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Biomedical subjects

S Kojima

Publications and source records attributed to S Kojima.

At least 487 records · Page 27Linked to original sources
PubMed1994

Secretory production of chicken ovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificity toward proteases by substitution of the P1 site residue.

Ovomucoids are commonly present in bird egg white and exhibit inhibitory activity toward various serine proteases. To investigate the structure-function relationship of ovomucoid domain 3, we established a secretory expression system for the chicken ovomucoid domain 3 (OMCHI3)-encoding gene in Escherichia coli by ligating it downstream from the tac promoter and signal peptide of E. coli alkaline phosphatase. E. coli JM105 was transformed with the resulting plasmid and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The mature OMCHI3 was detected in the culture supernatant, and was purified to homogeneity by three-step chromatography. Amino-acid sequence analysis showed that processing by the signal peptidase was carried out exactly at the expected site. Measurements of circular dichroism spectra and inhibitory activity indicated that OMCHI3 was produced in the properly folded form. Furthermore, site-specific replacement of the Ala residue at the P1 site with Met or Lys resulted in acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively, indicating that the P1 site is the predominant determinant for inhibitory specificity.

Amino Acid Sequence↗
PubMed1994

Comparative effects of chelating agents on distribution, excretion, and renal toxicity of gold sodium thiomalate in rats.

The effects of various chelating agents, such as (2S)-1-(3-mercaptopropionyl)-L-proline (captopril), N-(2-mercaptopropionyl)-glycine (tiopronin), L-cysteine (L-Cys), D-cysteine (D-Cys), N-acetyl-L-cysteine (L-NAC), N-benzyl-D-glucamine dithiocarbamate (BGD), and ethylenediaminetetraacetate (EDTA), on the distribution, excretion, and renal toxicity of gold sodium thiomalate (AuTM) in rats were investigated. Rats were intraperitoneally injected with the chelating agents (1.2 mmol/kg each) immediately after intravenous injection of AuTM (0.026 mmol/kg). Treatment with captopril or tiopronin significantly prevented increases in the urinary excretion of protein, aspartate aminotransferase (AST), and glucose and the blood urea nitrogen (BUN) level after AuTM injection. L-NAC and D-Cys significantly prevented increases in the urinary excretion of protein, AST, and glucose after AuTM injection, but did not reduce to control levels. Treatment with BGD, EDTA, or L-Cys did not prevent AuTM-induced increases in the urinary excretion of protein, AST, and glucose and BUN level. Tiopronin significantly increased the urinary excretion of gold. Captopril slightly promoted both the urinary and fecal excretion of gold, resulting in the significant increase in the total excretion of the metal. Tiopronin and captopril significantly decreased the gold concentration in the kidney and liver. L-Cys, D-Cys, L-NAC, BGD, and EDTA had no significant effect on the excretion or distribution of gold at 7 days after AuTM injection. These results indicate that tiopronin and captopril can ameliorate the renal toxicity induced by AuTM. In addition, the comparative effects of 2,3-dimercaptopropane sulfonate (DMPS), N-(2-mercapto-2-methylpropanoyl)-L-cysteine (bucillamine), captopril, and tiopronin at various dose levels (1.2, 0.4 or 0.2 mmol/kg) on the distribution and renal toxicity of gold were studied. DMPS was effective in removing gold from the kidney and in protecting against the renal toxicity after AuTM injection at the even lower dose level (0.2 mmol/kg). Bucillamine and tiopronin protected against the renal toxicity of gold at dose levels of 0.4 and 1.2 mmol/kg and captopril ameliorated the gold toxicity only at higher dose level (1.2 mmol/kg).

Animals↗
PubMed1994

Effects of neopterin on ischemic neuronal damage in gerbils.

The effects of neopterin on ischemic neuronal damage were examined. Cerebral ischemia was produced in the gerbil by bilateral common carotid occlusion for 8 min or unilateral occlusion for 30 min, which resulted in delayed neuronal death in the CA1 region of the hippocampus. However, preischemic treatment with neopterin (3 mg/kg i.p.) markedly reduced hippocampal neuronal damages in both cases. Since neopterin serves both as an antioxidant and as an oxidant depending on its redox state, these findings indicate that neopterin attenuates the ischemic neuronal injury by scavenging oxygen free radicals and/or by inhibiting their generation.

Animals↗
PubMed1994

A reverse transcriptase-polymerase chain reaction detects heterogeneous chimeric mRNAs in leukemias with 11q23 abnormalities.

The MLL gene involved in 11q23 translocations found in the majority of infantile leukemias and some secondary leukemias makes fusion transcripts with genes such as LTG4 (chromosome 4), LTG9 (chromosome 9), and LTG19 (chromosome 19) as a result of reciprocal translocation. We have examined 25 cases of leukemias with 11q23 abnormalities by Southern blot analysis and the reverse transcriptase-polymerase chain reaction (RT-PCR). Using various primer pairs, chimeric mRNAs could be amplified in 6 of 7 leukemias with t(4;11), 6 of 8 leukemias with t(9;11) including secondary leukemia, 8 of 9 leukemias with t(11;19), and 1 with a deletion at 11q23. The chimeric mRNAs were heterogeneous and differential usage of the MLL exons was found, irrespective of the partner chromosomes. Sensitivity studies showed that a single clone with chimeric mRNA in 10(4) to 10(5) cells could be detected. These findings show that the present RT-PCR settings provide a rapid, accurate, and sensitive tool for diagnosing leukemias with 11q23 translocations and for monitoring response to therapy in these patients.

Adult↗
PubMed1994

Prevention of diabetes by thymic hormone in alloxan-treated rats.

We investigated the effects of facteur thymique sérique (FTS), a thymic peptide hormone, on alloxan- and streptozotocin-induced diabetes in rats. Pretreatment with intravenous injection of FTS significantly suppressed both alloxan- and streptozotocin-induced hyperglycemia. The effects of FTS were time dependent. FTS suppressed hyperglycemia in a dose range of 1-6600 micrograms/kg. Alloxan-induced hyperglycemia was completely prevented when FTS was injected in doses of 40-50 micrograms/kg 1 min before injection of alloxan. Histological examination of islet areas showed that alloxan-induced destruction of beta-cells was inhibited by FTS. FTS had no significant effects on lymphocyte subsets and immunity-related cells or on plasma superoxide dismutase activity and total glutathione level. The blood half-life time of exogenously injected FTS was short (2-3 min), indicating acute internalization of FTS into pancreatic beta-cells. Our results suggested that FTS acutely and directly blocks some initial effect of alloxan, preventing the destruction of beta-cells.

Amino Acid Sequence↗
PubMed1994

Mutations of the N-ras gene in juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML), a myeloproliferative disorder of childhood, is distinct from adult-type chronic myelogenous leukemia (CML) and bears resemblance to chronic myelomonocytic leukemia (CMMoL). Since mutations in the N-ras gene have been found at high frequencies in CMMoL, but only rarely in CML, we analyzed mutations activating the N-ras gene in 20 patients with JCML. We used the strategy for analysis of gene mutations based on in vitro DNA amplification by polymerase chain reaction (PCR) followed by single-strand conformation polymorphism (SSCP) analysis and/or direct sequence analysis. Nucleotide sequence analysis showed single nucleotide substitutions involving codons 12, 13, or 61 in six of 20 patients (30%). Four of six patients with mutations were in chronic phase and the other two in blast crisis, indicating no apparent correlation with disease stage. Most of the patients with mutations were in the older age group with poor prognosis, although one patient in the younger age group also harbored the mutation. These data suggest that N-ras gene mutations may be involved in the pathogenesis and/or prognosis of JCML and provide further evidence that JCML is an entity distinct from CML.

Base Sequence↗
PubMed1994

Achievement of renaturation of subtilisin BPN' by a novel procedure using organic salts and a digestible mutant of Streptomyces subtilisin inhibitor.

The pro-sequences of proteases have been considered to be required for the refolding of denatured proteases. However, here we report achievement of almost complete restoration of enzymatic activity of subtilisin BPN' in the absence of its pro-sequence. The presence of 2 M potassium acetate in the folding medium enhanced the refolding efficiency of guanidine hydrochloride (GdnHCl)-denatured subtilisin BPN' by up to 28%, and other organic salts were also found to be useful, suggesting that general contribution of the bulky hydrophobic moieties of the salts to the formation of a favorable environment required for folding. This finding will provide new insights into the folding mechanisms not only of proteases but also of various other proteins. Almost complete restoration of enzymatic activity of denatured subtilisin in the organic salt solution was accomplished by further addition of mutated Streptomyces subtilisin inhibitor (SSI), which had been converted to a digestible temporary inhibitor by removal of the disulfide bridge near the reactive site.

Acetates↗
PubMed1994

Comparative studies on the primary structures and inhibitory properties of subtilisin-trypsin inhibitors from Streptomyces.

Three novel proteinaceous inhibitors of serine proteases which had been identified as Streptomyces subtilisin inhibitor-like (SIL) inhibitors were isolated from culture supernatant of Streptomyces; SIL2 from Streptomyces parvulus, SIL3 from Streptomyces coelicolor and SIL4 from Streptomyces lavendulae. They exhibited not only strong inhibitory activity toward subtilisin BPN' but also less strong inhibition of trypsin. Their primary sequences were determined by sequence analysis of peptides obtained by specific cleavage at the reactive site and subsequent proteolytic digestion. Each inhibitor consisted of about 110 amino acids, and was considered to form a dimer. The reactive site of the inhibitors was identified as Arg-Glu for SIL2 and SIL3, and Lys-Leu for SIL4, from sequence analysis of modified forms of the inhibitors produced from the inhibitor-subtilisin complex under acidic conditions. The presence of an arginine/lysine residue at the P1 site was in agreement with their trypsin-inhibition property. Sequence comparison with other members of the Streptomyces subtilisin inhibitor family revealed that amino acid replacements in the three isolated SIL inhibitors were frequently localized on the surface region, and many of the amino acid residues in beta-sheets and the hydrophobic core were highly conserved. Values of the inhibitor constant (Ki) toward subtilisin BPN' and trypsin were also measured, and the differences were discussed on the basis of the determined structures of the inhibitors.

Amino Acid Sequence↗
PubMed1994

Stimulation of granulopoiesis by high-dose recombinant human granulocyte colony-stimulating factor in children with aplastic anemia and very severe neutropenia.

We investigated the efficacy and safety of high-dose recombinant human granulocyte colony-stimulating factor (rhG-CSF) in treating 10 children with severe aplastic anemia and fewer than 0.05 x 10(9)/L neutrophils. Doses of rhG-CSF ranging from 400 to 2,000 micrograms/m2/d were administered as a 30-minute intravenous infusion daily for 4 weeks. In 6 of the 10 children, treatment increased the neutrophil count by 10-fold to greater than 60-fold (range, 0.21 to 1.8 x 10(9)/L). Bacterial or fungal infections that were present at study entry resolved in all 6 responders, who are still alive with a median survival of more than 27 months (range, 15 to 54 months) since the initiation of treatment. Three of 4 nonresponders died of infection, whereas 1 nonresponder received a bone marrow transplant and is alive. No serious toxicity was attributable to rhG-CSF. It was well tolerated at doses up to 2,000 micrograms/m2/d and effectively stimulated granulopoiesis. This agent thus offers promise as adjuvant treatment for severe infections in children with aplastic anemia and very severe neutropenia.

Adolescent↗
PubMed1994

Novel FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F). Cloning and expression of its gene in Escherichia coli.

A gene encoding a novel FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F) was cloned, and its expression system was constructed in Escherichia coli. The 1.4-kilobase pair DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with KpnI and SmaI was found to express a protein binding FMN as a prosthetic group under control of the lac promoter in E. coli. This DNA fragment contained several putative open reading frames. The partial amino acid sequence of the polypeptide portion of the purified FMN-binding protein and its tryptic peptides were completely consistent with those deduced from the nucleotide sequence of the third open reading frame in the cloned SmaI-SmaI fragment of D. vulgaris (Miyazaki F) DNA, which may include promoter and regulatory sequences. The nucleotide sequence of FMN-binding protein indicated that the protein is composed of 122 amino acids including an initiator Met residue and lacks a signal peptide for secretion. The main redox potential of the FMN-binding protein was measured as -325 mV using direct current cyclic and differential pulse voltammetric techniques and an electroreflectance method, suggesting that this FMN-binding protein functions as a redox protein like other FMN-binding proteins. Immunoblot analysis of the whole proteins from D. vulgaris (Miyazaki F) clearly indicated that this protein is expressed in this bacteria. However, the protein was found to have a primary structure distinct from those of other FMN-binding proteins and to be the smallest FMN-binding protein yet reported.

Amino Acid Sequence↗
PubMed1994

Mechanism of mobilization of renal and hepatic cadmium by dithiocarbamates in mice.

To clarify the mechanism of mobilization of renal and hepatic cadmium (Cd) by N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD) in mice exposed to Cd, the effects of pretreatment with probenecid, an organic anion transport inhibitor, or with acivicin, a gamma-glutamyltranspeptidase (gamma-GTP) inhibitor and ureter-ligation were investigated on the excretion and distribution of chelating agents and Cd. The renal contents of BGD and HBGD were increased by ureter-ligation and decreased by acivicin pretreatment. The mobilizing effect of BGD on the renal Cd was inhibited by probenecid pretreatment. The action of HBGD in removing Cd from the kidney was inhibited by both probenecid pretreatment and ureter-ligation. These results suggest that BGD and HBGD are mainly taken up into the renal tubular cells through the basolateral membrane which is dependent on the action of gamma-GTP; that the Cd-BGD complex formed in the tubular cells is secreted by a probenecid-sensitive organic anion transport system through the basolateral membrane; and that the Cd-HBGD complex formed in the tubular cells is secreted to the tubular lumen by an organic anion transport system through the brush border membrane. Probenecid pretreatment increased the hepatic contents of BGD and HBGD and also promoted the effects of these chelating agents in removing Cd from the liver, indicating an inhibitory effect of probenecid on the glucuronidation of BGD and the secretion of HBGD from the kidney. These results suggest that BGD and HBGD are taken up into the liver and secreted from the organ to the bile by a transport system other than a probenecid-sensitive transport mechanism.

Animals↗
PubMed1994

Contribution of salt bridge in the protease inhibitor SSI (Streptomyces subtilisin inhibitor) to its inhibitory action.

The tertiary structure of proteinaceous protease inhibitors is considered to be maintained by various interactions in the molecule that prevent degradation by protease. In this study, the Arg29 of Streptomyces subtilisin inhibitor (SSI) forming a salt bridge with the carboxyl group of carboxyl-terminal Phe113 was replaced with Ala, Met or Lys by cassette mutagenesis to clarify the role of Arg29 in the function of SSI. The inhibitory activity of each mutated SSI decreased with increasing incubation time after mixing with subtilisin, indicating that the SSI was changed into a temporary inhibitor upon mutation. This decrease was shown by SDS polyacrylamide gel electrophoresis to be due to cooperative degradation of the mutated SSI by subtilisin. In addition, the denaturation temperature of the Ala or Met mutant was decreased by ten degrees and that of the Lys mutant by 1.5 degrees, suggesting that the destabilization of SSI may be related to its temporary inhibition. Thus, interaction in the protease inhibitor molecule for maintaining the tertiary structure, such as that of Arg29 in SSI, was shown to be required for the inhibitory action.

Alanine↗
PubMed1994

Improvement of a useful enzyme (subtilisin BPN') by an experimental evolution system.

In order to improve a natural enzyme so as to fit industrial purposes, we have applied experimental evolution techniques comprised of successive in vitro random mutagenesis and efficient screening systems. Subtilisin BPN', a useful alkaline serine protease, was used as the model enzyme, and the gene was cloned to an Escherichia coli host-vector system. Primary mutants with reduced activities of below 80% of that of the wild type were first derived by hydroxylamine mutagenesis directly applied to subtilisin gene DNA, followed by screening of clear-zone non-forming transformant colonies cultured at room temperature on plates containing skim-milk. Then, secondary mutants were derived from each primary mutant by the same mutagenic procedure, but screened by detecting transformant colonies incubated at 10 degrees C with clear zones that were greater in size than that of the wild type. One such secondary mutant, 12-12, derived from a primary mutant with 80% activity, was found to gain 150% activity (kcat/Km value) of the wild-type when the mutant subtilisin gene was subcloned to a Bacillus subtilis host-vector system, expressed to form secretory mutant enzyme in the medium, and the activity measured using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as the substrate. When N-succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide was used, 180% activity was gained. Genetic analysis revealed that the primary and secondary mutations corresponded to D197N and G131D, respectively. The activity variations found in these mutant subtilisins were discussed in terms of Ca(2+)-binding ability. The thermostability was also found to be related to the activity.

Amino Acid Sequence↗
PubMed1994

Relationship between intracellular calcium and oxygen consumption: effects of perfusion pressure, extracellular calcium, dobutamine, and nifedipine.

All of the mechanisms that connect the cardiac mechanical work load with energy production have not been clearly defined. The purpose of this study was to evaluate the relationship between intracellular calcium and oxygen consumption in intact hearts, to further understand this relationship. Intracellular calcium was measured in isolated nonworking perfused rat hearts loaded with Indo-1 by means of a surface fluorometry technique. Glucose was used as a substrate. Myocardial contraction and oxygen consumption were modulated by perfusion pressure (80, 110, and 140 cm of water), extracellular calcium (1, 2, 3, and 4 mmol/L), dobutamine (10(-6) mol/L), and nifedipine (10(-6) mol/L). With all of these interventions there was a close correlation between intracellular calcium (systolic, diastolic, and amplitude) and oxygen consumption or left ventricular developed pressure. Observations in this study support the hypothesis that intracellular calcium plays a regulatory role in the link between cardiac mechanics and energy production.

Animals↗
PubMed1994

Intracellular calcium transients in potentiated contractions induced by multiple extrasystolic beats in isolated perfused rat hearts.

Mechanisms underlying contractile potentiation induced by multiple extrasystolic contractions (ESC) were evaluated with surface fluorometry in isolated perfused rat hearts loaded with Indo-1/AM. After baseline pacing with a 400 ms interval, 1-25 ESC were interposed with a regular 160 ms interval followed by the postextrasystolic beat with a 400 ms interval. With an increase in the ESC number, left ventricular developed pressure and peak positive dP/dt increased in an exponential manner, reaching a plateau, that was the same for 3 extracellular Ca2+ ([Ca2+]o; 0.55 (n = 9), 1.25 (n = 11) and 2.75 mM (n = 7). Increased [Ca2+]o shifted this relationship left and upward, and with 2.75 mM [Ca2+]o developed pressure and dP/dt decreased after the maximum potentiation was obtained. The relationship between the ESC number and the amplitude of the Indo-1 fluorescence (F400/F510; an index of intracellular Ca2+ ([Ca2+]i)) was also exponential and was shifted left and upward by high [Ca2+]o; however, it lacked the declining phase. Thus, the relationship between the amplitude of F400/F510 and developed pressure or dP/dt consisted of a positively linear part until the maximum potentiation was obtained and a negatively linear part with a further increase in the amplitude of F400/F510. This observation suggests that although contractile potentiation is mediated by increased [Ca2+]i transients, the maximum response might be determined by the responsiveness of the sarcomere.

Animals↗
PubMed1994

Sequence comparison between the flavoprotein subunit of the fumarate reductase (complex II) of the anaerobic parasitic nematode, Ascaris suum and the succinate dehydrogenase of the aerobic, free-living nematode, Caenorhabditis elegans.

Complex II in adult mitochondria of the parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron-transport observed in these organelles. In the present study, cDNAs for the flavoprotein (Fp) subunits of complex II have been isolated, cloned and sequenced from both A. suum and the aerobic, free-living nematode, Caenorhabditis elegans. Additional sequence at the 3' end of the mRNAs was determined by the Rapid Amplification of cDNA Ends (RACE). Nucleotide sequence analysis of the A. suum cDNAs revealed a 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1935 nucleotides and a 3' untranslated region of 616 nucleotides including a poly (A) tail from a polyadenylation signal (AATAAA). The open reading frame encoded a 645 amino acid sequence, including a 30 amino acid mitochondrial presequence. The amino acid sequences for the Fp subunits from both organisms were very similar, even though the ascarid enzyme functions physiologically as a fumarate reductase and the C. elegans enzyme a succinate dehydrogenase. The ascarid sequence was much less similar to the Escherichia coli fumarate reductase. The sensitivity of other Fp subunits to sulfhydryl reagents appears to reside in a cysteine immediately preceding a conserved arginine in the putative active site. In both nematode sequences, this cysteine is replaced by serine even though the succinate dehydrogenase activity of both enzymes is still sensitive to sulfhydryl inhibition. A cysteine six residues upstream of the serine may be involved in the sulfhydryl sensitivity of the nematode enzymes. Surprisingly, in contrast to succinate dehydrogenase activity, the fumarate reductase activity of the ascarid enzyme was not sensitive to sulfhydryl inhibition, suggesting that the mechanism of the two reactions involves separate catalytic processes.

Amino Acid Sequence↗