Ethylene as a potent inducer of sexual development

A novel and critical function of ethylene, a potent plant hormone, has been well documented in Dictyostelium, because it leads cells to the sexual development (macrocyst formation) by inducing zygote formation. Zygote formation (sexual cell fusion) and the subsequent nuclear fusion are the characteristic events occurring during macrocyst formation. A novel gene, zyg1 was found to be predominantly expressed during the sexual development, and its enforced expression actually induces zygote formation. As expected, the zygote inducer, ethylene enhances the expression of zyg1. Thus the function of ethylene has been verified at all of individual (macrocyst formation), cellular (zygote formation), and molecular levels (zyg1 expression). Based on our recent studies concerning the behavior and function of the zyg1 product (ZYG1 protein), the signal transduction pathways involved in zygote formation are proposed in this review.     

The structure of starch can be manipulated by changing the expression levels of starch branching enzyme IIb in rice endosperm

When the starch branching enzyme IIb (BEIIb) gene was introduced into a BEIIb-defective mutant, the resulting transgenic rice plants showed a wide range of BEIIb activity and the fine structure of their amylopectins showed considerable variation despite having the two other BE isoforms, BEI and BEIIa, in their endosperm at the same levels as in the wild-type. The properties of the starch granules, such as their gelatinization behaviour, morphology and X-ray diffraction pattern, also changed dramatically depending on the level of BEIIb activity, even when this was either slightly lower or higher than that of the wild-type. The over-expression of BEIIb resulted in the accumulation of excessive branched, water-soluble polysaccharides instead of amylopectin. These results imply that the manipulation of BEIIb activity is an effective strategy for the generation of novel starches for use in foodstuffs and industrial applications.     

Isolation and characterization of light-dependent hemolytic cytotoxin from harmful red tide phytoplankton Chattonella marina

Chattonella marina (C. marina), a raphidophycean flagellate, is a causative organism of red tide, and highly toxic to fish. In this study, we found that the cell-free methanol extract prepared from this flagellate exhibited potent hemolytic activity against rabbit erythrocytes. Interestingly, the hemolytic activity of the extract was absolutely light-dependent, and no hemolytic activity was detected in the dark even at very high concentration. Gel filtration chromatography of the methanol extract on a column of Sephadex LH-20 revealed that the extract contained hemagglutinin as well as hemolytic agents, and the substances responsible for these activities were separately eluted. These results suggest that the hemagglutinating and hemolytic activities were derived from distinct compounds. The hemolytic fraction obtained after gel filtration (F4) caused marked inhibition of the growth of C. marina itself and other species of phytoplanktons. Furthermore, F4 showed a potent cytotoxicity toward various mammalian cultured cell lines including human tumor cells (HeLa cells) in a dose-dependent manner. The cytotoxicity was also light-dependent, and no cytotoxic effect was exhibited in any cell lines tested in the dark. After further purification procedures via preparative thin-layer chromatography and subsequent HPLC, a major hemolytic agent was obtained as highly purified form. Since the methanol extracts prepared from other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica showed light-dependent hemolytic activity toward rabbit erythrocytes, it was suggested that the light-dependent hemolytic agents commonly exist at least in these raphidophycean flagellates.     

Homologous subunits of 1,3-beta-glucan synthase are important for spore wall assembly in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-beta-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-beta-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-beta-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-beta-glucan synthase.     

A high temperature-sensitive mutant of Synechococcus sp. PCC 7002 with modifications in the endogenous plasmid, pAQ1

To study thermal adaptations in the cyanobacterium, Synechococcus sp. PCC 7002, we screened about 3,000 mutants for their tolerance to high temperature, and found one, SHT1, that is sensitive to high-temperature stress. The mutant had a modified gene construct in the endogenous plasmid, pAQ1. One of the four ORFs, ORF93, was duplicated, and its mRNA level was higher than in the wild type. At 38 degrees C, the growth of SHT1 was retarded as compared with the wild type, and above 38 degrees C, almost all the cells of SHT1 died. This temperature is much lower than that required for induction of heat shock proteins. Interestingly, in both the wild type and SHT1, the thermal stability of oxygen-evolving machinery increased upon acclimation to high temperatures. These findings indicate that the lack of thermal tolerance in the SHT1 strain is likely independent of the adaptation of the PSII complex and heat shock responses, whereas there are essential contributions of genes in the endogenous plasmid to the adaptation to high temperature. Thus, understanding the role of pAQ1 in the adaptation of Synechococcus sp. PCC 7002 to high-temperature environments is the first step in elucidating the function of this plasmid.     

Gas phase acidities of N-substituted amine-boranes

Complexation energies and acidities of 19 primary, secondary and tertiary amine-boranes were investigated using MP2/6-311+G(d,p) and B3LYP/6-311+G(d,p) methods. Gas phase acidities for free amines were also calculated. Acidity values for studied complexes range from 327.3 to 349.1 kcal mol^?1 and the most acidic are the ones with direct connection between deprotonation center and a π-system. Results obtained by both computational methods are in good agreement with each other and with known experimental data. Addition of BH₃ increases the acidity of amines by 30 to 50 kcal mol^?1. This enhancement effect was compared to the respective effect witnessed in phosphine-boranes and traced back to changes of charge delocalization on nitrogen. A question about the structural stability of several deprotonated amine-borane anions in the gas phase was also raised.     

Preliminary characterization of the hemolysin of Rhizopus nigricans

A hemolysin was extracted from fungous mats ofRhizopus nigricans cultured for two weeks in modified Sabouraud broth. Preliminary characterization indicated that the active substance is water soluble, stable after heating to 100°C, not destroyed by proteolytic enzymes, not dialyzable, and not precipitable with ethanol. It is however precipitable with ammonium sulfate and extractable with lipid solvents. Lipid fractionation revealed activity in the non-acidic phospholipid fraction. The cumulative findings suggest that the active hemolytic substance is a lipid perhaps attached to a protein.

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Zusammenfassung Das Hämolysin war vom Pilzmyzelium vonRhizopus nigricans extrahiert worden, das in Sabouraud's Brühe für zwei Wochen gezüchtet worden ist. Vorläufige Charakteristik zeigte, daß die aktive Substanz wasserlöslich, hitzeresistent ist und sie durch proteolytische Fermente nicht zerstört wird. Sie ist dialysierbar, und wird durch Ethanol nicht prezipitiert. Jedoch ist sie durch Ammoniumsulfate prezipitiert und durch Fettlösungsmittel extrahierbar Lipoidfraktionierung zeigte eine Aktivität in der nich-saueren Phospholipoidfraktion. Kumulative Befunde legen es nahe, daß die aktive, hämolytische Substanz ein an Protein gebundenes Lipoid ist.

     

Galectin-8 modulates neutrophil function via interaction with integrin alphaM

The members of the galectin family are associated with diverse cellular events, including immune response. We investigated the effects of galectin-8 on neutrophil function. Human galectin-8 induced firm and reversible adhesion of peripheral blood neutrophils but not eosinophils to a plastic surface in a lactose-sensitive manner. Other human galectins, galectins-1, -3, and -9, showed low or negligible effects on neutrophil adhesion. Confocal microscopy revealed actin bundle formation in the presence of galectin-8. Cytochalasins inhibited both actin assembly and cell adhesion induced by galectin-8. Affinity purification of galectin-interacting proteins from solubilized neutrophil membrane revealed that N-terminal carbohydrate recognition domain (CRD) of galectin-8 bound promatrix metalloproteinase-9 (proMMP-9), and C-terminal CRD bound integrin alphaM/CD11b and proMMP-9. A mutant galectin-8 lacking the carbohydrate-binding activity of N-terminal CRD (galectin-8R69H) retained adhesion-inducing activity, but inactivation of C-terminal CRD (galectin-8R233H) abolished the activity. MMP-3-mediated processing of proMMP-9 was accelerated by galectin-8, and this effect was inhibited by lactose. Galectins-1 and -3 did not affect the processing. Superoxide production, an essential event in bactericidal function of neutrophils, was stimulated by galectin-8 to an extent comparable to that induced by fMLP. Galectin-8R69H but not galectin-8R233H could stimulate superoxide production. Taken together, these results suggest that galectin-8 is a novel factor that modulates the neutrophil function related to transendothelial migration and microbial killing.