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Biomedical subjects

K Arai

Publications and source records attributed to K Arai.

At least 487 records · Page 27Linked to original sources
PubMed1993

Purification and characterization of lysosomal H(+)-ATPase. An anion-sensitive v-type H(+)-ATPase from rat liver lysosomes.

Lysosomal H(+)-ATPase was purified to homogeneity from rat liver lysosomes. It is a bafilomycin A1-sensitive Mg(2+)-ATPase, which reacts with antibodies against the 16- and 70-kDa subunits of vacuolar H(+)-ATPase (Nezu, J., Motojima, K., Tamura, H., and Ohkuma, S. (1992) J. Biochem. (Tokyo) 112, 212-219), and has been separated from both the N-ethylmaleimide (NEM)-sensitive/bafilomycin A1-insensitive Mg(2+)-ATPase (ATPase I) and the NEM-insensitive Mg2+/Ca(2+)-ATPase (ATPase II) (Hayashi, H., Arai, K., Sato, O., Shimaya, A., Sai, Y., and Ohkuma, S. (1992) Chem. Pharm. Bull. 40, 2783-2786). The purified enzyme had the subunit structure of vacuolar H(+)-ATPase, consisting of 110-, 70-, 56-, 42-, 39-, 34-, (32-,) and 16-kDa proteins. It had optimal activity at a pH of 7.0-8.0, with an apparently single Km value for ATP of 95 microM. It hydrolyzed ATP > or = dATP >> GTP, ITP >> UTP, but not CTP, and was inhibited by ADP. It demonstrated divalent cation specificity in the order of Mg2+, Mn2+ > Fe2+, Co2+ > Ca2+. Among various anions, Cl-, Br-, and F- activated ATPase activity, whereas NO3- inhibited activity. It was inhibited not only by bafilomycin A1 but also by NEM, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin, and dicyclohexylcarbodi-imide. The purified enzyme was incorporated into proteoliposomes where its proton pump activity was reconstituted. This suggested that the isolated enzyme maintains its H+ translocation activity. These findings suggest that the isolated enzyme is an anion-sensitive vacuolar type H(+)-ATPase that functions as a lysosomal proton pump.

Animals↗
PubMed1993

Mutations on 170Glu, a substrate recognition residue in mouse cAMP-dependent protein kinase, generate enzymes with altered substrate affinity and biological functions.

Site-directed mutations in the catalytic subunit of mouse cAMP-dependent protein kinase (PKAcat) were generated to assess the residue(s) important for the recognition of the substrate peptide and its biological functions. Since the region, 165R-166D-167L-168K-169P-170E-171 N of PKAcat has been shown to be located near the substrate analogue inhibitor peptide binding site (Knighton et al. (1991) Science 253, 414-420), we initially constructed three PKAcat mutants, D166A, K168A, and E170A, in which 166D, 168K, and 170E, respectively, were altered to alanine. When expressed in COS7 cells, D166A and K168A were insoluble, whereas E170A was soluble but had lower in-vitro kinase activity than the wild-type PKAcat. E170A and other 170E mutants, E170Q, E170V, E170R and E170D were equally soluble and displayed various catalytic activities with increased Km and decreased Vmax with regard to Kemptide substrate. Most prominently, E170R did not phosphorylate Kemptide, suggesting that 170E is important for the interaction with Kemptide. The in-vivo activities of the PKAcat mutants were examined in two independent biological assays. First, in Jurkat cells, overexpression of all the 170E mutants except E170R activated the c-fos promoter at various levels lower than the wild-type PKAcat, suggesting that these mutants retain at least partial biological activity. Second, progesterone-induced germinal vesicle break-down in Xenopus oocytes, inhibited by expression of wild-type PKAcat, was inhibited to a similar extent by all the 170E mutants except E170R. All these results support the idea that 170E is a peptide-recognition residue.

Amino Acid Sequence↗
PubMed1993

c-fps/fes protein-tyrosine kinase is implicated in a signaling pathway triggered by granulocyte-macrophage colony-stimulating factor and interleukin-3.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) are hematopoietic growth factors which stimulate the proliferation and differentiation of myeloid progenitor cells. There is a considerable degree of overlap in target cell specificity and the functional effects of GM-CSF and IL-3. GM-CSF and IL-3 induce a nearly identical pattern of protein-tyrosine phosphorylation in certain cell lines, although their receptors have no kinase domains. Furthermore, their receptor complexes share one subunit (designated as beta). These observations raise the possibility that GM-CSF and IL-3 have a common signaling pathway. Here we show that both GM-CSF and IL-3 induce tyrosine phosphorylation and kinase activity of the c-fps/fes proto-oncogene product (p92c-fes), a non-receptor protein-tyrosine kinase, in a human erythro-leukemia cell line, TF-1, which requires GM-CSF or IL-3 for growth. In addition, GM-CSF induces physical association between p92c-fes and the beta chain of the GM-CSF receptor. p92c-fes is therefore a possible signal transducer of several hematopoietic growth factors including GM-CSF and IL-3 through the common beta chain.

Amino Acid Sequence↗
PubMed1993

Signal transduction by the high-affinity GM-CSF receptor: two distinct cytoplasmic regions of the common beta subunit responsible for different signaling.

The high-affinity receptors for granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and IL-5 consist of two subunits, alpha and beta. The alpha subunits are specific to each cytokine and the same beta subunit (beta c) is shared by these three receptors. Although none of these receptor subunits has intrinsic kinase activity, these cytokines induce protein tyrosine phosphorylation, activation of Ras, Raf-1 and MAP kinase, and transcriptional activation of nuclear proto-oncogenes such as c-myc, c-fos and c-jun. In this paper, we describe a detailed analysis of the signaling potential of the beta c subunit by using a series of cytoplasmic deletion mutants. The human beta c consists of 881 amino acid residues. A C-terminal deletion mutant of beta c at amino acid 763 (beta 763) induced phosphorylation of Shc and activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, whereas a deletion at amino acid 626 (beta 626) induced none of these effects. The beta 763 mutant, as well as the full-length beta c, induced transcription of c-myc, c-fos and c-jun. Deletions at amino acid 517 (beta 517) and 626 (beta 626) induced c-myc and pim-1, but no induction of c-fos and c-jun was observed. GM-CSF increased phosphatidylinositol 3 kinase (PI3-K) activity in anti-phosphotyrosine immunoprecipitates from cells expressing beta 763 as well as beta c, whereas it was only marginally increased from cells expressing beta 517 or beta 626. Thus, there are at least two distinct regions within the cytoplasmic domain of beta c that are responsible for different signals, i.e. a membrane proximal region of approximately 60 amino acid residues upstream of Glu517 is essential for induction of c-myc and pim-1, and a distal region of approximately 140 amino acid residues (between Leu626 and Ser763) is required for activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, as well as induction of c-fos and c-jun.

Amino Acid Sequence↗
PubMed1993

An endoplasmic reticulum protein, calreticulin, is transported into the acrosome of rat sperm.

Recently, we purified a Ca(2+)-binding protein from rat spermatogenic cells [Biochem. Biophys. Res. Commun. 176, 135-1364, 1991]. In the present study, this protein was identified as calreticulin, which is a resident protein of the endoplasmic reticulum (ER). Immunohistochemical studies revealed that calreticulin was present in the acrosome of both round spermatids and mature sperm. However, under immunoelectron microscopy, gold-particles were seen over other subcellular structures of spermatocytes, spermatids, and Sertoli cells. When the labeling density in subcellular structures of spermatids was analyzed, the acrosome was found to be most heavily labeled and the Golgi apparatus was second. The complete amino acid sequence of calreticulin, deduced from the cDNA sequence, shares a high degree of identity with that of the analogous mouse protein. The cDNA encoded a protein of 416 amino acids, including a 17-residue NH2-terminal signal sequence. The mature protein contains a KDEL sequence as an ER signal at the COOH terminus. Sperm calreticulin contained no glycosyl moiety. Northern blot analysis of RNAs from purified populations of rat spermatogenic cells indicated that the calreticulin mRNA was present in both pre- and postmeiotic cells. Immunoblot analysis of calreticulin during developmental stages showed that calreticulin was detected in the testis between the ages of 5 and 50 days. Furthermore, purified rat calreticulin contained two Ca(2+)-binding sites, a low affinity/high capacity site and a high affinity/low capacity site. These results suggest that calreticulin, which is not specific to testis, is closely associated with spermatogenesis of rats. This ER protein may be incorporated into the acrosomal vesicle via the Golgi apparatus, without glycosylation, during spermiogenesis, and may play an important role in the regulation of cell functions such as sperm motility and acrosome reaction.

Acrosome↗
PubMed1993

Successful long-term storage of rat limbs. The use of simple immersion in Euro-Collins solution.

The effect of simple cold storage in Euro-Collins solution on prolonging successful preservation time was examined on an established rat limb transplant model. Limbs amputated at mid-thigh level were simply immersed in the solution and stored in a refrigerator at 4 degrees C. After varying periods of cold storage, the limb was orthotopically grafted to another isogeneic rat using microsurgical techniques. The survival rates for 12, 24, 48 and 72 hours' storage were 100%, 90%, 67% and 50% respectively, and the longest time of successful preservation achieved was 78 hours. Survival rates were greatly decreased when amputated limbs were initially perfused with Euro-Collins solution and immersed in the same solution. In a third set of experiments, the limbs were preserved by a conventional method of cold storage in air, and the survival rates were decreased even further. Evaluation by measuring postoperative paw swelling and microscopic examination of the grafts further confirmed the effectiveness of the simple immersion method without perfusion.

Amputation, Surgical↗
PubMed1993

Renal cell carcinoma extending into left side of double inferior vena cava.

A case of renal cell carcinoma extending into the left side of a double inferior vena cava is presented. En bloc excision of the left inferior vena cava and left kidney was performed without complications. Recognition of various venous anomalies, including double inferior vena cava, is important in the evaluation and surgical treatment of retroperitoneal disease.

Aged↗
PubMed1993

Pitfall in clipping of unruptured cerebral aneurysms: narrowing of the parent artery.

Two patients with unruptured cerebral aneurysms are presented, who were complicated by transient ischaemic neurological deficits after seemingly uneventful neck clipping. Postoperative angiography disclosed narrowing of the parent arteries. Arteriosclerotic thickening of the wall of the aneurysmal neck and the parent artery was presumed to be the cause of the narrowing. To minimize the operative morbidity in patients with unruptured aneurysms, the possibility of this phenomenon should always be borne in mind. Neck clipping procedures avoiding this pitfall are discussed.

Aged↗
PubMed1993

Differential regulation of early response genes and cell proliferation through the human granulocyte macrophage colony-stimulating factor receptor: selective activation of the c-fos promoter by genistein.

Granulocyte macrophage colony-stimulating factor (GM-CSF) binds to the high-affinity GM-CSF receptor (GMR) consisting of alpha and beta subunits and induces rapid tyrosine phosphorylation, activation of early response genes, and proliferation of hematopoietic cells. The alpha subunit is the primary cytokine binding component and the beta subunit is required for high-affinity binding as well as for signal transduction. Using tyrosine kinase inhibitors and cytoplasmic deletion mutants of the beta subunit, we obtained evidence that there are at least two distinct pathways downstream of the GMR in BA/F3 cell, one which is essential for proliferation, leads to the c-myc gene activation, and is sensitive to herbimycin and genistein. Activation of this pathway depends on the cytoplasmic region between amino acid positions 455 and 517 of the beta subunit. The second pathway, which leads to activation of c-fos and c-jun genes, is only partially sensitive to herbimycin, is resistant to genistein and depends on the region between amino acid positions 626 and 763 of the beta subunit. Unexpectedly, the c-fos mRNA induction was augmented by genistein. The enhanced expression of c-fos mRNA by genistein also occurred with stimulation with cAMP, PMA, or EGF in NIH3T3 cells. It thus seems likely that genistein affects a common pathway downstream of these signals.

Animals↗
PubMed1993

Reconstitution of the functional granulocyte macrophage colony stimulating factor promoter: evidence for distinct activation mechanisms that mediate the response to phorbol ester/calcium and human T cell leukemia virus type I Tax signals.

Functional elements in the promoter region of the mouse granulocyte-macrophage colony stimulating factor (GM-CSF) gene were assessed by constructing chimeric promoters linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and by employing a transient transfection assay of human T cell leukemia Jurkat cells. We previously reported that CLE2/GC-box (at positions -95 to -73, which is homologous to the NF-kappa B binding site) and CLE0 (at positions to -40) of the mouse GM-CSF promoter are essential for transcriptional activation in response to phorbol-12-myristate-13-acetate (PMA)/calcium ionophore (A23187). Here we show that CLE2/GC-box and the NF-kappa B binding motif are functionally interchangeable and that CLE2/GC-box and CLE0 as a unit activate the basic GM-CSF promoter in response to PMA/calcium signals. This unit is also capable of activating heterologous promoters in response to PMA/calcium signals. In addition, we show that Tax, the trans-activator encoded by human T cell leukemia virus type I (HTLV-I), activates the GM-CSF promoter via CLE2/GC-box without the involvement of CLE0. These results indicate that PMA/A23187-dependent and Tax-dependent activation of the GM-CSF gene proceeds through distinct mechanisms.

Base Sequence↗
PubMed1993

Comparison of haemoconcentration induced by big endothelin-1 and endothelin-1 in mice.

1. The profile of haemoconcentration induced by big endothelin-1(big ET-1), a precursor of endothelin-1 (ET-1), was compared with that induced by endothelin-1 in mice. 2. ET-1(1.5 nmol kg-1, i.v.) increased haematocrit in mice, which reached a maximum at 5 min and then returned to the control value within 30 min after the administration, this occurred at the same time as changes in the plasma immunoreactive endothelin-1 and rat atrial natriuretic peptide (rANP)-like activities (IR-ET-1 and IR-rANP, respectively). 3. Big ET-1(2.5-15 nmol kg-1, i.v.) also caused a significant and dose-dependent increase in haematocrit, that lasted over 3 h although elevated plasma IR-ET-1 and IR-rANP had almost been restored to the initial levels within 10 min after big ET-1 injection. 4. A metalloproteinase inhibitor, phosphoramidon (10 mg kg-1, i.v.), which inhibits the activity of endothelin converting enzyme (ECE), delayed the onset of big ET-1-induced haemoconcentration, but failed to alter the maximal value and the duration of the haemoconcentration. 5. Pretreatment with phosphoramidon (10 mg kg-1, i.v.) did not affect the big ET-1-induced change in plasma IR-ET-1, while significant delay of the disappearance of plasma IR-rANP and significant suppression of a sustained increase in tissue IR-ET-1 were observed. 6. These results suggest that ET-1, not in plasma but in tissue, plays an important role in the pathogenesis of big ET-1-induced long-lasting haemoconcentration, in which unknown factors besides rANP are involved.

Animals↗
PubMed1993

[Methicillin resistance, production of toxic shock syndrome toxin-1 and types of enterotoxin, protease and coagulase at vaginal isolates of Staphylococcus aureus].

The vaginal colonization with Staphylococcus aureus, including strains that have methicillin resistance and production of toxic shock syndrome toxin-1 (TSST-1), enterotoxins and protease was studied in 308 women at the time of delivery. Thirty-six of these women were colonized with S. aureus in the vagina. All of the isolates were methicillin sensitive S. aureus (MSSA), except one women who had both methicillin resistant S. aureus (MRSA) and MSSA. The strains, included MRSA, did not elaborate the TSST-1 and produced protease. The distribution of protease pattern (types) observed by MSSA strains on standard methods caseinate agar plates was type A, 1 strain; type B, 15 strains; type C, 6 strains; type D, 14 strains. Sixteen of the 36 strains produced non of the enterotoxins and 20 strains produced enterotoxins and the type distribution was enterotoxin type A, 6 strains; type B, 10 strains; type C, 1 strain; A and B, 1 strain; A, B and C, 2 strains. These results provide the possibility of pathogenic S. aureus colonized in vagina transmit to neonates at the time of delivery.

Adolescent↗