Anti-microbial action against verotoxigenic Escherichia coli O157:H7 of nitric oxide derived from sodium nitrite

The levels of verotoxin-1 and verotoxin-2 released by verotoxigenic Escherichia coli O157:H7 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were evaluated. Of the three strains of E. coli O157:H7 used in this study, two strains produced both verotoxin-1 and verotoxin-2, and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6000 mg/l, minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with a treatment by sodium chloride or antibiotics. When the electron paramagnetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77 K to clarify the mechanism for the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron-nitric oxide complexes. It appears that the dinitrosyl iron complexes in the E. coli O157:H7 cells were generated from the reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. The amount of ATP was decreased by the presence of sodium nitrite in the cell suspension. These findings indicate that nitric oxide derived from sodium nitrite penetrated the cells and inactivated enzymes related to the respiratory chain.     

Identification of the rat IgA Fc receptor encoded in the leukocyte receptor complex

Human FcRI (CD89) is a myeloid-specific IgA Fc receptor encoded in the leukocyte receptor complex. Thus far, no gene coding for FcRI has been identified in mice. Here, we show that, unlike mice, rats have the gene (Fcar) coding for FcRI. The rat Fcar gene has an exon-intron structure essentially identical to that of the human counterpart and is encoded in the leukocyte receptor complex on Chromosome 1. Southern blot analysis using the rat Fcar as a probe revealed hybridizing bands in Chinese and Syrian hamsters and gerbils, but not in mice, indicating that Fcar was lost in the lineage leading to mice after the divergence of rats and mice. Identification of FcRI in rats should facilitate the elucidation of the in vivo role of this receptor.The sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers: AB109766, AB109767, and AB109768     

Dominant microbial composition and its vertical distribution in saline meromictic Lake Kaiike (Japan) as revealed by quantitative oligonucleotide probe membrane hybridization

Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two delta-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the delta-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.     

Deactivation of photosynthetic activities is triggered by loss of a small amount of water in a desiccation-tolerant cyanobacterium, Nostoc commune

Changes in photosynthetic activities under hypertonic conditions were studied in a terrestrial, highly desiccation-tolerant cyanobacterium, Nostoc commune, and in some desiccation-sensitive cyanobacteria. The amounts of water sustained in the colony matrix outside the N. commune cells and the cellular solute concentration were estimated by measuring the water potential, and the solute concentration was supposed to correspond to around 0.22 M sorbitol. Incubation of the colonies in 0.8 M sorbitol solution inhibited the energy transfer from the phycobilisome (PBS) anchor to PSII core complexes. At higher sorbitol concentrations, light energy absorbed by PSI, PSII, and PBS was dissipated to heat. PSI and cyclic electron flow around PSI was also deactivated by hypertonic treatment. Fv/Fm and (Fm'-F)/Fm' values started to decrease at 0.6 and 0.3 M sorbitol and reached zero at 1.0 and 0.8 M, respectively. Decreases in these two fluorescence parameters corresponded to the decreases in PSII fluorescence (F695) and photosynthetic CO2 fixation, respectively. The intensity of delayed light emission started to decrease at 1.0 M sorbitol and became negligible at 4.0 M. Comparing these changes in N. commune with those in desiccation-sensitive species, we found that N. commune cells actively deactivates photosynthetic systems on sensing water loss.     

Repair machinery of symbiotic photosynthesis as the primary target of heat stress for reef-building corals

In a coral-algae symbiotic system, heat-dependent photoinhibition of photosystem II (PSII) leads to coral bleaching. When the reef-building coral Acropora digitifera was exposed to light, a moderate increase of temperature induced coral bleaching through photobleaching of algal pigments, but not through expulsion of symbiotic algae. Monitoring of PSII photoinhibition revealed that heat-dependent photoinhibition was ascribed to inhibition of the repair of photodamaged PSII, and heat susceptibility of the repair machinery varied among coral species. We conclude that the efficiency of the photosynthesis repair machinery determines the bleaching susceptibility of coral species under elevated seawater temperatures.     

Mice lacking inducible nitric oxide synthase show strong resistance to anti-Fas antibody-induced fulminant hepatitis

Although nitric oxide (NO) plays important roles in pathogenesis of various liver diseases, the role of NO in the in vivo mechanism of Fas-mediated fulminant hepatitis is not known well. The effect of anti-Fas antibody (Jo2) on the survival, liver function, and histology was analyzed in wild-type (WT) and inducible NO synthase (iNOS)-deficient (iNOS(-/-)) mice. Upon intravenous injection of a lethal dose of Jo2, WT mice died on fulminant hepatitis within 12h. Under identical conditions, however, iNOS(-/-) mice showed strong resistance to Jo2 and survived without revealing liver injury. In conclusion, these observations suggest that regulation of NO metabolism may have therapeutic potential in the treatment of patients with fulminant hepatitis.     

Characterization of short-chain dehydrogenase/reductase homologues of Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C)

Short-chain dehydrogenase/reductase homologues from Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C) show high sequence similarity to serine dehydrogenase from Agrobacterium tumefaciens. We cloned each gene encoding YdfG and YMR226C into E. coli JM109 and purified them to homogeneity from the E. coli clones. YdfG and YMR226C consist of four identical subunits with a molecular mass of 27 and 29 kDa, respectively. Both enzymes require NADP(+) as a coenzyme and use L-serine as a substrate. Both enzymes show maximum activity at about pH 8.5 for the oxidation of L-serine. They also catalyze the oxidation of D-serine, L-allo-threonine, D-threonine, 3-hydroxyisobutyrate, and 3-hydroxybutyrate. The k(cat)/K(m) values of YdfG for L-serine, D-serine, L-allo-threonine, D-threonine, L-3-hydroxyisobutyrate, and D-3-hydroxyisobutyrate are 105, 29, 199, 109, 67, and 62 M(-1) s(-1), and those of YMR226C are 116, 110, 14600, 7540, 558, and 151 M(-1) s(-1), respectively. Thus, YdfG and YMR226C are NADP(+)-dependent dehydrogenases acting on 3-hydroxy acids with a three- or four-carbon chain, and L-allo-threonine is the best substrate for both enzymes.     

Catalytic mechanism of the maloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26

Haloalkane dehalogenases are bacterial enzymes capable of carbon-halogen bond cleavage in halogenated compounds. To obtain insights into the mechanism of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), we studied the steady-state and presteady-state kinetics of the conversion of the substrates 1-chlorohexane, chlorocyclohexane, and bromocyclohexane. The results lead to a proposal of a minimal kinetic mechanism consisting of three main steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond with simultaneous formation of an alkyl-enzyme intermediate, and (iii) hydrolysis of the alkyl-enzyme intermediate. Release of both products, halide and alcohol, is a fast process that was not included in the reaction mechanism as a distinct step. Comparison of the kinetic mechanism of LinB with that of haloalkane dehalogenase DhlA from Xantobacter autotrophicus GJ10 and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 shows that the overall mechanisms are similar. The main difference is in the rate-limiting step, which is hydrolysis of the alkylenzyme intermediate in LinB, halide release in DhlA, and liberation of an alcohol in DhaA. The occurrence of different rate-limiting steps for three enzymes that belong to the same protein family indicates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolutionary closely related proteins. The differences in the rate-limiting step were related to: (i) number and size of the entrance tunnels, (ii) protein flexibility, and (iii) composition of the halide-stabilizing active site residues based on comparison of protein structures.