Purification,structure-function analysis,and molecular characterization of novel linear peptides from scorpion Opisthacanthus madagascariensis

In the previous report [Biochem. Biophys. Res. Commun. 286 (2001) 820], we described a novel short linear peptide, IsCT, with cytolytic activity isolated from the venom of scorpion Opisthacanthus madagascariensis. From the same scorpion venom, we further purified and characterized three short linear peptides named IsCT2, IsCTf, and IsCT2f that shared high homology with IsCT, while with different C-terminal areas between IsCT/IsCT2 and IsCTf/IsCT2f. Structure-activity relationship was analyzed by performing vivo and vitro assays and circular dichroism spectroscopy. Like IsCT, IsCT2 showed broad activity spectra against microbes (Gram positive and negative bacteria as well as fungi) and relatively weak hemolytic activity against sheep red blood cells. It adopts an amphipathic alpha-helical structure in aqueous TFE and is able to disrupt the artificial membrane. However, the other two peptides IsCTf and IsCT2f showed no activity in antimicrobial or hemolytic assay. Furthermore, IsCTf and IsCT2f cannot form amphipathic alpha-helix while demonstrating random coil structure in aqueous TFE, which might result in their lost cytolytic activity. IsCTf and IsCT2f both exist in the crude venom and are proved to be enzymatic products from IsCT and IsCT2. Whether they have some other biological activity is still unclear. In addition, we got the cDNAs encoding the precursors of IsCT and IsCT2. Besides the signal peptide, they still contain an unusual acidic pro-peptide at the C-terminal that was quite different from other known precursors of scorpion venom peptides. The novel structure and biological activity of these peptides proposed them to be a new class in scorpion venom.     

The X-ray crystal structure of pyrrolidone–carboxylate peptidase from hyperthermophilic archaea Pyrococcus horikoshii

The crystal structure of pyrrolidone–carboxylate peptidase (PCP) from hyperthermophilic archaea Pyrococcus horikoshii (PhoPCP) has been determined at 1.6-A resolution by X-ray crystallography. PCP belongs to the C15 family of cysteine protease, and specifically removes the amino terminal pyroglutamate residue from a wide range of N-terminal-blocking peptides. The crystal structure is very similar to that of other hyperthermophiles, Pyrococcus friosus and Thermococcus litoralis, and even that from the mesophile, Bacillus amyloliquefacience. The inter-subunit disulfide bonds, which have been proposed as one of the thermostabilizing factors of the PCP from such hyperthermophiles, was not present in PhoPCP. The result suggests that the thermostability of PhoPCP may be obtained by the accumulation of many weak factors. Abbreviations: NCS – non-crystallographic symmetry; PCP – pyrrolidone-carboxylate peptidase; rmsd – root mean square deviation     

Isolation and characterization of a rice homebox gene, OSH15

In many eukaryotic organisms including plants, homeobox genes are thought to be master regulators that establish the cellular or regional identities and specify the fundamental body plan. We isolated and characterized a cDNA designated OSH15 (Oryza sativa homeobox 15) that encodes a KNOTTED-type homeodomain protein. Transgenic tobacco plants overexpressing the OSH15 cDNA showed a dramatically altered morphological phenotype caused by disturbance of specific aspects of tobacco development, thereby indicating the involvement of OSH15 in plant development. We analyzed the in situ mRNA localization of OSH15 through the whole plant life cycle, comparing the expression pattern with that of another rice homeobox gene, OSH1. In early embryogenesis, both genes were expressed as the same pattern at a region where the shoot apical meristem would develop later. In late embryogenesis, the expression pattern of the two genes became different. Whereas the expression of OSH1 continued within the shoot apical meristem, OSH15 expression within the shoot apical meristem ceased but became observable in a ring shaped pattern at the boundaries of some embryonic organs. This pattern of expression was similar to that observed around vegetative or reproductive shoots, or the floral meristem in mature plants. RNA in situ localization data suggest that OSH15 may play roles in the shoot organization during early embryogenesis and thereafter, OSH15 may be involved in morphogenetic events around the shoot apical meristem.     

Cloning and nucleotide sequence of carbazole catabolic genes from Pseudomonas stutzeri strain OM1, isolated from activated sludge

A new carbazole (CAR)-degrading bacterium, called strain OM1, was isolated from activated sludge obtained from sewage disposal plants in Fukuoka Prefecture, and it was identified as Pseudomonas stutzeri. Anthranilic acid (AN), 2'-aminobiphenyl-2,3-diol and its meta-cleavage product, 2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4-dienoic acid, were identified as metabolic intermediates of CAR in the ethyl acetate extract of the culture broth. Therefore, the CAR catabolic pathway to AN in strain OM1 was indicated to be identical to those found in the Pseudomonas sp. strains CA06 and CA10. The strain OM1 degraded catechol (CAT) via a meta-cleavage pathway in contrast to strains CA06 and CA10, which transform catechol into cis, cis-munonic acid. Clones containing a 6.9-kb EcoRI fragment and a 3-kb PstI-SphI fragment were isolated from colonies, forming a clear zone of CAR and a yellow ring-cleavage product from CAT, respectively. Recombinant E. coli carrying the 6.9-kb fragment degraded CAR in the L-broth and produced AN. Cell-free extract from the clone carrying a 3-kb PstI-SphI fragment had high meta-ring-cleavage dioxygenase activity for CAT. The nucleotide sequences of these fragments were determined. The 6.9-kb fragment showed a very high degree of homology with the CAR catabolic genes of strain CA10. The amino acid and nucleotide sequences of the 3-kb fragment were found to exhibit significant homology with the genes for the CAT-catabolic enzymes of TOL plasmid pWW0, plasmid NAH7, and plasmid pVI150.     

Does troop size of wild Japanese macaques influence birth rate and infant mortality in the absence of predators?

For the wild Japanese macaques of Yakushima and Kinkazan Islands, we analyzed the relationship between the troop size or the number of adult females of each troop, infant/adult female ratio (IFR; crude birth rate), and infant mortality (IM) in habitats with no predators. In Yakushima, IFR was positively correlated to troop size and the number of adult females. In Kinkazan, however, IFR tended to decrease with the number of adult females. This difference may be due to the difference in troop size; i.e. in Yakushima, where troop size was small, IFR may increase with that of troop size, because a relatively larger troop is likely to the advantage in intertroop competition. In Kinkazan, where troop size was large, however, IFR is likely to decrease with troop size, because intratroop competition may increase. Thus, the present data roughly supportWrangham's model of the social structure of female-bonded primates, and suggests that there is an optimal troop size for birth rate (BR). On the other hand, there was no clear correlation between IM and the troop size or number of adult females of each troop.     

Denitrification by Actinomycetes and Purification of Dissimilatory Nitrite Reductase and Azurin from Streptomyces thioluteus

Many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide (N₂O). As a representative of actinomycete denitrification systems, the system of Streptomyces thioluteus was investigated in detail. S. thioluteus attained distinct cell growth upon anaerobic incubation with nitrate or nitrite with concomitant and stoichiometric conversion of nitrate or nitrite to N₂O, suggesting that the denitrification acts as anaerobic respiration. Furthermore, a copper-containing, dissimilatory nitrite reductase (CuNir) and its physiological electron donor, azurin, were isolated. This is the first report to show that denitrification generally occurs among actinomycetes.     

Heterogeneity of Light Availability and its Effects on Simulated Carbon Gain of Tree Leaves in a Small Gap and the Understory in a Tropical Rain Forest1

To clarify the small-scale heterogeneity of light regimes in a rain forest, photosynthetic photon flux density (PFD) was measured at 1-min intervals during six days at 12 microsites in each of two plots, a small gap and an understory in Pasoh Forest Reserve, Peninsular Malaysia. Frequency distribution of microsite PFD was unimodal with the peak value between 16 and 32 μmol/m²/sec in the small gap, but between 8 and 16 μmol/m²/sec in the understory. In the small gap, PFD was more variable among microsites; total daily PFD and daily sunfleck PFD exceeding 10 μmol/ m²/sec tended to be higher (P <0.05; t-test) compared to those in the understory. Sunfleck PFD exceeding 50 μmol/ m²/sec, however, showed no difference between the two plots. Diffuse PFD transmittance, defined as the ratio of PFD in the forest to that measured at 43 m above ground during the periods 0800-0810 and 1750-1800 h, was significantly higher in the small gap than in the understory plot. Diffuse PFD transmittance was also positively correlated with microsite total daily PFD. To examine the effects of the subtle heterogeneity of light regimes on leaf carbon gain, we simulated carbon gain by sun and shade leaves in a typical shade-tolerant species, Brosimum aticastrum Sw. (Moraceae). Despite the similarity in total daily PFD, total daily carbon gain was considerably higher in the gap than in the understory for both sun and shade leaves. This study suggests that frequency distribution of PFD is critical in describing microsite PFD regimes and determining leaf carbon gain in the tropical forest floor.     

Peroxidase-mediated chlorophyll degradation in horticultural crops

One of the symptoms of senescence in harvested horticultural crops is the loss of greenness that comes with the degradation of chlorophyll (Chl). With senescence, peroxidase, which is involved in Chl degradation, increased greatly in stored horticultural crops. C13²-hydroxychlorophyll a, an oxidized form of Chl a, is formed in vitro through Chl oxidation by peroxidase. Peroxidase mediates Chl degradation in the presence of phenolic compounds such as p-coumaric acid and apigenin, which have a hydroxyl group at the p-position. Apparently, not all phenolic compounds are able to degrade Chl in this system, and their effectiveness appears to depend on their molecular configuration. In peroxidase-mediated Chl degradation, peroxidase oxidizes the phenolic compounds with hydrogen peroxide and forms phenoxy radical; then, the phenoxy radical oxidizes Chl and its derivatives to colorless low molecular weight compounds through the formation of C13²-hydroxychlorophyll a,a fluorescent Chl catabolite and a bilirubin-like compound as an intermediate. In addition to the phenoxy radical, superoxide anion, which is formed in the peroxidase-catalyzed reaction, might be involved in Chl oxidation. Moreover, Chl degradation by peroxidase seems to occur in the chloroplast and/or the vacuole. The involvement of peroxidase in Chl degradation in senescing horticultural crops is also discussed.     

Group IID heparin-binding secretory phospholipase A(2) is expressed in human colon carcinoma cells and human mast cells and up-regulated in mouse inflammatory tissues.

Group IID secretory phospholipase A(2) (sPLA(2)-IID), a heparin-binding sPLA(2) that is closely related to sPLA(2)-IIA, augments stimulus-induced cellular arachidonate release in a manner similar to sPLA(2)-IIA. Here we identified the residues of sPLA(2)-IID that are responsible for heparanoid binding, are and therefore essential for cellular function. Mutating four cationic residues in the C-terminal portion of sPLA(2)-IID resulted in abolition of its ability to associate with cell surface heparan sulfate and to enhance stimulus-induced delayed arachidonate release, cyclooxygenase-2 induction, and prostaglandin generation in 293 cell transfectants. As compared with several other group II subfamily sPLA(2)s, which were equally active on A23187- and IL-1-primed cellular membranes, sPLA(2)-IID showed apparent preference for A23187-primed membranes. Several human colon carcinoma cell lines expressed sPLA(2)-IID and sPLA(2)-X constitutively, the former of which was negatively regulated by IL-1. sPLA(2)-IID, but not other sPLA(2) isozymes, was expressed in human cord blood-derived mast cells. The expression of sPLA(2)-IID was significantly altered in several tissues of mice with experimental inflammation. These results indicate that sPLA(2)-IID may be involved in inflammation in cell- and tissue-specific manners under particular conditions.