Biomedical subjects

H Tsujibo

Publications and source records attributed to H Tsujibo.

At least 19 recordsLinked to original sources

PubMed2005

Role of the N-terminal polycystic kidney disease domain in chitin degradation by chitinase A from a marine bacterium, Alteromonas sp. strain O-7.

AIMS: The aim of study was to clarify whether the polycystic kidney disease (PKD) domain of chitinase A (ChiA) participates in the hydrolysis of powdered chitin. METHODS AND RESULTS: Site-directed mutagenesis of the conserved aromatic residues of PKD domain was performed by PCR. The aromatic residues, W30, Y48, W64 and W67, were replaced by alanine, and single- and double-mutant chitinases were produced in Escherichia coli XL10 and purified with HisTrap column. Single mutations were not quite effective on the hydrolysing activities against chitinous substrates when compared with wild-type ChiA. However, mutations of W30 and W67 decreased the activities against powdered chitin by 87.6%. Wild-type and mutant PKD domains were produced in E. coli TOP10 and purified with glutathione-Sepharose 4B column. Wild-type PKD domain showed significant binding activity to powdered chitin, whereas mutations of W30 and W67 reduced the binding activity to powdered chitin drastically. These results suggest that PKD domain of ChiA is essential for effective hydrolysis of powdered chitin through the interaction between two aromatic residues and chitin molecule. CONCLUSIONS: PKD domain of ChiA participates in the effective hydrolysis of powdered chitin through the interaction between two aromatic residues (W30 and W67) and chitin molecule. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study provide important information on chitin degradation by microbial chitinases.

Adsorption↗

PubMed2001

Cytotoxicity of the hinokitiol-related compounds, gamma-thujaplicin and beta-dolabrin.

Gamma-thujaplicin and beta-dolabrin, the constituents of the wood of Thujopsis dolabrata Sieb. et Zucc. var. hondai showed strong in vitro cytotoxic effects against the human stomach cancer cell lines KATO-III and Ehrlich's ascites carcinoma. The cytotoxic effects of the two compounds against both tumor cell lines were clear when cell growth was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Gamma-thujaplicin and beta-dolabrin at 0.32 microg/ml inhibited cell growth of human stomach cancer KATO-III by 85 and 67%, and Ehrlich's ascites carcinoma by 91 and 75%, respectively. There is no large difference in cytotoxicity between these compounds, but the activity of gamma-thujaplicin was slightly more potent than that of beta-dolabrin. On the other hand, hinokitiol acetate did not show a cytotoxic effect, suggesting that at least a part of the mechanism of the cytotoxic effect of hinokitiol-related compounds is due to metal chelation between the carbonyl group at C-1 and the hydroxyl group at C-2 in the tropolone skeleton of these molecules. The acute toxicities [50% lethal dose (LD50) value: intraperitoneal injection, Van der Waedem] of gamma-thujaplicin and beta-dolabrin in mice were 277 mg/kg and 232 mg/kg, respectively.

Animals↗

PubMed2001

Biological activity of alpha-thujaplicin, the minor component of Thujopsis dolabrata SIEB. et ZUCC. var. hondai MAKINO.

Alpha-thujaplicin, a minor component of Thujopsis dolabrata SIEB. et ZUCC. var. hondai MAKINO, which was synthesized, showed the antibacterial activity, phytogrowth-inhibitory effect, inhibition of carboxypeptidase A and cytotoxic effect. Antibacterial activity of alpha-thujaplicin on Enterococcus faecalis IFO-12965 [minimum inhibitory concentration (MIC): 1.56 microg/ml] was higher than that of gentamicin (MIC: 6.25 microg/ml) used as a positive control. Inhibitory activity of alpha-thujaplicin on carboxypeptidase A [50% inhibitory concentration (IC50): 3.24 x 10(-5) M] was higher than that of 1,10-phenanthroline used as a positive control. Alpha-thujaplicin showed germination inhibition toward the seed of Echinochloa utilis Ohwi et Yabuno even at the low concentration of 10 ppm and its growth inhibitory effect was stronger than that of sodium 2,4-dichlorophenoxyacetate used as a standard. Alpha-thujaplicin at 1.25 microg/ml inhibited cell growth of human stomach cancer KATO-IIl by 86%, and Ehrlich's ascites carcinoma by 87%, respectively. This compound even at the low concentration of 0.32 microg/ml also inhibited cell growth of the former by 66%, and the latter by 75%, respectively. The acute toxicity of alpha-thujaplicin [50% lethal dose (LD50) value: 256 mg/kg] in mice was as strong as those of beta-dolabrin (LD50 value: 232 mg/kg) and gamma-thujaplicin (LD50 value: 277 mg/kg).

Animals↗

PubMed2001

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520.

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.

Amino Acid Sequence↗

PubMed2000

A serine protease-encoding gene (aprII) of Alteromonas sp. Strain O-7 is regulated by the iron uptake regulator (Fur) protein.

The ferric uptake regulator (Fur) box-like sequence was located upstream of the serine protease-encoding gene (aprII) from a marine bacterium, Alteromonas sp. strain O-7. To clarify whether the production of AprII (the gene product of aprII) is regulated by the environmental iron concentrations, this strain was cultured under iron-depleted or iron-rich conditions and the level of AprII in the culture supernatant was analyzed by Western blotting. The production of AprII was significantly repressed under iron-rich conditions. Northern hybridization analysis demonstrated that AprII biosynthesis was regulated by iron through the control of transcription. These results indicate that aprII is a new member of the iron regulon and plays an important role in the iron acquisition system of the strain. Furthermore, the gene encoding Fur was cloned and sequenced. The deduced amino acid sequence of the cloned Fur showed high sequence similarity with that from gram-negative bacteria.

Alteromonas↗

PubMed2000

Biological activity of alkyl 2-(acylthio)benzoates.

Of a series of synthetic alkyl 2-(acylthio)benzoate (1-20), all the derivatives except for n-butyl 2-butyrylthiobenzoate (18) and n-butyl 2-n-valerylthiobenzoate (20) showed clear phytogrowth-inhibitory activity. All the compounds tested except for methyl 2-butyrylthiobenzoate (3) exhibited cytotoxic activity on mouse splenic T cells. Strong phytogrowth-inhibition and cytotoxic activity were found with 1, 6, 11 and 16 with an acetylthio group at C-2, suggesting that the acetyl group seems to play an important role in both activities of alkyl 2-(acylthio)benzoates. Among them, methyl 2-acetylthiobenzoate (1) was the strongest inhibitor. On the other hand, potent inhibition of prolyl endopeptidase was exhibited by 2, 7, 12 and 17 with a propionylthio group at C-2. These findings imply that a propionyl group might be useful for increasing the inhibitory activity against on prolyl endopeptidase.

Animals↗

PubMed2000

Phytogrowth-Inhibitory activities of beta-dolabrin and gamma-thujaplicin, hinokitiol-related compounds and constituents of Thujopsis dolabrata Sieb. et Zucc. var hondai Makino.

Beta-dolabrin and gamma-thujaplicin isolated from Thujopsis dolabrata Sieb. et Zucc. var hondai Makino, like hinokitiol, showed strong phytogrowth-inhibitory activities, and their growth-inhibitory activities were as high as that of sodium 2,4-dichlorophenoxyacetate used as a positive control. In particular, the phytogrowth-inhibitory activity of gamma-thujaplicin was strong and it completely inhibited the germination of this seed of Brassica campestris L. subsp. rapa Hook f. et Anders at the concentration of 30 ppm. Both compounds exhibited inhibitory activities on B. campestris L. subsp. rapa Hook f. et Anders and Sesamum indicum Linne, even at the low concentration of 10 ppm. At 7 d after treatment with beta-dolabrin and gamma-thujaplicin, the amount of chlorophyll in the cotyledons of B. campestris L. subsp. rapa Hook f. et Anders treated with both compounds was greatly decreased as compared with the control. The findings indicate that the phytogrowth-inhibitory action might be a common biological activity of hinokitiol-related compounds, suggesting that at least a part of their phytogrowth-inhibitory actions seems to be related to a decrease in chlorophyll content.

Brassica↗

PubMed2000

Antifungal activity of Hinokitiol-related compounds on wood-rotting fungi and their insecticidal activities.

Hinokitiol (beta-thujaplicin), beta-dolabrin and gamma-thujaplicin isolated from Thujopsis dolabrata SIEB. et ZUCC var hondai MAKINO showed antifungal activities against all of the wood-rotting fungi examined. The antifungal activity of three compounds on Daedalea dickinsii IFO-4979 was especially strong, their minimum inhibitory concentration (MIC) values being 0.2 microg/ml. Their antifungal activities on D. dickinsii IFO-4979 were as high as that of amphotericin B used as a positive control. Three compounds had strong insecticidal activities on Tyrophagus putrescentiae [50%-lethal concentration (LC50 : g/m2) 0.25 in hinokitiol, 0.02 in beta-dolabrin and gamma-thujaplicin. Their insecticidal activities were higher than that of N,N-diethyl-m-toluamide (DEET, LC50 : 1.46 g/m2) used as a positive control. Three compounds also showed strong insecticidal activity on Coptotermes formosanus [LC50 (g/m2) 0.07 in hinokitiol, 0.05 in beta-dolabrin and gamma-thujaplicin], although their insecticidal activities were much lower than that of commercial chloropyrifos (LC50 : 0.00016 g/m2).

Antifungal Agents↗

PubMed2000

Family 19 chitinases from Streptomyces thermoviolaceus OPC-520: molecular cloning and characterization.

Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced. The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively. Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain. Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases. The cloned Chi35 and Chi25 were purified from E. coli and S. lividans as a host, respectively. The optimum pH of Chi35 and Chi25 were 5-6, and the optimum temperature of Chi35 and Chi25 were 60 and 70 degrees C, respectively. Chi35 bound to chitin, Avicel, and xylan. On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35. These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain. Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25. In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain. However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains.

Amino Acid Sequence↗

PubMed2000

Molecular cloning of the gene encoding an outer-membrane-associated beta-N-acetylglucosaminidase involved in chitin degradation system of Alteromonas sp. strain O-7.

The gene encoding beta-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97kDa. A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane. The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial beta-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases.

Acetylglucosaminidase↗

PubMed2000

Purification and characterization of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520 and cloning of the encoding gene.

When Streptomyces thermoviolaceus OPC-520 was grown in a minimal medium with 1% chitin, three activity bands corresponding to proteins of 40 kDa (Chi40), 30 kDa (Chi30), and 25 kDa (Chi25) were detected. Among them, Chi30 was purified from the culture filtrate of the strain. The molecular mass was estimated to be 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its isoelectric point was 3.8. The optimum pH and temperature of Chi30 were 4.0 and 60 degrees C, respectively. Chi30 was stable at pH 6-8 up to 60 degrees C. The gene encoding Chi30 (chi30) was cloned and its nucleotides sequenced. The open reading frame of chi30 encoded a protein consisting of 347 amino acids with a calculated molecular weight of 35,621. The mature Chi30 consisted of only a catalytic domain and showed a significant similarity with ChiA from S. coelicolor and ChiA from S. lividans. The existence of a 12-bp direct repeat sequence in the promoter region of chi30 was detected, which have been suggested to be involved in both chitin induction and glucose repression.

Base Sequence↗

PubMed1999

Synthesis of chitinase in Streptomyces thermoviolaceus is regulated by a two-component sensor-regulator system.

The chiS and chiR genes located upstream of the chitinase locus (chi40) on the chromosome of Streptomyces thermoviolaceus OPC-520 were cloned and sequenced. The deduced amino acid sequences revealed that ChiS (390 amino acids, 40.9 kDa) and ChiR (213 amino acids, 22 kDa) show significant sequence similarities to histidine kinases and response regulators, respectively, of typical prokaryotic two-component regulatory systems. The extracellular chitinase activity of Streptomyces lividans 66 (pTSR2 (bearing chiS, chiR and chi40)) was significantly enhanced by a high dosage of the chiS and chiR genes.

Amino Acid Sequence↗

PubMed1999

Molecular analysis of the gene encoding a novel transglycosylative enzyme from Alteromonas sp. strain O-7 and its physiological role in the chitinolytic system.

We purified from the culture supernatant of Alteromonas sp. strain O-7 and characterized a transglycosylating enzyme which synthesized beta-(1-->6)-(GlcNAc)2, 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2- deoxyglucopyranose from beta-(1-->4)-(GlcNAc)2. The gene encoding a novel transglycosylating enzyme was cloned into Escherichia coli, and its nucleotide sequence was determined. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 99,560 Da which corresponds very closely with the molecular mass of the cloned enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the cloned enzyme was much larger than that of enzyme (70 kDa) purified from the supernatant of this strain. These results suggest that the native enzyme was the result of partial proteolysis occurring in the N-terminal region. The enzyme showed significant sequence homology with several bacterial beta-N-acetylhexosaminidases which belong to family 20 glycosyl hydrolases. However, this novel enzyme differs from all reported beta-N-acetylhexosaminidases in its substrate specificity. To clarify the role of the enzyme in the chitinolytic system of the strain, the effect of beta-(1-->6)-(GlcNAc)2 on the induction of chitinase was investigated. beta-(1-->6)-(GlcNAc)2 induced a level of production of chitinase similar to that induced by the medium containing chitin. On the other hand, GlcNAc, (GlcNAc)2, and (GlcNAc)3 conversely repressed the production of chitinase to below the basal level of chitinase activity produced constitutively in medium without a carbon source.

Amino Acid Sequence↗

PubMed1999

Inhibitory activities of 2-pyridinecarboxylic acid analogs on phytogrowth and enzymes.

Five 2-pyridinecarboxylic acid-related compounds (1, 2 and 5-7) showed germination inhibition against the seed of Brassica campestris L. subsp. rapa HOOK fil et ANDERS at a concentration of 5.0 x 10(-4) M. These compounds also demonstrated inhibitory activity on the growth of the root of this plant at a concentration of 3.0 x 10(-4) M; among these compounds, 2-pyridylacetic acid (5) showed the strongest inhibitory activity, and the effect was slightly stronger than that of sodium 2,4-dichlorophenoxyacetate (2,4-D) used as a positive control. The amounts of chlorophyll in the cotyledons of this plant treated with these active compounds was lower than that of the control group. Four compounds (1 and 5-7) with germination inhibition also showed inhibitory activities against alpha-amylase and carboxypeptidase A, and 5 was the strongest inhibitor toward both enzymes.

Brassica↗

PubMed1999

Antimicrobial activity and metalloprotease inhibition of hinokitiol-related compounds, the constituents of Thujopsis dolabrata S. and Z. hondai MAK.

Gamma-thujaplicin, beta-dolabrin and hinokitiol(beta-thujaplicin), hinokitiol-related compounds isolated from the wood of Thujopsis dolabrata S. and Z. hondai MAK have antimicrobial activity. In particular, strong antibacterial activity of hinokitiol and beta-dolabrin on Staphylococcus epidermidis IFO-12993 was found, with a minimum inhibitory concentration (MIC) of 0.2 microg/ml. This activity was higher than that of gentamicin, used as a positive control, and so the strong antibacterial activity of both compounds on this bacterium is of considerable interest. Of the three compounds, gamma-thujaplicin showed the strongest antifungal activity and its MIC was found to be around 1.5 microg/ml. The three compounds also inhibited metalloproteases. The inhibitory activity of hinokitiol on carboxypeptidase A was especially strong, its 50%-inhibitory concentration (IC50) being 2.76x10(-6) M. Considering that metalloproteases are involved in inflammation, the strong inhibitory activity of hinokitiol could be important. On the other hand, hinokitiol-acetate did not show any antimicrobial activity and metalloprotease inhibition, suggesting that at least part of the activity is due to metal chelation between the carbonyl group at C-1 and the hydroxyl group at C-2 in the tropolone skeleton.

Anti-Bacterial Agents↗

PubMed1998

Cloning, characterization and expression of beta-N-acetylglucosaminidase gene from Streptomyces thermoviolaceus OPC-520(1).

The nagB gene encoding beta-N-acetylglucosaminidase from S. thermoviolaceus OPC-520 was cloned and sequenced. The nagB gene could encode a protein of 541 amino acids with a calculated molecular mass of 58274. NagB revealed significant similarities to beta-N-acetylhexosaminidases and chitobiases from bacteria, which are classified into family 20 glycosyl hydrolases. NagB effectively hydrolyzed all of the chitin oligosaccharides from dimer to hexamer.

Acetylglucosaminidase↗

PubMed1998

Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure.

One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.

Amino Acid Sequence↗

PubMed1998

A novel beta-N-acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: gene cloning, expression, and assignment to family 3 of the glycosyl hydrolases.

A beta-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an Mr of 66, 329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned beta-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical beta-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable beta-glucosidase activity, revealed homology with microbial beta-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of beta-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60 degreesC and pH 5.0, and the apparent Km and Vmax values for p-nitrophenyl-beta-N-acetylglucosamine were 425.7 microM and 24.8 micromol min-1 mg of protein-1, respectively.

Acetylglucosaminidase↗