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Biomedical subjects

K Oda

Publications and source records attributed to K Oda.

At least 127 records · Page 7Linked to original sources
PubMed1999

The G10BP-1 gene encoding a GC box binding protein, is a target of Myc and Jun/Fos.

BACKGROUND: G10BP, a serum-inducible factor, represses the transcription of the fibronectin gene through binding to the G-rich sequences in the promoter excluding Sp1 from binding to these sequences. RESULTS: The 5' flanking sequence of the G10BP-1 gene was isolated by polymerase chain reaction of the adaptor-ligated genomic DNA library using the adaptor primer and the G10BP-1 cDNA primer. The elements required for activation of the G10BP-1 promoter following serum stimulation were analysed by transfection of quiescent rat 3Y1 cells with G10BP-1 promoter-luciferase cDNA constructs containing 5' sequential deletions or base substitutions. The results showed that the promoter was activated by Myc and Jun through the E box and AP1 sites. The formation of DNA-protein complexes with 32P-labelled oligonucleotides containing the E box or AP1 site with cell extracts prepared during G1 progression was correlated with the promoter activation and greatly reduced by immunodepletion of Myc or c-Jun from the extracts. CONCLUSION: These results indicate that the G10BP-1 gene is a target of Myc and Jun/Fos and that these factors repress the fibronectin gene expression through induction of G10BP-1 during G1-to-S phase progression.

Animals↗
PubMed1999

Cloning and sequence of a cDNA for a highly basic protease from the digestive juice of the silkworm, Bombyx mori.

A serine protease of the silkworm, Bombyx mori, with an isoelectric point of pH 10-11 and a pH optimum for succinyl-Leu-Leu-Val-Tyr-MCA degrading activity of about 10, was found in a 0.33 M NaCl-eluted fraction obtained from cation-exchange chromatography of digestive juice. The activity of the enzyme was strongly inhibited by chymostatin and PMSF, indicating that the protease is a chymotrypsin-like serine protease. The N-terminal amino acid sequence of the protease was determined, and a full-length cDNA clone (0.92 kbp) which was isolated from a midgut cDNA library was sequenced. The cDNA encodes a pre-proenzyme of 284 amino acids with a pro-segment of 50 amino acids and mature protein of 234 amino acids. From its primary structure, the predicted molecular mass of the mature protein is 24.5 kDa. A sequence comparison of the Bombyx highly basic protease with other serine proteases revealed that this enzyme is a mammalian-type serine protease with a catalytic triad consisting of His45, Asp92 and Ser186. A large number of Arg residues are encoded by the cDNA which may be responsible for its stability and/or function in the alkaline condition, by remaining charged at high pH.

Amino Acid Sequence↗
PubMed1999

Studies on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor and thermolysin.

The effects of certain physicochemical parameters on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor (SMPI) and thermolysin were investigated. SMPI had its lowest Ki value at a pH of around 6.5 (similar to the pH dependence of the kcat/K(m) of thermolysin catalysis), reflecting the splitting mechanism of the SMPI inhibition of thermolysin. This Ki increased with an increase in pressure, and in (Ki-1) was almost linear with respect to pressure. The volume of the reaction (delta Vcomp), which is the volume change accompanying enzyme-inhibitor complex formation, was calculated as +8.1 +/- 0.3 mL.mol-1, which has a sign opposite to delta Vcomp for neutral peptide inhibitors and acyl-peptide substrates. The temperature dependence of Ki-1 gave the reaction enthalpy (delta Hcomp) and reaction entropy (delta Scomp) of the complex formation as 34.6 +/- 1.4 kJ.mol-1 and 298 +/- 5 J.mol-1.K-1, respectively. These positive reaction volumes and reaction entropies were related to the electrostatic interactions and ionic strength dependence of Ki which corresponded to the key ionic interaction during complex formation. Complex formation with SMPI stabilized thermolysin against pressure perturbation as observed by the changes in the Trp fluorescence of thermolysin with increasing pressure. Thermal stability, however, was affected very little by complex formation with SMPI. Phosphoramidon, Cbz-Phe-Gly-NH2 and Cbz-Phe also positively affected the pressure-tolerance of thermolysin, in the following order: Cbz-Gly-Phe-NH2 < Cbz-Phe << phosphoramidon. The third compound exhibited stabilizing effects comparable with those of SMPI, which suggests that the interaction between SMPI and thermolysin was localized to the reactive site.

Bacterial Proteins↗
PubMed1999

Mutational analysis of the reactive site loop of Streptomyces metalloproteinase inhibitor, SMPI.

Streptomyces metalloproteinase inhibitor (SMPI) is the only inhibitor to show "standard mechanism inhibition" against metalloproteinases. SMPI is a globular protein with an exposed loop containing the reactive site, C64-V65. To analyze the importance of basic residues in the reactive site loop of SMPI, mutants were constructed for R60, K61, and R66 (R60A, K61A, R66A, R60/K61A, 60/61/66A, and 60/61/66E). The mutants involving only R60, K61, and R60/K61 residues, respectively, showed strong inhibitory activity and were stable against enzyme activity. Both the triple mutants showed very weak inhibitory activity and underwent rapid degradation. The addition of basic residues to the loop (V62R and T63R) did not cause any further increase in inhibitory activity. These results suggest that basic residues in the reactive site loop play some role in maintaining a stable enzyme-inhibitor complex. The R66 mutant showed reduced activity and was rapidly degraded by enzymes. It was concluded that R66 is essential for maintaining a strong hydrophobic interaction with the S1' hydrophobic pocket of the enzyme. To investigate the roles of the disulfide bridge and the P68 residue near the reactive site, C64/69S and P68T mutants were constructed. These mutants showed very weak inhibitory activity and were rapidly degraded by enzymes. These results suggest that the disulfide bridge and P68 residue are very essential for SMPI to function as an inhibitor.

Bacterial Proteins↗
PubMed1999

Identification of carboxyl residues in pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 that participate in catalysis and substrate binding.

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, is the first example from a prokaryote of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. To identify the catalytic residue(s) of PCP, chemical modification was carried out using carboxyl residue-specific reagents, carbodiimides. PCP was inactivated effectively by N,N'-dicyclohexylcarbodiimide (DCCD) with pseudo-first-order kinetics. For the inactivation, 0.7 mol DCCD was involved per 1 mol PCP. The effects of pH and methanol on the inactivation showed that two carboxyl residues (Asp and/or Glu) were involved in the reaction. The inactivation by DCCD was prevented by a competitive inhibitor, tyrostatin, or a synthetic substrate in a concentration-dependent manner. Based on these data, differential labeling of PCP with DCCD was carried out: Firstly, PCP was treated with cold DCCD in the presence of tyrostatin. After removal of the tyrostatin, which covered the substrate binding site, by dialysis, the PCP was treated with [14C]DCCD to label carboxyl residue(s) essential for its function. Two labeled peptides were isolated by HPLC from a trypsin digest of cold- and [14C]DCCD modified PCP. On analysis of their amino acid sequences, it was revealed that the [14C]DCCD was bound to Asp140 and Glu222 of PCP, respectively. Based on these data, it was strongly suggested that Asp140 and Glu222 of PCP were involved in its catalytic function or substrate binding.

Amino Acid Sequence↗
PubMed1999

Subsite preferences of pepstatin-insensitive carboxyl proteinases from bacteria.

Pseudomonas sp. 101 carboxyl proteinase (PCP) and Xanthomonas sp. T-22 carboxyl proteinase (XCP), the first and second unique carboxyl proteinases from prokaryotes to be isolated and characterized, are not inhibited by the classical carboxyl proteinase inhibitor pepstatin. In this study, we elucidated their subsite preferences by using a series of synthetic chromogenic substrates, Lys-Pro-Ile(P3)-Glu(P2)-Phe*Nph-Arg(P2')-Leu(P3') (Nph is p-nitrophenylalanine, Phe*Nph is the cleavage site) with systematic substitutions at the P3, P2, P2', and P3' positions. Among 45 substrates tested, the best substrate for PCP had a Leu replacement at the P2 position (kcat = 27.2 s-1, Km = 4.22 microM, kcat/Km = 6.43 microM-1.s-1), and that for XCP had an Ala replacement at the P3 position (kcat = 79.4 s-1, Km = 6.05 microM, kcat/Km = 13.1 microM-1. s-1). PCP and XCP preferred such charged amino acid residues as Glu, Asp, Arg, or Lys at the P2' position. This suggested that the S2' subsites of PCP and XCP are occupied by hydrophilic residues, similar to that of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [Shibata et al. (1998) J. Biochem. 124, 642-647]. In contrast, the S2' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydophilic nature of the S2' subsite appears to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases.

Aspartic Acid Endopeptidases↗
PubMed1999

Basement membrane heparan sulfate proteoglycan (perlecan) synthesized by ACC3, adenoid cystic carcinoma cells of human salivary gland origin.

The biosynthesis of basement membrane heparan sulfate proteoglycan (HSPG), known as perlecan, in ACC3 cells established from a adenoid cystic carcinoma of the human salivary gland was studied using metabolic labeling and immunoprecipitation with discriminative antibodies specific for HSPG core protein. Treatment of immunoprecipitated HSPG with HNO2, heparitinase, and chondroitinase ABC revealed that ACC3 cells synthesized HSPG molecules composed of 470-kDa core protein and heparan sulfate but not of chondroitin sulfate. The core protein was shown to contain complex type N-linked oligosaccharides by digestion with N-glycanase and endoglycosidase H. Pulse-chase experiments showed that the mature form of HSPG was formed in the cells in 30 min and released into the medium thereafter. Degradation of HSPG was also found in the chase period of 3 h. In time course experiments, HSPG was found to be synthesized maximally at day 4 after plating, deposited in the cell layer maximally at day 6, and secreted maximally at day 8. This was also confirmed by immunofluorescence, Northern blotting, and in-situ hybridization. The results indicate that ACC3 cells synthesize, secrete and degrade basement membrane type HSPG, which is analogous to those produced by other cell types, and that the biosynthesis and secretion of HSPG in ACC3 cells are strictly regulated by the cell growth, that may be reflected in the characteristic histology of adenoid cystic carcinomas.

Basement Membrane↗
PubMed1999

A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspecific alkaline phosphatase.

A soluble form of tissue-nonspecific alkaline phosphatase was purified to apparent homogeneity from the culture media of Sf9 cells which had been infected with recombinant baculoviruses encoding human tissue-nonspecific alkaline phosphatase (TNSALP). To facilitate purification, an oligonucleotide consisting of 6 tandem codons for histidine and a stop codon was engineered into the TNSALP cDNA. The molecular mass of the enzyme purified through a nickel-chelate column was estimated to be 54 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. That of the native enzyme was 90 kDa as estimated by gel filtration, indicating that the purified soluble TNSALP is dimeric. The enzyme was used for production of antibodies specific for human TNSALP. Immunoblotting analysis showed a single 80-kDa band in the cell homogenate prepared from Saos-2 (human osteosarcoma) cells. However, upon digestion with peptide: N-glycosidase F, the 80-kDa TNSALP of human origin and the soluble enzyme of insect origin migrated to the same position on SDS-polyacrylamide gel, indicating that the size difference between the two enzymes is ascribed to N-linked oligosaccharides. The antibodies prepared against the purified TNSALP were found to be useful also for immunoprecipitation and immunofluorescence studies.

Alkaline Phosphatase↗
PubMed1999

[Steroid pulse therapy and vital prognosis among pediatric cancer patients with sepsis].

The usefulness of glucocorticosteroid therapy in patients with sepsis has been controversial. We investigated the effect of steroid pulse therapy on the vital prognosis of pediatric patients with sepsis and followed the vital status up to one month after the use of pulse therapy. We reviewed the medical records of 89 pediatric cancer patients with sepsis treated at our hospital between 1988 and 1996. The risks of potential predictors were estimated by calculating crude and adjusted relative risk (RR). The total cumulative death was 33/89 (25%). All patients treated with steroid pulse therapy died (12/12). Patients with either interstitial pneumonia (IP), infection-associated hemophagocytic syndrome (IAHS) and graft-versus-host disease (GVHD) showed a significantly higher cumulative death (57% (12/21) vs 31% (21/68), p = 0.03). Increased risk of cumulative death was suggested for the older age group, inappropriate antimicrobial therapy, and the conditions requiring steroid pulse therapy (IP, IAHS, and GVHD), (crude RR were 1.6, 1.6, and 1.9, respectively). However, when adjusted for pulse therapy, these three factors no longer indicated risk elevation, (adjusted RR = 1.2, 1.2, and 0.3, respectively). On the other hand, steroid pulse therapy per se was independently associated with increased risk of cumulative death (crude RR = 3.6, adjusted RR = 10). Thus, the risk of the conditions requiring steroid pulse therapy (IP, IAHS, and GVHD) firstly observed could be regarded as an apparent association due to steroid pulse therapy.

Child↗
PubMed1999

[Plasmodium falciparum infection in a 8-year old boy].

A 8-year old Japanese boy who returned from Tanzania was admitted to our hospital because of fever, vomiting, and headache. He was diagnosed as a Plasmodium falciparum infection verified by a blood smear. He was treated with quinine and halofantrine, and recovered completely. Malaria infection should be considered when patients return from Malaria endemic areas.

Child↗
PubMed1999

A rabbit model of abdominal aortic aneurysm associated with intimal thickening.

A model of abdominal aortic aneurysm with intimal thickening was developed in the rabbit. A segment of the abdominal aorta just proximal to the bifurcation (1 or 2 cm in length) was dissected and isolated with clamps. This segment was perfused by injecting physiologic saline or 100 U/ml of hog pancreatic elastase from the lumen. Perfusion was performed manually for 5 min and the peak pressure in the segment was between 300 and 400 mm Hg in order to cause aortic wall injury. After 4 weeks, animals that had received perfusion with elastase had aneurysms in the perfused segment on arteriography. None of the other animals developed aortic aneurysms. Histologically, the segments of aorta perfused with saline exhibited intimal hyperplasia. In addition to the intimal hyperplasia, the segments of aorta perfused with elastase solution showed lysis of the elastic lamellae in the media that resulted in aneurysm formation.

Animals↗
PubMed1999

Identification of BAIAP2 (BAI-associated protein 2), a novel human homologue of hamster IRSp53, whose SH3 domain interacts with the cytoplasmic domain of BAI1.

BAI1 (brain-specific angiogenesis inhibitor 1) was originally isolated as a p53-target gene specifically expressed in brain. To clarify its function, we have been searching for cellular proteins that associate with the cytoplasmic domain of BAI1. Using its intracellular carboxyl terminus as "bait" in a yeast two-hybrid system, we isolated a cDNA clone named BAIAP2 whose nucleotide sequence would encode a 521-amino acid protein showing significant homology to a 58/53-kDa substrate of insulin-receptor kinase in the hamster. As the expression profile of BAIAP2 examined by Northern blot analysis was almost identical to that of BAI1, BAIAP2 appears to be active mainly in neurons. In vitro binding assays confirmed that a proline-rich cytoplasmic fragment of BAI1 interacted with the Src homology 3 (SH3) domain of BAIAP2. Double-color immunofluorescent analysis revealed that BAIAP2 was localized at the cytoplasmic membrane when it was coexpressed with BAI1 in COS-7 cells; BAIAP2 not associated with BAI1 was diffused in the cytoplasm. Predominant localization of BAI1 protein in a sub-cellular fraction enriched in growth cones indicated a possible role of BAI1 as a cell adhesion molecule inducing growth cone guidance. As a protein partner of BAI1, BAIAP2 may represent an important link between membrane and cytoskeleton in the process of neuronal growth.

Amino Acid Sequence↗
PubMed1999

[Pharmacological studies of Reiousan, a drug containing bezoar and ginseng--effects on the blood rheology].

Effects of Reiousan, a crude drug preparation consisting of bezoar and ginseng, on blood rheology were studied. Reiousan improved the deformability of rat erythrocytes exposed to hyperosmorality and treated with phenylhydrazine. The ATP depletion in erythrocytes, the polybrene-induced erythrocyte aggregation and the oxidation of low density lipoprotein were suppressed by Reiousan. Oral administration of Reiousan also improved the erythrocyte deformability in phenylhydrazine-treated rats and delayed the thromboembolic death induced by arachidonic acid in mice. These results suggest that Reiousan has an ameliorative effect on blood rheology related to "Oketsu" syndrome in Kampo diagnostics.

Adenosine Triphosphate↗
PubMed1999

Isolation and some properties of a novel killer toxin-like protein produced by Streptomyces sp. F-287.

A killer toxin-like protein was found in the culture supernatant of a strain isolated from soil. The strain was classified and designated as Streptomyces sp. F-287. The molecular weight of the purified killer toxin-like protein was estimated to be 9,500 by SDS-PAGE. The purified protein was heat stable (100 degrees C, 5 min), pH stable (pH 6.0-9.0, 60 degrees C, for 30 min), and had a relatively wide action spectra. The SKLP showed a cytocidal effect on both budding yeast, Saccharomyces cerevisiae W303 (IC50 = 15.6 micrograms/ml) and on fission yeast, Schizosaccharomyces pombe SP870 (IC50 = 20.0 micrograms/ml). The SKLP also caused morphological changes on some sensitive yeasts and filamentous fungi. These characteristics are apparently different from known killer toxins. These results suggest that this is a novel killer toxin-like protein from Streptomyces sp. strain F-287.

Amino Acid Sequence↗
PubMed1999

Apolipoprotein A-I of hyperlipidemia atherosclerosis prone (LAP) quail: cDNA sequence and tissue expression.

Apolipoprotein A-I (apo A-I) has an important role in the transport of cholesterol. This study describes the complete nucleotide and deduced amino acid sequence for apo A-I of LAP quail. A full length apo A-I cDNA clone for hyperlipidemia atherosclerosis prone (LAP) quail was isolated from a lambda gt10 liver cDNA library. The DNA sequence of LAP apo A-I cDNA was similar to that of normal Japanese quail. The deduced amino acid sequence of LAP apo A-I was hence identical to that of normal Japanese quail. LAP apo A-I mRNA is about 1.4 kilobases in length and expressed in a variety of tissues including small intestine, liver, lung, breast muscle, testis, and heart. Although the tissue distribution of apo A-I was similar between strains, LAP quail expressed more apo A-I mRNA than normal Japanese quail in all tissues examined. This tendency was pronounced with the small intestine. Although the concentration of serum apo A-I did not correlate with the tissue expression of mRNA, the observation may suggest that the increased apo A-I expression in LAP strain had some relevance to the susceptibility of this strain to the experimental atherosclerosis.

Amino Acid Sequence↗
PubMed1999

Aspergillus osteomyelitis in a child who has p67-phox-deficient chronic granulomatous disease.

Here we describe Aspergillus osteomyelitis of the tibia in a 9-year-old boy who has an autosomal recessive form of chronic granulomatous disease (CGD). The patient showed a p67-phagocyte oxidase (phox) deficiency, which is rare type of CGD in Japan. The initial treatment which consisted of surgical debridement and antibiotic therapy with amphotericin B (AMPH), did not control the infection. Aspergillus fumigatus (A. fumigatus) pure isolated from drainage fluid and necrotic bone tissue demonstrated less susceptible to antifungal agents, including AMPH, fluconazole and flucytosine. Recombinant interferon gamma was then administrated, and it was effective in controlling the course of severe invasive aspergillosis. This report indicates the use of interferon gamma might be helpful in control for Aspergillus osteomyelitis of the tibia in a child with CGD demonstrated p67-phox deficiency refractory to conventional therapy with AMPH.

Amphotericin B↗
PubMed1999

Type-3 dengue viruses responsible for the dengue epidemic in Malaysia during 1993-1994.

To characterize the dengue epidemic that recently occurred in Malaysia, we sequenced cDNAs from nine 1993-1994 dengue virus type-3 (DEN-3) isolates in Malaysia (DEN-3 was the most common type in Malaysia during this period). Nucleic acid sequences (720 nucleotides in length) from the nine isolates, encompassing the precursor of membrane protein (preM) and membrane (M) protein genes and part of the envelope (E) protein gene were aligned with various reference DEN-3 sequences to generate a neighbor-joining phylogenetic tree. According to the constructed tree, the nine Malaysian isolates were grouped into subtype II, which comprises Thai isolates from 1962 to 1987. Five earlier DEN-3 virus Malaysian isolates from 1974 to 1981 belonged to subtype I. The present data indicate that the recent dengue epidemic in Malaysia was due to the introduction of DEN-3 viruses previously endemic to Thailand.

Amino Acid Sequence↗