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Toshihiro Watanabe

Publications and source records attributed to Toshihiro Watanabe.

At least 37 records · Page 2Linked to original sources
PubMed2004

Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes.

Clostridium botulinum type C 16S progenitor toxin consists of a neurotoxin (NTX), a non-toxic non-HA (NTNH), and a haemagglutinin (HA). The HA acts as an adhesin, allowing the 16S toxin to bind to intestinal epithelial cells and erythrocytes. In type C, these bindings are dependent on sialic acid. The HA consists of four distinct subcomponents designated HA1, HA2, HA3a and HA3b. To identify the binding subcomponent(s) of HA of type C 16S toxin, all of the HA-subcomponents and some of their precursor forms were produced as recombinant proteins fused to glutathione S-transferase (GST). These proteins were evaluated for their capacity to adhere to intestinal epithelial cells of guinea pig and human erythrocytes. GST-HA1, GST-HA3b and GST-HA3 (a precursor form of HA3a and HA3b) bound intestinal epithelial cells and erythrocytes, whereas GST alone, GST-HA2 and GST-HA3a did not. GST-HA3b and GST-HA3 showed neuraminidase-sensitive binding to the intestinal epithelial cells and erythrocytes, whereas GST-HA1 showed neuraminidase-insensitive binding. TLC binding assay revealed that GST-HA3b and GST-HA3 recognized sialosylparagloboside (SPG) and GM3 in the ganglioside fraction of the erythrocytes, like native type C 16S toxin [Inoue, K. et al. (1999). Microbiology 145, 2533-2542]. On the other hand, GST-HA1 recognized paragloboside (PG; an asialo- derivative of SPG) in addition to SPG and GM3. Deletion mutant analyses of GST-HA3b showed that the C-terminal region of HA3b is important for its binding activity. Based on these data, it is concluded that the HA component contains two distinct carbohydrate-binding subcomponents, HA1 and HA3b, which recognize carbohydrates in different specificities.

Amino Acid Sequence↗
PubMed2004

Autolysis of Porphyromonas gingivalis is accompanied by an increase in several periodontal pathogenic factors in the supernatant.

Porphyromonas gingivalis autolyzes in the culture media. To examine in more detail the molecular components of the autolysate, two-dimensional gel electrophoresis was performed. Many protein spots varied both in number and volume. One of these spots included Arg-gingipain (Rgp) as determined by N-terminal amino acid sequencing. Corresponding to the increase in spot volume, Rgp activity also increased during autolysis. The results of this study suggested that Rgp and other proteins in the P. gingivalis autolysate may be involved with the prolongation of periodontal disease, even after the death of P. gingivalis cells.

Adhesins, Bacterial↗
PubMed2004

Distribution of Prx-linked hydroperoxide reductase activity among microorganisms.

Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670-27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.

Animals↗
PubMed2003

Synthesis and pharmacological evaluation of 1-oxo-2-(3-piperidyl)-1,2,3,4- tetrahydroisoquinolines and related analogues as a new class of specific bradycardic agents possessing I(f) channel inhibitory activity.

A series of 1-oxo-2-(3-piperidyl)-1,2,3,4-tetrahydroisoquinolines and related analogues were prepared and evaluated for their bradycardic activities in isolated right atrium and in anesthetized rats. (+/-)-6,7-Dimethoxy-2-[1-[3-(3,4-methylenedioxyphenoxy)propyl]-3-piperidyl]-1,2,3,4-tetrahydroisoquinoline (4) was chosen as a lead, and structural modifications were performed on the tetrahydroisoquinoline ring and the terminal aromatic ring. The modifications on the tetrahydroisoquinoline ring revealed that the 1-oxo-1,2,3,4-tetrahydroisoquinoline ring system was optimum structure for both in vitro potency and in vivo efficacy. Furthermore, methoxy, ethoxy, and methoxycarbonyl groups were identified as preferable substituents on the terminal aromatic ring. One of the 1-oxo-1,2,3,4-tetrahydroisoquinoline derivatives, (R)-10a, was further evaluated for its bradycardic activity and inhibitory activity against I(f) currents. Compound (R)-10a demonstrated potent bradycardic activity in rats with minimal influence on blood pressure after oral administration. The compound also showed inhibition of I(f) currents (IC(50) = 0.32 muM) in guinea pig pacemaker cells.

Animals↗
PubMed2003

Complete subunit structure of the Clostridium botulinum type D toxin complex via intermediate assembly with nontoxic components.

Clostridium botulinum serotype D strains usually produce two types of stable toxin complex (TC), namely, the 300 kDa M (M-TC) and the 660 kDa L (L-TC) toxin complexes. We previously proposed assembly pathways for both TCs [Kouguchi, H., et al. (2002) J. Biol. Chem. 277, 2650-2656]: M-TC is composed by association of neurotoxin (NT) and nontoxic nonhemagglutinin (NTNHA); conjugation of M-TC with three auxiliary types of hemagglutinin subcomponents (HA-33, HA-17, and HA-70) leads to the formation of L-TC. In this study, we found three TC species, 410, 540, and 610 kDa TC species, in the culture supernatant of type D strain 4947. The 540 and 610 kDa TC species displayed banding patterns on SDS-PAGE similar to that of L-TC but with less staining intensity of the HA-33 and HA-17 bands than those of L-TC, indicating that these are intermediate species in the pathway to L-TC assembly. In contrast, the 410 kDa TC species consisted of M-TC and two molecules of HA-70. All of the TC species, except L-TC, demonstrated no hemagglutination activity. When the intermediate TC species were mixed with an isolated HA-33/17 complex, every TC species converted to 650 kDa L-TC with full hemagglutination activity and had the same molecular composition of L-TC. On the basis of titration analysis with the HA-33/17 complex, the stoichiometry of the HA-33/17 complex molecules in the L-TC, 610 kDa, and 540 kDa TC species was estimated as 4, 3, and 2, respectively. In conclusion, the complete subunit composition of mature L-TC is deduced to be a dodecamer assembled by a single NT, a single NTNHA, two HA-70, four HA-33, and four HA-17 molecules.

Amino Acids↗
PubMed2003

Effect of Porphyromonas gingivalis vesicles on coaggregation of Staphylococcus aureus to oral microorganisms.

Vesicles from the outer membrane of Porphyromonas gingivalis have the ability to aggregate a wide range of Streptococcus spp., Fusobacterium nucleatum, Actinomyces naeslundii, and Actinomyces viscosus. We found that in the presence of P. gingivalis vesicles, Staphylococcus aureus coaggregated with Streptococcus spp., and the mycelium-type Candida albicans, but not the yeast type. Autoaggregation of S. aureus in the presence of P. gingivalis vesicles is inhibited by L-arginine, L-lysine, and L-cysteine. Both the methicillin-sensitive (MSSA) and -resistant (MRSA) strains of S. aureus were able to coaggregate with Streptococcus spp., A. naeslundii, and A. viscosus when they were treated with P. gingivalis vesicles. P. gingivalis vesicle-treated mycelium-type C. albicans coaggregated with S. aureus, but the yeast-type did not. These results indicate that strains of S. aureus, including MRSA, could adhere to oral biofilms in dental plaque on the tooth surface or in the gingival crevice when P. gingivalis is present.

Actinomyces↗
PubMed2003

Molecular characterization of GroES and GroEL homologues from Clostridium botulinum.

We report novel findings of significant amounts of 60- and 10-kDa proteins on SDS-PAGE in a culture supernatant of the Clostridium botulinum type D strain 4947 (D-4947). The N-terminal amino acid sequences of the purified proteins were closely related to those of other bacterial GroEL and GroES proteins, and both positively cross-reacted with Escherichia coli GroEL and GroES antibodies. Native GroEL homologue as an oligomeric complex is a weak ATPase whose activity is inhibited by the presence of GroES homologue. The 2634-bp groESL operon of D-4947 was isolated by PCR and sequenced. The sequence included two complete open reading frames (282 and 1629 bp), which were homologous to the groES and groEL gene family of bacterial proteins. Southern and Northern blot analyses indicate that the groESL operon is encoded on the genomic DNA of D-4947 as a single copy, and not on that of its specific toxin-converting phage.

Amino Acid Sequence↗
PubMed2003

Nutritional characteristics of the leaves of native plants growing in adverse soils of humid tropical lowlands.

Acid sulfate, peat, sandy podzolic, and saline soils are widely distributed in the lowlands of Thailand and Malaysia. The nutrient concentrations in the leaves of plants grown in these type of soils were studied with the aim of developing a nutritional strategy for adapting to such problem soils. In sago and oil palms that were well-adapted to peat soil, the N, P, and K concentrations were the same in the mature leaves, while the Ca, Mg, Na, and Fe concentrations were higher in the mature leaves of the oil palm than of the sago palm. Melastoma malabathricum and Melaleuca cajuputi plants that were well-adapted to low pH soils, peat. and acid sulfate soils were also studied. It was observed that a high amount of Al accumulated in the M. marabathricum leaves, while Al did not accumulate in M. cajuputi leaves. M. cajuputi plants accumulated large amounts of Na in their leaves or stems regardless of the exchangeable Na concentration in the soil, while M. malabathricum that was growing in saline-affected soils excluded Na. Positive relationships between macronutrients were recognized between P and N, between K and N, and between P and K. Al showed antagonistic relationships with P, K, Ca, Mg, Fe, Zn, Cu, and Na. Na also showed antagonistic relationships with P, K, Zn, Mn, Cu, and Al. Fe showed weak antagonistic relationships with Zn, Mn, Cu, and Al.

Aluminum↗
PubMed2003

A comparative study of metal levels in leaves of some Al-accumulating Rubiaceae.

Concentrations of Al, Si, Fe, Mn, Cu and Ca were analysed in leaves of ten Rubiaceae species, most of which are Al accumulators, and these were compared with concentrations in one species of Melastomataceae. Quantitative data confirmed the distribution of Al accumulation as previously determined by semi-quantitative tests, and suggest that there is an apparent congruency between the shoot Al concentration and the number of accumulators within a certain genus or tribe. Al accumulators within the Rubiaceae are most characteristic of the Rubioideae subfamily, although a second origin is likely in at least a few members of the tribes Vanguerieae and Alberteae. While the leaf Si concentration in Melastomata malabathricum L. (Melastomataceae) was negligible, all Rubiaceae studied showed relatively high Si levels (mostly >3000 mg kg(-1)). It is hypothesized that an Al-Si complex is formed in the shoot tissues of Al-accumulating Rubiaceae and that this may contribute to Al detoxification. However, the Si : Al mole ratio tended to differ widely among species. There was no significant correlation between Al and the other metals analysed. A remarkably high Mn concentration was found in Coptosapelta olaciformis Elm.

Aluminum↗
PubMed2003

Adhesins encoded by the gingipain genes of Porphyromonas gingivalis are responsible for co-aggregation with Prevotella intermedia.

Co-aggregation among bacterial cells caused by the adherence of one bacterial species to another is a potential colonization mechanism. Several putative aggregation factors for co-aggregation between Porphyromonas (Por.) gingivalis and Prevotella (Pre.) intermedia were partially purified from Por. gingivalis vesicles by gel filtration and affinity chromatography. Antisera against the aggregation factors were made. Analysis using these antisera revealed that 18 and 44 kDa proteins might be responsible for Por. gingivalis vesicle-mediated aggregation of Pre. intermedia. Using antiserum against the 18 kDa protein, the DNA region encoding it was cloned from Por. gingivalis genomic DNA. Sequence analysis revealed that the DNA region was located within the rgpA and kgp genes, encoding Arg-gingipain (Rgp) and Lys-gingipain (Kgp), respectively, and it encoded non-catalytic adhesin domain regions, namely a C-terminal portion of HGP15, the entire HGP17 sequence and an N-terminal portion of HGP27. A portion of the DNA sequence was also found in the haemagglutinin A (hagA) gene. A recombinant glutathione S-transferase (GST)-HGP17 fusion protein reacted to antiserum against the 18 kDa protein and Pre. intermedia cells could adhere to GST-HGP17-conjugated Sepharose 4B beads, indicating that the HGP17 domain protein is responsible for Por. gingivalis vesicle-mediated aggregation of Pre. intermedia.

Adhesins, Bacterial↗
PubMed2003

Purification of fully activated Clostridium botulinum serotype B toxin for treatment of patients with dystonia.

Clostridium botulinum serotype B toxins 12S and 16S were separated by using a beta-lactose gel column at pH 6.0; toxin 12S passed through the column, whereas toxin 16S bound to the column and eluted with lactose. The fully activated neurotoxin was obtained by applying the trypsin-treated 16S toxin on the same column at pH 8.0; the neurotoxin passed through the column, whereas remaining nontoxic components bound to the column. The toxicity of this purified fully activated neurotoxin was retained for a long period by addition of albumin in the preparation.

Amino Acid Sequence↗
PubMed2003

Synthesis and pharmacological evaluation of 2-(3-piperidyl)-1,2,3,4-tetrahydroisoquinoline derivatives as specific bradycardic agents.

Novel 1,2,3,4-tetrahydroisoquinoline derivatives bearing directly a cyclic amine at the 2-position were prepared and examined for their bradycardic activities in isolated right atria and in anesthetized rats. The structure-activity relationships (SAR) study revealed that the 2-(3-piperidyl)-1,2,3,4-tetrahydroisoquinoline skeleton is essential for the appearance of potent in vitro activity, and that the presence of at least one methoxy group at the 6- or 7-position of the 1,2,3,4-tetrahydroisoquinoline ring is important to exert potent in vitro activity. In vivo tests of selected compounds demonstrated that 2-(1-benzyl-3-piperidyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (6c) exhibited potent bradycardic activity with negligible influence on mean blood pressure in rats, although its potency is a half of that of Zatebradine.

Animals↗
PubMed2002

Spontaneous nicking in the nontoxic-nonhemagglutinin component of the Clostridium botulinum toxin complex.

The nontoxic-nonhemagglutinin (NTNHA) component, in both isolated form and the neurotoxin (NT)/NTNHA complexed form, was prepared protease-free from toxin complexes produced by Clostridium botulinum type D strain 4947. NTNHA in both preparations was found to be spontaneously converted to the nicked NTNHA form leading to 15- and 115-kDa fragments with the excision of several amino acid residues at specific sites on SDS-PAGE during long-term incubation, while that of the NT/NTNHA/hemagglutinin complexed form remained unnicked single-chain polypeptides under the same conditions. Considering that the NTNHA preparation contained small amounts of the nicked form of NTNHA and the addition of trypsin accelerated the cleavage, it is speculated that a nicked form of NTNHA remaining after the purification and/or NTNHA itself catalyzes the cleavage of intact NTNHA.

Amino Acid Sequence↗
PubMed2002

Aluminium accumulation in leaves of 127 species in Melastomataceae, with comments on the order Myrtales.

The distribution and systematic significance of aluminium accumulation is surveyed based on semi-quantitative tests of 166 species, representing all tribes and subfamilies of the Melastomataceae as well as a few members of related families within the Myrtales. The character is strongly present in nearly all members of the Memecylaceae and in most primitive taxa of the Melastomataceae, while non-accumulating taxa are widespread in the more derived tribes of the Melastomataceae. The variable distribution of Al accumulation in advanced clades of the latter family is probably associated with the tendency to herbaceousness, although it is unclear whether the more herbaceous representatives have developed more specialized Al-response mechanisms that may exclude high Al levels from the shoot. It is hypothesized that Al accumulation is symplesiomorphic for Melastomataceae and Memecylaceae, and that the feature characterizes the most primitive families in the Myrtales. Indeed, Al accumulation is also characteristic of Crypteroniaceae, Rhynchocalycaceae and Vochysiaceae. Crypteroniaceae and Rhynchocalycaceae probably take a basal position in a sister clade of the Memecylaceae and Melastomataceae, while Al accumulation suggests a basal position for Vochysiaceae in the Myrtaceae clade.

Aluminum↗
PubMed2002

Role of organic acids in aluminum accumulation and plant growth in Melastoma malabathricum.

Melastoma malabathricum L. (melastoma) is an Al-accumulating woody plant that grows in tropical Southeast Asia in acid soils with high aluminum (Al) concentrations and low nutrient concentrations. Because oxalate serves as a ligand for Al accumulation in melastoma leaves and citrate is the ligand associated with Al translocation from roots to shoots, we investigated the role of organic acids in the adaptation of melastoma to growth on these soils. Phosphorus starvation increased oxalate concentration in the rhizosphere, enabling melastoma to solubilize insoluble aluminum phosphate in the rhizosphere. Increased availability of P and Al in the rhizosphere enhanced growth. In the xylem sap, the concentration of citrate increased with increasing Al concentration. In contrast, the concentrations of malate, succinate and alpha-ketoglutarate in the xylem sap decreased with increasing Al concentration, suggesting that tricarboxylic acid cycle enzymes were affected by Al treatment.

Aluminum↗
PubMed2002

Mucosal immunisation with Clostridium botulinum type C 16 S toxoid and its non-toxic component.

Clostridium botulinum types C and D produce a 16 S (500 kDa) toxin that is formed by conjugation of neurotoxin with a non-toxic component (nonTox). The amino acid sequences of type C and D nonTox components are almost identical. In a previous report it was proposed that nonTox is necessary for the effective absorption of the toxin from the small intestine. This suggested the hypothesis that mucosal immunity against nonTox in the small intestine might prevent the absorption of both C- and D-16 S toxins. The nonTox was purified from a mutant strain, (C)-N71, that does not produce neurotoxin. This nonTox or detoxified C-16 S toxin were mixed with adjuvant (a mutant form of heat-labile toxin of Escherichia coli), and inoculated into mice via the nasal or oral route, or both. The mice inoculated nasally four times with nonTox or toxoid produced high levels of antibodies (including IgA) against the immunogens, both in intestinal fluids and sera. When these nonTox-immunised mice were challenged orally with 2 and 20 oral minimum lethal doses (MLD) of C- or D-16 S toxins, the same results were obtained with both C and D; the mice survived after challenge with 2 MLD of either C or D but were killed by 20 MLD of either toxin although the time to death was significantly longer than in the control non-immunised mice. These results indicate that the local anti-nonTox antibodies reduce absorption of both C- and D-16 S toxins from the small intestine. The C-16 S toxoid-immunised mice showed similar behaviour with type D toxin challenge, probably due to the same mechanism, but were protected against 20 MLD of C-16 S toxin.

Administration, Intranasal↗
PubMed2001

In vitro reconstitution of the Clostridium botulinum type D progenitor toxin.

Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing. The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (HA-70, HA-17, and HA-33). The HA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents. We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17. The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice. M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents. We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17.

Amino Acid Sequence↗