PubMed Health⌕ Search

Biomedical subjects

K Fukudome

Publications and source records attributed to K Fukudome.

34 records · Page 2Linked to original sources
PubMed1995

Molecular cloning and expression of murine and bovine endothelial cell protein C/activated protein C receptor (EPCR). The structural and functional conservation in human, bovine, and murine EPCR.

Recently, we identified and cloned a human endothelial cell protein C/activated protein C receptor (EPCR). EPCR was predicted to be a type 1 transmembrane glycoprotein and a novel member of the CD1/major histocompatibility complex superfamily with 28% identity with CD1d. Even greater homology (62% identity) was detected with the murine protein, CCD41, which was previously characterized as a centrosome-associated, cell cycle-dependent protein. This raised the possibility that CCD41 was the murine homologue of EPCR. To address this possibility, to better understand structure-function relationships, and to facilitate physiological experiments on EPCR function, we cloned and sequenced murine and bovine EPCR from endothelial cell cDNA libraries. The nucleotide sequence of murine EPCR and CCD41 exhibited five differences corresponding to one base change, three single-base insertions, and one base deletion in the protein coding region. As a result, the predicted structures of EPCR and CCD41 differed in their amino and carboxyl termini but were identical in the central portion of the coding sequence. Based on comparison of the murine, bovine, and human EPCR sequences and the regions where discrepancies between murine EPCR and CCD41 were detected, we believe that CCD41 is probably identical to murine EPCR and that the reported sequence differences are likely the result of compression on the sequencing gel. Compared with human EPCR, the murine and bovine sequences were 69 and 73% identical, respectively, and 57% of the residues were identical between all three species. Both bovine and murine EPCR could bind human activated protein C when the cDNA clones were transfected into 293T cells. Like human EPCR, of the cell lines tested, the murine EPCR message was restricted to endothelium. Cloning of the murine and bovine homologue of EPCR will facilitate in vivo and in vitro studies of the role of EPCR in the protein C pathway.

Amino Acid Sequence↗
PubMed1995

Induction of ICAM-1 and LFA-3 by Tax1 of human T-cell leukemia virus type 1 and mechanism of down-regulation of ICAM-1 or LFA-1 in adult-T-cell-leukemia cell lines.

The present study was undertaken to determine the role of HTLV-I TaxI in the up-regulation of ICAM-I and LFA-3 in human T cells transformed with HTLV-I and the mechanism of down-regulation of ICAM-I and LFA-I in ATL-derived cell lines. Induction of TaxI in a human T-cell line Jurkat carrying the TaxI gene under the metallothionein promoter led to increases in mRNA and surface expression of ICAM-I. The response of LFA-3 to TaxI induction was, on the other hand, relatively slow and weak, and might be indirect. Transactivation of the ICAM-I promoter by TaxI was further shown by co-transfection of a CAT reporter construct with the ICAM-I promoter and a plasmid expressing TaxI. The mechanism of down-regulation of ICAM-I or LFA-I in 4 ATL cell lines was next examined. ICAM-I mRNA was quite low in MT-I, but no genomic changes were found. The CAT reporter with the ICAM-I promoter was inactive in MT-I. Finally, combined treatment of MT-I with 5-azacytidine and IFN-gamma induced re-expression of ICAM-I. Collectively, (a) transcriptional factor(s) necessary for expression of ICAM-I gene may be repressed in MT-I through DNA methylation. Three other ATL cell lines (TL-OmI, H582, HuT102) were found to have little mRNA for the LFA-I beta chain (CD18). H582 and HuT102 were also negative for the LFA-I alpha chain (CDIIa) mRNA. No genomic changes were found, and a CAT reporter gene with the CD18 promoter was inactive in the 3 of them, again suggesting lack of (a) transcriptional factor(s) necessary for CD18 expression.

Antigens, CD↗
PubMed1995

Cellular regulation of the protein C pathway.

The protein C anticoagulant pathway regulates thrombin formation. The pathway is triggered when thrombin binds to the endothelial cell proteoglycan, thrombomodulin. Unlike thrombin, this complex is a potent activator of the protein C zymogen, but it cannot clot blood. Activated protein C binds to protein S on cell surfaces where it proteolytically inactivates coagulation factors Va and VIIIa. Activated protein C also binds to a newly identified endothelial protein C receptor. Congenital deficiencies in this pathway are associated with thrombotic disease, and inflammation can cause acquired deficiencies. Activated protein C appears to inhibit inflammation. Thus, this pathway modulates both coagulation and inflammation.

Animals↗
PubMed1994

Activation of prothrombin by a novel membrane-associated protease. An alternative pathway for thrombin generation independent of the coagulation cascade.

We herein report that a novel membrane-associated protease capable of activating prothrombin is present in several mammalian cells. This protease can directly convert prothrombin to active thrombin and induces blood clotting both in vivo and in vitro but is apparently different from coagulation factor Xa, which has been thought to be the only physiological activator of prothrombin. This protease activity was initially found and was very high in 8C feline kidney fibroblast cells, and we characterized its enzymological features using this cell line. Activity was detected in neither the cytosolic fraction nor the culture medium but found in the membranes and identified on the surface of intact cells. The activation of prothrombin required Ca2+ ions, and the apparent Km value for prothrombin was 0.2 microM. The activity was irreversibly inhibited by exposure to EDTA, but various inhibitors for serine proteases including antithrombin III were without effect. Based on these results, we propose that this novel enzyme, membrane-associated prothrombin activator, catalyzes an alternative pathway for generation of thrombin, which is independent of the blood coagulation cascade, and that the thrombin generated is involved in certain pathological states and/or in activation of cells that are spatially separated from the bloodstream.

Amino Acid Sequence↗
PubMed1994

Identification, cloning, and regulation of a novel endothelial cell protein C/activated protein C receptor.

Human protein C and activated protein C are shown to bind to endothelium specifically, selectively and saturably (Kd = 30 nM, 7000 sites per cell) in a Ca(2+)-dependent fashion. Expression cloning revealed a 1.3-kilobase pair cDNA that coded for a novel type 1 transmembrane glycoprotein capable of binding protein C. This protein appears to be a member of the CD1/major histocompatibility complex superfamily. Like thrombomodulin, the receptor involved in protein C activation, the endothelial cell protein C receptor function and message are both down-regulated by exposure of endothelium to tumor necrosis factor. Identification of endothelial cell protein C receptor as a member of the CD1/major histocompatibility complex superfamily provides insights into the role of protein C in regulating the inflammatory response.

Amino Acid Sequence↗
PubMed1993

Enhanced expression of LFA-3 on human T-cell lines and leukemic cells carrying human T-cell-leukemia virus type 1.

Previously, we studied the surface expression of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in a panel of 16 human T-cell lines and found that those carrying HTLV-1 pro-viruses expressed high levels of ICAM-1. Enhanced expression of ICAM-1 was also seen in fresh leukemic cells from ATL patients. In the present study, we systematically examined the surface expression of 19 different cell-adhesion molecules (CAMs) (the immunoglobulin superfamily, the homing receptors, the integrins, etc.) in a panel of 20 human T-cell lines (6 HTLV-1-negative lines and 14HTLV-1-positive lines). The surface expression of LFA-3 (CD58) was constantly enhanced in HTLV-1-positive T-cell lines except for MT-1, an ATL-derived cell line, which was devoid of a number of CAMs including ICAM-1. LFA-3 proteins and mRNA were found at strongly increased levels in HTLV-1-positive T-cell lines.

Antigens, CD↗
PubMed1993

Proteolytic enhancement of human rotavirus infectivity.

Rotaviruses are the most important etiologic agents of severe diarrhea worldwide. Despite great advances in vaccine development, little is known about host protection mechanisms other than immunity. This presentation focuses on the proteolytic enhancement of rotavirus infection, with emphasis on the functions of VP4, an outer capsid protein. The in vitro growth of human rotavirus is enhanced by trypsin, which selectively cleaves VP4. Treatment with trypsin increases the infectivity of human rotavirus while decreasing its hemagglutination activity. There are two modes of rotavirus internalization: direct penetration with the aid of trypsin and endocytosis without trypsin. Direct penetration via VP4 cleaved by trypsin is essential for the replication of the virus, whereas endocytotic internalization does not give rise to viral replication.

Animals↗
PubMed1992

C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including CD9, CD37, CD53, and CD63.

C33 Ag was originally identified by mAb inhibitory to syncytium formation induced by human T cell leukemia virus type 1. The Ag was shown to be a highly heterogeneous glycoprotein consisting of a 28-kDa protein and N-linked oligosaccharides ranging from 10 to 50 kDa. In the present study, cDNA clones were isolated from a human T cell cDNA expression library in Escherichia coli by using mAb C33. The identity of cDNA was verified by immunostaining and immunoprecipitation of transfected NIH3T3 cells with mAb. The cDNA contained an open reading frame of a 267-amino acid sequence which was a type III integral membrane protein of 29.6 kDa with four putative transmembrane domains and three putative N-glycosylation sites. The C33 gene was found to belong to a newly defined family of genes for membrane proteins, such as CD9, CD37, CD53, CD63, and TAPA-1, and was identical to R2, a cDNA recently isolated because of its strong up-regulation after T cell activation. Availability of mAb for C33 Ag enabled us to define its distribution in human leukocytes. C33 Ag was expressed in CD4+ T cells, CD19+ B cells, CD14+ monocytes, and CD16+ granulocytes. Its expression was low in CD8+ T cells and mostly negative in CD16+ NK cells. PHA stimulation enhanced the expression of C33 Ag in CD4+ T cells by about 5-fold and in CD8+ T cells by about 20-fold. PHA stimulation also induced the dramatic size changes in the N-linked sugars previously shown to accompany human T cell leukemia virus type 1-induced transformation of CD4+ T cells.

Amino Acid Sequence↗
PubMed1992

Strong induction of ICAM-1 in human T cells transformed by human T-cell-leukemia virus type 1 and depression of ICAM-1 or LFA-1 in adult T-cell-leukemia-derived cell lines.

Sixteen human T-cell lines were studied for the expression of a cell-adhesion molecule ICAM-1 and its counter-receptor LFA-1. The cell lines included 3 human T-cell-leukemia-virus-type-I (HTLV-1)-negative cell lines derived from acute lymphoblastic leukemia (ALL) and 13 HTLV-1-positive cell lines, 7 of them established from cord- or peripheral-blood T cells by in vitro transformation with HTLV-1, 2 derived from HTLV-1 carriers, and 4 derived from patients with adult T-cell leukemia (ATL). In sharp contrast to a basal level of ICAM-1 in 3 HTLV-1-negative ALL cell lines, strong induction of ICAM-1 was seen in all HTLV-1-positive T-cell lines except for MT-1, one of the 4 ATL cell lines used in the present study. On the other hand, the expression of LFA-1 (CD11a and CD18) was more or less similar among the cell lines with and without HTLV-1. Interestingly, however, 3 out of 4 ATL cell lines (TL-Om1, H582, HUT102) revealed striking depression of LFA-1 expression. Several lines of evidence strongly argued against direct involvement of the viral transactivator p40tax or some autocrine cytokines in the induction of ICAM-1 in HTLV-1-positive T-cell lines. It was also found that ICAM-1 and LFA-1 were involved in syncytium formation induced in the co-culture of HTLV-1-positive and HTLV-1-negative human T-cell lines. Implications of constitutive expression of ICAM-1 for certain clinical manifestations of ATL and of depression of either ICAM-1 or LFA-1 during progression of ATL are discussed.

Animals↗
PubMed1992

Induction of strong homotypic adhesion in human T cell lines positive with human T-cell leukemia virus type 1 by monoclonal antibodies to MHC class I and beta 2-microglobulin.

To identify the cellular receptors and other cell surface molecules playing essential roles in the transmission of human T-cell leukemia virus type 1 (HTLV-1), we have been isolating monoclonal antibodies (mAbs) that are capable of inhibiting HTLV-1-induced syncytium formation. In the present study, we isolated two mAbs, H11 (IgM) and H14 (IgG1), inhibitory to syncytium formation in the coculture of TOM-1 or C91/PL (both HTLV-1-positive human T-cell lines) and MOLT-4/8 (HTLV-1-negative human T-cell line) by immunizing the membrane fraction of human osteosarcoma line HOS. By immunoprecipitation and immunoblotting, H11 and H14 were found to be specific for MHC class I heavy chain and beta 2-microglobulin (beta 2 M), respectively. Among the four commercially obtained mAbs, two mAbs for MHC class I antigen and two mAbs to beta 2 M, one mAb to MHC class I antigen and one mAb to beta 2 M were also found to be inhibitory to the syncytium formation. The functional comparison of these mAbs revealed that the syncytium-inhibitory mAbs induced strong homotypic cell adhesion particularly in the HTLV-1-positive T-cell lines. This cell adhesion was dependent on temperature, energy metabolism, and microfilament function but not on the activity of protein kinase C or divalent cations. These results suggest a novel type of LFA-1-independent cell adhesion induced by signal transduction via MHC class I antigen.

Antibodies, Monoclonal↗
PubMed1992

Identification of membrane antigen C33 recognized by monoclonal antibodies inhibitory to human T-cell leukemia virus type 1 (HTLV-1)-induced syncytium formation: altered glycosylation of C33 antigen in HTLV-1-positive T cells.

We isolated four monoclonal antibodies (MAbs), M38, M101, M104, and C33, which were capable of inhibiting syncytium formation induced in a human T-cell line, MOLT-4-#8, by coculture with human T-cell leukemia virus type 1 (HTLV-1)-positive human T-cell lines. The MAbs had, however, no inhibitory activity on syncytium formation induced in a human osteosarcoma line, HOS, by HTLV-1-positive T-cell lines. They also did not inhibit syncytium formation induced in MOLT-4-#8 by human immunodeficiency virus type 1-positive MOLT-4. All MAbs reacted with various human cell lines of lymphoid and nonlymphoid origins, including HTLV-1-positive T-cell lines. Furthermore, they all reacted with a murine A9 clone containing human chromosome 11 fragment q23-pter. Two MAbs, M104 and C33, immunoprecipitated a membrane antigen with the same molecular size. The antigen (henceforth called C33 antigen) was about 40 to 55 kDa in HTLV-1-negative Jurkat, CEM, MOLT-4, and normal peripheral blood CD4-positive human T cells and about 40 to 75 kDa in HTLV-1-positive C91/PL, TCL-Kan, MT-2, and in fresh HTLV-1-transformed CD4-positive human T-cell lines. Pulse-chase experiments revealed that C33 antigen was synthesized as a 35-kDa precursor that was then processed to 41 to 50 kDa in MOLT-4 and to 44 to 70 kDa in C91/PL. In the presence of tunicamycin, a 28-kDa protein was synthesized. The conversion from 35 kDa to 41 to 50 kDa in MOLT-4 and to 44 to 70 kDa in C91/PL was inhibited by monensin. Treatment with N-glycanase alone, but not with sialidase and O-glycanase in combination, completely removed the sugar moiety of C33 antigen from both HTLV-1-negative Jurkat and HTLV-1-positive C91/PL. Therefore, C33 antigen has only N-linked carbohydrates, the modification of which appears to be substantially altered in the presence of the HTLV-1 genome.

Antibodies, Monoclonal↗
PubMed1991

gamma-Carboxyglutamic acid (Gla)-domainless blood coagulation factor IXa species: preparation and properties.

To investigate the function of the gamma-carboxyglutamic acid (Gla) residues of factor IXa in the activation of factor X, a new species of bovine factor IXa, designated "factor IXa beta'," and its corresponding Gla-domainless form, designated "Gla-domainless factor IXa beta'," were prepared under controlled conditions and characterized. First, bovine factor IXa alpha was converted by alpha-chymotrypsin in the presence of calcium ions to factor IXa beta' (Mr 47,000). Compared with factor IXa beta, factor IXa beta' had essentially identical activities towards a synthetic substrate, benzoyl-L-arginine ethylester (BAEE), towards an active site titrant, p-nitrophenyl-p'-guanidinobenzoate, and towards protein substrate, namely, factor X. Next, the Gla-rich region (residues 1-41) of the light chain was removed from factor IXa beta' by additional selective cleavage by alpha-chymotrypsin in the absence of calcium ions. Gla-domainless factor IXa beta' was purified to homogeneity on a column of DEAE-Sepharose CL-6B. The heavy chain was not altered by either chymotryptic digestion. Functional comparisons of the three activated forms, namely, factor IXa alpha, factor IXa beta', and Gla-domainless factor IXa beta', with factor IXa beta revealed that all four activated forms of factor IX had one active-site residue per molecule and essentially identical specific esterase activity towards BAEE. However, the clotting activity of Gla-domainless factor IXa beta' was less than 0.5% of that of factor IXa beta'.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Carboxyglutamic Acid↗
PubMed1989

Comparison of human, simian, and bovine rotaviruses for requirement of sialic acid in hemagglutination and cell adsorption.

Human rotaviruses (Wa, KUN, MO) showed hemagglutination (HA) only with fixed 1-day-old chicken erythrocytes, and their HA activities were completely destroyed by trypsin activation of virions. Simian SA-11 and bovine NCDV had HA activities not only against fixed erythrocytes but also against fresh erythrocytes from various species. Their HA activities against fixed erythrocytes were also inhibited by trypsin activation, but those against fresh erythrocytes were not. Neuraminidase treatment of fixed erythrocytes did not inhibit HA by trypsin-untreated rotaviruses. In contrast, HA of fresh human erythrocytes by SA-11 and NCDV was completely inhibited by neuraminidase treatment of erythrocytes or glycophorin A, the major erythrocyte sialoglycoprotein. Adsorption and infection of SA-11 and NCDV to monkey kidney MA104 cells were also inhibited by neuraminidase treatment of cells. Adsorption and infection of human rotaviruses were not, however, affected by treatment of cells with neuraminidase from Vibrio cholerae or Arthrobacter ureafaciens or with potassium periodate. Therefore, HA of fixed chicken erythrocytes by trypsin-untreated human and animal rotaviruses may be independent of sialic acids, whereas that of fresh erythrocytes by SA-11 and NCDV is sialic acid dependent and probably mediated by glycophorin A. Sialic acids also constitute an essential part of the cellular receptors for SA-11 and NCDV, whereas those for human rotaviruses were quite resistant to treatments known to destroy major types of sialic acids.

Animals↗
PubMed1988

Differences in the release of cholesterol from taurocholate versus taurochenodeoxycholate micellar solutions.

The maximal micellar solubility, distribution and apparent monomer activity of cholesterol in taurine-conjugated cholate and chenodeoxycholate micellar solutions were studied to clarify the different modulating effect of these bile salt species on cholesterol uptake in an intestinal lumen. The maximal micellar solubility was significantly greater in taurochenodeoxycholate. The intermicellar cholesterol monomer concentration was not significantly different between the two kinds of micellar solution. However, the apparent cholesterol monomer activity determined using an artificial organic phase (polyethylene disc) was significantly higher in taurocholate than that in taurochenodeoxycholate. A linear relationship between the intermicellar cholesterol concentration and the apparent cholesterol monomer activity was found, with the slope depending upon the bile salt species. It is concluded that the difference in partitioning of cholesterol from taurocholate and taurochenodeoxycholate micelles into a fixed organic phase may contribute in part to the different regulating effects of these bile salts on the uptake of cholesterol in the intraluminal phase.

Chenodeoxycholic Acid↗
PubMed1987

Effect of albumin on the solubility of cholesterol in bile.

Despite the fact that a considerable amount of albumin is present in bile, little is known about the effect of albumin on micellar solubility of cholesterol. The effect of albumin on solubility of cholesterol in various micellar bile salt solutions was studied using Millipore filtration after equilibration. In addition, partitioning of cholesterol from micellar solution was studied using a polyethylene disc method. Decrease of the solubility of cholesterol by the presence of albumin was observed only in unconjugated bile salt solution. The lowering effect of albumin on the cholesterol solubility was found to be proportional to the hydrophobicity of bile salt. In contrast, albumin had almost no effect on cholesterol solubility, either in conjugated bile salt solution or in micellar bile salt solution containing phosphatidylcholine. Addition of albumin enhanced the partitioning of cholesterol out of the micelles in sodium chenodeoxycholate solution as a result of decreased micellar solubility and increased the aqueous solubility of cholesterol in the presence of albumin. Therefore, conjugated bile salt and phosphatidylcholine exert a buffering action on the albumin-induced adverse effect on cholesterol solubility, thus stabilising bile against inadvertent precipitation of cholesterol.

Bile↗