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Biomedical subjects

M Hatanaka

Publications and source records attributed to M Hatanaka.

At least 109 records · Page 6Linked to original sources
PubMed1995

Blocking measles virus infection with a recombinant soluble form of, or monoclonal antibodies against, membrane cofactor protein of complement (CD46).

Human membrane cofactor protein (MCP, CD46) functions as an inhibitor of the complement (C) cascade to protect host cells from C attack, and as a receptor for measles virus (MV). Normal human sera contains 10-60 ng/ml of naturally produced soluble forms of MCP, which is also a cofactor for the factor I-mediated inactivation of C3b. We produced monoclonal antibodies (mAb) against MCP and a recombinant soluble form of MCP similar to the natural soluble forms, and tested their ability to block MV infection. Vero cells and CHO cells expressing human MCP were the targets. Of the antibodies tested, M75 and M177, which blocked the C regulatory activity of MCP, efficiently blocked MV infection. More than 50 micrograms/ml of the soluble form moderately blocked MV infection of CHO cells expressing MCP, but barely blocked that of Vero cells. The two mAb and the soluble form also inhibited MV H protein-mediated green monkey erythrocyte rosette formation. A quantitative analysis suggested that 30 micrograms/ml of the soluble form functionally corresponded to 0.2 microgram/ml of M177 or M75. These data established that the C regulatory function and the MV receptor function of MCP were blocked simultaneously by the individual mAb, and that soluble forms of MCP could inhibit MV infection in cells expressing human MCP, although doses far higher than the natural concentration of soluble MCP were required.

Animals↗
PubMed1994

Tumor necrosis factor alpha-induced phosphorylation of I kappa B alpha is a signal for its degradation but not dissociation from NF-kappa B.

Activation of the NF-kappa B/Rel family of transcription factors is regulated by a cytoplasmic inhibitor, I kappa B alpha. Activity of I kappa B alpha is in turn modulated by phosphorylation and proteolysis. It has been postulated that phosphorylation of I kappa B alpha leads to its dissociation from NF-kappa B, and free I kappa B alpha is targeted for rapid degradation. However, this phosphorylation-mediated dissociation event has not been demonstrated in vivo. We demonstrate that, contrary to this hypothesis, phosphorylation of I kappa B alpha induced by tumor necrosis factor alpha in HeLa cells does not induce dissociation. We propose a model in which (i) induced phosphorylation of I kappa B alpha does not result in its dissociation from NF-kappa B, (ii) phosphorylation of I kappa B alpha serves as a signal for degradation, and (iii) degradation of I kappa B alpha occurs while it is still complexed with NF-kappa B.

Calpain↗
PubMed1994

Unphosphorylated and tyrosine-phosphorylated forms of a focal adhesion protein, paxillin, are substrates for calpain II in vitro: implications for the possible involvement of calpain II in mitosis-specific degradation of paxillin.

Cell-to-substratum adhesion becomes weakened during mitosis of the cell cycle in fibroblasts. The level of one focal adhesion protein, paxillin, is greatly reduced in mitotic-arrested cells. We show here the possible involvement of calpain II, known to be localized in focal adhesion plaques, in the degradation of paxillin. Paxillin is tyrosine-phosphorylated during interphase of the cell cycle by protein tyrosine kinases (PTK) such as c-Src and Csk, and becomes dephosphorylated during mitosis. Our data, however, indicate that tyrosine phosphorylation of paxillin does not affect the rate of paxillin degradation by calpain in vitro.

3T3 Cells↗
PubMed1994

The functions of the ninth component of human complement are sustained by disulfide bonds with different susceptibilities to reduction.

Purified C9 with expected hemolytic and polymerizing activities was found to contain approximately 0.2 mol of sulfhydryl groups/mol of C9. By proteolysis of C9 with labeled SH groups, the SH residues on intact C9 were mapped to Cys-359 and Cys-384 which, presumably, form an intra-domain disulfide bond in the intact molecule. The blocking of these sulfhydryl residues by alkylation, however, had minimal influence on the functions of C9. On the other hand, reduction of C9 by 1 mM dithiothreitol (DTT) (6-fold molar excess over Cys residues) followed by alkylation resulted in a complete block of polymerization activity and a 50% loss of C9 hemolytic activity. In contrast, the ability of C9 to bind EAC1-8 remained largely unaffected. The loss of poly-C9 formation activity correlated with the alkylation of approx. 6 liberated sulfhydryl groups. Hemolytic activity was abolished by treatment with > 5 mM DTT which allowed the liberation of approximately 18 sulfhydryl groups. Most of the DTT-susceptible disulfides were within the C9a fragment (an N-terminal peptide derived by thrombin). Thus, three major functions of C9, EAC1-8 binding, polymerization, and hemolytic activity, are sustained by disulfide bond-dependent conformational motifs with different susceptibility to reducing reagents. The maintenance of the N-terminal C9a region is essential for polymerization, but not EAC1-8 binding activity of C9. Taken together, the results of the present study differentiate in molecular terms several of the functional portions of C9, and stress the significance of intra-chain disulfide linkages in maintaining the structural components necessary for the functions of C9.

Animals↗
PubMed1994

Histamine as an activator of cell growth and extracellular matrix reconstruction for human vascular smooth muscle cells.

Atherosclerosis is characterised by unusual growth of vascular smooth muscle cells (VSMCs) in the intima. We examined the effects of histamine on human VSMCs and the VSMC-derived cell line, ISS10. Histamine enhanced phosphoinositide hydrolysis, increased cytoplasmic Ca2+ level and stimulated the transcription of c-fos protooncogene, which resulted in DNA synthesis and the enhancement of proMMP-1 expression. These results indicate that histamine may play some roles in the pathological process of atherosclerosis and raise the possibility that mast cells migrating into the atherosclerotic foci are involved in the process of atherosclerogenesis.

Arteriosclerosis↗
PubMed1994

Amino-terminal conserved region in proteinase inhibitor domain of calpastatin potentiates its calpain inhibitory activity by interacting with calmodulin-like domain of the proteinase.

Calpastatin is a widely distributed endogenous inhibitor protein specifically acting on calpain (Ca(2+)-dependent proteinase) and is known to interact with the calmodulin-like domain (CaMLD) of the proteinase in a Ca(2+)-dependent fashion. The calpastatin molecule consists of four inhibitory domains (domains 1-4) with mutually homologous sequences in three regions designated as A, B, and C. Acidic amphiphilic alpha-helical motifs are found in both regions A and C. We investigated the correlation between the calpain inhibition potency and the ability of calpastatin to bind to recombinant CaMLD of the mu-calpain large subunit using various mutant proteins of pig calpastatin domain 1 expressed in Escherichia coli. Substitution of conserved Leu-161 with Pro in region A caused a reduction in activity of both calpain inhibition and CaMLD binding. Additional substitution of Leu-236 with Pro in region C further decreased the calpain inhibitory activity and caused a loss of CaMLD binding ability. The effects of mutation in region C alone on the above activities were smaller than those in region A. Although a mutant of deletion in the entire region B had no calpain inhibitory activity, it retained the CaMLD binding ability. On the other hand, although a region B oligopeptide had a moderate inhibitory activity, it had no CaMLD binding ability. These results suggest that region A has a role in potentiating the inhibitory activity of calpastatin by interacting with CaMLD of calpain to form a tighter complex where region B exerts the inhibitory function.

Amino Acid Sequence↗
PubMed1994

Real-time-analysis of the calcium-dependent interaction between calmodulin and a synthetic oligopeptide of calcineurin by a surface plasmon resonance biosensor.

The calcium-dependent interaction between calmodulin (CaM) and the synthetic oligopeptide of a predicted CaM-binding region of human calcineurin A-2 was analysed with an automated surface plasmon resonance biosensor, BIAcore. The oligopeptide was immobilized to a biosensor chip via the amino-terminal cysteine residue by a thiol-disulphide exchange method. The biosensor chip was regenerated by an EGTA-containing buffer after each analysis. Kinetics experiments showed that CaM bound with a high affinity to the oligopeptide in a Ca(2+)-dependent manner. The estimated rate constants of association (kass) and dissociation (kdiss) were 2.3 x 10(5) M-1.s-1 and 3.9 x 10(-3)s-1, respectively. The ratio of kdiss/kass, 1.7 x 10(-8) M, was in good agreement with the dissociation constant (Kd) of 2.4 x 10(-8) M determined from the equilibrium phase.

Amino Acid Sequence↗
PubMed1994

Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and hepatocellular carcinoma cells.

Mutations of the p53 gene are found in various human cancers. The frequency of its mutation is reported to increase during tumor progression in most tumors. In human gliomas, mutations of the p53 gene are found in about one-third of the malignant forms and in few of the benign ones, indicating their possible involvement in tumor progression. On the other hand, we have recently shown that basic fibroblast growth factor (basic FGF) plays a crucial role in tumor progression as an autocrine growth factor in tissues of human gliomas. Therefore, we hypothesized that p53 might regulate the promoter activity of the basic FGF gene, which has several GC boxes and no typical TATA box. In this study, cotransfection assays using human glioblastoma and hepatocellular carcinoma cells and establishment of stable cell lines expressing mutant-type p53 were performed. The basic FGF gene promoter was demonstrated to be regulated by p53 at the transcriptional level and its basal core promoter was found to be responsive to p53. Expression of endogenous basic FGF was also demonstrated to be activated by mutant type p53. Wild-type p53 repressed gene expression of the basic FGF and its mutant activated it in vitro, implying one of the possible pathways in tumor progression.

Carcinoma, Hepatocellular↗
PubMed1994

Analysis of calcium-dependent interaction between amino-terminal conserved region of calpastatin functional domain and calmodulin-like domain of mu-calpain large subunit.

Calpain requires Ca2+ both for proteolysis of its substrates and for interaction with its endogenous inhibitor, calpastatin. Although calmodulin-like domains (CaMLDs) of large and small subunits of calpain have been suggested to be the sites for Ca(2+)-dependent interaction with calpastatin, specificity and molecular basis of the interaction have remained unclear. We investigated the interaction between the CaMLD of human mu-calpain large subunit expressed in Escherichia coli and a 19-residue synthetic oligopeptide corresponding to the region A (the amino-terminal conserved acidic region) of one of the four repetitive functional domains of calpastatin. The recombinant CaMLD bound to the oligopeptide immobilized on Sepharose beads in a Ca(2+)-dependent manner. The CaMLD failed in binding to a mutant oligopeptide with one amino acid substitution. Enhancement of fluorescence intensity of a hydrophobic probe, 2-(p-toluidino)naphthalene-6-sulfonate, was observed upon incubating with the CaMLD and further increased by Ca2+. The Ca(2+)-dependent enhancement of fluorescence intensity was strongly suppressed by the wild type oligopeptide, but not by the mutant one. Kinetic experiments were performed with BIAcore where binding of the CaMLD to the oligopeptide immobilized on a biosensor chip was detected as real time signals of surface plasmon resonance. The determined dissociation constant (KD) was 3.1 x 10(-9) M. These results suggest that the region A of calpastatin binds to the CaMLD in a specific manner similar to interactions between calmodulin-binding peptides and calmodulin where hydrophobic properties are known to be important.

Amino Acid Sequence↗
PubMed1994

Immunohistochemical study for basic fibroblast growth factor and fibroblast growth factor receptor I in pituitary adenomas.

Immunohistochemistry and RT-PCR of basic fibroblast growth factor (bFGF) and one of its receptors (FGFR-I) were performed in pituitary adenomas. Sixty percent of pituitary adenomas showed strong or moderate immunoreactivity to bFGF. The immunoreactivity for FGFR-I in tumor tissues showed positive correlation to that for bFGF (X2 = 6.176, P = 0.0456). Basic FGF-positive cells consisted of pituitary adenoma cells as well as folliculostellate cells and, their distribution was heterogeneous. Expressions of bFGF and FGFR-I were not related to cell proliferation of pituitary adenomas or hormones produced, suggesting that bFGF plays some role other than progression of pituitary adenomas.

Adenoma↗
PubMed1994

The origin of human immunodeficiency virus type-1 rev gene. An evolutionary hypothesis.

The Rev protein of human immunodeficiency virus type-1 is an RNA-binding posttranscriptional transregulator encoded by an accessory gene that is distinct from retroviral oncogenes and whose origin is unclear. We hypothesize that the rev gene was generated by duplication of a viral RNA segment having a secondary-structure that evolved into the Rev-responsive element (RRE). This hypothesis is based on the following findings. First, accumulated data on functional mapping of Rev, Tat, and the transmembrane protein of Env suggested that the major coding exon of rev should have been inserted into the transmembrane region of env during the course of its evolution. Experiments with equine infectious anemia virus, another complex retrovirus, also indicate that a large portion of rev is located within the dispensable transmembrane region of env. Second, base usage analysis suggests the same origin for rev and RRE. Our hypothesis may provide a new insight into the evolutionary aspect of RNA-binding transactivators.

Base Sequence↗
PubMed1994

Identification and characterization of membrane cofactor protein (CD46) in the human kidneys.

Membrane cofactor protein (MCP, CD46) is an integral protein that serves as a cofactor for factor I in inactivating C3b/C4b deposited on the same cell membrane as C3bi/C4c+C4d. This C3b/C4b inactivation is closely associated with self-protection of host cells from autologous complement attack. We have studied the distribution and properties of MCP in the normal human kidney by immunohistochemical and immunoblotting methods using monoclonal antibodies against MCP. MCP was predominantly expressed on the juxtaglomerular apparatus. Glomerular capillary walls, mesangial areas, and tubulus were also MCP positive. Glomerulus MCP was composed of two major bands of 45-65 kDa, which were similar to those of lymphocyte MCP. The proportion of the high and low molecular weight components in glomerulus MCP, however, was considerably different from that of lymphocyte MCP among the individual samples tested. Glomerular epithelial cells and mesangial cells from an individual having equal amounts of high and low molecular weight components in the lymphocytes were cultured separately and the properties of their MCP investigated. MCP in the mesangial cells and glomerular epithelial cells showed profiles in which the upper band was predominant. The results may explain the unique distribution of the high and low molecular weight forms in the glomerulus. These forms of MCP together with factor I were all capable of inactivating C3b to C3bi. Message analysis suggested that glomerular epithelial cells and mesangial cells synthesized a single species of mRNA of 4.2 kb from which the polymorphic MCP species were generated. Flow cytometric analysis suggested that MCP was minimal in mesangial cells. These results, taken together with the previous reports on the distribution of other complement regulatory proteins, infer that the distribution profile of MCP is rather similar to that of DAF but differs from those of CD59 and CR1 in the normal human kidney; this may reflect the differences between their roles or functional properties in renal tissue.

Antigens, CD↗
PubMed1994

Suppression of the transformed phenotype in hybrids of human T-cell leukemia virus type I tax-transformed rat fibroblasts and normal human fibroblasts.

We have isolated and characterized hybrid cell lines derived from Rat-2 cells transformed by the human T-cell leukemia virus type-I (HTLV-I) Tax protein fused with WI-38 normal human fibroblasts. These hybrid cells (designated as RTW cells) showed contact inhibition after growing to confluency, loss of anchorage-independent growth in 0.33% soft agar, and inability to form tumors in athymic mice. Assays of transcription from the HTLV-I long terminal repeat (LTR) demonstrated that Tax-mediated trans-activation of its own promoter was fully maintained in RTW cells, implying that functional Tax is expressed in these cells. Our results indicate that WI-38 cells contain a suppressor gene(s) which can block Tax-mediated cell transformation without interfering its trans-activation of the HTLV-I LTR.

Animals↗
PubMed1994

Spontaneous non-traumatic rupture of the thoracic aorta.

Spontaneous non-traumatic rupture of the aorta is a very rare condition that requires immediate surgery. However, correct preoperative diagnosis is difficult or impossible to establish. We report a case of spontaneous aortic rupture which was successfully treated by emergency surgical exploration following a transesophageal echographic diagnosis of hemopericardium. This case, as well as other reports, suggests that hemopericardium along with symptoms which suggest aortic dissection or rupture (e.g., acute chest or back pain) should raise a strong suspicion of spontaneous aortic rupture even in the absence of intimal flap or aortic dilatation, and the emergency surgery may be life-saving.

Aorta, Thoracic↗
PubMed1994

Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.

Amino Acid Sequence↗
PubMed1994

The human T-cell leukemia virus type I Tax protein induces apoptosis which is blocked by the Bcl-2 protein.

The Tax protein of human T-cell leukemia virus type I activates transcription of cellular and viral genes and can immortalize primary T lymphocytes. We have previously reported that the Tax protein transforms Rat-1 cells. Here we show that Tax-transformed Rat-1 cells detach from plates to undergo apoptotic cell death by serum deprivation. These cells exhibit DNA fragmentation into oligonucleosomal fragments and chromatin condensation. Constitutive expression of a proto-oncogene, bcl-2, effectively blocks Tax-mediated apoptosis caused by serum deprivation without affecting the levels of Tax expression and the transformed phenotype of the cells.

Animals↗
PubMed1994

Expression of fibroblast growth factor receptor-1 in human glioma and meningioma tissues.

We examined the expression of fibroblast growth factor receptor-1 (FGFR-1), namely FLG, in tissues of 18 human gliomas, 10 human meningiomas, 3 human metastatic brain tumors, and 2 normal human brains by means of immunohistochemistry. All tissues were positively stained for FGFR-1. Primary brain tumors were more abundantly immunoreactive than normal brain tissues (Mann-Whitney U test, P < 0.05). There was significant correlation between the expression level of basic fibroblast growth factor (basic FGF) and that of FGFR-1 in tissues of human glioma (Spearman's test, P < 0.05). The expression level of FGFR-1 of tumor cells increased in correlation with that of endothelial cells in glioma tissues (Spearman's test, P < 0.001). We previously reported that basic FGF is produced in more than 90% of human glioma and meningioma tissues. Together with these data, it is suggested that basic FGF is involved in autonomous cell growth and tumorigenesis of gliomas and meningiomas as an autocrine growth factor in vivo.

Adenocarcinoma↗