Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate

Hydroxyl radical (·OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO₃) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the ·OH generation after injection of Fe-NTA or KBrO₃ in the kidneys. A significant increase in 8-hydroxy-2'-deoxyguanosine in nuclei of the kidney was detected only in the KBrO₃ treated mice, while the comet assay showed that the Fe-NTA and KBrO₃ treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by ·OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of ·OH generation in cellular injury induced by chemicals in vivo.     

Polyamines upregulate the mRNA expression of cationic amino acid transporter-1 in human retinal pigment epithelial cells

We previously showed that ornithine was mainly transported via cationic amino acid transporter (CAT)-1 in human retinal pigment epithelial (RPE) cell line, human telomerase RT (hTERT)-RPE, and that CAT-1 was involved in ornithine cytotoxicity in ornithine--aminotransferase (OAT)-deficient cell produced by a OAT specific inhibitor, 5-fluoromethylornithine (5-FMO). We showed here that CAT-1 mRNA expression was increased by ornithne in OAT-deficient RPE cells, which was reversed by an inhibitor of ornithine decarboxylase (ODC), -difluoromethylornithine (DFMO). Polyamines, especially spermine, one of the metabolites of ODC, also enhanced the expression of CAT-1 mRNA. ODC mRNA expression was also increased by ornithine and polyamines, and gene silencing of ODC by siRNA decreased ornithine transport activity and its cytotoxicity. In addition, the mRNA of nuclear protein c-myc was also increased in 5-FMO- and ornithine-treated hTERT-RPE cells, and gene silencing of c-myc prevented the induction of CAT-1 and ODC. Increases in expression of CAT-1, ODC, and c-myc, and the inhibition of these stimulated expression by DFMO were also observed in primary porcine RPE cells. These results suggest that spermine plays an important role in stimulation of mRNA expression of CAT-1, which is a crucial role in ornithine cytotoxicity in OAT-deficient hTERT-RPE cells. ornithine transport; ornithine decarboxylase; c-myc     

Distribution of virulence profiles related to new toxins and putative adhesins in Shiga toxin-producing Escherichia coli isolated from diverse sources in Brazil

The distribution of virulence markers related to cytolethal distending toxin-V (CDT-V), subtilase cytotoxin (SubAB), the enterohemorrhagic Escherichia coli factor for adherence (Efa1), the adhesin similar to IrgA (Iha), the long polar fimbriae (LpfO113), the autoagglutinating adhesin (Saa), and the protein required for full expression of adherence of O157:H7 Sakai strain (ToxB) was investigated in 121 Shiga toxin-producing E. coli (STEC) strains isolated in Brazil. STEC strains were isolated from human infections (n=49), cattle (n=68) and ground meat samples (n=4). Overall, the lpfA(O113), iha, efa1, saa, and toxB sequences were observed in 89.2%, 87.6%, 47.1%, 43%, and 13.2% of the strains, respectively. The genes efa1 (96.6%) and toxB (27%) were only identified among eae-positive strains, while saa (83.8%), cdt-V (12.9%), and subAB (48.4%) just occurred in eae-negative STEC strains. STEC strains harboring cdt-V and subAB were for the first time described in the South American subcontinent. In addition, the simultaneous presence of cdt-V and subAB has not been previously reported, nor the presence of subAB in STEC O77, O79, O105, O174, and O178 serogroups. A diversity of virulence profiles was observed among the STEC strains studied. The most prevalent profile observed among eae-positive STEC strains mainly isolated from humans was eae efa1 iha lpfA(O113), whereas iha lpfA(O113) saa ehxA subAB prevailed among eae-negative STEC strains, mostly isolated from cattle and foods.     

Evaluation of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the identification of Campylobacter strains isolated from poultry in Thailand

We have recently developed a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for identifying Campylobacter jejuni, C. coli and C. fetus. In the present study, the applicability of this assay was evaluated with 34 Campylobacter-like organisms isolated from poultry in Thailand for species identification and was compared with other assays including API Campy, 16S rRNA gene sequence, and hippuricase (hipO) gene detection. Of the 34 strains analyzed, 20, 10 and 1 were identified as C. jejuni, C. coli, and Arcobacter cryaerophilus, respectively, and 3 could not be identified by API Campy. However, 16S rRNA gene analysis, showed that all 34 strains are C. jejuni/coli. To discriminate between these 2 species, the hipO gene, which is specifically present in C. jejuni, was examined by PCR and was detected in 20 strains, which were identified as C. jejuni by API Campy but not in the remaining 14 strains. Collective results indicated that 20 strains were C. jejuni whereas the 14 strains were C. coli. When the cdt gene-based multiplex PCR was employed, however, 19, 20 and 19 strains were identified as C. jejuni while 13, 14 and 13 were identified as C. coli by the cdtA, cdtB and cdtC gene-based multiplex PCR, respectively. Pulsed-field gel electrophoresis revealed that C. jejuni and C. coli strains analyzed are genetically diverse. Taken together, these data suggest that the cdt gene-based multiplex PCR, particularly cdtB gene-based multiplex PCR, is a simple, rapid and reliable method for identifying the species of Campylobacter strains.     

Cloning and characterization of the gene encoding endo-beta-1,3-glucanase from Arthrobacter sp. NHB-10

The gluA gene, encoding an endo-beta-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-beta-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-beta-1,3-glucanases, but GluA was partially similar to two fungal exo-beta-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two beta-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-beta-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.     

Molecular cloning and functional expression of soybean allene oxide synthases

A plant allene oxide synthase (AOS) reacting with 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid (13-HPOT), a lipoxygenase product of alpha-linolenic acid, provides an allene oxide which functions as an intermediate for jasmonic acid (JA) synthesis, making AOS a key enzyme regulating the JA level in plants. Although AOSs in various plants have been investigated, there is only limited information about AOSs in soybean (Glycine max). In this study, we cloned and characterized two soybean AOSs, GmAOS1 and GmAOS2, sharing 95% homology in the predicted amino acid sequences. GmAOS1 and GmAOS2 were composed of 564 and 559 amino acids respectively, with predicted N-terminal chloroplast-targeting signal peptides. Both AOSs expressed in Escherichia coli were selective for 13S-hydroperoxides of alpha-linolenic and linoleic acids, suggesting the potential of GmAOS1 and GmAOS2 to contribute to JA synthesis. GmAOS1 and GmAOS2 were expressed in leaves, stems, and roots, suggesting broad distribution in a soybean plant.     

Flies without Trehalose

Living organisms adapt to environmental changes through metabolic homeostasis. Sugars are used primarily for the metabolic production of ATP energy and carbon sources. Trehalose is a nonreducing disaccharide that is present in many organisms. In insects, the principal hemolymph sugar is trehalose instead of glucose. As in mammals, hemolymph sugar levels in Drosophila are regulated by the action of endocrine hormones. Therefore, the mobilization of trehalose to glucose is thought to be critical for metabolic homeostasis. However, the physiological role of trehalose as a hemolymph sugar during insect development remains largely unclear. Here, we demonstrate that mutants of the trehalose-synthesizing enzyme Tps1 failed to produce trehalose as expected but survived into the late pupal period and died before eclosion. Larvae without trehalose grew normally, with a slight reduction in body size, under normal food conditions. However, these larvae were extremely sensitive to starvation, possibly due to a local defect in the central nervous system. Furthermore, Tps1 mutant larvae failed to grow on a low-sugar diet and exhibited severe growth defects on a low-protein diet. These diet-dependent phenotypes of Tps1 mutants demonstrate the critical role of trehalose during development in Drosophila and reveal how animals adapt to changes in nutrient availability.