutase and glutathione peroxidase were observed prior to the increase in 8-oxo-2'-deoxyguanosine content, while the catalase activity decreased gradually during in vitro cellular aging at late-passage. Furthermore, the activities of 8-oxo-2'-deoxyguanosine endonuclease and DNA polymerases decreased with the progression of proliferation. These results indicate that defense systems against oxidative stress in late-passage cells remain sufficiently active before the cessation of cell division, but that repair systems against oxidative damage decay at late-passage. Oxidative stress beyond the antioxidant capacity and/or repair activity seems to result in an accumulation of 8-oxo-2'-deoxyguanosine in late-passage cells.     

Functional analysis of the C-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a "shuttle" peptide to help secretion

Thrombopoietin (TPO) is a cytokine that primarily stimulates megakaryocytopoiesis and thrombopoiesis. TPO has a unique C-terminal tail peptide of about 160 amino acids that consists mostly of hydrophilic residues and contains six N-linked sugar chains. In order to investigate the biological function of the C-terminal domain, two series of mutations were performed. One is systematic truncation from the C terminus. Another is elimination of N-glycosylation sites in the C-terminal domain by Asn to Gln mutations. After the mutant proteins were expressed by mammalian cells, it was found that the elimination of the N-linked sugar sites did not affect the biological activity, whereas truncation of the C-terminal domain resulted in elevation of in vitro activity up to 4-fold. The C-terminal peptide itself was found to inhibit the in vitro activity. Moreover, both the C-terminal truncation and the elimination of the N-glycosylation sites decreased the secretion level progressively down to (1)/(10) that of wild type, and the amount of the mutant left in the cell increased. The N-glycosylation in the C-terminal region was found to be important for secretion of TPO. Among six N-glycosylation sites in the C-terminal region, two locations, Asn-213 and Asn-234, were found to be critical for secretion, and two other locations, Asn-319 and Asn-327, did not affect the secretion.     

Identification of Pip4k2β as a mechanical stimulus responsive gene and its expression during musculoskeletal tissue healing

To investigate the mechano-transduction system of cells, we identified genes responsive to a cyclic mechanical stimulus. MC3T3.E1 cells were cultured on a computer-controlled vacuum-pump-operated device designed to provide a cyclic mechanical stimulus. A maximum elongation of 15% of membrane at 10 cycles/min (3 s extension followed by 3 s relax per cycle) was repeated for 48 h. By means of a differential display, the gene expression pattern of cells exposed to the stimulus was compared with that of unexposed cells. As a result, a gene fragment that was exclusively expressed in mechanically stressed cells was identified. By using expressed sequence tag walking together with the oligo-capping method, this gene was identified as phosphatidylinositol 4-phosphate 5-kinase type II β (initially known as Pip5k2β but now reclassified as Pip4k2β). The specific up-regulation of Pip4k2β upon mechanical stimulus was also confirmed by using another apparatus, viz. a computer-controlled linearized-stepping motor system. To examine the involvement of the cyclic mechanical stimulus in the regulation of Pip4k2β expression in musculoskeletal tissue, we created an Achilles tendon transection model in rabbits. The temporal expression of Pip4k2β was assessed by means of a quantitative reverse-transcribed polymerase chain reaction. In the gastrocnemius muscle, expression of Pip4k2β rapidly decreased 1 week after transection but was restored to normal levels at 4 weeks. In the Achilles tendon, however, expression remained decreased until 4 weeks after transection. We suggest that the expression of Pip4k2β can be used as a marker for cells receiving a suitable mechanical stimulus. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.