Helicobacter pylori in Japanese river water and its prevalence in Japanese children

AIMS: The major transmission route of Helicobacter pylori remains unclear. In this study, we examined H. pylori in the environmental waters in Japan. METHODS AND RESULTS: A total of 24 water samples were collected from the upper, middle and downstream reaches of four Japanese rivers. Helicobacter pylori-specific DNA was examined using nested PCR. In addition, 224 children who lived near one river were studied by the stool antigen test for H. pylori prevalence. Helicobacter pylori DNA was detected in the water from the middle and downstream reaches of all four rivers, but not in the upper reaches. Helicobacter pylori was not found in cultured water samples with positive PCR results. Helicobacter pylori prevalence in the children examined was 9.8% for those living near the middle reaches and 23.8% nearby downstream, both of which were higher than the value in an area distant from the river (0%) (both, P < 0.01). CONCLUSIONS: Difference in H. pylori prevalence in the children may be related to the presence of H. pylori in the river. The results of this study showed that H. pylori DNA is frequently present in river water from the middle and downstream reaches in which the human biosphere is embedded. SIGNIFICANCE AND IMPACT OF THE STUDY: It is suggested that river water in the natural environment could be a risk factor for H. pylori transmission.     

Spontaneous transformation and its use for genetic mapping in Bacillus subtilis

Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores. Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment. We also found evidence that the spontaneous transformation system can be used for genetic mapping in B. subtilis.     

Accumulation and degradation of thiamin-binding protein and level of thiamin in wheat seeds during seed maturation and germination

Changes in the levels of thiamin-binding globulin and thiamin in wheat seeds during maturation and germination were studied. The thiamin-binding activity of the seed proteins increased with seed development after flowering. The thiamin content of the seeds also increased with development. Thiamin-binding activity decreased during seed germination. On the other hand, immunological analysis using an antibody directed against the thiamin-binding protein isolated from wheat seeds showed that the thiamin-binding globulin accumulated in the aleurone layer of the seeds during maturation, and then the protein was degraded and disappeared during seed germination. These results suggested that the thiamin-binding globulin of wheat seeds was synthesized and accumulated in the aleurone layer of the seeds with seed development, similar to the thiamin-binding albumin in sesame seeds, and that thiamin bound to the thiamin-binding globulin in the dormant wheat seeds for germ growth during germination.     

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO₂) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from ¹⁵N-labeled NO₂ taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with ¹⁵NO₂. Secondly, tobacco (Nicotiana tabacum L.) plants fed with ¹⁵N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.Abbreviations NO Nitric oxide - NO₂ Nitrogen dioxide - RNS Reactive nitrogen species - UN Unidentified nitrogen - TNNAT, RNNAT, INNAT and UNNAT Total, Kjeldahl, inorganic and unidentified nitrogen in naturally fed plants, respectively - TNNIT, RNNIT, INNIT and UNNIT Total, Kjeldahl, inorganic and unidentified nitrogen derived from nitrate, respectively - TNNO2, RNNO2, INNO2 and UNNO2 Total, Kjeldahl, inorganic and unidentified nitrogen derived from NO₂, respectively     

Characterization of the portal signal during 24-h glucose delivery in unrestrained, conscious rats

To characterize the "portal signal" during physiological glucose delivery, liver glycogen was measured in unrestrained rats during portal (Po) and peripheral (Pe) constant-rate infusion, with minimal differences in hepatic glucose load (HGL) and portal insulin between the delivery routes. Hepatic blood flows were measured by Doppler flowmetry during open surgery. Changes in hepatic glucose, portal insulin, glucagon, lactate, and free fatty acid concentrations were generally similar in either delivery except for glucagon at 4 h. Hepatic glycogen, however, increased continuously in Po and was higher than Pe at 8 and 24 h, although it decreased to the level of Pe upon the removal of Po at 8 h. There was a near-linear relationship between hepatic glycogen and HGL in either delivery, with the slope being twice as high in Po and the intercepts converging to basal HGL. The hepatic response to Po did not alter upon 80% replacement by Pe. These results suggest that negative arterial-portal glucose gradients increase the rate of hepatic glycogen synthesis against the incremental HGL in an all-or-nothing mode.     

Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments

Despite recent progress in sequencing the complete genome of rice (Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60–98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Jürgens     

Neuropeptide Y (NPY) suppresses experimental autoimmune encephalomyelitis: NPY1 receptor-specific inhibition of autoreactive Th1 responses in vivo

Prior studies have revealed that the sympathetic nervous system regulates the clinical and pathological manifestations of experimental autoimmune encephalomyelitis (EAE), an autoimmune disease model mediated by Th1 T cells. Although the regulatory role of catecholamines has been indicated in the previous works, it remained possible that other sympathetic neurotransmitters like neuropeptide Y (NPY) may also be involved in the regulation of EAE. Here we examined the effect of NPY and NPY receptor subtype-specific compounds on EAE, actively induced with myelin oligodendrocyte glycoprotein 35-55 in C57BL/6 mice. Our results revealed that exogenous NPY as well as NPY Y(1) receptor agonists significantly inhibited the induction of EAE, whereas a Y(5) receptor agonist or a combined treatment of NPY with a Y(1) receptor antagonist did not inhibit signs of EAE. These results indicate that the suppression of EAE by NPY is mediated via Y(1) receptors. Furthermore, treatment with the Y(1) receptor antagonist induced a significantly earlier onset of EAE, indicating a protective role of endogenous NPY in the induction phase of EAE. We also revealed a significant inhibition of myelin oligodendrocyte glycoprotein 35-55-specific Th1 response as well as a Th2 bias of the autoimmune T cells in mice treated with the Y(1) receptor agonist. Ex vivo analysis further demonstrated that autoimmune T cells are directly affected by NPY via Y(1) receptors. Taken together, we conclude that NPY is a potent immunomodulator involved in the regulation of the Th1-mediated autoimmune disease EAE.     

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).     

Initiation and transduction of stretch-induced RhoA and Rac1 activation through caveolae: cytoskeletal regulation of ERK translocation

The Rho family small GTPases play a crucial role in mediating cellular responses to stretch. However, it remains unclear how force is transduced to Rho signaling pathways. We investigated the effect of stretch on the activation and caveolar localization of RhoA and Rac1 in neonatal rat cardiomyocytes. In unstretched cardiomyocytes, RhoA and Rac1 were detected in both caveolar and non-caveolar fractions as assessed using detergent-free floatation analysis. Stretching myocytes for 4 min activated RhoA and Rac1. By 15 min of stretch, RhoA and Rac1 had dissociated from caveolae, and there was decreased coprecipitation of RhoA and Rac1 with caveolin-3. To determine whether compartmentation of RhoA and Rac1 within caveolae was necessary for stretch signaling, we disrupted caveolae with methyl beta-cyclodextrin (MbetaCD). Treatment with 5 mm MbetaCD for 1 h dissociated both RhoA and Rac1 from caveolae. Under this condition, stretch failed to activate RhoA or Rac1. Stretch-induced actin cytoskeletal organization was concomitantly impaired. Interestingly the ability of stretch to activate extracellular signal-regulated kinase (ERK) was unaffected by MbetaCD treatment, but ERK translocation to the nucleus was impaired. Stretch-induced hypertrophy was also inhibited. Actin cytoskeletal disruption with cytochalasin-D also prevented stretch from increasing nuclear ERK, whereas actin polymerization with jasplakinolide restored nuclear translocation of activated ERK in the presence of MbetaCD. We suggest that activation of RhoA or Rac1, localized in a caveolar compartment, is essential for sensing externally applied force and transducing this signal to the actin cytoskeleton and ERK translocation.     

Molecular mechanisms of DNA damage induced by procarbazine in the presence of Cu(II)

Procarbazine [N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide], a hydrazine derivative, which has been shown to have effective antineoplastic activity, induces cancer in some experimental animals and humans. To clarify a new mechanism for its carcinogenic effect, we examined DNA damage induced by procarbazine in the presence of metal ion, using 32P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Procarbazine plus Cu(II) induced piperidine-labile and formamidopyrimidine-DNA glycosylase-sensitive lesions at the 5'-ACG-3' sequence, complementary to a hotspot of the p53 gene, and the 5'-TG-3' sequence. Catalase partially inhibited DNA damage, suggesting that not only H(2)O(2) but also other reactive species are involved. Procarbazine plus Cu(II) significantly increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, which was completely inhibited by calatase. Electron spin resonance spin-trapping experiments revealed that methyl radicals were generated from procarbazine and Cu(II). On the basis of these findings, it is considered that procarbazine causes DNA damage through non-enzymatic formation of the Cu(I)-hydroperoxo complex and methyl radicals. In conclusion, in addition to alkylation, oxidative DNA damage may play important roles in not only antitumor effects but also mutagenesis and carcinogenesis induced by procarbazine.