Biomedical subjects

K Soda

Publications and source records attributed to K Soda.

At least 19 recordsLinked to original sources

PubMed2001

Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.

Alanine Racemase↗

PubMed2001

Chemo-enzymatic D-enantiomerization of DL-lactate.

We investigated the total conversion of racemic lactate, L-lactate, and pyruvate into D-lactate, which is very useful as a starting material for the synthesis of chiral compounds and much more valuable than the L-enantiomer by means of coupling of L-specific oxidation of the racemate with L-lactate oxidase and non-enantiospecific reduction of pyruvate to DL-lactate with sodium borohydride. In this one-pot system, L-lactate was enantiospecifically oxidized to an achiral product, pyruvate, which was chemically reduced to DL-lactate leading to a turnover. Consequently, either DL-lactate, L-lactate, or pyruvate was fully converted to the D-enantiomer. We optimized the reaction conditions: DL-lactate was converted to D-lactate in 99% of the theoretical yield and with more than 99% enantiomeric excess. DL-alpha-Hydroxybutyrate and alpha-ketobutyrate were converted also to D-alpha-hydroxybutyrate in the same way, though slowly.

Borohydrides↗

PubMed2001

Assay of human platelet guanylate cyclase activity by high-performance liquid chromatography with fluorescence derivatization.

A selective and sensitive high-performance liquid chromatography (HPLC) method with fluorescence derivatization for the assay of guanylate cyclase (GC) activity is described. GTP and cGMP, which are the substrate and the product of GC, respectively, and other guanine-containing compounds are selectively converted by the reaction with (3,4-dimethoxyphenyl)glyoxal to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. The limit of detection at a signal-to-noise ratio of 3 for cGMP was 10 fmol on the column. The sensitivity of this method was less than that of the conventional radioisotopic method, but this method is simple and convenient. Human platelet GC activity was measured, and the effects of some compounds were investigated.

Blood Platelets↗

PubMed2001

Magnetic circular dichroism at transition metal L2,3 edges in D03-type (Fe(1-x)Mn(x))3Al alloys.

We have measured magnetic circular dichroism (MCD) spectra at the transition-metal L2,3 edges in D03-type (Fe(1-x)Mn(x))3Al in order to investigate their local magnetic moments. The analysis of the spectra shows that Fe has moments much larger than Mn, whose moment is ferromagnetically coupled with the Fe one. This does not lend support to the antiferromagnetic mechanism proposed for the reduction in magnetization as well as a large Mn moment predicted for x = 1/3. The evolution of satellites found in the Mn spectrum with x increased suggests that the change in the electronic state may result in the magnetization reduction.

Journal Article↗

PubMed2001

Magnetic circular dichroism at transition metal L2,3 edges in D03-type (Fe(1-x)Vx)3Al alloys.

Magnetic circular dichroism (MCD) spectra have been measured at the Fe and V L2,3 edges of DO3-type (Fe(1-x)Vx)3Al in order to investigate their local magnetic moments and electronic structures. Large MCD is observed at the Fe L2,3 edges, while the V L2,3 MCD shows relatively small intensity with complicated features. Signs of these MCD spectra indicate an antiferromagnetic coupling between the magnetic moments on Fe and V. According to the analysis based on the magneto-optical sum rules, the magnetic moment decreases with x, but remains fairly large for Fe2VAl, which might arise from its marginally magnetic nature.

Journal Article↗

PubMed2001

Fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus: its construction and reconstitution of active fragmentary enzyme.

X-ray crystallographic studies revealed that various amino acid dehydrogenases fold into two domains in each subunit, a substrate-binding domain and an NAD(P)(+)-binding domain (Baker, P. J., Turnbull, A. P., Sedelnikova, S. E., Stillman, T. J., and Rice, D. W. (1995) Structure 3, 693-705). To elucidate the function and folding process of these two domains, we have genetically constructed a fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus consisting of an N-terminal polypeptide fragment corresponding to the substrate-binding domain including an N-terminus, and a C-terminal fragment corresponding to the NAD(+)-binding domain. The two peptide fragments were expressed in separate host cells and purified. When both fragments were mixed, the leucine dehydrogenase activity with a specific activity of 1.4% of that of the wild-type enzyme appeared. This suggests that both peptide fragments mutually recognize each other, associate and fold correctly to be catalytically active, although the activity is low. However, the fragmentary form of enzyme produced catalyzed the oxidative deamination of l-leucine, l-isoleucine, and l-valine with broad substrate specificity compared to that of the wild-type enzyme. The fragmentary enzyme retained more than 75% of the initial activity after heating at 50 degrees C for 60 min. The fragmentary enzyme was more stable on heating than separate peptide fragments. These results suggest that the two domains of leucine dehydrogenase probably fold independently, and the two peptide fragments interact and associate with each other to form a functional active site.

Amino Acid Oxidoreductases↗

PubMed2001

Detection of pinpoint tenderness on the appendix under ultrasonography is useful to confirm acute appendicitis.

HYPOTHESIS: Ultrasonography can be efficiently performed using new criteria for the diagnosis of acute appendicitis. DESIGN: Prospective trial. PATIENTS: Eighty-nine patients admitted to the hospital with suspected appendicitis between March 1998 and November 2000. INTERVENTION: At hospital admission, a staff surgeon evaluated each patient and determined whether the patient had appendicitis requiring immediate surgery or another disease. Patients then underwent ultrasonography. A sonographic transducer was placed on the area of maximal tenderness. When the pathological manifestation was depicted, the examiner slipped a fingertip between the transducer and the patient's skin and then pressed the area of depicted pathological manifestation to find pinpoint tenderness. When maximal pinpoint tenderness was noted on the appendix or on pathological manifestations contiguous to the appendix, we diagnosed the condition as appendicitis. MAIN OUTCOME MEASURES: Sensitivity, specificity, positive and negative predictive values, and overall accuracy. RESULTS: The diagnosis of appendicitis by this criteria had a sensitivity of 86.7%, a specificity of 89.7%, a positive predictive value of 94.5%, a negative predictive value of 76.5%, and overall accuracy of 87.6%. All 50 patients with pinpoint tenderness noted on the appendix had appendicitis. The surgeon's initial clinical impression had a sensitivity of 83.3%, a specificity of 44.8%, a positive predictive value of 75.8%, a negative predictive value of 56.5%, and overall accuracy of 70.8%. CONCLUSIONS: The efficacy of ultrasonography using the simple criteria was superior to that of the surgeon's initial clinical impression (P<.001). Our ultrasonographic criteria for the diagnosis of appendicitis are simple to use and efficient.

Acute Disease↗

PubMed2001

Stereospecificity for the hydrogen transfer of pyridoxal enzyme reactions.

We have studied the stereospecificities of various pyridoxal 5'-phosphate dependent enzymes for the hydrogen transfer between the C-4' of a bound coenzyme and the C-2 of a substrate in the transamination catalyzed by the enzymes. Prior to our studies, pyridoxal enzymes so far studied were reported to catalyze the hydrogen transfer only on the si-face of the planar imine intermediate formed from substrate and coenzyme. This finding had been considered as the evidence that pyridoxal enzymes have evolved divergently from a common ancestral protein, because identity in the stereospecificity reflects the similarity in the active-site structure, in particular in the geometrical relationship between the coenzyme and the active site base participating in the hydrogen transfer. However, we found that D-amino acid aminotransferase, branched-chain L-amino acid aminotransferase, and 4-amino-4-deoxychorismate lyase catalyze the re-face specific hydrogen transfer, and that amino acid racemases catalyze the nonstereospecific hydrogen transfer. These findings suggest the convergent evolution of pyridoxal enzymes. Crystallographical studies have shown that the stereospecificity reflects the active-site structure of the enzymes, and that the enzymes with the same fold exhibit the same stereospecificity. The active site structure with the catalytic base being situated on the specific face of the cofactor has been conserved during the evolution among the pyridoxal enzymes of the same family.

Binding Sites↗

PubMed2001

Isolation of Bacillus subtilis (chungkookjang), a poly-gamma-glutamate producer with high genetic competence.

A bacterium with high poly-gamma-glutamate (PGA) productivity was isolated from the traditional Korean seasoning, Chung-Kook-Jang. This bacterium could be classified as a Bacillus subtilis, but sporulation in culture was infrequent in the absence of Mn2+. It was judged to be a variety of B. subtilis and designated B. subtilis (chungkookjang). L-Glutamate significantly induced PGA production, and highly elongated PGAs were synthesized. The volumetric yield reached 13.5 mg ml(-1) in the presence of 2% L-glutamate. The D-glutamate content was over 50% in every PGA produced under the conditions used. During PGA production, glutamate racemase activity was found in the cells, suggesting that the enzyme is involved in the D-glutamate supply. Molecular sizes of PGAs were changed by the salt concentration in the medium; PGAs with comparatively low molecular masses were produced in culture media containing high concentrations of NaCl. B. subtilis (chungkookjang) harbors no plasmid and is the first B. subtilis strain reported with both naturally high PGA productivity and high genetic competence.

Alanine Transaminase↗

PubMed2001

Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis.

An enzymatic system for poly gamma-glutamate (PGA) synthesis in Bacillus subtilis, the PgsBCA system, was investigated. The gene-disruption experiment showed that the enzymatic system was the sole machinery of PGA synthesis in B. subtilis. We succeeded in achieving the enzymatic synthesis of elongated PGAs with the cell membrane of the Escherichia coli clone producing PgsBCA in the presence of ATP and D-glutamate. The enzyme preparation solubilized from the membrane with 8 mM Chaps catalyzed ADP-forming ATP hydrolysis only in the presence of glutamate; the D-enantiomer was the best cosubstrate, followed by the L-enantiomer. Each component of the system, PgsB, PgsC, and PgsA, was translated in vitro and the glutamate-dependent ATPase reaction was kinetically analyzed. The PGA synthetase complex, PgsBCA, was suggested to be an atypical amide ligase.

Adenosine Triphosphatases↗

PubMed2001

Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC-1: purification, molecular characterization and expression.

We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC-1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be approximately 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD+ as a coenzyme, and catalyzed the oxidative deamination of L-valine, L-isoleucine, L-leucine and the reductive amination of alpha-ketoisovalerate, alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketocaproate. The reaction proceeds through an iso-ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment and the half-life at 45 degrees C was estimated to be 2.4 min. The kcat/Km (micro(-1).s(-1)) values for L-valine and NAD+ at 20 degrees C were 27.48 and 421.6, respectively. The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino-acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.

Amino Acid Oxidoreductases↗

PubMed2001

Relationship between cytocidal activity and glutathione-S-transferase inhibition using doxorubicin coupled to stereoisomers of glutathione with different substrate specificity.

To determine the cytotoxic mode of action of a glutathione (GSH)--doxorubicin (DXR) conjugate, which exhibited potent cytotoxicity against various multidrug-resistant as well as DXR-sensitive cell lines, the molecular interaction between covalent GSH--DXR conjugates and glutathione-S-transferase (GST), a possible molecular target of the conjugates, was investigated. The following four GSH molecules with stereoisomeric forms were prepared: L-Glu--L-Cys--Gly (LL-GSH), D-Glu--L-Cys--Gly (DL-GSH), L-Glu--D-Cys--Gly (LD-GSH) and D-Glu--D-Cys--Gly (DD-GSH). The enzymic activity of GST against each GSH stereoisomer was 88, 38, 8 and 4 nmol/mg/min, respectively, suggesting that the L-form cysteine residue in the molecule was an important substrate of GST. Addition of DXR conjugated with each isomer (10 microM) to a GSH-containing GST assay mixture inhibited the GST activity to 32% for LL-GSH--XR, 16% for DL-GSH-DXR and 61% for LD-GSH-DXR as compared with the solvent control. Moreover, IC50 values for these conjugates were 30, 20 and 250 nM, respectively. The cytocidal activity of each conjugate corresponded to the substrate specificity of GST activity for the GSH isomer. These conjugates bound to the GST molecule, and the binding ability was 0.746, 0.627 and 0.462 mol/mol of GST for LL-GSH--XR, DL-GSH-DXR and LD-GSH--XR, respectively. These findings suggested that GSH--DXR interacted with the substrate-binding site of the GST molecule and inhibition of GST activity exhibited potent cytotoxicity.

Animals↗

PubMed2001

A sensitive assay of human blood platelet cyclic nucleotide phosphodiesterase activity by HPLC using fluorescence derivatization and its application to assessment of cyclic nucleotide phosphodiesterase inhibitors.

A selective and sensitive HPLC measurement of 3',5'-cyclic nucleotide phosphodiesterase (PDE) activity in human platelets using (3,4-dimethoxyphenyl)glyoxal (DMPG) as a fluorogenic reagent for guanine and its nucleosides and nucleotides is described. cGMP, a substrate for PDE, and GMP, which was produced by the enzyme reaction, are selectively converted by the reaction with DMPG to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. Human platelet PDE activity was measured and the inhibitory effects of several compounds were investigated.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2001

Increased transglycosylation activity of Rhodotorula glutinis endo-beta-glucanase in media containing organic solvent.

The transglycosylation of p-nitrophenyl-beta-D-cellotrioside to cellotetraose catalyzed by endo-1,4-beta-glucanase (cellulase, EC 3.2.1.4) from a psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, was increased by addition of a miscible organic solvent in the reaction mixture. Among various organic solvents tested, acetone was most effective. The transglycosylation activity increased with an increase in acetone concentrations, while hydrolysis activity was suppressed. The transglycosylation preferably occurred at acidic pH with the optimum pH at 2 in 10 mM Gly-HCl buffer. The optimum temperature of transglycosylation was found to be 50 degrees C in the presence of 40% acetone.

Carbohydrate Sequence↗

PubMed2000

High catalytic activity of alanine racemase from psychrophilic Bacillus psychrosaccharolyticus at high temperatures in the presence of pyridoxal 5'-phosphate.

We examined the effect of the pyridoxal 5'-phosphate (PLP) cofactor on the activity and stability of the psychrophilic alanine racemase, having a high catalytic activity at low temperature, from Bacillus psychrosaccharolyticus at high temperatures. The decrease in the enzyme activity at incubation temperatures over 40 degrees C was consistent with the decrease in the amount of bound PLP. Unfolding of the enzyme at temperatures above 40 degrees C was suppressed in the presence of PLP. In the presence of 0.125 mM PLP, the specific activity of the psychrophilic enzyme was higher than that of a thermophilic alanine racemase, having a high catalytic activity at high temperature, from Bacillus stearothermophilus even at 60 degrees C.

Alanine Racemase↗

PubMed2000

Pseudogap formation in the intermetallic compounds (Fe1-xVx)3Al

Optical conductivity data of the intermetallic compounds (Fe1-xVx)3Al ( 0</=x</=0.33) reveal that their density of states around the Fermi energy ( E(F)) is strongly reduced as x is increased. In particular, Fe2VAl ( x = 0.33) has a deep, well-developed pseudogap of 0.1-0.2 eV at E(F) and a small density ( approximately 5x10(20) cm(-3)) of carriers, which is highly unusual for intermetallic compounds. It is shown that the pseudogap results from the band structure of Fe2VAl, rather than from temperature-dependent correlation effects. Based on the present results, we propose a simple model that consistently explains both the semiconductorlike transport and the metallic photoemission results previously observed for Fe2VAl.

Journal Article↗

PubMed2000

Cross-sectional study on risk factors of HIV among female commercial sex workers in Cambodia.

To describe epidemiological features on HIV prevalence among female commercial sex workers (CSWs), a cross-sectional study on sexual behaviour and serological prevalence was carried out in Cambodia. The CSWs were interviewed on their demographic characters and behaviour and their blood samples were taken for testing on sexually transmitted diseases, including HIV, Chlamydia trachomatis, syphilis, and hepatitis B. Associations between risk factors and HIV seropositivity were analysed. High seroprevalence of HIV and Chlamydia trachomatis IgG antibody (CT-IgG-Ab) was shown among the CSWs (54 and 81.7%, respectively). Univariate logistic regression analyses showed an association between HIV seropositivity and age, duration of prostitution, the number of clients per day and CT-IgG-Ab. Especially, high-titre chlamydial seropositivity showed a strong significant association with HIV prevalence. In multiple logistic regression analyses, CT-IgG-Ab with higher titre was significantly independently related to HIV infection. These suggest that existence of Chlamydia trachomatis is highly related to HIV prevalence.

Adolescent↗

PubMed2000

Behavioural and serological human immunodeficiency virus risk factors among female commercial sex workers in Cambodia.

BACKGROUND: The spread of human immunodeficiency virus (HIV) in Cambodia is mainly caused by sexual transmission and the high-risk group in this country are female commercial sex workers (CSW). There are two types of CSW, direct CSW (DCSW) and indirect CSW (IDCSW), who are different from each other in sexual activities. This study was conducted in order to describe the risk factors on HIV for each type of CSW, and to establish effective preventive strategies against the HIV epidemic among CSW. METHODS: The participants, 143 DCSW and 94 IDCSW, were interviewed using a questionnaire to determine their demographic characteristics and behaviour. Blood samples were taken for serological tests on HIV, Chlamydia trachomatis and syphilis. The association between their behavioural pattern and their serological results was analysed. RESULTS: The questionnaire study showed that IDCSW had a riskier behavioural pattern than DCSW. The HIV seroprevalence rates of the DCSW and the IDCSW were 52.4% and 22.3%, respectively. Univariate logistic analyses showed a significant association between HIV antibody (HIV-Ab) and current age, age at commencement of commercial sex work, duration of commercial sex work, and the seropositivity of Chlamydia trachomatis-IgG antibody (CT-IgG-Ab) among the DCSW. The analyses also showed a significant relationship between HIV-Ab and CT-IgG-Ab among the IDCSW. CONCLUSIONS: Improving condom use rate is very important in order to prevent an HIV epidemic among the two types of CSW. This study also suggests it is important to prevent sexually transmitted disease (STD) such as Chlamydia trachomatis infection. The STD control programme could be efficient for HIV prevention, especially among DCSW.

Adolescent↗