Relationship between gastric ulcer and Helicobacter pylori VacA detected in gastric juice using bead-ELISA method

Background. VacA is an important pathogenetic factor produced by Helicobacter pylori. VacA has often been detected in supernatants of liquid cultures or lysates of whole bacterial cells. However, no studies have ever tried to assay VacA produced in the human stomach. We applied a very sensitive and simple method, bead‐ELISA, to detect VacA in gastric juice. Materials and Methods. Forty‐eight H. pylori‐positive patients (16 nonulcer dyspepsia, 16 gastric ulcer, and 16 duodenal ulcer) and four H. pylori‐negative nonulcer dyspepsia patients had endoscopy performed and gastric juice were aspirated. Polystyrene beads coated with the antibody to VacA, were used in this bead‐ELISA method. The nucleotide sequences of vacA in the signal and middle regions were investigated. Results. Of the 48 samples that were positive for H. pylori, 21 [43.8%] were found to be VacA positive in gastric juice. The average and maximum concentrations of detected VacA in gastric juice were 143.2 ± 216.5 and 840 pg/ml, respectively. The average density of VacA from gastric ulcer patients (227.5 ± 276.7 pg/ml) was higher than that found in nonulcer dyspepsia (51.8 ± 39.8 pg/ml) and duodenal ulcer (49.2 ± 21.5 pg/ml) patients. There was no relationship between VacA in gastric juice and vacA genotype. Conclusions. VacA in gastric juice could be directly detected by bead‐ELISA. In this study, the diversity of disease outcome was associated with not the quality but the quantity of VacA. Therefore, not only the quality but also the quantity of VacA is important etiological factors in the pathogenesis of mucosal damage.     

PPARgamma ligands suppress proliferation of human urothelial basal cells in vitro

Expression of peroxisome proliferator-activated receptor (PPAR) gamma in the human urinary tract through embryonic development suggests its possible roles in the development, proliferation, and differentiation of uroepithelium. Little is known, however, about physiological roles of PPARgamma in the urinary tract. We investigated effects of PPARgamma ligands on the proliferation of normal human urothelial cells and stromal cells cultivated from surgical specimens. Active proliferation in vitro as well as high molecular weight cytokeratin expression indicated that cultured urothelial cells possess basal cell phenotype. PPARgamma protein, expressed predominantly in the epithelial layer of the normal human urinary tract in vivo, was abundantly expressed in urothelial cells but barely detectable in stromal cells in vitro. Natural ligand for PPARgamma, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), as well as synthetic ones, troglitazone and pioglitazone, suppressed proliferation of the urothelial cells dose-dependently. These effects were PPARgamma specific because clofibrate or PGF(2alpha) did not affect proliferation of urothelial cells. Neither 9-cis retinoic acid or all-trans retinoic acid (ATRA) at 1 microM showed any synergism on the antiproliferative effects of PPARgamma ligands. Urothelial cells treated with PPARgamma ligands showed drastic morphologic changes and cell cycle arrest at G0/G1 phase accompanied with increased mRNA level of a cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Since 15d-PGJ(2) is present in vivo during the resolution phase of inflammation, these results indicated that PPARgamma might be involved in the terminal phase of urothelial re-epithelialization processes.     

Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing

In an effort to clarify the responses of a wide range of plant cells to freezing, we examined the responses to freezing of the cells of chilling-sensitive and chilling-resistant tropical and subtropical plants. Among the cells of the plants that we examined, those of African violet ( Saintpaulia grotei Engl.) leaves were most chilling-sensitive, those of hypocotyls in mungbean [ Vigna radiata (L.) R. Wilcz.] seedlings were moderately chilling-sensitive, and those of orchid [ Paphiopedilum insigne (Wallich ex Lindl.) Pfitz.] leaves were chilling-resistant, when all were chilled at -2 degrees C. By contrast, all these plant cells were freezing-sensitive and suffered extensive damage when they were frozen at -2 degrees C. Cryo-scanning electron microscopy (Cryo-SEM) confirmed that, upon chilling at -2 degrees C, both chilling-sensitive and chilling-resistant plant cells were supercooled. Upon freezing at -2 degrees C, by contrast, intracellular freezing occurred in Saintpaulia leaf cells, frost plasmolysis followed by intracellular freezing occurred in mungbean seedling cells, and extracellular freezing (cytorrhysis) occurred in orchid leaf cells. We postulate that chilling-related destabilization of membranes might result in the loss of the ability of the plasma membrane to act as a barrier against the propagation of extracellular ice in chilling-sensitive plant cells. We also examined the role of cell walls in the response to freezing using cells in which the plasma membrane had been disrupted by repeated freezing and thawing. In chilling-sensitive Saintpaulia and mungbean cells, the cells with a disrupted plasma membrane responded to freezing at -2 degrees C by intracellular freezing. By contrast, in chilling-resistant orchid cells, as well as in other cells of chilling-resistant and freezing-resistant plant tissues, including leaves of orchard grass ( Dactylis glomerata L.), leaves of Arabidopsis thaliana (L.) Heynh. and cortical tissues of mulberry ( Morus bombycis Koids.), cells with a disrupted plasma membrane responded to freezing by extracellular freezing. Our results indicate that, in the chilling-sensitive plants cells that we examined, not only the plasma membrane but also the cell wall lacked the ability to serve as a barrier against the propagation of extracellular ice, whereas in the chilling-resistant plant cells that we examined, not only the plasma membrane but also the cell wall acted as a barrier against the propagation of extracellular ice. It appears, therefore, that not only the plasma membrane but also the cell wall greatly influences the freezing behavior of plant cells.     

Unique fluorescence properties of a cyanobacterium Gloeobacter violaceus PCC 7421: reasons for absence of the long-wavelength PSI Chl a fluorescence at -196 degrees C

We investigated the reason for the absence of the long-wavelength PSI Chl a fluorescence at -196 degrees C in the cyanobacterium Gloeobacter violaceus using two methods: p-nitrothiophenol (p-NTP) treatment and time-resolved fluorescence spectra. The p-NTP treatment showed that PSII Chl a fluorescence was specifically affected in a manner similar to that for Synechocystis sp. PCC 6803 and spinach chloroplasts, although there were no components modified by the p-NTP treatment, indicating an absence of the long-wavelength PSI Chl a fluorescence. The time-resolved fluorescence spectra with a time resolution of 1.3 ps and spectral resolution of 1.0 nm gave no indication of the presence of the long-wavelength PSI fluorescence in the wavelength region between 700 nm and 760 nm, indicating that a very fast energy transfer among Chl a molecules could not account for the absence of the long-wavelength PSI fluorescence. From these data, it seems that the absence of the long-wavelength PSI fluorescence is due to a lack of the formation of a component responsible for the fluorescence at -196 degrees C, which may originate from a difference in the amino acid sequence. We discuss the significance of this phenomenon and interpret our findings in terms of the evolution of cyanobacteria.     

Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene

Autophagy is an intracellular process for vacuolar bulk degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has recently begun to be elucidated at the cellular level, but the role that autophagy plays at the organismal level has yet to be determined. In this study, a genome-wide search revealed significant conservation between yeast and plant autophagy genes. Twenty-five plant genes that are homologous to 12 yeast genes essential for autophagy were discovered. We identified an Arabidopsis mutant carrying a T-DNA insertion within AtAPG9, which is the only ortholog of yeast Apg9 in Arabidopsis (atapg9-1). AtAPG9 is transcribed in every wild-type organ tested but not in the atapg9-1 mutant. Under nitrogen or carbon-starvation conditions, chlorosis was observed earlier in atapg9-1 cotyledons and rosette leaves compared with wild-type plants. Furthermore, atapg9-1 exhibited a reduction in seed set when nitrogen starved. Even under nutrient growth conditions, bolting and natural leaf senescence were accelerated in atapg9-1 plants. Senescence-associated genes SEN1 and YSL4 were up-regulated in atapg9-1 before induction of senescence, unlike in wild type. All of these phenotypes were complemented by the expression of wild-type AtAPG9 in atapg9-1 plants. These results imply that autophagy is required for maintenance of the cellular viability under nutrient-limited conditions and for efficient nutrient use as a whole plant.     

Transforming growth factor alpha protects against Fas-mediated liver apoptosis in mice

The Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibody to mice causes massive hepatic apoptosis and fulminant hepatic failure. Several growth factors have recently been found to function in preventing apoptosis. In this study, we demonstrated that overexpression of transforming growth factor alpha (TGFalpha) has a dramatic protective effect on Fas-mediated hepatic apoptosis at the biochemical and histological levels. Moreover, 85.7% (six out of seven) of TGFalpha transgenic mice survived the lethal liver damage, whereas all wild-type mice died. Expression of Bcl-xL, an anti-apoptotic protein, was greatly increased in the transgenic mice. Taken together, our findings suggest that TGFalpha protects against Fas-mediated liver apoptosis in vivo and up-regulation of Bcl-xL may participate in protective effect of TGFalpha.     

Allosteric activation of cGMP-specific,cGMP-binding phosphodiesterase (PDE5) by cGMP

The effects of cGMP binding on the catalytic activity of cGMP-specific, cGMP-binding phosphodiesterase (PDE5) are unclear because cGMP interacts with both allosteric and catalytic sites specifically. We studied the effects of cGMP on the hydrolysis of a fluorescent substrate analogue, 2'-O-anthraniloyl cGMP, by PDE5 partially purified from rat cerebella. The preparation contained PDE5 as the major cGMP-PDE activity and was not contaminated with cAMP- or cGMP-dependent protein kinases. The Hill coefficients for hydrolysis of the analogue substrate were around 1.0 in the presence of cGMP at concentrations <0.3 microM, while they increased to 1.5 at cGMP concentrations >1 microM, suggesting allosteric activation by cGMP at concentrations close to the bulk binding constant of the enzyme. Consistent with an allosteric activation, increasing concentrations of cGMP enhanced the hydrolysis rate of fixed concentrations of 2'-O-anthraniloyl cGMP, which overcame competition between the two substrates. Such activation was not observed with cAMP, cyclic inosine 3',5'-monophosphate, or 2'-O-monobutyl cGMP, indicating specificity of cGMP. These results demonstrate that cGMP is a specific and allosteric activator of PDE5, and suggest that in cells containing PDE5, such as cerebellar Purkinje cells, intracellular cGMP concentrations may be regulated autonomously through effects of cGMP on PDE5.     

Cyclooxygenase-2 inhibitor inhibits the fracture healing

We investigated the effects of cyclooxigenase-2 (cox-2) on fracture healing. After closed non-displaced fractures were created at the middle of both femoral shafts in 12-week-old Wister rats, a cox-2 specific inhibitor, etodolac (20 mg/day; intra-peritoneal) was administered every day for three weeks (E group). Bone union and callus formation were evaluated by weekly radiographs. Three weeks after surgery, the mechanical strength of the fractured femur was evaluated by a three-point-bending test. These results were compared with those of a vehicle control group (V group). The fracture healing score on radiographs in the E group three weeks after the surgery was 3.3 +/- 0.9, and in the V group it was 5.8 +/- 1.5, indicating that fracture healing was significantly poorer in the E than the V group (p < 0.05). From the three point bending test, the ultimate strength and stiffness of etodolac-treated fractured femurs were shown to be significantly lower than those in vehicle control group (p < 0.05). Mechanically, femurs of etodolac treated rats were weaker than those of control rats. Thus, it was concluded that etodolac, a cox-2 specific inhibitor, inhibited fracture healing.     

Identification of thermoacidophilic bacteria and a new Alicyclobacillus genomic species isolated from acidic environments in Japan

Sixty strains of thermoacidophilic bacteria have been isolated from soil and water samples obtained from various acidic environments in Japan. An initial comparative sequence analysis of the hypervariable regions of the 16S rDNA revealed that all strains could be assigned to the Alicyclobacillus acidocaldarius- Alicyclobacillus genomic species 1 group, which could be further subdivided into three clusters (Clusters I-III). On the basis of phenotypic characteristics, chemotaxonomic profiles, and phylogenetic data of six selected strains, five strains were identified as either A. acidocaldarius or Alicyclobacillus genomic species 1; however, one strain (MIH 332) could not be determined to belong to either of these species. 16S rDNA sequence homology values between strain MIH 332 and the reference strains of A. acidocaldarius (ATCC 27009(T)) and Alicyclobacillus genomic species 1 (DSM 11984) were 98.8% and 99.1%, respectively, which were higher than the corresponding similarity between the reference strains (98.4%). On the other hand, DNA-DNA hybridization levels between strain MIH 332 and the reference strains were 39% and 44%, respectively, which were lower than the value between the reference strains (59% or 65%). However, the phenotype of strain MIH 332 was also similar to those of the reference strains, and a typical phenotype could not be found for the strain, thus indicating that the strain may be a new genomic species of A. acidocaldarius, for which the name Alicyclobacillus genomic species 2 is tentatively proposed. The results of this study suggest that A. acidocaldarius and its related species are widely distributed in acidic environments in Japan, with slight regional variations in morphological and genotypic characteristics.