Biomedical subjects
Publications and source records attributed to H Tsujibo.
Chitinase (Chi85) from Alteromonas sp. strain O-7 contains the two conserved regions common to microbial and plant chitinases. We did site-directed mutagenesis of Chi85 to investigate the effects of the conserved amino acid residues on chitinase activity. We suggest that Asp-290 and Glu-292 of Chi85 may be the essential amino acid residues for the cleavage of beta-glycosidic linkage of chitin.
Two types of xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were isolated from the culture filtrate of a thermophilic actinomycete, Streptomyces thermoviolaceus OPC-520. The enzymes (STX-I and STX-II) were purified by chromatography with DEAE-Toyopearl 650 M, CM-Toyopearl 650 M, Sephadex G-75, Phenyl-Toyopearl 650 M, and Mono Q HR. The purified enzymes showed single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of STX-I and STX-II were 54,000 and 33,000, respectively. The pIs were 4.2 (STX-I) and 8.0 (STX-II). The optimum pH levels for the activity of STX-I and STX-II were pH 7.0. The optimum temperature for the activity of STX-I was 70 degrees C, and that for the activity of STX-II was 60 degrees C. The enzymes were completely inhibited by N-bromosuccinimide. The enzymes degraded xylan, producing xylose and xylobiose as the predominant products, indicating that they were endoxylanases. STX-I showed high sequence homology with the exoglucanase from Cellulomonas fimi (47% homology), and STX-II showed high sequence homology with the xylanase from Bacillus pumilus (46% homology).
Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 degrees C, respectively. Chi-A was stable in the range of pH 5-10 up to 40 degrees C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulans WL-12.
We examined the inhibitory activities of 3,3'-dihydroxy-alpha,beta-diethylstilbene (DDS) and 3,3',4,5'-tetrahydroxystilbene (THS) on microtubule assembly in vitro and their effects on chromosome number and cellular microtubule networks in Chinese hamster V79 cells. DDS showed half the inhibitory activity of diethylstilbestrol (DES) on microtubule assembly in vitro, while THS had none of the inhibitory activity. DDS induced tetraploid at 30 microM, whereas THS was found to be inactive. Furthermore, DDS disturbed cellular microtubule networks at 100 microM. We also examined the effects of DES, DDS and THS on cellular actin networks in mouse BALB 3T3 cells. DES induced a change of actin stress fiber distribution and THS had similar activity, while DDS showed no activity.
Two chalcones, xanthoangelol (I) and 4-hydroxyderricin (II), isolated from the root of Angelica keiskei KOIDZUMI (Umbelliferae) showed antibacterial activity against gram-positive pathogenic bacteria. The activity of I on Micrococcus luteus IFO-12708 (minimum inhibitory concentration (MIC), 0.76 microgram/ml) was the same potency as that of gentamicin, which is used as a standard. Although the activity of both chalcones on plant-pathogenic bacteria was lower than that of streptomycin sulfate, used as a positive control, they also exhibited growth-inhibitory effects. The antibacterial activity of I isolated from Angelica keiskei KOIDZUMI is being reported here for the first time. The growth-inhibitory effect of II on plant-pathogenic bacteria is also reported for the first time in this paper.
3,4'-Dihydroxy-alpha,beta-diethylstilbene (I), like diethylstilbestrol (II), showed phytogrowth-inhibitory and antimicrobial activities. First, compound I showed strong growth-inhibitory activity against the roots of two kinds of plants. The inhibitory activity of I was almost equal to that of sodium 2,4-dichlorophenoxyacetate used as a positive control. The phytogrowth-inhibitory activity of I was much higher than that of II. Next, unlike II, I had broad antifungal spectrum against phathogenic fungi. Compound I showed antifungal activity against six kinds of Fusarium oxysporum sp. This compound also had antibacterial activity against pathogenic and plant-pathogenic bacteria. These antibacterial activities of I were as high as those of II, the isomer of I. It should be emphasized that by shifting one of the phenolic hydroxyl groups of II to meta-position, phytogrowth-inhibitory activity was largely increased, while antimicrobial activity was unchanged.
3,3',4,5'-Tetrahydroxystilbene (I) and 3,3',4,5'-tetrahydroxybibenzyl (II), isolated from the heartwood of Cassia garrettiana Craib (Leguminosae), showed inhibitory effects on antigen-induced histamine release from rat peritoneal mast cells in vitro. The inhibitory effect of I (IC50 = 30.2 microM) was much stronger than that of II (greater than 100 microM). Compound II, as well as I (IC50 = 7.3 microM) reported previously, also inhibited the histamine release from human peripheral basophils induced by anti-immunoglobulin E (IgE) in vitro, and its IC50-value was 68.0 microM. These results suggest that the trans-olefin structure in the molecule may be necessary for I to have an inhibitory effect on histamine release. Considering that disodium cromoglycate did not show any significant inhibitory effect on anti-IgE-induced histamine release from human basophils, the strong inhibitory effects of I in both tests are of considerable interest.
3,3',4,5'-Tetrahydroxystilbene (I), a constituent of Cassia garrettiana, strongly inhibited the anti-IgE-induced histamine release from human basophils in vitro at concentrations of 3 to 30 microM. Considering that disodium cromoglycate showed no significant inhibitory activity in this assay method, the strong effect of I should be emphasized.
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Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21,000 and 36,000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10-12 (protease I) and pH 10.5 (protease II). The optimum temperture for the activity of protease I was 70 degrees C and that for protease II was 60 degrees C. Protease I was stable in the range of pH 4.0-8.0 up to 60 degrees C and protease II was stable in the range of pH 6.0-12.0 up to 50 degrees C.
Angiotensin-converting enzyme (ACE) inhibitors were excised from the molecule of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preparation of baker's yeast by heating at 120 degrees C in 1 M AcOH-20 mM HCl. Three inhibitors were then purified by gel-permeation and reverse-phase chromatographies. One of the yeast ACE inhibitors, YG-3, was GAPDH peptide 79-89 (Pro-Ala-Asn-Leu-Pro-Trp-Gly-Ser-Ser-Asn-Val, IC50:18 microM), and contained the sequence homologous to vertebrate ACE inhibitors (GAPDH peptides 79-86 or 81-88). Other inhibitors, YG-1 (Gly-His-Lys-Ile-Ala-Thr-Phe-Gln-Glu-Arg, IC50: 0.4 microM) and YG-2 (Gly-Lys-Lys-Ile-Ala-Thr-Tyr-Gln-Glu-Arg, IC50: 2 microM), corresponded to amino acid residues 68-77 in two different forms of yeast GAPDH, respectively. Their sequences were quite different from those of the venom peptide family. YG-1 was the most potent ACE inhibitor among yeast and vertebrate GAPDH peptides excised by acid-limited proteolysis. Thus, yeast GAPDH seems to be an excellent source of naturally occurring ACE inhibitors.
Four hypotensive peptides called elapherine-A, -B, -C and -D were isolated from the body of Elaphe climacophora after removal of the internal organs. Elapherine-A, which had the lowest molecular weight, exhibited a prolonged fall for 5 min in the blood pressure of spontaneously hypertensive rats, whereas elapherin-B, -C and -D displayed a transient fall in the blood pressure. The molecular weights of elapherine-A---D estimated from sodium dodecyl sulfate (SDS) gel disk electrophoresis were 6600, 6900, 7000 and 7200, respectively. The isoelectric points of these peptides were 10.4, 10.6, 10.7 and 10.5, respectively. They possessed a single polypeptide chain of 34-35 (elapherine-A), 56-58 (elapherine-B), 56-57 (elapherine-C) and 52-53 (elapherine-D) amino acid residues.
The diethylstilbestrol-related compounds 3,3'-dihydroxy-alpha, beta-diethyldiphenylethane (I), diethylstilbestrol (II) and hexestrol (III) showed hypotensive effects on spontaneously hypertensive rats (SHR) and antifungal activities against all Fusarium oxysporum sp. tested. As previously reported, I had strong hypotensive action on normotensive rats at the dose of 10 mg/kg, while II and III showed weak hypotensive effects on these rats at the same dose. In this work, all three compounds also had hypotensive actions on SHR at the same dose. I showed the strongest hypotensive effect (-80.0 +/- 5.0 mmHg, 10 mg/kg, i.v.) on both SHR and normotensive rats. The three compounds also had antifungal activities against five kinds of Fusarium oxysporum sp. tested. Especially, II strongly inhibited the growth of Fusarium oxysporum f. sp. raphani IFO-9972 (minimum inhibitory concentration (MIC): 1.0 micrograms/ml).
Racemomycin-B (RM-B), the main component of Streptomyces lavendulae OP-2 which is the basis of 50% of the antibiotics produced, is a streptothricin antibiotic which contains three beta-lysine moieties in the molecule. RM-B had antimicrobial activity against plant-pathogenic microorganisms and growth-inhibitory activity against the root of Brassica rapa L. at the concentration of 50 ppm. It strongly inhibited the growth of Pseudomonas syringae pv. tabaci IFO-3508 (minimum inhibitory concentration (MIC): 0.4 microgram/ml), and also showed antifungal activity against six kinds of Fusarium oxysporum species (MIC: 0.1-2.0 micrograms/ml). The antimicrobial activity of RM-B was much stronger than those of RM-A and -C which contain, respectively, one and two beta-lysine moieties in their molecules. The above activities of RM-A, -C and -B were thus in the order of -B greater than -C greater than -A: namely, the biological activity of racemomycin compounds tended to be stronger with increase in the number of beta-lysine moieties in the molecule.
An alkalophilic actinomycete, strain OPC-553 regarded as Nocardiopsis dassonvillei subsp. prasina, produced the cytotoxic substance, TS-1, which showed a marked inhibitory activity against L5178Y mouse leukemic cell in vitro. The cytotoxicity of TS-1 on this cell was very strong and its ID50 was 0.018 micrograms/ml. Through direct comparison of its spectral data with those of an authentic sample, TS-1 was identified as the antifungal antibiotic, kalafungin, already isolated from the culture broth of Streptomyces tanashiensis. However, the isolation of kalafungin from an alkalophilic actinomycete and its cytotoxicity are reported for the first time in this paper.
2,5-Dihydroxy-1,4-dithiane (I) and its derivatives (II-IV) showed rather marked inhibitory activities on the growth of the roots of two plant species. All compounds tested had phytogrowth-inhibitory activities. These compounds markedly inhibited the growth of the two plant species at the concentration of 1.0 x 10(-3) M. Seeds of Brassica rapa treated with 2,5-dihydroxy-2,5-dimethyl-1,4-dithiane (III) and its diacetate (IV) at the same concentration failed to germinate. Among these compounds, IV showed the most potent inhibitory activity on the two plant species. The radicles of both plant species treated with these compounds at concentrations higher than 1.0 x 10(-4) M showed negative geotropism, even though germination occurred. The compounds except for 2,5-diacetoxy-1,4-dithiane (II) also had antibacterial activities. In particular, III had rather marked antibacterial activity and its minimal inhibitory concentration (MIC) for Staphylococcus aureus IFO-3060 and Escherichia coli IFO-12734 was 4.0 micrograms/ml.
The more potent inhibitory activity against angiotensin-converting enzyme (ACE) was excised from a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preparation of Bacillus stearothermophilus by heating at 120 degrees C in 1 M AcOH-20 mM HCl, as compared with GAPDH preparations of yeast and pig. Sufficient excision of B. stearothermophilus ACE inhibitors required a longer proteolysis time of 60 min. Two inhibitors were then purified by gel-permeation and reverse-phase chromatographies. One of the B. stearothermophilus ACE inhibitors BG-1, was the GAPDH peptide 68-77 (Gly-Lys-Glu-Ile-Ile-Val-Lys-Ala-Glu-Arg, IC50: 32 microM). Another inhibitor, BG-2 (Gly-Lys-Met-Val-Lys-Val-Val-Ser-Trp-Tyr, IC50: 6 microM), correspond to GAPDH peptide 304-313. These sequences were quite different from those of vertebrate GAPDH peptides and the venom peptide family with ACE inhibitory activity. BG-2 was found to be a non-competitive type inhibitor, differing from many natural peptide inhibitors. Thus, B. stearothermophilus GAPDH seemed to be a good source of new type ACE inhibitors, in addition to the advantages due to its thermophilic property.
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