Degradation of polyaromatic hydrocarbons by Burkholderia cepacia 2A-12

A new strain of bacterium degrading polyaromatic hydrocarbons (PAHs), Burkholderia cepacia 2A-12, was isolated from oil-contaminated soil. Of three PAHs, the isolated strain could utilize naphthalene (Nap) and phenanthrene (Phe) as a sole carbon source but not pyrene (Pyr). However, the strain could degrade Pyr when a cosubstrate such as yeast extract (YE) was supplemented. The PAH degradation rate of the strain was enhanced by the addition of other organic materials such as YE, peptone, glucose, and sucrose. YE was a particularly effective additive in stimulating cell growth as well as PAH degradation. When 1 g YE l^–1, an optimum concentration, was supplemented into the basal salt medium (BSM) with 215 mg Phe l^–1, the specific growth rate (0.30 h^–1) and Phe-degrading rate (29.6 mol l^–1 h^–1) were enhanced approximately ten and three times more than those obtained in the BSM with 215 mg Phe l^–1, respectively. Both cell growth and PAH degradation rates were increased with increasing Phe and Pyr concentrations, and B. cepacia 2A-12 had a tolerance against Phe and Pyr toxicity at the high concentration of 730–760 mg l^–1. Through kinetic analysis, the maximum specific growth rate ( _max) and PAH degrading rate ( _max) for Phe were obtained as 0.39 h^–1 and 300 mol l^–1 h^–1, respectively. Also, _max and _max for Pyr were 0.27 h^–1 and 52 mol l^–1 h^–1, respectively. B. cepacia 2A-12 could simultaneously degrade crude oil as well as PAHs, indicating that this bacterium is very useful for the removal of oils and PAHs contaminants.     

Regulation of apoptosis by somatostatin and substance P in peritoneal macrophages

Recent studies have shown that somatostatin (SOM) inhibits interleukin 6 (IL-6) and interferon gamma (IFNgamma) production by lymphocytes and peritoneal macrophages, whereas substance P (SP) enhances these cytokines production. To define the mechanism of the cytokine production enhancements and inhibitions by SOM and SP, we examined the expression of apoptosis modulator, p53, Bcl-2, Bax, inducible nitric oxide synthase (iNOS), Fas, caspase-8 and nitric oxide (NO) in thioglycolate-elicited peritoneal macrophages. SOM caused up-regulation of p53, Bcl-2, Fas and caspase-8 activities, and down-regulation of iNOS expression and NO production. On the other hand, SP slightly induces p53 and highly induces Bcl-2, iNOS expression and NO production. These data suggest that apoptosis by SOM may occur by a Bax- and NO-independent p53 accumulation, and through Fas and caspase-8 activation pathways, and that the inducible expression of Bcl-2 and NO production by SP may contribute to prevent the signals of apoptosis by Bax, and via Fas and caspase-8 activation.     

Cooperative roles of Bozozok/Dharma and Nodal-related proteins in the formation of the dorsal organizer in zebrafish

In vertebrates, specification of the dorso-ventral axis requires Wnt signaling, which leads to formation of the Nieuwkoop center and the Spemann organizer (dorsal organizer), through the nuclear accumulation of beta-catenin. Zebrafish bozozok/dharma (boz) and squint (sqt), which encode a homeodomain protein and a Nodal-related protein, respectively, are required for the formation of the dorsal organizer. The zygotic expression of boz and sqt in the dorsal blastoderm and dorsal yolk syncytial layer (YSL) was dependent on the maternally derived Wnt signal, and their expression at the late blastula and early gastrula stages was dependent on the zygotic expression of their own genes. The dorsal organizer genes, goosecoid (gsc) and chordin (din), were ectopically expressed in wild-type embryos injected with boz or sqt RNA. The expression of gsc strictly depended on both boz and sqt while the expression of din strongly depended on boz but only partially depended on sqt and cyclops (cyc, another nodal-related gene). Overexpression of boz in embryos defective in Nodal signaling elicited the ectopic expression of din but not gsc and resulted in dorsalization, implying that boz could induce part of the organizer, independent of the Nodal proteins. Furthermore, boz; sqt and boz;cyc double mutants displayed a severely ventralized phenotype with anterior truncation, compared with the single mutants, and boz;sqt;cyc triple mutant embryos exhibited an even more severe phenotype, lacking the anterior neuroectoderm and notochord, suggesting that Boz/Dharma and the Nodal-related proteins cooperatively regulate the formation of the dorsal organizer.     

Inhibitory effects of collagen on the PCR for detection of Clostridium perfringens

It is essential to identify specific food components that inhibit PCR in order to increase the sensitivity of the PCR method for rapid detection of pathogens contaminating a food. We found that collagen, a major component of several foods, inhibited PCR. The inhibitory action of collagen on PCR could be partially reversed by adjusting the concentration of magnesium ion in the reaction mixture and by the use of various DNA extraction methods to remove the collagen from the DNA. Also, the source of thermostable DNA polymerase was affected by the presence of collagen. These results suggest the need to optimize the extraction and assay conditions for rapid detection of enterotoxigenic Clostridium perfringens by PCR with respect to the kind of food being analyzed.     

Phospholipase D1 is phosphorylated and activated by protein kinase C in caveolin-enriched microdomains within the plasma membrane

Activities of phospholipase D (PLD) in diverse subcellular organelles have been identified but the details of regulatory mechanisms in such locations are unknown. Protein kinase C (PKC) is a major regulator of PLD. Serine 2, threonine 147, and serine 561 residues of phospholipase D1 (PLD1) were determined as sites of phosphorylation by PKC (Kim, Y., Han, J. M., Park, J. B., Lee, S. D., Oh, Y. S., Chung, C., Lee, T. G., Kim, J. H., Park, S. K., Yoo, J. S., Suh, P. G., Ryu, S. H. (1999) Biochemistry 38, 10344-10351). In our present study, a triple mutation of these phosphorylation sites diminished markedly phorbol 12-myristate 13-acetate (PMA)-induced PLD1 activity in COS-7 cells. We looked at the location of the PLD1 phosphorylation by PKC by observing PMA induced band shifts and by use of anti-phospho-PLD1 monoclonal antibody. The shifted PMA-induced proteins and the immunoreactivity of the anti-phospho-PLD1 antibody were mainly found in the caveolin-enriched membrane (CEM) fraction. Depletion of cellular cholesterol led to a loss of this compartmentalization of phosphorylated PLD1 in the CEM. Replacement of the cellular cholesterol led to the restoration of phosphorylated PLD1 in the CEM. Immunocytochemical studies of COS-7 cells revealed that PLD1 was localized in the plasma membrane as well as in the vesicular structures in the cytoplasm, but the phosphorylation of PLD1 occurred only in the plasma membrane. Our results, therefore, show that phosphorylation, and thereby activation, of PLD1 by PKC occurs in the caveolin and cholesterol-enriched low density domain of the plasma membrane in COS-7 cells.     

A Novel Technique for the Effective Production of Short Peptide Analogs from Concatameric Short Peptide Multimers

We designed a basic unit of the modified chicken gonadotropin releasing hormone II (cGnRH-II) peptide containing a trypsin cleavable linker peptide at both ends of the original peptide. We made a synthetic DNA coding for the modified cGnRH-II peptide with asymmetric and complementary cohesive ends of linker nucleotides. A tandemly repeated DNA cassette for the expression of concatameric short peptide multimers was constructed by ligating the basic units. The expressed peptide multimers were purified and subject to amino-terminal sequence analysis, which displayed the amino acid sequences expected from the designed nucleotides of the expression cassette. The monomeric cGnRH-II peptide analogs were generated after trypsin digestion. The present results showed that the technique developed for the production of the concatameric peptide multimers with cleavable linker peptides can be generally applicable to the production of short peptide analogs.     

Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae

In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1. Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation. The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation. The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development. However, the plants recovered from these negative growth effects when grown in a soil mixture. The TPS1 transgenic potato plants showed significantly increased drought resistance. These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.     

Dual requirement for rho and protein kinase C in direct activation of phospholipase D1 through G protein-coupled receptor signaling

G protein-coupled and tyrosine kinase receptor activation of phospholipase D1 (PLD1) play key roles in agonist-stimulated cellular responses such as regulated exocytosis, actin stress fiber formation, and alterations in cell morphology and motility. Protein Kinase C, ADP-ribosylation factor (ARF), and Rho family members activate PLD1 in vitro; however, the actions of the stimulators on PLD1 in vivo have been proposed to take place through indirect pathways. We have used the yeast split-hybrid system to generate PLD1 alleles that fail to bind to or to be activated by RhoA but that retain wild-type responses to ARF and PKC. These alleles then were employed in combination with alleles unresponsive to PKC or to both stimulators to examine the activation of PLD1 by G protein-coupled receptors. Our results demonstrate that direct stimulation of PLD1 in vivo by RhoA (and by PKC) is critical for significant PLD1 activation but that PLD1 subcellular localization and regulated phosphorylation occur independently of these stimulatory pathways.