Biomedical subjects

Kouhei Ohnishi

Publications and source records attributed to Kouhei Ohnishi.

At least 19 recordsLinked to original sources

PubMed2025

Sucrose and malic acid in the tobacco plant induce hrp regulon in a phytopathogen Ralstonia pseudosolanacearum.

Genes encoding a type III secretion system in Ralstonia pseudosolanacearum are regulated by HrpB as an hrp regulon and induced only in plants. This study aimed to identify the plant signals that induce the hrp regulon and confirm the signal recognition mechanism. Signaling molecules that induce hrpB expression were screened using resting cells of the hrpB-lacZ reporter strain. Only the soluble fraction of smashed tobacco seedlings induced hrpB expression. The heated soluble fraction retained its hrpB-inducing activity, indicating that the signaling molecules were not proteins. When the soluble fraction was fractionated into acidic, neutral, and basic components, both the acidic and neutral fractions induced hrpB expression. As neutral compounds, sucrose, glucose, and fructose have been found to induce hrpB expression. Sucrose-induced hrpB expression was greatly reduced in the prhA mutant, indicating that the TonB-dependent receptor PrhA perceives sugars. Among the organic acids found in the acidic fractions, malic acid most efficiently induced hrpB expression, which was reduced by the mutation of a hybrid histidine kinase gene of a two-component system, rsc1598, indicating that Rsc1598 may sense malic acid. We demonstrated direct binding of Rsc1598 to malic acid using isothermal titration calorimetry.IMPORTANCESimilar to other Gram-negative plant pathogens, the type III secretion system (T3SS) is the most important virulence factor in Ralstonia pseudosolanacearum. The genes for the T3SS are regulated as an hrp regulon, activated only when the pathogen encounters the plants, indicating that the pathogen must sense plant signals. For the first time, we identified two signaling compounds, sucrose and malic acid, that are abundantly found in tobacco roots. The hrp operon was induced even in non-host plants, possibly because sucrose and malic acid are common in plants. We also found that R. pseudosolanacearum membrane proteins received sucrose and malic acid independently. As a next step, antagonists of signaling molecules can be screened.

Malates↗

PubMed2006

Amino acid substitutions in GyrA of Burkholderia glumae are implicated in not only oxolinic acid resistance but also fitness on rice plants.

Oxolinic acid (OA) resistance in field isolates of Burkholderia glumae, a causal agent of bacterial grain rot, is dependent on an amino acid substitution at position 83 in GyrA (GyrA83). In the present study, among spontaneous in vitro mutants from the OA-sensitive B. glumae strain Pg-10, we selected OA-resistant mutants that emerged at a rate of 5.7 x 10(-10). Nucleotide sequence analysis of the quinolone resistance-determining region in GyrA showed that Gly81Cys, Gly81Asp, Asp82Gly, Ser83Arg, Asp87Gly, and Asp87Asn are observed in these OA-resistant mutants. The introduction of each amino acid substitution into Pg-10 resulted in OA resistance, similar to what was observed for mutants with the responsible amino acid substitution. In vitro growth of recombinants with Asp82Gly was delayed significantly compared to that of Pg-10; however, that of the other recombinants did not differ significantly. The inoculation of each recombinant into rice spikelets did not result in disease. In inoculated rice spikelets, recombinants with Ser83Arg grew less than Pg-10 during flowering, and growth of the other recombinants was reduced significantly. On the other hand, the reduced growth of recombinants with Ser83Arg in spikelets was compensated for under OA treatment, resulting in disease. These results suggest that amino acid substitutions in GyrA of B. glumae are implicated in not only OA resistance but also fitness on rice plants. Therefore, GyrA83 substitution is thought to be responsible for OA resistance in B. glumae field isolates.

Amino Acid Substitution↗

PubMed2006

Identification of a new tetrameric pyridoxal 4-dehydrogenase as the second enzyme in the degradation pathway for pyridoxine in a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti.

We have found for the first time that a chromosomal gene, mlr6807, in Mesorhizobium loti encodes a new tetrameric pyridoxal 4-dehydrogenase (PLDH). The recombinant enzyme expressed in Escherichia coli cells was homogenously purified and characterized. The enzyme consisted of four subunits each with a molecular weight of 26,000+/-1000, and exhibited Km and kcat values of 91+/-2 microM and 149+/-1s(-1), respectively. PLDH used NAD+ as a cosubstrate, showed no activity toward sugars, and belonged to a short-chain dehydrogenase/reductase family. The mlr6807 gene-disrupted M. loti cells could grow in a nutrient-rich TY medium but not in a synthetic one containing pyridoxine or pyridoxamine as the sole carbon and nitrogen source. Thus, it was found that PLDH is essential for the assimilation of vitamin B6 compounds and the second step enzyme in their degradation pathway in M. loti.

Alcohol Oxidoreductases↗

PubMed2006

Molecular cloning, expression and characterization of pyridoxamine-pyruvate aminotransferase.

Pyridoxamine-pyruvate aminotransferase is a PLP (pyridoxal 5'-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine-pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the alpha family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429-432]. The K(d) value for pyridoxal determined by means of CD was 100-fold lower than the K(m) value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed.

Alphaproteobacteria↗

PubMed2006

Comparative analysis of induction pattern of programmed cell death and defense-related responses during hypersensitive cell death and development of bacterial necrotic leaf spots in eggplant.

Pseudomonas cichorii causes necrotic leaf spots (NLS), while Pseudomonas syringae pv. tabaci induces a hypersensitive response (HR) in eggplant. P. cichorii induced cell death at 9 h after inoculation (HAI), reaching a maximum of around 24-30 HAI. On the other hand, cell death was induced 6 HAI with P. syringae pv. tabaci, reaching a maximum of around 12-18 HAI. Superoxide generation was observed in eggplant inoculated with both bacteria. DNA fragmentation, cytochrome c release into the cytosol and expression of defense-related genes such as PR-1 and hsr203J was also induced by inoculation with both bacteria, but these plant reactions were more rapidly induced in eggplant inoculated with P. syringae pv. tabaci rather than those with P. cichorii. Lipid peroxidation and induction of lipoxygenase (LOX) was drastically induced in eggplant inoculated with P. syringae pv. tabaci compared to P. cichorii-inoculated eggplant. Pharmacological studies showed that induction of the cell death, and the NLS or the HR in response to both bacteria was commonly associated with de novo protein synthesis, reactive oxygen species and caspase III-like protease. Interestingly, involvement of lipid peroxidation, LOX, serine protease, and DNase differed between induction of NLS and HR. These results suggest that programmed cell death might be closely associated not only with the HR but also NLS. However, there may be differences not only in the induction kinetics and level of plant responses but also in the infection-related responses between HR and NLS.

Cell Death↗

PubMed2006

Directed evolution of extradiol dioxygenase by a novel in vivo DNA shuffling.

RecA-dependent homologous recombination in Escherichia coli is a very effective way to construct chimeras between two homologous genes. The disadvantage of in vivo method is a small library size of chimeric genes in comparison with in vitro DNA shuffling. In order to overcome the disadvantage, we have developed novel in vivo DNA shuffling methods with successive homologous recombinations. Linearized DNA molecules with two homologous genes were made with ligation rather than the conventional restriction enzyme cleavage between two genes. The three-way ligation of a vector and two homologous bphC genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenases or the two-way ligation of the donor bphC gene and an acceptor plasmid carrying the homologous bphC gene generated a variety of linearized DNA molecules. The homologous recombination between the genes on the linearized DNA molecules created the large chimeric bphC gene libraries in a recBC sbcA E. coli strain. After three rounds of recombinations, chimeric bphC genes with four-part gene fragments by triple-crossover were easily obtained. By employing a 96-well microtiter plate high-throughput screening, thermally stable chimeric 2,3-dihydroxybiphenyl 1,2-dioxygenases were selected from chimeric bphC gene libraries. This opens up a new way for directed evolution of proteins in vivo.

DNA Shuffling↗

PubMed2006

Induction of apoptotic cell death leads to the development of bacterial rot caused by Pseudomonas cichorii.

Pseudomonas cichorii is the major causal agent of bacterial rot of lettuce. Collapse and browning symptoms were observed in lettuce leaf tissue from 15 to 24 h after inoculation (HAI) with P. cichorii; superoxide anion generation was detected at 1 to 6 HAI; and cell death was induced at 6 HAI, reaching a maximum at approximately 9 and 12 HAI. Heterochromatin condensation and DNA laddering also were observed within 3 HAI. Pharmacological studies showed that induction of cell death and DNA laddering was closely associated with de novo protein synthesis, protein kinase, intracellular reactive oxygen species, DNase, serine protease, and caspase III-like protease. Moreover, chemicals, which inhibited the induction of cell death and DNA laddering, also suppressed the development of disease symptoms. These results suggest that apoptotic cell death might be closely associated with the development of bacterial rot caused by P. cichorii.

Anions↗

PubMed2006

Phylogenetic study and multiplex PCR-based detection of Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli using gyrB and rpoD sequences.

In order to develop a detection method for the rice pathogens Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli, the phylogeny of six plant-pathogenic Burkholderia species was analysed using the combined nucleotide sequences of gyrB and rpoD. B. plantarii, B. glumae and B. gladioli formed tight monophyletic branches supported by high bootstrap probabilities. The high sequence similarity revealed a close phylogenetic relationship between B. glumae and B. plantarii. B. plantarii strains were divided into three subclusters comprising rice strains, whereas the single Vanda strain occupied a unique position in the phylogenetic tree. The gyrB and rpoD sequences of all B. glumae strains examined were highly conserved. In contrast, B. gladioli strains demonstrated a far greater sequence diversity, but this diversity did not correlate with pathovar, host plant or geographical origin of the strains. A multiplex-PCR protocol using specific primers from the gyrB sequences was designed that allowed the specific detection and identification of B. plantarii, B. glumae and B. gladioli in rice seeds infected with these pathogenic species.

Bacterial Proteins↗

PubMed2006

Combined use of dendritic cells enhances specific antileukemia immunity by leukemia cell-derived heat shock protein 70 in a mouse model with minimal residual leukemia cells.

We have reported that immunotherapy using leukemia cell-derived heat shock proteins (HSPs) is effective against minimal residual disease (MRD) after syngeneic stem cell transplantation (SCT) in mice. However, leukemia patients after SCT are usually immunocompromised and immunologically tolerant to leukemia cells. We investigated whether the use of dendritic cells (DCs) in combination with HSP70 enhances cytotoxicity against B-cell leukemia cell line A20 in mice after syngeneic SCT. All unimmunized mice died of leukemia early after A20 cell inoculation, whereas mice immunized with HSP70 or HSP70-pulsed DCs survived significantly longer. Although only 60% of the HSP70-immunized mice survived, all mice immunized with HSP70-pulsed DCs survived without MRD. In addition, the cytotoxicities against A20 cells for splenocytes from mice immunized with HSP70-pulsed DCs were significantly higher than those of HSP70-immunized mice, and the cytotoxicities against A20 cells were significantly blocked by anti-CD8 antibody and by major histocompatibility complex class I antibody, but not by anti-CD4 antibody. Moreover, abnormalities were detected in neither the biochemical data nor the histopathologic findings. These findings indicate that the combined use of DCs and leukemia cell-derived HSP70 enhances the antileukemia effect by inducing the specific cytotoxicities of CD8+ cytotoxic T-cells, thereby eradicating MRD effectively and safely, even in an immunocompromised state after syngeneic SCT. This approach may thus be useful for further application of HSP in leukemia patients after autologous SCT.

Animals↗

PubMed2006

[A neuron specific enolase-producing multiple myeloma].

A 53-year-old woman was admitted to our hospital with left chest-wall pain. Computed tomography scans showed a homogenous mass on the left chest-wall with pleural effusion. Laboratory data showed anemia, hypercalcemia, and high levels of serum IgG. An IgG-lambda monoclonal protein was detected with serum immunoelectrophoresis. In addition, the serum level of neuron specific enolase (NSE) was elevated. A chest-wall tumor biopsy and a bone marrow aspiration revealed diffuse proliferation of atypical plasma cells, which were positive for cytoplasmic CD38 and IgG-lambda. The patient was diagnosed as having IgG-lambda type multiple myeloma with a chest-wall plasmacytoma. Immunostaining revealed diffuse NSE staining in the cytoplasm of the atypical plasma cells. These findings suggested that the myeloma cells produced NSE. The left chest-wall tumor and bone marrow myeloma cells disappeared following several courses of chemotherapy and radiotherapy and the serum levels of IgG and NSE also normalized. No recurrence of the multiple myeloma was seen after an autologous peripheral blood stem cell transplantation. This is the second report of an NSE-producing multiple myeloma. Interestingly, our case has similar clinical phenotypes with the previously reported case, such as chest-wall plasmacytoma, pleural effusion and hypercalcemia.

Female↗

PubMed2005

4-Pyridoxolactonase from a symbiotic nitrogen-fixing bacterium Mesorhizobium loti: cloning, expression, and characterization.

4-Pyridoxolactonase is involved in the degradation pathway for pyridoxine, a free form of vitamin B6. The gene (mlr6805) encoding the putative 4-pyridoxolactonase of nitrogen fixing symbiotic microorganism Mesorhizobium loti MAFF303099 has been identified based on the genome database. The gene was cloned and overexpressed in a cotransformant Escherichia coli cell. The recombinant enzyme was dimeric protein and contained one mole of Zn2+ per mole of subunit. The enzyme showed about 30% identity with various N-acylhomoserine lactone lactonases and metallo-beta-lactamases. The phylogram made with ClustalW shows that 4-pyridoxolactonase makes a cluster with Agrobacterium tumefaciens acyl-homoserine lactone lactonase. The alignment of amino acid sequences suggests that 4-pyridoxolactonase has three histidine residues probably involved in binding of Zn2+.

Agrobacterium tumefaciens↗

PubMed2005

Vaccine efficacy of recombinant GAPDH of Edwardsiella tarda against Edwardsiellosis.

Thirty-seven kilodalton glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Edwardsiella tarda was suggested to be an effective vaccine candidate against E. tarda infection in previous research. For developing a vaccine, obtaining GAPDH in large quantities is necessary. In this study, we determined the complete nucleotide sequence of the gene that encodes GAPDH of E. tarda, and overexpressed the GAPDH of E. tarda by using the Escherichia coli expression system. We immunized Japanese flounder with recombinant GAPDH (rGAPDH) and evaluated its vaccine efficacy. Our results showed that rGAPDH effectively protected Japanese flounder from experimental E. tarda infection, and will contribute to the development of a vaccine against E. tarda.

Animals↗

PubMed2005

N-terminal signal region of FliK is dispensable for length control of the flagellar hook.

The length of the flagellar hook is regulated; it is 55 +/- 6 nm long in Salmonella. Five genes involved in hook-length regulation are fliK, flhB, fliG, fliM and fliN. The last four genes encode structural components of the protein export apparatus in the flagellar base, whereas FliK is soluble and secreted during flagellar assembly. The role of FliK, however, remains ambiguous. We constructed two kinds of FliK variants: N-terminally truncated FliK protein and FliK N-terminally fused with cyan fluorescent protein (CFP-FliK). Both N-terminally truncated FliK missing the first 99 amino acids (aa) and CFP-FliK fusion variants partially complemented a fliK null (polyhook) mutant to produce cells with filaments, allowing cells to swim; the hooks, however, were not normal but were polyhooks. When the N-terminally defective FliK variants were expressed at high levels, the average polyhook length was shortened coming close to the length of the wild-type hook, independently of the sizes of the FliK variants. These FliK variants were not secreted. CFP-FliK fusion proteins were observed to homogeneously distribute in the cytoplasm. We conclude that FliK does not need to be exported to control hook length and is unlikely to be a ruler; instead, we conclude that FliK controls hook length by the timely switching of secretion modes of the flagellar type III secretion system by the FliK C-terminal domain, and that the N-terminal region is dispensable for hook length control.

Amino Acid Sequence↗

PubMed2005

Contribution of folate biosynthesis to Ralstonia solanacearum proliferation in intercellular spaces.

The vigorous proliferation of Ralstonia solanacearum OE1-1 in host intercellular spaces after the invasion of host plants is necessary for the virulence of this bacterium. A folate auxotroph, RM, in which a mini-Tn5 transposon was inserted into pabB encoding para-aminobenzoate synthase component I, lost its ability to vigorously proliferate in intercellular spaces along with its systemic infectivity and virulence after inoculation into roots and infiltration into leaves of tobacco plants. Complementation of RM with the pabB gene allowed the mutant to multiply in intercellular spaces and to cause disease. In tobacco plants that were pretreated with folate, RM was able to vigorously proliferate in the intercellular spaces and cause disease. Interestingly, when it was inoculated through cut stems, the mutant multiplied in the plants and was virulent. Moreover, the mutant multiplied well in stem fluids but not in intercellular fluids, suggesting that the folate concentration within intercellular spaces may be a limiting factor for bacterial proliferation. Therefore, folate biosynthesis contributes to the vigorous proliferation of bacteria in intercellular spaces and leads to systemic infectivity resulting in virulence.

DNA Transposable Elements↗

PubMed2005

Directed evolution of bacterial alanine racemases with higher expression level.

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the interconversion of L-alanine and D-alanine. It can be classified into two groups: biosynthetic enzymes with low catalytic activity and catabolic enzymes with high catalytic activity. It can react with serine to a limited extent. Two biosynthetic alanine racemase genes in Escherichia coli and Salmonella typhimurium were DNA shuffled, and a very diverse chimeric gene library was constructed. An E. coli serine auxotroph was transformed with the shuffled genes, and the recombinant clones were screened on selective media supplemented with 0.5-5 mM D-serine as an L-serine supplier. Selected clones were expected to contain racemases exhibiting higher catalytic activities toward alanine as well as serine. Three independent clones that grew on selective media were isolated. The specific activities of crude extracts prepared from cells expressing the chimeric racemases were increased up to approximately three times more than those expressing the parental enzymes. The best chimera Ser15 racemase was expressed at a level approximately twofold higher than the parental alanine racemases. This high protein expression was demonstrated to be posttranscriptionally achieved.

Alanine Racemase↗

PubMed2005

Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of gene products expressed in Escherichia coli.

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, alanine racemases are classified into two types, monomeric and homodimeric. Alanine racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli alanine racemase-deficient mutant. From the cloning results, both strains are likely to possess only one alanine racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D-amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX alanine racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli alanine racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.

Alanine Racemase↗

PubMed2005

[Case of intraspinal epidural tumor developing after systemic mastocytosis with marked osteosclerosis and myelofibrosis].

A 64-year-old man was diagnosed as having urticaria pigmentosa in 1998, and treated with PUVA therapy. In January 2002, X-ray imaging revealed osteosclerosis was detected in the systemic bone and bone scintigraphy. A bone marrow aspiration sample was not obtained due to a dry tap. CT scans showed hepatosplenomegaly and mesenteric lymphadenopathy. Myelofibrosis and diffuse mast cell infiltration were revealed by a bone marrow biopsy, and a diagnosis of systemic mastocytosis with severe osteosclerosis and myelofibrosis was made. In October 2003, he was admitted to our hospital because of mid back pain. A neurological examination showed muscle weakness in the upper and lower limbs, sensory disturbance below the level of Th4 and urinary obstruction. T1 and T2 weighted images of MRI demonstrated a high intensity epidural mass lesion extending from the vertebral level of C5 to Th2 and severely compressing the spinal cord. We considered the possibility of the invasion of the spinal canal by the mastocytosis. The patient was treated with interferon alpha-2b (IFN-alpha2b) and prednisolone. Subsequently, the motor and sensory disturbances were gradually alleviated, and spinal MRI confirmed a marked reduction in the size of the epidural tumor. However, the patient became resistant to interferon, and died of multiple organ failure in spite of steroid pulse and cladribine therapies. Multiple organ infiltration by mast cells was revealed at autopsy.

Antineoplastic Agents↗

PubMed2004

A conserved 37 kDa outer membrane protein of Edwardsiella tarda is an effective vaccine candidate.

An effective vaccine against Edwardsiella tarda has not been reported, one of main reasons is the variation in its serotypes. This study aimed to develop an effective vaccine against different serotypes of E. tarda. A conserved 37 kDa outer membrane protein (OMP) of E. tarda was obtained by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). It showed comprising several proteins by two-dimensional (2D) PAGE analysis and showed a high similarity to glyceraldehyde-3-phosphate dehydrogenase by N-terminal amino acid sequence analysis. Japanese flounder (Paralichthys olivaceus) vaccinated with the 37 kDa OMP was significantly protected against the infections by different serotypes of E. tarda. A specific antibody was also detected by using ELISA. This study suggests that the 37 kDa OMP is an effective potent vaccine candidate against different serotypes of E. tarda.

Amino Acid Sequence↗