Search PubMed⌕ Search

Biomedical subjects

S Iwai

Publications and source records attributed to S Iwai.

At least 127 records · Page 7Linked to original sources
PubMed1995

Recognition of 2'-hydroxyl groups by Escherichia coli ribonuclease HI.

In order to investigate the hydrogen-bonding interactions between Escherichia coli ribonuclease HI and the 2'-hydroxyl functions of the substrate, oligonucleotide duplexes containing 2'-amino-2'-deoxyuridine or 2'-fluoro-2'-deoxyuridine at a specific site were used, and their affinities for the enzyme were determined by kinetic analyses. The results indicate that the hydroxyl groups of the nucleoside 3'-adjacent to the cleaved phosphodiester linkage and the second nucleoside 5' to the cleaved phosphodiester act as both a proton donor and an acceptor and as a proton acceptor, respectively, in the enzyme-substrate complex. A molecular model was constructed using the interactions derived from the results.

Base Sequence↗
PubMed1995

Reaction mechanism of T4 endonuclease V determined by analysis using modified oligonucleotide duplexes.

The reaction mechanism of bacteriophage T4 endonuclease V was investigated using modified oligodeoxyribonucleotide duplexes containing a cis-syn thymine dimer. For the pyrimidine dimer glycosylase step, the formation of a covalent intermediate has been proposed. A fluorine atom was attached to the 2'-position of the 5'-component of the thymine dimer site, which could stabilize the covalent complex and prevent the ring opening of the sugar moiety. The strand cleavage of the 12 base pair substrate analog did not occur, although the glycosyl bond was cleaved by this enzyme. A covalent enzyme--substrate complex was separated by gel electrophoresis under denaturing conditions. It was shown that the enzyme molecules were completely converted to a stable complex in the reaction mixture. Two mechanisms have been proposed for the beta-elimination step. A 12-mer containing a phosphorothioate linkage between adjacent thymidines was prepared. The diastereomers were separated, and the absolute configurations were determined. After formation of the thymine dimer and 32P-labeling of the 5'-terminus, these oligonucleotides were annealed to the complementary 12-mer, and the reaction rates of the pyrimidine dimer glycosylase step and the overall reaction for each duplex were measured under the substrate-saturation conditions. The rate constants indicated that the chemical reaction at the beta-elimination step was rate-limiting. Since no difference was observed in the rate constants for the Rp- and Sp-phosphorothioate substrates, it is concluded that the beta-elimination reaction is catalyzed, not by the internucleotide phosphate, but by an amino acid residue of the enzyme.

Base Sequence↗
PubMed1995

[Pharmacokinetic study of intraperitoneal cisplatin chemotherapy for gastric cancer as an adjuvant setting].

The pharmacokinetic studies of intraperitoneal cisplatin (CDDP) for gastric cancer were discussed elsewhere, but those studies were investigated in patients with ascites. The purpose of this study is to compare the difference in pharmacokinetics between patients with malignant ascites and those curatively resected without ascites. One hundred mg of CDDP and 300 ml of saline were administered intraperitoneally for 9 curatively resected patients by catheter just after operation, and the same doses of CDDP were administered for 3 advanced or recurrent patients with ascites just after removal of whole fluid. Blood samples were corrected at 6 points after administration. Results were as follows: The 0-t area under the curve (AUC) and the Cmax of both total and free CDDP in the patients without ascites was higher than in the patients with ascites. The 0-infinity AUC and MRT of the ascites patients were higher than in the patients without ascites. These data suggest that intraperitoneal CDDP chemotherapy for gastric cancer as an adjuvant setting is more effective than chemotherapy for advanced malignant ascites patients.

Ascitic Fluid↗
PubMed1995

Metabolism of 3-acetylcoumarin in rat 9000xg supernatant fraction (S-9)and baker's yeast.

In the incubation of 3-acetylcoumarin in rat liver supernatant fraction (S-9), four metabolites were isolated, and two of which were inseparable diastereomeric mixture as a major product. However, when 3-acetylcoumarin was fermented with baker's yeast (Saccharomyces cerevisiae), only two compounds were obtained as the same as the above mixture. The structures of those metabolites were confirmed by NMR and Mass spectra. However, the above metabolites in rat 9000xg supernatant fraction were not induced by phenobarbital, nor inhibited with SKF-525A at all.

Animals↗
PubMed1995

[Bacteriological and clinical evaluation tazobactam/piperacillin in infectious diseases in surgical field].

We carried out bacteriological and clinical studies on tazobactam/piperacillin (TAZ/PIPC), a combination drug of piperacillin with the new beta-lactamase inhibitor tazobactam, in various infectious diseases in surgical field such as intra-abdominal infections (peritonitis and intra-abdominal abscess), hepatobiliary infections (cholecystitis, cholangitis and hepatic abscess) and secondary infections in wound, etc. The total number of cases treated with the combination drug was 164. Of these cases, 141 cases were assessable for clinical responses including 60 cases with intra-abdominal infections, 38 cases with hepatobiliary infections, and 43 cases with secondary infections. Clinical efficacy rates of the drug were 83.3% in cases with intra-abdominal infections, 86.8% in cases with hepatobiliary infections, and 95.3% in those with secondary infections, hence the overall efficacy rate was 87.9%. In the cases from which beta-lactamase producing strains were isolated, clinical efficacy rates were 84.8% in cases with intra-abdominal infections, 84.6% in those with hepatobiliary infections, and 96.2% in those with secondary infections, hence the overall efficacy rate was 88.9%. Bacteriological efficacy rates were 92.9% in cases with Gram-positive bacterial infections, 64.3% in those with Gram-negative bacterial infections, and 100% in those with anaerobic bacterial infections. Bacteriological efficacy rates were 84.2% in cases with single bacterial infections and 56.5% in those with multi-bacterial infections, and the overall bacteriological efficacy rate was 69.0%. In the cases of infections with beta-lactamase producing strains, bacteriological efficacy rates were 80.0% in cases with Gram-positive bacterial infections, 75.0% in those with Gram-negative bacterial infections, and 100% in those with anaerobic bacterial infections. They were 82.6% in cases with single bacterial infections and 57.9% in those with multi-bacterial infections; the overall bacteriological efficacy rate was 67.2%. The bacterial eradication rate was 79.9% against all the isolates, and it was 79.2% against beta-lactamase producing isolates. In addition, we compared the sensitivity distribution of the isolates to TAZ/PIPC with those to control drugs piperacillin (PIPC), cefotiam (CTM), ceftazidime (CAZ), sulbactam/cefoperazone (SBT/CPZ). The MIC50 and MIC90 values of TAZ/PIPC against all strains were 3.13 micrograms/ml and 50 micrograms/ml, respectively. MIC50 values show that TAZ/PIPC was two times less effective than CAZ and SBT/CPZ but four times more effective than CTM; furthermore, from the MIC90 values, TAZ/PIPC was four times more effective than PIPC, CTM and CAZ. The MIC50 and MIC90 values of TAZ/PIPC against beta-lactamase producing strains were 3.13 micrograms/ml and 50 micrograms/ml, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Abdominal Abscess↗
PubMed1995

[Two patients with far advanced gastric cancer responding to combination chemotherapy with 5-FU and CDDP].

Combination chemotherapy with 5-FU and CDDP was given to two patients with far advanced gastric cancer. One patient was associated with metastases of lung, liver, pancreas and Virchow and periaortic lymph nodes, and the other was associated with metastases of periaortic lymph nodes and malignant ascitis. The regimen consisted of 5-FU 1,000 mg/m2 (day 1-5, continuous infusion) and CDDP 100 mg/m2 (day 3, 1 hr drip infusion). The interval was from the 6th to the 21st day. The response to chemotherapy showed shrinking of primary gastric lesions and metastases of liver, pancreas and periaortic lymph nodes, and disappearance of Virchow lymph nodes and malignant ascitis. Adverse reactions were thrombocytopenia (Grade 4), leukocytopenia (Grade 3), stomatitis (Grade 1, 3), vomiting (Grade 1, 2) and peripheral neuropathy (Grade 3). This therapy is thought to be effective against far advanced gastric cancer.

Adenocarcinoma↗
PubMed1995

Regio- and stereo-selective oxidation of phenylbutane by rat liver.

Regio- and stereo-selective oxidation of butylbenzene (1) has been examined in vitro by rat liver supernatant fraction(S-9). When phenylbutane (1 ) was incubated at 37 degrees C for 1 hr with S-9, asymmetric oxidation occurred regioselectively at benzylic and omega-1 positions to afford preferentially (R)- and (S)-alcohol (2, 4), respectively. This enzymatic propensity was the case for the production of 1, 3-diols. (1R, 3S)- or (1R, 3R)-Butanediols(3a, 3b) were also obtained at 87% and 27%, respectively. This oxidation was induced by phenobarbital (PB) or beta-napthoflavone(beta-NF), and significant sex-related differences in control and PB pre-treated rats have been observed. Since these oxidations were inhibited with SKF-525A and CO, it was inferred that cytochrome P-450 was responsible for the oxidation.

Animals↗
PubMed1995

Expression of IL-8, TNF-alpha and IFN-gamma m-RNA in ulcerative colitis, particularly in patients with inactive phase.

OBJECTIVE: The etiology of ulcerative colitis (UC) remains uncertain. It has been said that in patients with an active phase of UC the levels of the proinflammatory cytokines increased. Paying particular attention to patients with inactive UC, we investigated several cytokines' m-RNA transcripts. METHODS: In 27 patients with UC, the mucosal cytokine expression of m-RNA including IL-8, TNF-alpha, IFN-gamma and IL-6 were examined using the reverse transcription-coupled polymerase chain reaction (RT-PCR) and oligonucleotide hybridization. RESULTS: 1) The expression ratios of m-RNA of IL-8, IFN-gamma and TNF-alpha in cases with UC (92.6%, 70.4% and 77.8%, respectively) were significantly higher than in normal controls (17.6%, 0.0% and 5.9%, respectively) (p < 0.01). Regarding IL-8 and IFN-gamma, those in UC cases were also significantly higher than in cases with other types of colitis (p < 0.05). 2) No significant difference was noted between active and inactive cases with UC in terms of the expression ratios of m-RNA of IL-8, TNF-alpha and IFN-gamma. 3) Only the expression ratio of IL-6 m-RNA correlated with the macroscopic score according to Matts. In addition, expression of IL-6 m-RNA in the colon tissue was observed in 9 out of 10 cases with other types of colitis (90.0%). CONCLUSION: The expression of IL-8, TNF-alpha and IFN-gamma m-RNA increased even in inactive UC, suggesting that these three cytokines play an important role in the pathogenesis of repeated inflammation of UC.

Adult↗
PubMed1994

Studies of the interactions between Escherichia coli ribonuclease HI and its substrate.

Ribonuclease H (RNase H) recognizes a DNA-RNA hybrid duplex and catalyzes the hydrolysis of the phosphodiester linkages in only the RNA strand. Previously, we developed a method to cleave RNA in a sequence-dependent manner using RNase H and a complementary oligonucleotide containing 2'-O-methylribonucleosides. Since cleavage is restricted to a single site by the modified complementary strand, this system allows kinetic analysis of the RNase H reaction. We describe an investigation of the interactions between RNase HI from Escherichia coli and its substrate, and between the substrate and a metal ion using synthetic oligonucleotide duplexes modified at the cleavage site in combination with the 2'-O-methylribonucleotides. Firstly, the base moiety was changed to interfere with enzyme binding in either the major or minor groove. When 2-N-methylguanine was incorporated into the cleavage site, the Km value for this substrate, containing a methyl group in the minor groove, was 20-fold larger than that for the unmodified substrate, whereas 5-phenyluracil, with a phenyl group residing in the major groove of the duplex, did not affect the affinity. Secondly, the phosphodiester linkage at the cleavage site was changed into a phosphorothioate with a defined configuration. Only the Rp isomer was cleaved at this site in the presence of Mg2+ or Cd2+. These results suggest that the enzyme, but not the metal ion, interacts with the phosphate residue at the cleavage site. Thirdly, the 2'-position of the nucleoside on the 5'-side of the scissile phosphodiester was modified. Alteration of the 2'-hydroxyl function into an amino, fluoro or methoxy group, or removal of this 2'-hydroxyl group, did not affect the affinity for the enzyme, but reduced the reaction rate. An outer sphere interaction of a metal ion with the 2'-hydroxyl group is suggested.

Base Sequence↗
PubMed1994

Endonuclease V from bacteriophage T4 interacts with its substrate in the minor groove.

The binding of bacteriophage T4 endonuclease V to its substrate has been studied using synthetic oligodeoxyribonucleotide duplexes containing a cis-syn thymine dimer. Substrate analogues containing a methylphosphonate linkage with a defined configuration at the thymine dimer site were prepared, and the binding of the enzyme to each diastereomer was analyzed by the filter-binding method. The duplex containing a methylphosphonate with the SP configuration formed a complex with the enzyme, although the dissociation constant for this substrate analogue was about 8 times larger than that for the 12-mer substrate containing a phosphodiester linkage at this site. In contrast, no binding was observed when a duplex containing the RP-methylphosphonate linkage was used. The glycosyl bond of the thymine dimer in the SP isomer was cleaved by the enzyme, while no incision was detected in the case of the RP isomer, even after alkali treatment. Another substrate analogue containing a sulfur atom in place of the 3'-oxygen of the 5'-component at the thymine dimer site showed a reduced affinity for the enzyme. These results suggest that T4 endonuclease V interacts with its substrate in the minor groove. This mode of binding was confirmed by methylation protection experiments.

Bacteriophage T4↗
PubMed1994

Nuclear magnetic resonance study of the interaction of T4 endonuclease V with DNA.

T4 endonuclease V catalyzes the DNA strand cleavage in the vicinity of a thymine dimer. In order to obtain insight into the specific recognition mechanism of this enzyme with a thymine photodimer within DNA, the conformations of five different DNA duplexes, [sequence: see text] with which the enzyme can interact, were studied by 1H NMR. DNA I, DNA IV, and DNA V do not contain the TT sequence or a thymine dimer and hence, are expected to bind the enzyme only in a nonspecific manner. DNA II includes a single TT sequence which does not form a thymine dimer. Only DNA III is expected to bind specifically to the enzyme through a thymine photodimer. The NMR spectra of these five DNA duplexes in the absence of the enzyme clearly show that the formation of a thymine dimer within the DNA induces only a minor distortion in the structure and that the overall structure of B-type DNA is retained. The photodimer formation is found to cause a large change in chemical shifts at the GC7 base pair, which is located at the 3'-side of the thymine dimer, accompanied by the major conformational change at the thymine dimer site. The effects of T4 endonuclease V binding on these DNA duplexes were also investigated by 1H NMR. The binding of this enzyme to DNA I, DNA IV, and DNA V causes no alteration in chemical shift values of the imino proton resonances, but the binding to DNA II induces a small downfield shift in the imino proton resonance of GC7.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1994

Role of the Mg2+ ion in the Escherichia coli ribonuclease HI reaction.

To study the interaction and the role of the metal ion in the reaction catalyzed by Escherichia coli ribonuclease HI (E. coli RNase HI), substrate analogues containing a phosphorothioate linkage or 2'-modified nucleosides at the cleavage site were used. In the presence of Mg2+, Mn2+, Co2+, Zn2+, or Cd2+, the phosphorothioate linkage with the RP-configuration was cleaved, while the SP-isomer was not. Kinetic studies showed that Mn2+ and Cd2+ facilitated the cleavage of the phosphorothioate to only a small extent, which indicated the absence of an interaction between the metal ion and this phosphate residue. The interaction of the metal ion with the 2'-functional group was analyzed by Mg(2+)-titration experiments using the -OH, -NH2, and -F substrates. From Hill plots, it was found that the KMg values were almost the same. These results are evidence of an interaction between Mg2+ and the 2'-functional group by the formation of an outer-sphere complex with a water molecule. The Hill coefficient of 1.0 for the -OH substrate indicated that a single Mg2+ ion is required for the catalysis.

Bacterial Proteins↗
PubMed1994

Cytopathological changes induced by cadmium-exposure in canine proximal tubular cells: a cytochemical and ultrastructural study.

A highly sensitive cytochemical method for demonstrating intracellular Cd using 8-hydroxyquinoline was developed and applied in the cytopathological study of primary-cultured renal tubular cells from beagle kidneys. The Cd-8-hydroxyquinoline emitted a yellowish-green fluorescence which first appeared in the cytoplasm within 30 min and in the nucleus about 60-90 min after exposure to 100 mM CdCl2. It was noteworthy that intranuclear Cd was stained in the nucleolar regions. The sensitivity of the cytochemical method for Cd was estimated to be about 1.0 pg Cd/cell. Ultrastructural features of the dead cells were consistent with those of apoptosis. We conclude that Cd absorbed by proximal tubular cells rapidly reaches to the nuclei and affects nuclear as well as cytoplasmic metabolism.

Animals↗