Biomedical subjects

K Muramoto

Publications and source records attributed to K Muramoto.

At least 55 records · Page 3Linked to original sources

PubMed1998

Molecular properties and activity of a carboxyl-terminal truncated form of xylanase 3 from Aeromonas caviae W-61.

Aeromonas caviae W-61 produces five species of xylanases, xylanases 1, 2, 3, 4, and 5 [Nguyen, V.D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993) and Appl. Environ. Microbiol., 57, 445-449 (1991)]. While preserving a purified xylanase 3 preparation from A. caviae in solution at 4 degrees C, the xylanase 3 was found to be proteolyzed to give a truncated form with a smaller molecular mass than that of the intact one. It appears likely that the truncated form of xylanase 3 was produced in this particular purification experiment by the action of a contaminating protease. We isolated the truncated form of xylanase 3 (Xyn3tr), of which the C-terminal 102-residue segment is missing. By the chemical analysis of the N- and C-terminal amino acid residues of Xyn3tr and the DNA sequencing analysis of the xylanase 3 gene (xyn3), the N-terminal 398th proline residue of xylanase 3 was found to be the C-terminus of Xyn3tr. Xyn3tr had the activity to form xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as main final products from oat spelt xylan. In contrast, intact xylanase 3 released X6 and higher xylo-oligosaccharides as main products. Xylanase 3 hydrolysed X4 through X6. However, Xyn3tr had no activity towards X4 and X5. The recombinant Xyn3tr and recombinant xylanase 3 (XYN3) were purified homogeneously from the periplasmic space of E. coli harboring the plasmids pXYN3 and pXYN3tr, which include xyn3 and xyn3tr genes, respectively, and their enzymatic activities were measured. The cleavage patterns of oat spelt and xylo-oligosaccharides by XYN3tr were identical with that by intact Xyn3tr. Thus, we conclude that the C-terminal region comprising a 102-residue segment in xylanase 3 is involved in governing the molecular size of xylo-oligosaccharides cleaved from beta-1,4-xylan by the enzyme and in the hydrolytic activity towards X4 and X5.

Aeromonas↗

PubMed1997

Up-regulation of ganglioside biosynthesis, functional synapse formation, and memory retention by a synthetic ceramide analog (L-PDMP).

To address the role of brain gangliosides in synaptic activity, the ceramide analogs, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and its enantiomer, L-PDMP, were used to inhibit and stimulate ganglioside biosynthesis in cultured cortical neurons. Prolonged treatment with both PDMP isomers exhibited opposite effects on functional synapse formation measured by spontaneous synchronized oscillatory activity of intracellular Ca2+ between the neurons: suppression by D-PDMP and facilitation by L-PDMP. Up-regulation of synaptic activity by L-PDMP could be correlated with the slow but robust stimulation of ganglioside biosynthesis through activating GM3, GD3 and GQ1b synthases. In a similar time course, the activity of p42 mitogen-activated protein kinase was also enhanced by L-PDMP. To evaluate the efficacy of this drug in long-term memory, rats were trained for 2 weeks using an 8-arm radial maze task, and then forebrain ischemia was induced by 4-vessel occlusion. Treatment with L-PDMP starting 24 hours after the transient ischemia ameliorated the deficit of a well-learned spatial memory, demonstrating the potential therapeutic intervention of the ceramide analog for neurodegenerative disorders.

Animals↗

PubMed1997

Vibrio alginolyticus mutants resistant to phenamil, a specific inhibitor of the sodium-driven flagellar motor.

The polar flagella of Vibrio alginolyticus are driven by sodium motive force and those motors are specifically and strongly inhibited by phenamil, an amiloride analog that is thought to interact with a sodium channel of the flagellar motor. To study the sodium ion coupling site, we isolated motility mutants resistant to phenamil and named the phenotype Mpa(r) for motility resistant to phenamil. The motility of the wild-type (Mpa(s)) was inhibited by 50 microM phenamil, whereas Mpa(r) strains were still motile in the presence of 200 microM phenamil. The Ki value for phenamil in the Mpa(r) strain was estimated to be five times larger than that in the Mpa(s) strain. However, the sensitivities to amiloride or benzamil, another amiloride analog, were not distinctly changed in the Mpa(r) strain. The rotation rate of the wild-type Na+-driven motor fluctuates greatly in the presence of phenamil, which can be explained in terms of a relatively slow dissociation rate of phenamil from the motor. We therefore studied the stability of the rotation of the Mpa(r) and Mpa(s) motors by phenamil. The speed fluctuations of the Mpa(r) motors were distinctly reduced relative to the Mpas motors. The steadier rotation of the Mpa(r) motors can be explained by an increase in the phenamil dissociation rate from a sodium channel of the motor, which suggests that a phenamil-specific binding site of the motor is mutated in the Mpa(r) strain.

Amiloride↗

PubMed1997

Purification and characterization of proteasome from ostrich liver.

The proteasome (EC 3.4.99.46) is a high molecular mass (approximately 700 kDa) multisubunit enzyme complex which is the focus of worldwide research in order to identify the structure, mechanism of action and specificity of the complex. The purpose of the present study was to investigate the tryptic, chymotryptic and peptidylglutamyl-peptide hydrolysing (PGPH) activities of ostrich liver proteasome. The proteasome was purified from ostrich liver by employing ammonium sulphate fractionation, followed by three sequential chromatographic steps on Toyopearl Super Q-650 S, Sephadex G-150 and phenyl-Toyopearl columns. Temperature and pH optima were examined and the effect of inhibitors, detergents, fatty acids and cations on the peptidase activities was determined. Ostrich proteasome exhibited a relative M(r) of approximately 665,000 using non-denaturing gradient PAGE and dissociated into the characteristic "ladder" associated with the proteasome subunits during SDS-PAGE. The pH optima for the peptidase activities were found to be slightly alkaline (tryptic activity) and neutral (chymotryptic-like and PGPH activities). Ostrich liver proteasome was found to be activated in terms of the PGPH activity by fatty acids and SDS, whereas the chymotryptic and tryptic-like activities were differentially inhibited. Ostrich proteasome, in its inhibition by monovalent cations, was similar to the proteasomes extracted from other sources. The specificity of the proteasome appears to be very broad, although it lacks aminopeptidase activity. The yield compared favourably with similar extraction procedures which have been reported. On the basis of the physicochemical and kinetic properties which ostrich liver proteasome exhibited, it can be safely concluded that it corresponds well with the proteasomes isolated from many other sources.

Amino Acid Sequence↗

PubMed1997

Purification and partial characterization of an ostrich alpha 1-antichymotrypsin-like serum inhibitor.

alpha 1-Antichymotrypsin, a member of the serpins, is the predominant plasma inhibitor of neutrophil cathepsin G. The aim of this study was to purify ostrich alpha 1-antichymotrypsin and to compare its biochemical properties with those of other species. Ostrich alpha 1-antichymotrypsin was purified from serum by ammonium sulphate fractionation, QAE-Sephadex C-50 and phenyl-Toyopearl chromatography. N-terminal sequence, amino acid composition, molecular mass, isoelectric point and reaction with cathepsin G, elastase and chymotrypsin were determined. SDS-PAGE revealed a M, of 55,000 for ostrich alpha 1-antichymotrypsin and pI values of 6.8 and 4.1-4.3 were obtained. The amino acid composition revealed 444 residues and the N-terminal sequence of the first 20 residues revealed a homology of 30% when compared with several other alpha 1-antichymotrypsin sequences. Total inhibition of cathepsin G by ostrich alpha 1-antichymotrypsin was found at a 4:1 molar ratio of inhibitor to enzyme which was similar to that found for commercial alpha 1-antichymotrypsin. Immunological studies highlighted the lack of cross-reactivity between ostrich and human alpha 1-antichymotrypsin. The study indicated that ostrich alpha 1-antichymotrypsin-like molecule exhibited similar properties to human alpha 1-antichymotrypsin although there were notable differences.

Amino Acid Sequence↗

PubMed1997

Gene of LukF-PV-like component of Panton-Valentine leukocidin in Staphylococcus aureus P83 is linked with lukM.

Staphylococcus aureus P83 (ATCC 31890) produces five components, I to V for synergistic toxins, leukocidin and gamma-hemolysin [Sudo et al., Biosci. Biotech. Biochem., 56, 1786-1789 (1995)]. We report here the identification of component II, which is designated LukF-PV(P83) and its gene (lukF-PV(P83)). The lukF-PV(P83) gene was found to be one base downstream of the stop codon of the lukM gene. The deduced amino acid sequence of LukF-PV(P83) showed 78.4% identity with that of LukF-PV. The lukM and lukF-PV(P83) genes were encoded as one operon like that of Panton-Valentine leukocidin.

Amino Acid Sequence↗

PubMed1997

[Rotavirus-associated gastroenteritis in adults].

The human rotavirus in adult diarrhea cases was evaluated in 109 cases during 12 months, 1996. Rotavirus antigen was identified, using the ELISA technique, in stool samples in 23 cases (21%). We arranged the positive cases as rotavirus-associated gastroenteritis in adults and analyzed them. Fourteen out of them (74%) occurred from June through September. Fourteen cases (74%) needed to be hospitalized. Colonoscopy revealed some inflammatory changes in 9 out of 16 cases (47%). Most of symptom was watery diarrhea, but bloody diarrhea was observed in 6 cases (32%). It was suggested we should introduce a concept of rotavirus-associated gastroenteritis in adults.

Adolescent↗

PubMed1996

Rotational fluctuation of the sodium-driven flagellar motor of Vibrio alginolyticus induced by binding of inhibitors.

Rotation of the Na(+)-driven flagellar motor of Vibrio alginolyticus was investigated under the influence of inhibitors specific to the motor, amiloride and phenamil. The rotation rate of a single flagellum on a cell stuck to a glass slide was examined using laser dark-field microscopy. In the presence of 50 mM NaCl, the average rotation rate (omega) was about 600 r.p.s. with a standard deviation (sigma omega) of 9% of omega. When omega was decreased to about 200 r.p.s. by the presence of 1.5 mM amiloride, sigma omega increased to 15% of omega. On the other hand, when omega was decreased to about 200 r.p.s. by the addition of 0.6 microM phenamil, a large increase in sigma omega up to 50% of omega, was observed. Similarly large fluctuations were observed at other concentrations of phenamil. These observations suggest that dissociation of phenamil from the motor was much slower than that of amiloride. A very low concentration of phenamil caused a transient but substantial reduction in rotation rate. This might suggest that binding of only a single molecule of phenamil strongly inhibits the torque generation in the flagellar motor.

Amiloride↗

PubMed1996

Effects of glucosylceramide synthase inhibitor and ganglioside GQ1b on synchronous oscillations of intracellular Ca2+ in cultured cortical neurons.

To evaluate the role of endogenous gangliosides in synapse formation, the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), was used to deplete glycosphingolipids (GSLs) of cultured rat cerebral cortical neurons. Synapse formation between the neurons was estimated by the synchronous oscillation of synaptic activity monitored by fura-2 calcium imaging. Treatment with D-PDMP resulted in dose- and time-dependent decreases in the frequency of synchronous oscillations. When a series of gangliosides (GM3, GM1, GD3, GD1b, GT1b, and GQ1b) was supplemented to the GSL depleted cells, only GQ1b was able to normalize the decreased frequency by D-PDMP. These results suggest that de novo synthesis of a particular molecular species of the gangliosides, GQ1b, is essential for synapse formation and synaptic activity.

Animals↗

PubMed1996

Ostrich (Struthio camelus) carboxypeptidase B: purification, kinetic properties and characterization of the pancreatic enzyme.

Carboxypeptidase B has been isolated from numerous mammalian and invertebrate species. In contrast, very little is known about carboxypeptidases of avian origin. To provide information for a comparative study, we have undertaken an investigation of the kinetic and physical properties of ostrich carboxypeptidase B. Carboxypeptidase B from the pancreas of the ostrich was purified by water extraction of acetone powder and aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. The effects of pH and temperature on CPB activity were examined. K(i)-values for numerous inhibitors (PCI, ABSA, hipp-D-lys, epsilon-aminocaproic acid, D-arg and 3-phenylproprionic acid) and kinetic parameters (K(m), k(cat) and k(cat)/K(m)) for several substrates (hipp-arg, hipp-lys, FAAA, FAAL and hipp-AA) were determined. N-terminal sequencing and amino acid analysis were also performed. Purified ostrich carboxypeptidase B was assessed to be homogeneous by SDS-PAGE with a M(r) value of approx. 35,000. For ostrich carboxypeptidase B the K(m) values for the different substrates were of the same order as those reported for other species, whereas the k(cat) values were 8- to 21-fold lower than the reported values. FAAA and hipp-AA were the preferred substrates. PCI was the most effective inhibitor, with a K(i) in the nM region, and no inhibition was shown with 3-phenylpropionic acid. The N-terminal sequence showed a high degree of homology when aligned with CPB from other species. Amino acid analysis showed significantly lower levels of Asx and Cyh and higher levels of Trp and Leu when compared with other species. Ostrich carboxypeptidase B would appear to show many physical, chemical and kinetic properties similar to those of other known carboxypeptidases.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of ostrich pancreatic secretory trypsin inhibitor.

Ostrich pancreatic secretory trypsin inhibitor was isolated and purified using acid extraction, salt fractionation. SP-Sephadex C-50 and QAE-Sephadex A-25 chromatography and RP-HPLC. The amino acid sequence of ostrich PSTI showed it is a single peptide chain containing 69 amino acid residues with the highest homology between ostrich and chicken PSTI. The molecular weight, as determined by electronspray mass spectrometry and from amino acid sequence data, is 7650 Da. The isoelectric point of ostrich PSTI was found to be 5.7. Ostrich PSTI specifically inhibited ostrich and commercial bovine trypsin with Ki values of 8.0 x 10(-9) and 2.4 x 10(-7) M, respectively, while no inhibitory effects were observed with other serine proteases.

Amino Acid Sequence↗

PubMed1996

Angiotensin I-converting enzyme inhibitors in autolysates of squid liver and mantle muscle.

Autolysis of squid liver and mantle muscle homogenates, and blends of both, each yielded inhibitory activity toward the angiotensin I-converting enzyme. The inhibitory activity and the amount of solubilized protein in each of these autolysates were examined over a period of 24 h. Inhibitory peptides were isolated from the mixed autolysate, their structures (IC50) being Tyr-Ala-Leu-Pro-His-Ala (9.8 microM) and Gly-Tyr-Ala-Leu-Pro-His-Ala (27.3 microM).

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1995

Molecular cloning of the defense factor in the albumen gland of the sea hare Aplysia kurodai.

Aplysianin-A, an antibacterial glycoprotein in the albumen gland of the sea hare Aplysia kurodai, inhibited the growth of both Gram-positive and Gram-negative bacteria. Aplysianin-A cDNA clones were isolated from an albumen gland cDNA library. Sequence analysis reveals that aplysianin-A is produced as a precursor protein of 556 amino acid residues with a signal peptide of 19 amino acid residues and contains 6 potential N-glycosylation sites. Aplysianin-A mRNA was expressed tissue-specifically in the albumen gland. Homology search reveals that aplysianin-A has a 50% overall amino acid sequence homology to achacin, an antibacterial glycoprotein of the giant African snail Achatina fulica.

Amino Acid Sequence↗

PubMed1995

High-speed rotation and speed stability of the sodium-driven flagellar motor in Vibrio alginolyticus.

The Na(+)-driven flagellar motor in Vibrio alginolyticus rotates very fast. Rotation of a single flagellum on a stuck cell was measured by laser darkfield microscopy with submillisecond temporal resolution. The rotation rate increased with increasing external concentration of NaCl, and reached 1000 r.p.s. at 300 mM NaCl. The Na+ influx through the motor should determine the rotation period (tau) and affect the speed stability. Fluctuation of the rotation period was analyzed at various rotation rates (from approximately 50 r.p.s. to approximately 1000 r.p.s.), which were changed by changing the external concentration of NaCl and the addition of a protonophore or a specific inhibitor. At high rotation rates (over 400 r.p.s.), the observed rotation was stable, and the standard deviation of tau (sigma tau) ranged from 7% to 16% of the average rotation period (< tau >). At low rotation rates (under 100 r.p.s), the rotation period tended to fluctuate, and the distributions of tau were non-Gaussian. The value of sigma tau ranged from 10 to 30% of < tau >. However, the observed minimum value of sigma tau at various rotation rates was approximately equal to the calculated standard deviation due to the rotational diffusion of the flagellar filament. These results suggest that the torque was stably generated at various Na+ influxes through the motor. We observed large fluctuations that cannot be explained by rotational diffusion. We discuss the factors that induce the large fluctuation.

Biophysical Phenomena↗

PubMed1995

Existence of a new protein component with the same function as the LukF component of leukocidin or gamma-hemolysin and its gene in Staphylococcus aureus P83.

Staphylococcal toxins, leukocidin and gamma-hemolysin, consist of two protein components, i.e. LukF and LukS for leukocodin and H gamma I and H gamma II for gamma-hemolysin. From a culture fluid of Staphylococcus aureus strain P83, a new protein component of leukocidin or gamma-hemolysin which was designated as LukM was isolated. This component showed the same biological activity as that of LukF component for leukocidin or H gamma I component of gamma-hemolysin in combination with LukS or H gamma II. However, the LukM component cross-reacted with the anti-LukS antibodies but not with the anti-LukF antibodies. On the basis of chemical analysis of the LukM component and the cloning and nucleotide sequencing of the lukM gene of S. aureus P83, we have demonstrated that LukM is an entirely new protein component of leukocidin or gamma-hemolysin. The deduced amino acid sequence of LukM from the lukM gene showed 66.7% and 67% identity to that of LukS and H gamma II, respectively. However, the amino acid sequence of LukM and LukF showed only 29% homology.

Amino Acid Sequence↗

PubMed1995

Ostrich pepsinogens I and II: purification, activation and chemical and immunochemical characterization of the enzymes from the proventriculus.

Pepsins are a series of gastric proteases secreted as inactive precursors (pepsinogens) which are active at acidic pH. The aim of this study was to purify ostrich pepsin(ogen)s and to compare their biochemical and immunological characteristics with those of pepsin(ogen)s of mammalian and avian origin. Ostrich pepsinogens were purified by ammonium sulphate fractionation, Toyopearl Super Q-650S chromatography and rechromatography, and hydroxylapatite chromatography of a pH 8.0 mucosal extract. Pepsins were obtained through acidification, and purified by chromatography on SP-Sephadex C-50. Amino acid compositions, N-terminal sequences, Ouchterlony double-diffusion as well as Western blot analysis were performed. Two pepsinogens were isolated and purified from the proventriculus of the ostrich, pepsinogens I and II. Both pepsinogens and pepsins were purified to homogeneity as shown by PAGE and SDS-PAGE, with SDS-PAGE revealing M(r) values of 40,400 and 41,900 for pepsinogens I and II, respectively. SDS-PAGE revealed M(r) values of 36,000 and 36,300 for ostrich pepsins I and II, respectively. Ostrich pepsinogens I and II were found to have identical N-terminal sequences, with Asp as N-terminal amino acid. Amino acid compositions were obtained for both pepsinogens, with ostrich pepsinogen I being slightly smaller in size with a total of 356 residues compared to 371 for ostrich pepsinogen II. Pepsinogen II showed a pI of 4.29. Ostrich pepsinogens I and II were found to be immunologically separate entities, and no cross-reactivity was observed between anti-(ostrich pepsinogen I/II) sera and porcine pepsin/pepsinogen. The study indicates that only two pepsinogens are present in the ostrich. They differ in terms of electrophoretic mobility, molecular mass and immunological reactivity, but have been found to have identical N-terminal sequences. It is concluded that both pepsinogens belong to the pepsinogen A class of aspartyl proteases (EC 3.4.23.1).

Amino Acid Sequence↗

PubMed1995

Simultaneous measurement of bacterial flagellar rotation rate and swimming speed.

Swimming speeds and flagellar rotation rates of individual free-swimming Vibrio alginolyticus cells were measured simultaneously by laser dark-field microscopy at 25, 30, and 35 degrees C. A roughly linear relation between swimming speed and flagellar rotation rate was observed. The ratio of swimming speed to flagellar rotation rate was 0.113 microns, which indicated that a cell progressed by 7% of pitch of flagellar helix during one flagellar rotation. At each temperature, however, swimming speed had a tendency to saturate at high flagellar rotation rate. That is, the cell with a faster-rotating flagellum did not always swim faster. To analyze the bacterial motion, we proposed a model in which the torque characteristics of the flagellar motor were considered. The model could be analytically solved, and it qualitatively explained the experimental results. The discrepancy between the experimental and the calculated ratios of swimming speed to flagellar rotation rate was about 20%. The apparent saturation in swimming speed was considered to be caused by shorter flagella that rotated faster but produced less propelling force.

Biophysical Phenomena↗

PubMed1995

Application of long-term cultured neurons in aging and neurological research: aluminum neurotoxicity, synaptic degeneration and Alzheimer's disease.

Dissociated hippocampal and cerebral cortical neurons from rat embryonic brain form many synapses in culture. The neuronal networks in culture fire spontaneously in synchronous oscillation and can be maintained for long periods of up to 6 months. To establish the relevance of this long-term culture to aging and neurodegenerative diseases, effects of long-term exposure to aluminum, a risk factor for Alzheimer's disease, on the cultured cortical neurons were investigated. It appeared that aluminium promoted the aggregation of amyloid beta-protein and enhanced its neurotoxicity.

Aluminum↗