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Biomedical subjects

K Nishikawa

Publications and source records attributed to K Nishikawa.

At least 181 records · Page 10Linked to original sources
PubMed1997

Apical enrichment of human EGF precursor in Madin-Darby canine kidney cells involves preferential basolateral ectodomain cleavage sensitive to a metalloprotease inhibitor.

EGF precursor (proEGF) is a member of the family of membrane-anchored EGF-like growth factors that bind with high affinity to the epidermal growth factor receptor (EGFR). In contrast to human transforming growth factor-alpha precursor (proTGFalpha), which is sorted basolaterally in Madin-Darby canine kidney (MDCK) cells (Dempsey, P., and R. Coffey, 1994. J. Biol. Chem. 269:16878-16889), we now demonstrate that human proEGF overexpressed in MDCK cells is found predominantly at the apical membrane domain under steady-state conditions. Nascent proEGF (185 kD) is not sorted but is delivered equally to the apical and basolateral membranes, where it is proteolytically cleaved within its ectodomain to release a soluble 170-kD EGF form into the medium. Unlike the fate of TGFalpha in MDCK cells, the soluble 170-kD EGF species accumulates in the medium, does not interact with the EGFR, and is not processed to the mature 6-kD peptide. We show that the rate of ectodomain cleavage of 185-kD proEGF is fourfold greater at the basolateral surface than at the apical surface and is sensitive to a metalloprotease inhibitor, batimastat. Batimastat dramatically inhibited the release of soluble 170-kD EGF into the apical and basal medium by 7 and 60%, respectively, and caused a concordant increase in the expression of 185-kD proEGF at the apical and basolateral cell surfaces of 150 and 280%, respectively. We propose that preferential ectodomain cleavage at the basolateral surface contributes to apical domain localization of 185-kD proEGF in MDCK cells, and this provides a novel mechanism to achieve a polarized distribution of cell surface membrane proteins under steady-state conditions. In addition, differences in disposition of EGF and TGFalpha in polarized epithelial cells offer a new conceptual framework to consider the actions of these polypeptide growth factors.

Animals↗
PubMed1997

Molecular cloning and expression of the Caenorhabditis elegans klp-3, an ortholog of C terminus motor kinesins Kar3 and ncd.

Common to all eukaryotes, kinesins are cytoskeletal motor proteins that mediate intracellular transport on microtubule tracks, using ATP hydrolysis. A Caenorhabditis elegans cDNA clone corresponding to the klp-3 gene, encoding a novel kinesin, was isolated, and mapped on LGII. Northern blot analysis using the klp-3 cDNA probe reveals a 1.9 kb mRNA that is transcribed at a low level during development. Temporal and spatial expression of the klp-3::lacZ fusion gene is limited to the marginal cells in the pharynx, and a group of muscle cells in the posterior gut region. The nucleotide sequence of klp-3 has been deduced from the cDNA and nematode genome sequencing consortium data. Conceptual translation of the klp-3 gene reveals a kinesin-like protein with its conserved motor domain containing the ATP binding and microtubule binding sites located in the C terminus. KLP-3 shares extensive homology with the yeast Kar3 and Drosophila ncd kinesins, which have previously been shown to mediate chromosomal movement and segregation during meiosis and mitosis. Overexpression of the klp-3 gene partially rescues the lethal phenotype of the maternal lethal him-14 ts(it44) mutants at non-permissive temperatures, and reduces the incidence of males caused by non-disjunction of the X-chromosome. Similarly, expression of a klp-3 antisense RNA, under the control of a heat shock promoter, causes embryonic arrest, dead eggs and polyploid cells in transgenic lines, suggesting a critical role for the klp-3 function in chromosome segregation. Further analysis of the klp-3 gene in C. elegans may elucidate diverse functions of the C terminus mitotic motor proteins during development.

Amino Acid Sequence↗
PubMed1997

Differences in dinucleotide frequencies of human, yeast, and Escherichia coli genes.

Nucleotide sequences coding proteins in human, yeast and Escherichia coli genes were analyzed in terms of dinucleotide occurrences. Every gene is plotted as a point in the dinucleotide space, which is spanned by 16 axes corresponding to the 16 components of the dinucleotide. The metric unit in the space is defined using the log-odds ratio of dinucleotide occurrences in a gene. The distribution of points showed that genes from the same organism are clustered in the space. The clusters of human and E. coli are completely separated, and the yeast cluster sits between, implying that individual genes are classified into the three sources from their location. In fact, they could be identified with accuracy of 90%, using the DNA data alone. Even genes encoding homologous proteins belonging to the same protein superfamily were discriminated by the DNA data, and were correctly identified into their sources with the same accuracy as above. DNA sequences of non-coding regions, including human introns, as well as human genes of GC-rich and GC-poor types, were also analyzed in the same manner. The most significant finding is that human genomic DNA sequences, including genes and introns together, exhibit the largest deviation of dinucleotide occurrence from the random expectation. Possible origins for this phenomenon are discussed.

Dinucleotide Repeats↗
PubMed1997

Role of a sialyl Lewis(x)-like epitope selectively expressed on vascular endothelial cells in local skin inflammation of the rat.

The role of the inducible L-selectin ligand was studied in complement-dependent acute dermatitis in rats. Although mAbs against typical sialyl Lewis(x) (CSLEX-1 and SNH-3) did not react with skin venules, a sialyl Lewis(x)-like epitope defined by mAb 2H5 (2H5-Ag) was de novo expressed on the endothelial cells of skin venules in the area of inflammation. Expression of 2H5-Ag increased concomitantly with the progression of inflammation. 2H5-Ag was identified at the 75-, 150-, and 180-kDa bands when inflammatory skin tissue was analyzed by Western blotting. In contrast, P- and E-selectins were not detectable. The role of 2H5-Ag in this model was studied in in vitro and in vivo methods. First, 2H5 was i.v. injected 15 min before induction of dermatitis. 2H5 bound to skin venules and significantly reduced the neutrophil infiltration and plasma protein leakage. In contrast, CSLEX-1, mAb ARP2-4 (P-selectin blocker), or mAb ARE-5 (E-selectin blocker) had no effects. Second, adhesion of isolated rat neutrophils to the inflammatory skin section was inhibited significantly when the sections, but not neutrophils, were preincubated with 2H5. Third, fluorescein-labeled normal rat neutrophils were injected into a rat 10 h after induction of dermatitis. The number of labeled neutrophils infiltrated into the inflammatory site was reduced significantly when they were preincubated with HRL-3 (blocking mAb against rat L-selectin), but not with 2H5 or HRL-4 (nonblocking mAb against rat L-selectin). These data show that de novo expressed 2H5-Ag/L-selectin adhesion pathway contributes to the development of acute complement-dependent inflammation in the skin.

Animals↗
PubMed1997

Only one nucleotide insertion to the long variable arm confers an efficient serine acceptor activity upon Saccharomyces cerevisiae tRNA(Leu) in vitro.

Several tRNA species have a long variable arm composed of over ten nucleotides, which are relevant to those specific to serine, leucine and tyrosine in prokaryotes, while there are only serine and leucine-specific tRNAs in eukaryotes. To clarify the evolutionary aspects of the identity determination mechanism of these tRNAs, the tRNA(Ser) recognition in Saccharomyces cerevisiae was studied. Unmodified tRNA(Leu) transcript had serylation ability of low efficiency, but native tRNA(Leu) did not, indicating that some modification of tRNA(Leu) serves as a negative identity determinant for seryl-tRNA synthetase. Changing the discriminator base did not seriously affect the serine accepting efficiency. The tRNA(Leu) transcript possessing the variable arm of tRNA(Ser) was efficiently aminoacylated with serine. Eventually, it was found that only one nucleotide insertion to the variable arm of tRNA(Leu) was sufficient to confer an efficient serine accepting activity. The mode of serine tRNA recognition is similar to that in Escherichia coli in that the end of the long variable arm, but not the anticodon or discriminator base, is important. However, S. cerevisiae seryl-tRNA synthetase adopts a substantially different mechanism for rejection of tRNA(Leu) from that of its E. coli counterpart.

Base Sequence↗
PubMed1997

Tertiary structural models for human interleukin-6 and evaluation by a sequence-structure compatibility method and NMR experimental information.

Tertiary structure models of Interleukin-6 were constructed using a routine prediction method based on the X-ray crystal structures of granulocyte colony-stimulating factor (GCSF) and leukemia inhibitory factor (LIF). The models were evaluated with the aid of the sequence-structure compatibility (3D-1D) method program compass and NMR experimental information. The model constructed from GCSF gained higher scores on compass examination than did that from LIF, and the NOE data [Nishimura et al. (1996) Biochemistry 35, 273-281] also turned to be more consistent with the former model.

Amino Acid Sequence↗
PubMed1997

Functional expression on human trophoblasts of interleukin 4 and interleukin 7 receptor complexes with a common gamma chain.

The gamma chain in the high-affinity receptor complex for interleukin 2(IL-2) is used in receptor complexes for IL-4 and IL-7. We examined expression and function of the gamma chain in trophoblast cells. Flow cytometric studies demonstrated that the human placental cell line and choriocarcinoma cell lines expressed the IL-2R gamma chain. None of these cell lines expressed the IL-2R alpha and beta chains, whereas the alpha chains of IL-4R and IL-7R were present. The gamma chain and IL-4R and IL-7R mRNA were detected in human placental trophoblasts by the in situ hybridization method. The release of human chorionic gonadotropin by trophoblasts was accelerated by IL-4 and IL-7 in a concentration-dependent manner. These findings indicate that IL-4 and IL-7 are involved in trophoblast function.

Antigens, CD↗
PubMed1997

Candesartan (CV-11974) dissociates slowly from the angiotensin AT1 receptor.

The mechanisms of the insurmountable antagonism of 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]1H-benzimid azole -7-carboxylic acid, candesartan (CV-11974), an angiotensin AT1 receptor antagonist, on angiotensin II-induced rabbit aortic contraction were examined in contraction and binding studies. Preincubation of the rabbit aorta with CV-11974 (0.1 nM) for 30 min reduced the maximal contractile response to angiostensin II by approximately 50%. This insurmountable antagonism of CV-11974 was reversed in the presence of losartan (1 microM), a surmountable angiotensin AT1 receptor antagonist. The inhibitory effect of CV-11974 on angiotensin II-induced contraction persisted longer after washing than did that of losartan but was irreversible. Scatchard analysis of [3H]CV-11974 binding in bovine adrenal cortical membranes indicated the existence of a single class of binding sites (Kd = 7.4 nM). Competition binding studies using angiotensin II receptor agonists and antagonists have demonstrated that [3H[CV-11974 binding sites may be identical to angiotensin AT1 receptors. The dissociation rate of [3H]CV-11974 binding (t1/2 = 66 min) was 5 times slower than that of [125I]angiotensin II binding (t1/2 = 12 min). These results suggest that the insurmountable antagonism by CV-11974 is due to its slow dissociation from angiotensin AT1 receptors.

Adrenal Cortex↗
PubMed1997

Determination of the specific substrate sequence motifs of protein kinase C isozymes.

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (alpha, betaI, betaII, gamma, delta, epsilon, eta, mu, and zeta) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position -3. All isozymes, except PKC mu, selected peptides with basic amino acids at positions -6, -4, and -2. PKC alpha, -betaI, -betaII, -gamma, and -eta selected peptides with basic amino acid at positions +2, +3, and +4, but PKC delta, -epsilon, -zeta, and -mu preferred peptides with hydrophobic amino acid at these positions. At position -5, the selectivity was quite different among the various isozymes; PKC alpha, -gamma, and -delta selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKC mu showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position -3 to +2 for PKC alpha, -betaI, -betaII, -gamma, -delta, and -eta were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKC alpha, -betaI,-delta, -zeta, and -mu were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.

Amino Acid Sequence↗
PubMed1997

Heat stress induces a glycosylation of membrane sterol in myxoamoebae of a true slime mold, Physarum polycephalum.

To know the very early events occurring after heat shock, the changes of membrane lipids were examined. Heat stress induced the production of a certain glycolipid in the myxoamoebae of Physarum polycephalum in a few minutes. The purified glycolipid was determined to be a poriferasterol monoglucoside by structural studies that was previously reported to be expressed during the differentiation of Physarum cells from haploid myxoamoebae to diploid plasmodia (Murakami-Murofushi, K., Nakamura, K., Ohta, J., Suzuki, M., Suzuki, A., Murofushi, H., and Yokota, T. (1987) J. Biol. Chem. 262, 16719-16723). The activity of UDP-glucose:poriferasterol glucosyltransferase (Murakami-Murofushi, K., and Ohta, J. (1989) Biochim. Biophys. Acta 992, 412-415) was also expressed rapidly after heat shock. Thus, the activation of sterol glucosyltransferase and the production of sterol-glucoside were considered to be important events that were involved in the signal transduction system to induce some succeeding heat-shock responses, such as the synthesis of heat-shock proteins.

Animals↗
PubMed1997

The effects of functional suppression of a membrane-bound complement regulatory protein, CD59, in the synovial tissue in rats.

OBJECTIVE: To investigate the roles of CD59 in the synovial tissue by functional suppression of CD59. METHODS: Rats treated with cobra venom factor to deplete complement or untreated rats were injected intraarticularly with 0.3 mg of the F(ab')2 fraction of a monoclonal antibody, 6D1, that inhibits the function of rat CD59. The circumference of knee joints was measured, and histologic changes in the synovium were studied. RESULTS: Joint swelling, thickening of the synovial tissues, infiltration of inflammatory cells into the synovium, and deposition of membrane attack complex (MAC) on the synovial surface were observed after intraarticular injection of 6D1. The inflammatory reaction reached its peak at 24 hours after injection, and finally subsided to normal within 3 days. It was suggested that functional suppression of CD59 in the synovium induced MAC formation followed by synovitis. Serum complement depletion did not completely suppress this reaction. This indicates that complement existing in the joint space is important for the formation of MAC on the synovial surface and for induction of synovitis. CONCLUSION: The membrane-bound complement regulatory protein, CD59, plays a key role in the protection of joints against MAC-mediated synovial injury and in maintaining the normal integrity of the joint.

Animals↗
PubMed1997

Predicted structure of the extracellular region of ligand-gated ion-channel receptors shows SH2-like and SH3-like domains forming the ligand-binding site.

Fast synaptic neurotransmission is mediated by ligand-gated ion-channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand-binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3 receptors) or anions (GABAA and glycine receptors and the invertebrate glutamate-binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D-3D structure mapping approach, we have modeled the extracellular ligand-binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium-resolution electron microscopy structures and with experimental structure/function data from ligand-binding, antibody-binding, mutagenesis, protein-labeling and subunit-linking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor alpha 1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand-binding domain with the activation of the M2 transmembrane helix is also suggested.

Amino Acid Sequence↗
PubMed1997

Comparison of protein structures using 3D profile alignment.

A novel method for protein structure comparison using 3D profile alignment is presented. The 3D profile is a position-dependent scoring matrix derived from three-dimensional structures and is basically used to estimate sequence-structure compatibility for prediction of protein structure. Our idea is to compare two 3D profiles using a dynamic programming algorithm to obtain optimal alignment and a similarity score between them. When the 3D profile of hemoglobin was compared with each of the profiles in the library, which contained 325 profiles of representative structures, all the profiles of other globins were detected with relatively high scores, and proteins in the same structural class followed the globins. Exhaustive comparison of 3D profiles in the library was also performed to depict protein relatedness in the structure space. Using multidimensional scaling, a planar projection of points in the protein structure space revealed an overall grouping in terms of structural classes, i.e., all-alpha, all-beta, alpha/beta, and alpha+beta. These results differ in implication from those obtained by the conventional structure-structure comparison method. Differences are discussed with respect to the structural divergence of proteins in the course of molecular evolution.

Algorithms↗
PubMed1997

KB-R7785, a novel matrix metalloproteinase inhibitor, exerts its antidiabetic effect by inhibiting tumor necrosis factor-alpha production.

It has been suggested that tumor necrosis factor-alpha (TNF-alpha) is a key mediator of insulin resistance in non-insulin-dependent diabetes mellitus (NIDDM). TNF-alpha is synthesized as a membrane-bound precursor; this is proteolytically processed to an active form by a matrix metalloproteinase (MMP)-like enzyme. In this study, we have used KKAy mice which show insulin resistance like NIDDM to investigate the effects of KB-R7785, a novel MMP inhibitor, on blood glucose and insulin levels. Subcutaneous administration of KB-R7785 at 100 mg/kg twice daily (i.e., 200 mg/kg/day) for 4 weeks resulted in a significant decrease in plasma glucose levels which was observed after 3 weeks. Oral administration of pioglitazone (20 mg/kg twice daily or 40 mg/kg/day for 4 weeks), an agent known to ameliorate insulin sensitivity, significantly decreased plasma glucose levels during the treatment period. KB-R7785, but not pioglitazone, also significantly decreased plasma insulin levels. Lipopolysaccharide (LPS) increased plasma TNF-alpha levels to a significantly greater degree in KKAy mice than in normal C57BL mice; this was inhibitable in KKAy mice by KB-R7785. In contrast, pioglitazone did not affect the LPS-induced increase in plasma TNF-alpha levels in KKAy mice. These results suggest that KB-R7785 exerts its antidiabetic effect by ameliorating insulin sensitivity through the inhibition of TNF-alpha production.

Animals↗
PubMed1997

Clinical application of hepatic venous hemodynamics by Doppler ultrasonography in chronic liver disease.

The phase differences of the hepatic vein (HV) Doppler waveform might be influenced by fibrosis and steatosis of the liver when using HV Doppler and pressure curve analysis. Because HV catheterization is invasive, we have advocated the noninvasive comparative index (QXs ratio) of the HV Doppler waveform phases recorded simultaneously with electrocardiography and phgonocardiography. Forty-three controls and 169 patients with a variety of liver diseases were enrolled in this study. The mean QXs ratio was 0.75 in the controls, 0.6 in the chronic hepatitis patients, 0.45 in the cirrhosis patients, and 0.90 in the fatty liver patients. There were significant differences between all pairs of these groups (p < 0.001). The QXs ratio may be used to assess the degree of hepatic fibrosis or steatosis.

Adult↗
PubMed1997

Phase shift of the hepatic vein flow velocity waveform in chronic liver disease: experimental and clinical studies.

We examined animal models and studied patients with chronic liver disease to evaluate phase shift of the flow-velocity waveform of the hepatic vein (HV). We decided the Doppler-Pressure (DP) ratio using electrocardiography, the HV Doppler waveform and HV pressure curves, and we calculated the DP ratios. In animal models, the mean DP ratio was 1.04 in controls, 0.68 in cirrhosis, and 1.22 in fatty liver. There were significant differences among these groups (p < 0.005). In the clinical study, the mean DP ratio was 1.03 in controls, 0.95 in chronic hepatitis and 0.79 in cirrhosis. The DP ratio in the Cirrhosis Group was significantly different from that in the Control Group (p < 0.001) and that in the Chronic Hepatitis Group (p < 0.05). We conclude that the DP ratio is decreased in certain hepatic disease states.

Adult↗
PubMed1997

Case-control study of leukemia and diagnostic radiation exposure.

A case-control study of leukemia and diagnostic X-ray exposure was conducted by a multi-institution co-operative study group. The subjects were 134 patients with acute myelogenous leukemia, 57 with chronic myelogenous leukemia, 56 with acute lymphocytic leukemia and 50 with myelodysplasia syndrome, who were between 15 and 79 years old, and diagnosed at one of 27 hospitals between September 1993 and August 1995. The controls were 479 first-visit patients seen at eight of these 27 hospitals. History of diagnostic X-ray tests between 1982 and 1991 was determined by an anonymous self-administered questionnaire. The total relative dose of radiation exposure was calculated by summing the products of given weights and frequencies of each test. The relative risk was 0.83 (95% confidence interval (C.I.), 0.58-1.19) for relative dose of 10-30 (equivalent to 4-11 times of UGI series), 0.76 (0.48-1.20) for relative dose of 30 or more (more than 12 times of UGI series), when compared with relative dose of 0-10 (0-3 times of UGI series). Analysis according to type of leukemia revealed that only acute myelogenous leukemia had an estimated relative risk above unity (1.08, 95% C.I. 0.69-1.69, for relative dose 10-30). This study did not support the hypothesis that diagnostic X-ray tests increases leukemia risk.

Adolescent↗