Trichomycetes (Zygomycota) in the digestive tract of arthropods in Amazonas, Brazil

Eight species of Harpellales and three species of Eccrinales (Zygomycota: Trichomycetes) were found associated with the digestive tract of arthropods from terrestrial and aquatic environments in the central Amazon region of Brazil. New species of Harpellales include: Harpella amazonica, Smittium brasiliense, Genistellospora tropicalis in Simuliidae larvae and Stachylina paucispora in Chironomidae larvae. Axenic cultures of S. brasiliense were obtained. Probable new species of Enterobryus (Eccrinales), Harpella, and Stachylina (Harpellales) are described but not named. Also reported are the previously known species of Eccrinales, Passalomyces compressus and Leidyomyces attenuatus in adult Coleoptera (Passalidae), and Smittium culisetae and Smittium aciculare (Harpellales) in Culicidae and Simuliidae larvae, respectively. Comments on the distribution of some of these fungi and their hosts in the Neotropics are provided.     

Putative functions of nucleoside diphosphate kinase in plants and fungi

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red–far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. In Neurospora crassa a Pro72His mutation in NDP kinase (ndk-1 ^Pro72His ) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as light-induced polarity of perithecia. In wild-type in darkness the beak was formed at random places on perithecia, and in ndk ^Pro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

Effects of benzo[a]pyrene on tissue activities of metabolizing enzymes and antioxidant system in normal and protein-malnourished rats

The effects of benzo[a]pyrene (B[a]P) on some drug-metabolizing and antioxidant systems in liver, lung, and stomach were investigated in normal and protein malnutrition (PM) rats. PM significantly inhibited tissue glutathione (GSH) content and increased hepatic lipid peroxidation. Cytochrome P450 isoform CYP1A1 was significantly increased in various tissues (42-73%). Also, lung glutathione S-transferase (GST) activity was significantly decreased (19%) in PM rats. On the other hand, B[a]P significantly induced tissue GSH of control and PM rats. Also, hepatic lipid peroxidation were significantly increased in control rats treated with B[a]P. Superoxide dismutase (SOD) activity was decreased by B[a]P treatment in PM rat stomach. B[a]P significantly induced both quinone reductase (QR) (in all tissues) and hepatic GST of control and PM rats. GST activity in PM rat liver was significantly higher than that of control rat liver after B[a]P treatment. Also, B[a]P induced hepatic CYP1A1 by 32-fold and 27-fold (P < or = 0.05) in control and PM rats, respectively. Stomach and hepatic UDP-glucuronosyltransferase activities were significantly decreased (34%) and increased (74%), respectively by B[a]P in PM rats. The results suggest that PM status has a modifying effect on the response of some antioxidant and metabolizing systems to a well-known carcinogen risk.     

The expression of platelet endothelial cell adhesion molecule-1 in mouse primordial germ cells during their migration and early gonadal formation

The platelet endothelial cell adhesion molecule-1 (PECAM-1), or CD31, a member of the immunoglobulin superfamily, is located on the plasma membrane of endothelial and hematopoietic cells and involved in vascular development and inflammation. In this study, by use of immunohistochemistry at light and electron microscopic levels in combination with enzyme histochemistry for alkaline phosphatase, we demonstrated that PECAM-1/CD31 is expressed in the mouse primordial germ cell (PGC). Up to 8 days postcoitum (dpc), PGCs with alkaline phosphatase activity showed no PECAM-1/CD31 immunoreactivity. At 9 dpc, PECAM-1/CD31 immunoreactivity was first detected with low intensity in some PGCs located in the hindgut. Between 10 and 11 dpc, intense immunoreactivity was shown on the entire surface of PGCs migrating along the dorsal wall. After arrival and settlement of PGCs in the genital ridges around 11.5 dpc, the intense immunoreactivity was maintained on the entire surface of PGCs. By electron microscopy, the immunoreactivity was localized exclusively on the plasma membrane of PGCs, being as strong at the portions adjacent to neighboring PGCs as those adjacent to somatic cells. As the male and female gonads began to differentiate, PECAM-1/CD31 immunoreactivity remained strong in germ cells until 13 dpc, after which it gradually decreased in intensity and disappeared by 16 dpc. These results suggested that cell-to-cell interaction through PECAM-1/CD31 plays roles in the development of PGCs during their migration on the dorsal wall and homing in the gonads.     

Ascorbic acid stimulation of production of a highly branched ,beta-1,3-glucan by Aureobasidium pullulans K-1--oxalic acid, a metabolite of ascorbic acid as the stimulating substance

The production of a highly branched beta-1,3-glucan by Aureobasidium pullulans K-1 in Czapek's medium has been found to be stimulated by ascorbic acid. When the culture supernatant, after removal of polysaccharide from the culture filtrate by ethanol precipitation, was concentrated, then added to a new medium and this strain was cultured in the medium, the polysaccharide production was stimulated the same as when L-ascorbic acid was added to the medium. The stimulating substance was partially purified from the supernatant, and was found to be oxalic acid; 0.03% oxalic acid was the most effective concentration for the stimulation of polysaccharide production. The stimulating substance, oxalic acid, was proved to be derived from ascorbic acid added to a medium in an experiment using L-[1-14C]ascorbic acid. We suggest that oxalic acid generated from the metabolism of ascorbic acid in cells of Aureobasidium pullulans K-1 participated in the stimulation of the polysaccharide production by ascorbic acid.     

Presence of two trans-o-hydroxybenzylidenepyruvate hydratase-aldolases in naphthalenesulfonate-assimilating Sphingomonas paucimobilis TA-2: comparison of some properties

Two trans-o-hydroxybenzylidenepyruvate hydratase-aldolases named tHBP HA A and tHBP HA B were purified from a cell-free extract of naphthalenesulfonate-assimilating Sphingomonas paucimobilis (formerly Pseudomonas sp.) TA-2 to an electrophoretically homogeneous state by successive column chromatographies on DEAE-cellulose, DEAE-Toyopearl 650M, Sephacryl S-100, Hydroxyapatite, and Mono Q. These enzymes were similar to each other in molecular mass (ca. 37 kDa on SDS-PAGE, ca. 110 kDa on ultracentrifugation), thermal stability (<50 degrees C) and optimum pH (pH 9.0). However, they differed from each other in N-terminal amino acid sequences, pH stability, K(m) values for trans-o-hydroxybenzylidenepyruvate (tHBP), and inhibition by p-chloromercuribenzoic acid (PCMB). tHBP HA B had a homologous N-terminal amino acid sequence with tHBP HAs from Pseudomonas vesicularis DSM 6383 (strain BN6) and Sphingomonas aromaticivorans F119, and tHBP HA A had a homologous sequence with tHBP HAs of Pseudomonas putida strain OUS82, Pseudomonas sp. strain C18 and NAH7 plasmid. tHBP HA B was inhibited by PCMB, but tHBP HA A was not. Their K(m) values for tHBP were 9 and 3 M, respectively. tHBP HA B was stable in the range of pH 7.1 to pH 10.7, and tHBP HA A was stable in the range of pH 6.0 to 9.3.