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Biomedical subjects

S Kojima

Publications and source records attributed to S Kojima.

At least 559 records · Page 31Linked to original sources
PubMed1993

Identification of amino acid residues responsible for the changes of absorption and fluorescence spectra on the binding of subtilisin BPN' and Streptomyces subtilisin inhibitor.

An ultraviolet absorption difference spectrum characteristic of the ionization change of a tyrosyl residue was observed on the binding of subtilisin BPN' with Streptomyces subtilisin inhibitor (SSI) at alkaline pH. This difference spectrum was considered to be induced by a pKa shift (from 9.7 to > or = 11.5) of a tyrosyl residue of subtilisin BPN' in the interaction with carboxyls of SSI [Inouye et al. (1979) J. Biochem. 85, 1115-1126]. In the present paper, the tyrosyl residue in subtilisin BPN' and the carboxyls in SSI were identified by analyzing the difference spectrum using mutants of subtilisin BPN' and SSI: naturally occurring mutants and those prepared by site-directed and cassette mutagenesis. The difference spectrum disappeared on the binding of a mutant subtilisin BPN' of which Tyr104 was replaced by Phe (S-BPN'Y104F) and SSI at pH 9.8. The magnitude of the absorption difference was much smaller when subtilisin BPN' was bound with a mutant SSI of which both Glu67 and Asp68 were replaced by Gly than with the wild-type SSI. These lines of evidence indicated that the difference spectrum was caused by Tyr104 of subtilisin BPN' interacting with Glu67 and Asp68 of SSI. The binding of subtilisin BPN' and SSI is accompanied by an increase of tryptophan fluorescence, which is pH-dependent in the range of pH 7-11 [Uehara et al. (1978) J. Biochem. 84, 1195-1202]. In the present study, this pH-dependence of the fluorescence diminished when SSI bound with S-BPN'Y104F. This suggested that the fluorescence increase was due to Trp106 of subtilisin BPN' and was influenced by the ionization of Tyr104.

Amino Acids↗
PubMed1993

Effects of deletion in the flexible loop of the protease inhibitor SSI (Streptomyces subtilisin inhibitor) on interactions with proteases.

The Streptomyces subtilisin inhibitor (SSI) is a proteinaceous protease inhibitor which inhibits serine proteases by forming a stable Michaelis complex. The flexible loop region (Thr64-Val69) is a very flexible region in an SSI molecule and its importance in interactions with proteases has been suggested, since conformational change of this loop was found to occur for the smooth binding of SSI with various proteases. In this study, mutated SSIs lacking one or two residues in this region were generated and the effects of deletions on the interaction with proteases were investigated. Deletion was introduced into mutated SSI(Lys73) and SSI(Gly70Lys73) both known to be trypsin inhibitors, to examine the effects of deletion on interactions with subtilisin BPN' or trypsin. The deletion of one residue (Gly66) caused increased inhibitory activity toward trypsin, indicating the protruding flexible loop hinders binding with trypsin. Reduction of such hindrance by one-residue shortening in this loop is shown to be effective for the interaction of SSI(Lys73) with trypsin. In contrast, one-residue shortening had virtually no effect on inhibition toward subtilisin BPN'. Differences in the subsite structures of these proteases may have been the reason for this contrast. The deletion of two residues (Thr64 and Gly66) in this region converted SSI into a temporary inhibitor. Structural analysis of the degradation intermediate showed that the peptide bond at the reactive site of doubly deleted SSI was cleaved by subtilisin BPN' after its binding with protease. Thus, the irreversibility of the cleaved peptide bond at the reactive site of mutated SSI in the complex with protease may possibly be the cause for its temporary inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Acute effects of alcohol ingestion on blood pressure and erythrocyte sodium concentration.

OBJECTIVE: To examine the acute effects of alcohol on blood pressure and erythrocyte cation concentrations in patients with essential hypertension. DESIGN: An alcoholic drink or an isocaloric control drink was given during supper in random order on different days, and blood pressure and erythrocyte cation concentrations were measured before and 2 h after the meal. METHODS: The subjects were 21 men with essential hypertension who habitually drank alcohol. Blood pressure was measured with a semi-automated sphygmomanometer, and erythrocyte cation concentrations were measured by flame photometry after haemolysis with distilled water. RESULTS: Blood pressure decreased after both drinks, but the decrease was significantly larger after the alcoholic drink than after the control drink. There was a significant difference between the changes in erythrocyte sodium caused by the alcoholic and the control drink. Furthermore, there were significant positive correlations between the fall in blood pressures and the decrease in erythrocyte sodium concentration. CONCLUSION: The predominant acute effect of alcohol ingestion in patients with hypertension is blood pressure reduction, and it may be associated with a decrease in intracellular sodium.

Adult↗
PubMed1993

The expression of invasive behavior of differentiated squamous carcinoma cell line evaluated by an in vitro invasion model.

In order to elucidate the factors contributory to the expression of invasiveness of oral squamous cell carcinoma, we conducted biochemical and morphological comparisons of well differentiated squamous carcinoma cell line OSC-19 (oral squamous cell carcinoma) and undifferentiated carcinoma cell line KB, both cultured on 3T3 cell-embedded collagen gel (in vitro invasion model). OSC-19 cells invaded 3T3 cell-embedded collagen gel, while KB cells and OSC-19 cells on 3T3 cell-free gel matrix were less invasive. Cultured OSC-19 cells were characterized by lower proliferating activity, lower secretion of laminin and higher secretion of fibronectin than those of KB cells. Although the basement membrane with deposition of laminin and type IV collagen was formed, it was discontinuous at the invasion front. Gelatin zymography and western blotting showed matrix metalloproteinases (MMP), i.e., 72 kDa gelatinase (MMP-2) and 92 kDa gelatinase (MMP-9). Gelatinolytic activity was assayed, and was higher in OSC-19 cells than in KB cells or OSC-19 cells of the 3T3 cell-free model. By immunohistochemical analysis, MMP-2-positive cells were found scattered in both cell lines without any preferential localization, and the positivity for MMP-9 was localized in the invasion front of OSC-19 cells. These results strongly suggest that the invasiveness of squamous cell carcinoma is well correlated with cell-matrix adhesion by fibronectin and with focal elaboration of metalloproteinases, especially MMP-9, which play a major role in degrading the extracellular matrix components.

3T3 Cells↗
PubMed1993

Improved detection rate of early breast cancer in mass screening combined with mammography.

A cohort study to compare mass screening with and without mammography was conducted in Miyagi Prefecture, Japan in order to establish whether the effectiveness of breast cancer screening would be improved when mammography was combined with physical examination. A trial of mass screening combined with mammography was carried out in 9634 women aged over 50. Lateral imaging of the breast using single-view film mammography was performed at the first stage of mass screening in addition to physical examination of the breast. Results in the trial were compared with those obtained in 35511 age-matched subjects without mammography. Thirty breast cancers were found in the trial with a detection rate of 0.31%, which was much higher than that (0.08%) obtained by physical examination without mammography. In 15 of the 30 patients the breast tumor was not palpated at the first screening, but abnormal findings were detected in the mammography. A higher rate (73%) of early breast cancer was obtained in the screening trial with mammography than that (39%) obtained in the screening with physical examination alone. Mass screening combined with mammography is superior to that without mammography for breast cancer screening, especially for the detection of non-palpable, early breast cancer.

Age Factors↗
PubMed1993

Clonal haematopoiesis in children with acquired aplastic anaemia.

The methylation pattern of three X-linked genes, phosphoglycerate kinase (PGK), hypoxanthine phosphoribosyl transferase (HPRT) and DXS255 detected by hypervariable M27 beta probe, was analysed to determine the proportion of aplastic anaemia (AA) with clonal haematopoiesis in Japanese children. Methylation analysis was performed on DNA from separated granulocytes and compared to that of bone marrow derived fibroblasts to exclude selective lyonization in all somatic cells. Of 20 female patients examined, the methylation pattern of at least one gene was informative in granulocyte DNA from 18 patients (90%). Of these, 8/20 patients (40%) were heterozygous for PGK, 8/18 (44%) were heterozygous for HPRT and 17/18 (94%) were heterozygous for DXS255. In 14/18 patients both alleles were equally methylated. Four patients exhibited a unilateral methylation pattern in their granulocytes. The same unilateral pattern was again demonstrated in fibroblasts from two of the four patients suggesting that in the latter one X chromosome was selectively inactivated in all of the somatic cells. The remaining two patients showed a unilateral methylation pattern that was restricted to their granulocytes, suggesting the existence of true clonal haematopoiesis. They responded well to antilymphocyte globulin (ALG) and presently have no evidence of a clonal disorder such as myelodysplastic syndrome (MDS) or paroxysmal nocturnal haemoglobinuria (PNH). Although these results indicate that some children with AA exhibit clonal haematopoiesis, analysis of a greater number of subjects will be required to establish the clinical value of clonal haematopoiesis in patients with AA.

Adolescent↗
PubMed1993

Detection of hepatitis C virus RNA in the liver by in situ hybridization.

To examine HCV infection histologically, we attempted non-radioactive in situ hybridization of HCV-RNA in the liver. We amplified cDNA probe (360 base pairs) by PCR using the primers deduced from the core region of the HCV genome. The probe was labelled with digoxigenin by PCR and used for in situ hybridization on paraformaldehyde-fixed frozen liver sections. The hybrids were visualized immunohistochemically with alkaline-phosphatase-conjugated anti-digoxigenin and alkaline-phosphatase substrates. HCV-RNA-cDNA hybrids were detected in 21 of 24 patients with positive serum HCV markers, whereas there were no positive signals in the liver of 12 cases without HCV infection. The signal intensity of HCV-RNA-cDNA hybrids was abolished after RNase treatment. Various other specificity experiments also verified specific hybridization of HCV-RNA-cDNA. HCV-RNA was visualized in liver cells and most of them were regarded as hepatocytes from their characteristic features. The infected hepatocytes were frequently associated with mononuclear cell infiltration. Hepatocytes positive for HCV-RNA were sometimes binuclear and distributed in various patterns among cases tested. The present in situ hybridization of HCV RNA is highly sensitive and specific and the results suggest the host immune response to HCV-infected cells.

Hepacivirus↗
PubMed1993

Streptomyces subtilisin inhibitor-like proteins are distributed widely in streptomycetes.

Streptomyces subtilisin inhibitor-like proteins were found to be distributed widely in streptomycetes by using the combination of the convenient, newly developed plate assay system and an established liquid culture assay. Almost all the strains formerly categorized as Streptoverticillium species produced proteins that exhibited inhibitory activity against both subtilisin BPN' and trypsin. N-terminal regions of three purified proteins showed high structural similarity to those of other previously reported SIL inhibitors.

Amino Acid Sequence↗
PubMed1993

Effects of perfusion pressure on intracellular calcium, energetics, and function in perfused rat hearts.

Effects of perfusion pressure in a range from 50 to 140 cmH2O on intracellular Ca2+ concentration ([Ca2+]i) were evaluated along with cardiac function, energy metabolism, and left ventricular geometry in a concentration of 2 or 4 mM of extracellular Ca2+ ([Ca2+]o) in isovolumic perfused rat hearts. [Ca2+]i was evaluated with a surface fluorometry technique in hearts loaded with indo-1/AM. The systolic and diastolic values and the amplitude (difference between systolic and diastolic values) of indo-1 fluorescence ratio (an index of [Ca2+]i) were linearly related to perfusion pressure. Changes in the fluorescence ratio were harmonious with rapid changes in left ventricular pressure and stabilized within 30-40 s after changes in perfusion pressure. Developed pressure and O2 consumption were closely, linearly correlated with the fluorescence ratio irrespective of [Ca2+]o. Left ventricular end-diastolic wall thickness, measured by 2-dimensional echocardiography, paralleled perfusion pressure and showed a good correlation with the fluorescence ratio. Diastolic myocardial adenosine 3',5'-cyclic monophosphate significantly decreased only at the lowest perfusion pressure. The ln[phosphocreatine]/[Pi] also changed with altered perfusion pressure. In conclusion, perfusion pressure modulates [Ca2+]i, which in turn regulates myocardial contraction and associated O2 utilization.

Animals↗
PubMed1993

Acquired resistance to Schistosoma japonicum in IgE-deficient SJA/9 mice immunized with irradiated cercariae.

Participation of IgE in protective immunity against Schistosoma japonicum was examined by comparing congenital IgE-deficient SJA/9 and IgE-producing SJL/J mice. Mice immunized with 100 irradiated cercariae 7 weeks previously were infected with 50 live cercariae. In SJL/J mice at 40 days after infection, a 3-to 4-fold increase of total IgE levels and anti-S, japonicum egg IgE antibody production were observed with no significant difference between immunized and nonimmunized mice. IgE was not detected in SJA/9 mice throughout the experiments. Protective immunity evaluated by recovery of adult worms was found in SJA/9 mice and was comparable to that of SJL/J mice. These results suggest that acquired immunity in mice with irradiated cercariae of S. japonicum was not dependent on IgE in these strains of mice.

Animals↗
PubMed1993

N-benzyl-D-glucamine dithiocarbamate and N-p-isopropylbenzyl-D-glucamine dithiocarbamate improve the protective effect of diethyldithiocarbamate against cadmium-induced testicular toxicity in rats.

The protective effects of combined treatment with diethyldithiocarbamate (DED) plus N-benzyl-D-glucamine dithiocarbamate (BGD) or DED plus N-p-isopropylbenzyl-D-glucamine dithiocarbamate (PBGD) against the testicular toxicity caused by acute exposure to cadmium (Cd) in rats were studied. Rats were injected subcutaneously with 109CdCl2 (3 mg Cd and 74 kBq of 109Cd/kg) and 30 min later, they were injected intraperitoneally with the chelating agents (1 mmol/kg each). Cd injection increased lipid peroxidation and concentrations of hemoglobin, Ca and Fe in the testes, decreased the testicular weight and nonprotein SH (NP-SH), and caused sterility. The coadministration of DED with BGD or PBGD significantly prevented the increase in the lipid peroxidation, hemoglobin, Ca and Fe in the testes, the decrease in the testicular weight and NP-SH, and the sterility caused by Cd injection. DED plus BGD or DED plus PBGD significantly decreased the Cd concentration in the testes without the redistribution of Cd to the brain and kidney, which is observed following treatment with DED alone. The coadministration of DED plus BGD or DED plus PBGD significantly increased the blood Cd concentration and the Cd distribution in the red blood cells compared to Cd alone. These results indicate that the coadministration of BGD or PBGD with DED prevents the accumulation of Cd in the testes on the basis of greater blood distribution of Cd, which results from the uptake of Cd by the red blood cells, without the redistribution of Cd to the brain, resulting in an improvement of the protective effect of DED against the Cd-induced testicular toxicity.

Animals↗
PubMed1993

Protective effects of dithiocarbamates against toxicity of cis-diamminedichloroplatinum in mice.

The protective effects of various dithiocarbamates such as N-benzyl-D-glucamine dithiocarbamate (BGD), N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD), N-p-carboxybenzyl-D-glucamine dithiocarbamate (CBGD), and diethyldithiocarbamate (DDTC) on cis-diamminedichloroplatinum (DDP)-induced toxicity in mice were studied. The mice were injected i.v. with a chelating agent (1 mmol/kg) immediately or 5 min after i.v. injection of DDP (15 or 20 mg/kg). The lethal toxicity of DDP (20 mg/kg) was completely prevented by treatment with HBGD or CBGD immediately after DDP. The survival time of mice treated with HBGD or CBGD 5 min after DDP tended to be longer than that treated with BGD or DDTC. Significant increases in blood urea nitrogen (BUN) level and plasma aspartate aminotransferase (AST) activity were observed 3d after DDP injection. The increase in BUN level was completely prevented only by HBGD and CBGD among these chelating agents, while increase in AST activity was significantly prevented by treatment with these two agents. Treatment with HBGD or CBGD immediately after DDP (20 mg/kg) completely protected against DDP-induced diarrhea. These chelating agents significantly decreased the platinum (Pt) contents in the kidney and liver after DDP administration. Treatment with HBGD or CBGD was the most effective in decreasing the renal Pt content, resulting in maximum protection against DDP-induced renal damage. The antitumor efficacy of DDP (15 mg/kg) in the colon 26 carcinoma-bearing mice was not affected by HBGD administration.

Animals↗
PubMed1993

Restrictive cardiomyopathy with complete atrioventricular block and distal myopathy with rimmed vacuoles.

Two-dimensional echocardiography, cardiac catheterization, electrophysiologic studies, and biopsies of cardiac and skeletal muscle were performed in a patient with complete atrioventricular block as the initial manifestation of restrictive cardiomyopathy. The patient also showed distal myopathy with rimmed vacuoles. Weakness of the distal leg muscles occurred several years after the initial cardiac manifestations and the patient developed congestive heart failure 1 year before the second admission. Endomyocardial biopsy showed patchy myocardial fibrosis without endomyocardial fibrosis or any other specific findings of myocardial disease. Histochemical examination of skeletal muscle showed the typical features of distal myopathy with rimmed vacuoles. In some patients with atrioventricular block as the initial manifestation of restrictive cardiomyopathy, the cardiomyopathy may be just 1 manifestation of a more generalized disorder affecting both cardiac and skeletal muscle.

Adult↗
PubMed1993

Importance of the nucleotide loop moiety coordinated to the cobalt atom of adenosylcobalamin for coenzymic function in the diol dehydrase reaction.

Three analogs of adenosylcobalamin were synthesized and their coenzymic properties in the diol dehydrase reaction were studied. Neither adenosylcobinamide nor adenosylcobinamide phosphate was active as coenzyme and showed very low affinity for apoenzyme, irrespective of the presence of nucleotide loop fragments, such as 5,6-dimethylbenzimidazole, alpha-D-ribazole, or alpha-D-ribazole-3'-phosphate. The coordination of pyridine to the cobalt atom neither confers the coenzymic function upon adenosylcobinamide nor strengthens the inhibitory effect of cyanoaquacobinamide and methylcobinamide significantly. The analog of adenosylcobalamin in which the N-3 position of 5,6-dimethylbenz-imidazole is methylated was also not active as coenzyme and showed very low affinity for apoenzyme. Since 3,5,6-trimethylbenzimidazole in this analog is no longer coordinated to the cobalt atom, these results show that at least a part of the nucleotide loop moiety coordinated to the cobalt atom of adenosylcobalamin is essential for tight binding to the apoenzyme and therefore for manifestation of coenzymic function.

Binding Sites↗
PubMed1993

Autologous BMT in high risk patients with CALLA-positive ALL: possible efficacy of ex vivo marrow leukemia cell purging with monoclonal antibodies and complement.

Autologous BMT (auto-BMT) has been conducted for 17 high-risk common ALL antigen (CALLA)-positive non-T cell type ALL patients. Ex vivo purging of leukemia cells from infused BM cells was performed using complement and three kinds of mouse monoclonal antibodies reactive to CALLA-positive leukemia cells: NL-1 (IgG2a), NL-22 (IgM), and HL-47 (IgM). Minimal residual leukemia cells in BM were examined by PCR method in Philadelphia chromosome (Ph1)-positive ALL cases. BCR/ABL chimeric transcript, which was positive in BM samples before purging, was shown to be absent after ex vivo purging in all three cases tested. Among four Ph1-positive cases transplanted in first CR, three cases survived in CR remission 77, 29 and 26 months after BMT, and one case died without relapse 4 months after BMT. The other four Ph1-negative cases transplanted in the first CR also remained in CR except one who relapsed. The results of minimal residual leukemia cell studies and clinical data indicate the effectiveness of our ex vivo leukemia cell purging method and auto-BMT in the early stage of CR for patients with high risk ALL.

Adolescent↗
PubMed1993

[An adult case of acute lymphoblastic leukemia preceded by aplasia resembling aplastic anemia].

Although aplastic anemia (AA) is well known to precede acute leukemia, commonly acute myeloblastic leukemia, an adult case of acute lymphoblastic leukemia (ALL) preceded by aplasia similar to severe AA is very rare. An 18-year-old female was admitted to our hospital for scrutiny of pancytopenia in October 3, 1991. She was diagnosed as severe AA because of severe bone marrow hypoplasia and no abnormal or dysplastic cell. Recovery of hematopoiesis was achieved by pulse therapy of methylprednisolone combined with granulocyte colony stimulating factor (G-CSF). 3 months after AA onset, she was readmitted because of high fever and paralytic ileus. Then, she was diagnosed as ALL (L1; common ALL) and was successfully treated by remission induction chemotherapy. Retrospectively, this case was atypical AA with respect to serum iron and LDH level.

Adolescent↗
PubMed1993

[Formation of 8-hydroxydeoxyguanosine from 2'-deoxyguanosine and its decomposition by active oxygen].

8-hydroxydeoxyguanosine (8-OHdG) was formed from dG and isolated DNA by photosensitization with rose bengal (RB) and methylene blue (MB). 8-OHdG formed from dG was decomposed by the photosensitization with these dyes. Singlet oxygen was concerned with the formation and decomposition of 8-OHdG by photosensitization. Fe++ oxidized dG to 8-OHdG, which was decomposed by the addition of H2O2. 8-OHdG was formed and decomposed by the treatment of dG with Fe++, EDTA and ascorbic acid. Hydroxy radical (.OH) participated in the formation and decomposition of 8-OHdG by Fe++.

8-Hydroxy-2'-Deoxyguanosine↗