Biomedical subjects

S Kojima

Publications and source records attributed to S Kojima.

At least 307 records · Page 17Linked to original sources

PubMed1997

The mechanisms of nickel uptake by rat primary hepatocyte cultures: role of calcium channels.

The present study was designed to clarify the mechanism of nickel (Ni) uptake in primary cultures of rat hepatocytes. Exposure of the hepatocytes to Ni (2-50 microM; as NiCl2) for up to 6 h was not cytotoxic, as assessed by the tetrazolium-based dye (MTT) assay. Hepatocytes were treated with 10 microM NiCl2 in the absence or presence of calcium (Ca) and magnesium (Mg) (1 mM). Ni uptake was increased by 19% in medium lacking Mg or Ca and was increased by 37% in Ca- and Mg-free medium. The role of Ca channels on Ni uptake by the hepatocytes was investigated. Pretreatment with nicardipine or verapamil (200 microM), potent inhibitors of Ca channels, decreased Ni uptake by 20%. This effect was only observed when the cells were incubated in the absence of Ca. Pretreatment with vasopressin (100 nM), a well-known Ca channel agonist, significantly increased Ni uptake by the hepatocytes (24%). To determine the involvement of carrier-mediated processes on Ni uptake, the effect of temperature was also investigated. At 4 degrees C the Ni uptake was decreased by 20% compared to uptake at 37 degrees C. These results indicate that Ni uptake by the hepatocytes occurs, at least in part, through the Ca channel transport processes. Further study will be required to assess what other mechanisms are involved.

Animals↗

PubMed1997

Vocal identification of speaker and emotion activates different brain regions.

Regional cerebral blood flow was measured in six healthy volunteers by positron emission tomography during identification of speaker and emotion from spoken words. The speaker identification task activated several audio-visual multimodal areas, particularly the temporal poles in both hemispheres, which may be involved in connecting vocal attributes with the visual representations of speakers. The emotion identification task activated regions in the cerebellum and the frontal lobe, suggesting a functional relationship between those regions involved in emotion. The results suggest that different anatomical structures contribute to the vocal identification of speaker and emotion.

Adolescent↗

PubMed1997

Myelodysplastic syndrome and acute myelogenous leukemia as a late clonal complication in children with acquired aplastic anemia.

The improved outcome of acquired aplastic anemia (AA) has revealed later complications, such as myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). We retrospectively analyzed 167 children with severe acquired AA. Eleven of 50 children treated with cyclosporin (CSA) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) developed MDS/AML; 8 of these were within 36 months of the diagnosis of AA, much earlier than previous reports. Six of the 11 children received rhG-CSF exceeding 10 microg/kg/d, and 9 received rhG-CSF therapy for over 1 year. Ten children showed monosomy 7 at diagnosis of MDS. All of the 11 children were administered both CSA and rhG-CSF. There was no development of MDS/AML among 41 children treated with either CSA or rhG-CSF or among 48 children who underwent bone marrow transplantation. A well-controlled clinical trial is warranted to determine whether therapeutic modalities affect the development of MDS/AML in children with severe acquired AA.

Acute Disease↗

PubMed1997

Enhancement of an inhibitory input to the feeding central pattern generator in Lymnaea stagnalis during conditioned taste-aversion learning.

To study the neuronal mechanism of a conditioned taste-aversion (CTA) learning in the pond snail Lymnaea stagnalis, we examined the synaptic connection between the neuron 1 medial (N1M) cell and the cerebral giant cell (CGC), the former is an interneuron in central pattern generator for the feeding response and the latter is a regulatory neuron to the central pattern generator. Inhibitory postsynaptic potential (IPSP) which was evoked in the N1M cell by activation of the CGC was larger and lasted longer in the conditioned animal than that in the control animal. The electrical properties of the cell body of CGC and the responses of the CGC to the chemosensory inputs were not changed during the CTA learning. These results, together with the previous report indicating the existence of excitatory projection from the N1M cell to the feeding motoneuron, suggest that enhanced IPSP in the N1M cell may underlie the suppression of feeding responses in the Lymnaea CTA learning.

Animals↗

PubMed1997

Identification of heparin-binding sites in midkine and their role in neurite-promotion.

Midkine (MK) is a heparin-binding growth factor, which promotes neurite outgrowth in embryonic neurons and enhances their survival. The three dimensional structure of MK clarified by NMR spectroscopy indicates that several basic amino acids are exposed on the surface of the C-terminal half domain, which retains heparin-binding and neurite-promoting activity. We performed site-directed mutagenesis of these amino acids, and found that mutation of arginine78 reduced the heparin-binding activity. Mutation of either lysine83 or lysine84 scarcely affected heparin-binding activity, while the double mutant involving both lysine residues showed reduction in the activity. Neurite-promoting activity of mutant MKs always correlated with their heparin-binding activity, illustrating the close relationship of the two activities. Thus, the present result verifies the occurrence of two distinct heparin-binding sites involved in neurite-promoting activity of MK molecule.

Animals↗

PubMed1997

9,13-di-cis-Retinoic acid induces the production of tPA and activation of latent TGF-beta via RAR alpha in a human liver stellate cell line, LI90.

We studied the mechanism by which 9,13-di-cis-retinoic acid (9,13dcRA), a novel and endogenous stereoisomer of all-trans-RA, induces TGF-beta formation in a human liver stellate cell line, LI90. 9,13dcRA induced the expression of RAR alpha and RARbeta, enhanced the production of tissue-type plasminogen activator (tPA), thereby, surface plasmin levels, and induced the activation of latent TGF-beta. Similar effects were obtained with RAR alpha-selective retinoid, but not with RARbeta- or RARgamma-selective retinoid, and the induction was inhibited by RAR alpha-selective antagonist. These results suggest that 9,13dcRA up-regulates tPA expression, resulting in the formation of TGF-beta by LI90 cells, at least in part, via induction and activation of RAR alpha.

Animals↗

PubMed1997

The propeptide of subtilisin BPN' as a temporary inhibitor and effect of an amino acid replacement on its inhibitory activity.

The propeptide of subtilisin-family proteases is known to exhibit inhibitory activity toward a cognate protease in addition to its function as an intramolecular chaperone. For detailed investigation of its inhibitory properties, the propeptide of subtilisin BPN' was produced in Escherichia coli. Inhibitory activity measurements and electrophoresis showed that the propeptide was a temporary inhibitor, which was initially potent but was gradually degraded by subtilisin BPN' through specific intermediates. The main cleavage site was identified as Glu53-Lys54, with minor sites at Thr17-Met18 and Met21-Ser22, which were located in turn regions of the propeptide in the complex with subtilisin BPN'. Since the isolated propeptide has been shown not to form a tertiary structure, these results indicate that main digestions proceed through proteolytic attack of subtilisin toward the accessible sites of the propeptide in the complex with subtilisin. Therefore, replacement of Glu53 at the main cleavage site by Asp, which is a less favorable amino acid than Glu for subtilisin, makes the propeptide a more resistant temporary inhibitor.

Binding Sites↗

PubMed1997

Disulfide bond formation is not involved in cap-binding activity of Xenopus translation initiation factor eIF-4E.

The eukaryotic initiation factor eIF-4E from Xenopus laevis was expressed in Escherichia coli and refolded in an active form. To define the cysteine residues forming a disulfide bond in Xenopus eIF-4E, each of the 3 cysteine residues was changed to serine by site-directed mutagenesis. Cap-binding activities of the mutant proteins were evaluated by 7-methyl-GTP(m7GTP)-affinity column chromatography. Even the mutant protein containing no cysteine showed an affinity for m7GTP. From the above results and the estimation of the sulfhydryl groups by Ellman's assay method, we concluded that a disulfide bond is not involved in the active Xenopus eIF-4E.

Amino Acid Sequence↗

PubMed1997

Molecular cloning and characterization of Drosophila genes encoding small GTPases of the rab and rho families.

We have isolated eight genes from Drosophila, small GTPases. They can be classified into three rab family genes (Drab2, Drab5, Drab11) and five rho family genes (Drac1a, Drac1b, Drac3, Dcdc42, DrhoA). While Drac3 is a novel type of rac gene, others are homologues of known mammalian genes for small GTPases. Northern blot analyses showed that all the genes are expressed throughout all developmental stages from embryo to adult. In situ hybridization to embryos revealed that Drab2, Drac1b, and Drac3 are highly expressed in the nervous system, in the trunk mesoderm, and in the cephalic mesoderm, respectively. Since hemocytes are derived from the cephalic mesoderm, we carried out double stainings using a hemocyte marker anti-peroxidasin antibody and Drac3 in situ hybridization. We found that Drac3 is expressed in hemocyte precursor cells. In the Drac3 deficiency embryos, the hemocyte precursor cells start to differentiate normally, but never develop into mature hemocytes, indicating that Drac3 is essential for their maturation. The DrhoA and Dcdc42 genes complemented S. cerevisiae rho1 and cdc42 mutations in the same manner as human rhoA and CDC42, respectively. These results suggest functional similarity between Drosophila and mammalian small GTPase genes.

Amino Acid Sequence↗

PubMed1997

Dimerization of midkine by tissue transglutaminase and its functional implication.

Midkine (MK), a retinoic acid-inducible growth/differentiation factor, serves as a substrate for tissue transglutaminase (Kojima, S. , Muramatsu, H., Amanuma, H., and Muramatsu, T. 1995. J. Biol. Chem. 270, 9590-9596). Upon incubation with transglutaminase MK forms multimers through cross-linkages. Here, we report the following results. 1) Heparin potentiated the multimer formation by MK. 2) The N- and C-terminal half domains each formed a dimer through the action of transglutaminase. 3) Gln42 or Gln44 in the N-terminal half and Gln95 in the C-terminal half served as amine acceptors in the cross-linking reaction, as judged from the incorporation of putrescine into whole MK or each half domain, and the competitive inhibition of the cross-linking by MK-derived peptides containing Gln residue(s). The strongest inhibition was obtained with Ala41-Pro51. 4) This peptide abolished the biological activity of MK to enhance the plasminogen activator activity in bovine aortic endothelial cells. The inhibition was limited against the MK monomer, and not seen against the MK dimer, separated by gel filtration chromatography. These results suggest that dimer formation through transglutaminase-mediated cross-linking is an important step as to the biological activity of MK.

Animals↗

PubMed1997

Functional transitions in myosin: role of highly conserved Gly and Glu residues in the active site.

It has been proposed from crystallographic comparisons [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960-8972] that in one of the important transitions of myosin head (M), M x ATP --> M x ADP x Pi, a rotation occurs in Gly468 (of chicken smooth muscle myosin). We find that mutation of this Gly to Ala does block the transition. Searching for proton acceptors in ATPase catalysis, we also find that mutation of a candidate (Glu470 of chicken smooth muscle myosin) blocks the transition. Interpretations of both findings are examined.

Actins↗

PubMed1997

Cloning and expression of the rubredoxin gene from Desulfovibrio vulgaris (Miyazaki F)--comparison of the primary structure of desulfoferrodoxin.

A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene (rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that are responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D. vulgaris rubredoxin. In vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its redox potential was determined as -5 mV.

Amino Acid Sequence↗

PubMed1997

Vibrio alginolyticus mutants resistant to phenamil, a specific inhibitor of the sodium-driven flagellar motor.

The polar flagella of Vibrio alginolyticus are driven by sodium motive force and those motors are specifically and strongly inhibited by phenamil, an amiloride analog that is thought to interact with a sodium channel of the flagellar motor. To study the sodium ion coupling site, we isolated motility mutants resistant to phenamil and named the phenotype Mpa(r) for motility resistant to phenamil. The motility of the wild-type (Mpa(s)) was inhibited by 50 microM phenamil, whereas Mpa(r) strains were still motile in the presence of 200 microM phenamil. The Ki value for phenamil in the Mpa(r) strain was estimated to be five times larger than that in the Mpa(s) strain. However, the sensitivities to amiloride or benzamil, another amiloride analog, were not distinctly changed in the Mpa(r) strain. The rotation rate of the wild-type Na+-driven motor fluctuates greatly in the presence of phenamil, which can be explained in terms of a relatively slow dissociation rate of phenamil from the motor. We therefore studied the stability of the rotation of the Mpa(r) and Mpa(s) motors by phenamil. The speed fluctuations of the Mpa(r) motors were distinctly reduced relative to the Mpas motors. The steadier rotation of the Mpa(r) motors can be explained by an increase in the phenamil dissociation rate from a sodium channel of the motor, which suggests that a phenamil-specific binding site of the motor is mutated in the Mpa(r) strain.

Amiloride↗

PubMed1997

Effects of dithiocarbamates on toxicity of cadmium in rat primary hepatocyte cultures.

The protective effects of N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD) on the toxicity of Cd in the rat primary hepatocyte cultures were studied. Cytotoxicity was assessed by measuring cell viability, extra cellular lactic dehydrogenase (LDH) activity, and intracellular lipid peroxidation and active oxygen species. Primary hepatocyte cultures were treated with 109CdCl2 (5, 10 or 50 microM Cd and 1.7 KBq of 109Cd/well) for 30 min or 4 h. BGD or HBGD was added to the culture medium to make the final concentration of 100 microM and incubated for 4.5 h in 30 min Cd exposure or 1 h in 4 h Cd exposure. Decreases in the hepatocyte viability caused by all Cd exposure concentrations were significantly prevented by treatment with BGD or HBGD. The treatment with the chelating agents for 4.5 h after Cd exposure for 30 min significantly prevented increases in extracellular LDH activity. Increases in the lipid peroxidation in hepatocytes exposed to Cd for 30 min or 4 h were prevented significantly by treatment with BGD or HBGD for 4.5 h or 1 h, respectively. Moreover, the increases in the level of active oxygen species caused by Cd exposure for 30 min were significantly prevented by treatment with the chelating agents for 1.5 h. These findings suggest that BGD and HBGD protect against the cytotoxicity of Cd in rat primary hepatocyte cultures and that the protective effects of chelating agents presumably result from a decrease in the Cd level, the effective sequestration of the reactive Cd ion, and the direct preventive effect on the active oxygen species in the hepatocytes.

Animals↗

PubMed1997

Molecular phylogenetic characterization of Streptomyces protease inhibitor family.

We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them "SSI-like proteins" (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338-4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised. The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the P1 site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site. Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites. It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins.

Amino Acid Sequence↗

PubMed1997

Mutations of the RAS genes in childhood acute myeloid leukemia, myelodysplastic syndrome and juvenile chronic myelocytic leukemia.

Using the polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, 12 acute myeloid leukemia (AML) cell lines and 108 fresh childhood myeloid tumor specimens, including 67 AML, 29 myelodysplastic syndrome (MDS), and 12 juvenile chronic myelocytic leukemia (JCML) were examined for mutation in H-, K-, and N-RAS genes. The mutation was found in eight of the 120 samples (6.7%), which consisted of four cell lines (33.3%) and four fresh myeloid tumors (3.7%). The frequency of the mutation in the cell lines was apparently higher than that in fresh myeloid tumors. K-RAS gene mutations were found in two of the 67 fresh AML specimens (3%). Interestingly, these two patients had 11q23 translocations. The N-RAS gene mutation was found in one of the 29 specimens (3.4%) of MDS and in one of the 12 specimens (8.3%) of JCML. All mutations were found in codon 12, 13 or 61 of the N-RAS and K-RAS genes. Frequency of mutation of RAS genes in fresh myeloid malignancies was very low. These findings suggest that mutation of RAS genes does not play an important role in the development of childhood myeloid malignancies.

Acute Disease↗

PubMed1997

The B cell epitope of paramyosin recognized by a protective monoclonal IgE antibody to Schistosoma japonicum.

Passive immunization of mice with a monoclonal IgE antibody to Schistosoma japonicum (SJ18 epsilon. 1) induces significant protection to a challenge infection and the target molecule of SJ18 epsilon. 1 is paramyosin. In the present study, we demonstrate the B cell epitope of paramyosin recognized by SJ18 epsilon. 1 by using a series of deletion mutants expressed in Escherichia coli. SJ18 epsilon 1 reacted with the recombinant paramyosin containing 113 amino acids (Glu301. Ala413) but not with a shorter peptide (Glu301-Asp343). Further epitope mapping carried out by a multi-pin system using heptameric peptides synthesized sequentially from 71 amino acids of paramyosin (Asp343-Ala413) demonstrated significant binding of SJ18 epsilon. 1 to the sequence, 359Ile-Arg-Arg-Ala362. Replacement set analysis of the pentameric peptide, 358Leu-Ile-Arg-Arg-Ala362, revealed that replacement of each residue with a hydrophobic or hydrophilic amino acid did not inhibit binding of SJ18 epsilon. 1, whereas replacement of positively charged Arg. or hydrophobic Ala with a negatively charged amino acid, Glu, showed reduction in binding of the antibody. Moreover, replacement of each amino acid including Arg with a positively charged amino acid, Lys, resulted in a drastic loss of the binding, indicating that binding of the antibody was markedly affected by the change of charges of the peptide as well as by the conformational alteration. The target epitope of SJ18 epsilon. 1 was common among paramyosins of S. mansoni, Taenia solium and Echinococcus granulosus but not among nematode paramyosins, suggesting that the epitope is specific for platyhelminthes.

Amino Acid Sequence↗

PubMed1997

Echocardiographic diagnosis of subepicardial aneurysm ruptured into the right ventricle after inferior myocardial infarction.

A subepicardial aneurysm is a rare complication of myocardial infarction that sometimes causes a left to right shunt at the ventricular level. We report on echocardiographic observations in two recent cases of inferior myocardial infarction complicated by rupture of a subepicardial aneurysm into the right ventricle (RV). Two-dimensional and color flow Doppler images in several planes offered diagnostic characteristics of the lesion, including discontinuity of the myocardium at the neck of the aneurysm, a narrow neck relative to the diameter of the aneurysm, and a back and forth flow at the neck of the aneurysm. These techniques also provided new findings, such as a two-chambered structure of the aneurysm and a circular flow pattern within the inner aneurysmal cavity. In addition, color flow Doppler imaging showed multiple shunt flows from the aneurysm to the RV. Echocardiographically diagnosed abnormalities were verified by and successfully treated with surgery in both cases. In conclusion, echocardiography with color flow Doppler imaging may have the potential to diagnose the anatomic and hemodynamic condition of a subepicardial aneurysm.

Aneurysm, Ruptured↗