Isolation and Characterization of 2-Nitroimidazole Produced by Streptomyces Species as an Inhibitor of Both Carbonic Anhydrase and Shell Formation in the Barnacle Balanus amphitrite

Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.     

Morphological studies on the bathyal ascidian,Megalodicopia hians Oka 1918 (Octacnemidae,Phlebobranchia), with remarks on feeding and tunic morphology

Megalodicopia hians Oka is a solitary ascidian belonging to the family Octacnemidae inhabiting the bathyal /abyssal zone as well as other octacnemid ascidians. The phylogenetic relationship of octacnemids is open to argument because of its extraordinary morphological features due to habitat adaptation, e.g., a pharynx lacking ciliated stigmata. Aggregations of M. hians were discovered by the manned submersible Shinkai 2000 in the bathyal seafloor of Toyama Bay, Japan Sea, in 2000; this was the first in situ observation of M. hians in the Japanese coastal waters. In 2001, a total of 36 M. hians specimens were collected from the bay (592 to approximately 978 m deep). In situ observation indicated that M. hians usually opens its large oral apertures to engulf the drifting food particles in the water current. Microscopical observation of the gut contents also showed that M. hians is a non selective macrophagous feeding on small crustaceans, diatoms, detritus, and so on. Along with the position of the intestinal loop and gonad, the morphological characteristics of the tunic (integument of ascidians) suggest that M. hians is closely related to Cionidae and/or Corellidae. Some symbiotic/parasitic organisms were occasionally found in the tunic, including rod-shaped bacteria, fungi-like multicellular structure, and spawns of unknown animals.     

Environmental,health and economic conditions perceived by 50 rural communities in Bangladesh

For randomly selected 50 villages in Bangladesh, an interview survey with a structured questionnaire was conducted to reveal their perception on the environmental, health and economic conditions at present and for the past 10-year change. The eight following items were analyzed in this paper: air pollution and water pollution, which represent environmental conditions with close relation to health conditions, soil degradation and deforestation, which represent environmental conditions with close relation to economic conditions, epidemic diseases and malnutrition, which represent health conditions, and poverty and jobless, which represent economic conditions. Among the 50 villages, deforestation was most frequently perceived serious at present and worsened in the past 10 years. Of the remaining seven items, those related to economic conditions were more seriously perceived than those related to health and environmental conditions. As revealed by the cluster analysis for the inter-item relations, epidemic diseases, which formed the same cluster with the environmental items, were recognized less serious whereas malnutrition, which formed the same cluster with the economic items, was recognized more serious. These findings are useful not only for rural development programs but also for mitigation programs toward health and environmental hazards in Bangladesh.     

Metalloproteinase with factor X activating and fibrinogenolytic activities from Vipera berus berus venom

We have previously shown that Vipera berus berus venom contains several factor X activating enzymes. In the present study we have investigated one of them. The enzyme was separated from venom by gel filtration on Sephadex G-100 superfine and chromatography on agarose HPS-7 and phenyl-agarose. The enzyme is a glycosylated metalloproteinase containing hexoses, hexosamines and neuraminic acid. The purified factor X activating enzyme consists of two equal chains (59 kDa). The specificity studies have shown that enzyme is nonspecific factor X activating proteinase hydrolysing also proteins such as azocasein, gelatin and fibrinogen. The enzyme hydrolyses oxidized insulin B-chain at the positions Ala¹⁴–Leu¹⁵ and Tyr¹⁶–Leu¹⁷ but it is inactive on fibrin, plasminogen and prothrombin. We used 8–10 amino acid residues containing peptides, which reproduce the sequence around the cleavage sites in factor X, factor IX and fibrinogen, as potential substrates for enzyme. Cleavage products of peptide hydrolysis were determined by MALDI-TOF MS. The peptide Asn–Asn–Leu–Thr–Arg–Ile–Val–Gly–Gly—factor X fragment was cleaved by enzyme at positions Leu³–Thr⁴ and Arg⁵–Ile⁶. The fibrinogen peptide fragment Glu–Tyr–His–Thr–Glu–Lys–Leu–Val–Thr–Ser was hydrolysed at position Lys⁶–Leu⁷.     

The cDNA microarray analysis using an Arabidopsis pad3 mutant reveals the expression profiles and classification of genes induced by Alternaria brassicicola attack

The hypersensitive response (HR) was induced in a wild-type Arabidopsis thaliana plant (Columbia) (Col-wt) by inoculation with Alternaria brassicicola that causes the development of small brown necrotic lesions on the leaves. By contrast, pad3-1 mutants challenged with A. brassicicola produced spreading lesions. The cell death in pad3-1 mutants could not inhibit the pathogen growth and development, although both production of H(2)O(2) and localized cell death were similar in Col-wt and pad3-1 plants after the inoculation. The difference between Col-wt and pad3-1 plants is defense responses after the occurrence of cell death. In other words, PAD3 is necessary for defense response to A. brassicicola. Therefore, we examined the changes in the expression patterns of ca. 7,000 genes by cDNA microarray analysis after inoculation with A. brassicicola. The cDNA microarrays were also done to analyze Arabidopsis responses after treatment with signal molecules, reactive oxygen species (ROS)-inducing compounds and UV-C. The results suggested that the pad3-1 mutation altered not only the accumulation of camalexin but also the timing of expression of many defense-related genes in response to the challenge with A. brassicicola. Furthermore, the plants integrate two or more signals that act together for promoting the induction of multiple defense pathways.     

Rapid increase of vacuolar volume in response to salt stress

Suspension-cultured cells of mangrove [Bruguiera sexangula (Lour.) Poir.] showed a rapid increase in vacuolar volume under salt stress, although there was no change in the cell volume. The rapid increase in the vacuolar volume was an active process, which followed the activation of the tonoplast H(+)-ATPase and the vacuolar acid phosphatase. The same phenomenon was observed in barley (Hordeum vulgare L. cv. Doriru) root meristematic cells under salt stress but not in pea ( Pisum sativum L.). Increases in vacuolar volume could potentially protect the cytoplasm by decreasing the cytoplasmic volume during the initial phases of salt stress.     

Determinants of human immunodeficiency virus (HIV) prevalence in homosexual and bisexual men screened for admission to a cohort study of HIV negatives in Belo Horizonte,Brazil: Project Horizonte

Project Horizonte, an open cohort of homosexual and bisexual human immunodeficiency virus (HIV-1) negative men, is a component of the AIDS Vaccine Program, in Belo Horizonte, Minas Gerais, Brazil. The objective of this study was to compare volunteers testing HIV positive at cohort entry with a sample of those who tested HIV negative in order to identify risk factors for prevalent HIV infection, in a population being screened for enrollment at Project Horizonte. A nested case-control study was conducted. HIV positive volunteers at entry (cases) were matched by age and admission date to three HIV negative controls each. Selected variables used for the current analysis included demographic factors, sexual behavior and other risk factors for HIV infection. During the study period (1994-2001), among the 621 volunteers screened, 61 tested positive for HIV. Cases were matched to 183 HIV negative control subjects. After adjustments, the main risk factors associated with HIV infection were unprotected sex with an occasional partners, OR = 3.7 (CI 95% 1.3-10.6), receptive anal intercourse with an occasional partner, OR = 2.8 (95% CI 0.9-8.9) and belonging to the negro racial group, OR = 3.4 (CI 95% 1.1-11.9). These variables were associated with an increase in the risk of HIV infection among men who have sex with men at the screening for admission to an open HIV negative cohort.     

Bone morphogenetic protein 4 mediates apoptosis of capillary endothelial cells during rat pupillary membrane regression

Programmed capillary regression is essential for development, but little is known about the mechanism behind this phenomenon. In this study, we characterized the molecular determinants of capillary regression utilizing the pupillary membrane (PM) in the newborn rat's eye. We observed in the 1-day-culture system that apoptotic endothelial cells decrease in number with the addition of a natural antagonist, Noggin, strongly suggesting the involvement of the bone morphogenetic protein (BMP) family in PM regression. In addition, the lens-conditioned medium (Lens-CM) induced apoptosis of HUVE cells and inhibited endothelial tubulogenesis, which were completely blocked by both Noggin and the BMP4-specific neutralizing antibody. Activation of BMP4 pathway in endothelial cells was confirmed by both the up-regulation of Msx genes correlated with apoptosis and the translocation of Smad1 into the nucleus. We showed a transient expression of BMP4 in Lens-CM by immunoprecipitation assay. Furthermore, the transcorneal injection of BMP4 in rats enhanced the apoptosis of PMs, while that of Noggin attenuated it. These results indicate that BMP4 pathways play pivotal roles in capillary regression in a paracrine manner between lens and PMs.