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Biomedical subjects

M Kimoto

Publications and source records attributed to M Kimoto.

At least 37 records · Page 2Linked to original sources
PubMed2001

Expression and anticoagulant function of the endothelial cell protein C receptor (EPCR) in cancer cell lines.

Induction of procoagulant factors in malignant cells is considered to be the major cause of coagulation disorders in cancer. Thrombomodulin (TM), a negative regulator of coagulation was also found to be expressed in cancer cells. We report here evidence for another anticoagulant, the endothelial cell protein C receptor (EPCR), in cancer cells. EPCR was detected in several cell lines derived from various types of cancer. Significant levels of protein C (PC) activation were detected only with cell lines expressed both EPCR and TM. Anti-EPCR monoclonal antibodies (mAbs) specifically inhibited the activation. Thus, EPCR function appears to be important for PC activation by cancer cells. In addition, we detected EPCR expression in tumor cells from breast cancer patients, with an extremely high frequency. EPCR function may contribute to progression or pathogenesis of some types of cancer, and may explain the complexity of coagulopathy in cancer patients.

Anticoagulants↗
PubMed2000

Antiadhesive function of 130-kd glycoform of CD43 expressed in CD4 T-lymphocyte clones and transfectant cell lines.

Conflicting findings regarding proadhesion and antiadhesion in cell-to-cell interactions were previously reported for CD43. We examined possible differences in the role of the 130-kd glycoform and the 115-kd glycoform of CD43 in cellular adhesion in vitro. We generated a monoclonal antibody (MFT3) that discriminates between helper and nonhelper murine T-cell clones. Characterization of MFT3 with use of biochemical analysis and complementary DNA (cDNA) transfection experiments showed that it is specific for the 130-kd glycoform of CD43. T-cell clones that expressed the 130-kd CD43 glycoform showed decreased homocytic aggregation and decreased adhesion to spleen cells, B-lymphoma cell lines, and fibroblastic cell lines compared with T-cell clones negative for the 130-kd glycoform. Expression of core 2 beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) cDNA together with CD43 cDNA resulted in expression of both the 130-kd CD43 glycoform and the 115-kd CD43 glycoform in fibroblastic cell lines. Using these cell lines, we showed that the 130-kd glycoform but not the 115-kd glycoform of CD43 has an antiadhesive function in cellular interactions. Our findings suggest that the antiadhesive function of CD43 is primarily carried out by the 130-kd glycoform. (Blood. 2000;96:4267-4275)

3T3 Cells↗
PubMed2000

The toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells.

The susceptibility to infections induced by Gram-negative bacteria is largely determined by innate immune responses to bacteria cell wall lipopolysaccharide (LPS). The stimulation of B cells by LPS enhances their antigen-presenting capacity and is accompanied by B cell proliferation and secretion of large quantities of LPS-neutralizing antibodies. Similar to macrophages and neutrophils, the LPS-induced activation of B cells is dependent on Toll-like receptor (TLR)4. Here, we demonstrate that the responses of B cells to LPS are also regulated by another TLR protein, RP105, which is predominantly expressed on mature B cells in mice and humans. The analysis of mice homozygous for the null mutation in the RP105 gene revealed impaired proliferative and humoral immune responses of RP105-deficient B cells to LPS. Using originally LPS-unresponsive Ba/F3 cells expressing exogenous TLR4 and RP105, we demonstrate the functional cooperation between TLR4 and RP105 in LPS-induced nuclear factor kappaB activation. These data suggest the existence of the TLR4-RP105 signaling module in the LPS-induced B cell activation.

Animals↗
PubMed2000

Comparison of whole genome sequences of Chlamydia pneumoniae J138 from Japan and CWL029 from USA.

Chlamydia pneumoniae is a widespread pathogen of humans causing pneumonia and bronchitis. There are many reports of an association between C.PNEUMONIAE: infection and atherosclerosis. We determined the whole genome sequence of C.PNEUMONIAE: strain J138 isolated in Japan in 1994 and compared it with the sequence of strain CWL029 isolated in the USA before 1987. The J138 circular chromosome consists of 1 226 565 nt (40.7% G+C) with 1072 likely protein-coding genes that is 3665 nt shorter than the CWL029 genome. Plasmids, phage- or transposon-like sequences were not identified. The overall genomic organization, gene order and predicted proteomes of the two strains are very similar, suggesting a high level of structural and functional conservation between the two unrelated isolates. The most conspicuous differences in the J138 genome relative to the CWL029 genome are the absence of five DNA segments, ranging in size from 89 to 1649 nt, and the presence of three DNA segments, ranging from 27 to 84 nt. The complex organization of these 'different zones' may be attributable to a unique system of recombination.

Base Composition↗
PubMed2000

Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages.

The human MD-2 molecule is associated with the extracellular domain of human Toll-like receptor 4 (TLR4) and greatly enhances its LPS signaling. The human TLR4-MD-2 complex thus signals the presence of LPS. Little is known, however, about cell surface expression and LPS signaling of the TLR4-MD-2 complex in vivo. We cloned mouse MD-2 molecularly and established a unique mAb MTS510, which reacted selectively with mouse TLR4-MD-2 but not with TLR4 alone in flow cytometry. Mouse MD-2 expression in TLR4-expressing cells enhanced LPS-induced NF-kappaB activation, which was clearly inhibited by MTS510. Thioglycolate-elicited peritoneal macrophages expressed TLR4-MD-2, which was rapidly down-regulated in the presence of LPS. Moreover, LPS-induced TNF-alpha production by peritoneal macrophages was inhibited by MTS510. Collectively, the TLR4-MD-2 complex is expressed on macrophages in vivo and senses and signals the presence of LPS.

Adjuvants, Immunologic↗
PubMed2000

Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression.

Monocytes/macrophages exposed to LPS show reduced responses to second stimulation with LPS, which is termed LPS tolerance. In this study, we investigated molecular mechanism of LPS tolerance in macrophages. Mouse peritoneal macrophages pre-exposed to LPS exhibited reduced production of inflammatory cytokines in a time- and dose-dependent manner. Activation of neither IL-1 receptor-associated kinase nor NF-kappaB was observed in macrophages that became tolerant by LPS pretreatment, indicating that the proximal event in Toll-like receptor 4 (TLR4)-MyD88-dependent signaling is affected in tolerant macrophages. Although TLR4 mRNA expression significantly decreased within a few hours of LPS pretreatment and returned to the original level at 24 h, the surface TLR4 expression began to decrease within 1 h, with a gradual decrease after that, and remained suppressed over 24 h. A decrease in inflammatory cytokine production in tolerant macrophages well correlates with down-regulation of the surface TLR4 expression, which may explain one of the mechanisms for LPS tolerance.

Animals↗
PubMed2000

Regulatory roles for CD14 and phosphatidylinositol in the signaling via toll-like receptor 4-MD-2.

The complex consisting of Toll-like receptor 4 (TLR4) and associated MD-2 signals the presence of lipopolysaccharide (LPS) when it is expressed in cell lines. We here show that normal human mononuclear cells express TLR4 and signal LPS via TLR4. CD14 is a molecule that binds to LPS and facilitates its signaling. Little is known, however, about the relationship of CD14 with TLR4-MD-2. We show that CD14 helps TLR4-MD-2 to sense and signal the presence of LPS. CD14 has also been implicated in recognition of apoptotic cells, which leads to phagocytosis without activation. Membrane phospholipids such as phosphatidylserine (PS) or phosphatidylinositol (PtdIns) are thought to serve as the ligands for CD14 in apoptotic cells. We find that PtdIns acts as an LPS antagonist in the signaling via TLR4-MD-2. TLR4-MD-2 seems to discriminate LPS from phospholipids. The signaling via TLR4-MD-2 is thus regulated by CD14 and phospholipid such as PtdIns.

Animals↗
PubMed2000

Adaptor protein Shc undergoes translocation and mediates up-regulation of the tyrosine kinase c-Src in EGF-stimulated A431 cells.

BACKGROUND: Shc is the adaptor protein that exists in three isoforms, P46, P52 and P66, and acts as a bridge between activated cell surface receptors and downstream signalling molecules which act in extracellular signal-regulated cell events such as cell cycle progression. In our previous studies, Shc was shown to be a substrate of the tyrosine kinase c-Src in vitro and in vivo. RESULTS: Using green fluorescent protein-fusion Shc (GFP-Shc), we have shown that following epidermal growth factor (EGF) stimulation of A431 cells, all Shc isoforms were rapidly recruited from the cytoplasm to the plasma membrane (within 5 min) and then redistributed to the cytoplasmic vesicle structures (in the next 10-20 min). Indirect immunofluorescent study demonstrated that all Shc isoforms co-localize with EGF receptor (EGFR) and activated c-Src in both plasma membranes and cytoplasmic vesicle structures. Our previous study has shown that EGF induces the indirect association of EGFR and c-Src and activation of c-Src in A431 cells. An immunoprecipitation study demonstrated that the EGFR-Src association and c-Src activation are augmented in cells expressing GFP-Shc P52 or P66, but not P46. In addition, P52 and P66, but not P46, are in association with EGFR-Src complex. We also found that EGFR and Shc can be dissociated from c-Src by the addition of a synthetic peptide that corresponds to the autophosphorylation site of c-Src. Interestingly, the peptide-induced dissociation of the complex was not affected by the tyrosine phosphorylation state of the peptide. CONCLUSION: These results demonstrated a dynamic subcellular movement of Shc in response to EGF, and suggested a hitherto unknown scheme whereby Shc can work not only as a substrate of c-Src but also as a mediator of the EGF-induced activation of c-Src in an isoform-specific manner.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Comparison of outer membrane protein genes omp and pmp in the whole genome sequences of Chlamydia pneumoniae isolates from Japan and the United States.

Chlamydia pneumoniae is a widespread pathogen of the respiratory tract that is also associated with atherosclerosis. The whole genome sequence was determined for a Japanese isolate, C. pneumoniae strain J138. The sequence predicted a variety of genes encoding outer membrane proteins (OMPs) including ompA and porB, another 10 predicted omp genes, and 27 pmp genes. All were detected in the whole genome sequence of strain CWL029, a strain isolated and sequenced in the United States. A comparative study of the OMPs of the two strains revealed a nucleotide sequence identity of 89.6%-100% (deduced amino acid sequence identity, 71.1%-100%). The overall genomic organization and location of genes are identical in both strains. Thus, a few unique sequences of the OMPs may be essential for specific attributes that define the differential biology of two C. pneumoniae strains.

Bacterial Outer Membrane Proteins↗
PubMed2000

Unnatural base pairs between 2-amino-6-(2-thienyl)purine and the complementary bases.

The unnatural base, 2-amino-6-(2-thienyl)purine (designated as s), instead of 2-amino-6-(N,N-dimethylamino)purine (designated as x), was designed in order to improve the specificity and efficiency of the base pairing with pyridin-2-one (designated as y). DNA fragments containing s were chemically synthesized, and the thermal stability and the enzymatic reactions involving the s-y pairing were examined. Thermal denaturation experiments showed that the DNA duplex (12-mer) containing the s-y pair was more stable than that containing the x-y pair. The incorporation of dyTP was also more advantageous to the s-y pairing than the x-y pairing in single-nucleotide insertion experiments using the Klenow fragment of Escherichia coli DNA polymerase I.

Base Pairing↗
PubMed2000

The dsbA-dsbB disulfide bond formation system of Burkholderia cepacia is involved in the production of protease and alkaline phosphatase, motility, metal resistance, and multi-drug resistance.

In a previous study, we isolated a dsbB mutant of Burkholderia cepacia KF1 and showed that phenotypes of protease production and motility are dependent on DsbB, a membrane-bound disulfide bond oxidoreductase. We have now isolated a dsbA mutant by transposon mutagenesis, cloned the dsbA gene encoding a periplasmic disulfide bond oxidoreductase, and characterized the function of the DsbA-DsbB disulfide bond formation system in B. cepacia. The complementing DNA fragment had an open reading frame for a 212-amino acid polypeptide with a potential redox-active site sequence of Cys-Pro-His-Cys that is homologous to Escherichia coli DsbA. The dsbA mutant, as well as the previously isolated dsbB mutant, was defective in the production of extracellular protease and alkaline phosphatase, as well as in motility. In addition, mutation in the DsbA-DsbB system resulted in an increase in sensitivity to Cd2+ and Zn2+ as well as a variety of antibiotics including beta-lactams, kanamycin, erythromycin, novobiocin, ofloxacin and sodium dodecyl sulfate. These results suggested that the DsbA-DsbB system might be involved in the formation of a metal efflux system as well as a multi-drug resistance system.

Alkaline Phosphatase↗
PubMed2000

Renal expression of constitutive NOS and DDAH: separate effects of salt intake and angiotensin.

BACKGROUND: Nitric oxide (NO) is generated from NO synthase (NOS) isoforms. These enzymes can be inhibited by asymmetric dimethylarginine, which is inactivated by N(G)-N(G)-dimethylarginine dimethylaminohydrolase (DDAH). The neuroneal (nNOS) type I and endothelial (eNOS) type III constitutive NOS isoforms are expressed predominantly in the macula densa and microvascular endothelium of the renal cortex, respectively. DDAH is expressed at sites of NOS expression. Since NO may coordinate the renal responses to angiotensin II (Ang II) and changes in salt intake, we tested the hypothesis that salt intake regulates the expression of nNOS, eNOS and DDAH by Ang II acting on type 1 (AT(1)) receptors. METHODS: Groups (N = 6) of rats were adapted to low-salt (LS) or high-salt (HS) intakes for 10 days. Other groups of LS and HS rats received the AT(1) receptor antagonist losartan for six days (to test the effects of salt independent of AT(1) receptors). A further group of HS rats received an infusion of Ang II for six days (to test the effect of Ang II independent of salt intake). RESULTS: Compared with HS rats, there was a significant (P < 0.05) increase in LS rats of nNOS protein in kidney and immunohistochemical expression in the macula densa, and of eNOS protein expression and immunohistochemical expression in the microvascular endothelium, and of DDAH protein expression. Losartan prevented these effects of salt on the expression of eNOS or DDAH, both of which were also increased by Ang II infusions in HS rats. In contrast, losartan did not prevent the effects of salt on nNOS expression, which was unresponsive to Ang II infusion. The generation of NO(2)(-) released by slices of renal cortex, in the presence of saturating concentrations of L-arginine, was increased by LS, compared to HS, independent of losartan and by Ang II during HS. CONCLUSION: The expressions of eNOS in cortical microvascular endothelium and DDAH in kidney are enhanced by Ang II acting on AT(1) receptors. The expression of nNOS in the macula densa is enhanced by salt restriction independent of Ang II or AT(1) receptors.

Amidohydrolases↗
PubMed2000

Structural and immunochemical homologies between foxtail millet glutelin 60 kDa and starch granule-bound starch synthase proteins from rice, barley, corn and wheat grains.

Foxtail millet glutelin 60 kDa (MG60) was purified by preparative SDS-PAGE, and the N-terminal amino acid sequence was determined within 20 residues. The result demonstrated that the primary structure at N-terminal of MG60 was almost identical to those of the granule-bound starch synthase (GBSS) proteins from rice, barley, corn, wheat and potato. The existence of common epitopes among MG60 and GBSS proteins from these starch-storing cereals were corroborated by immunoblot analysis using antisera raised against MG60. These facts strongly suggest a close relationship between MG60-like glutelins and GBSS proteins.

Amino Acid Sequence↗
PubMed2000

Efficient extraction and some properties of storage proteins (prolamin and glutelin) in ancient rice cultivars.

Two typical storage proteins (prolamin and glutelin) in a total of 14 samples of ancient rice cultivars were examined for their accumulation in the endosperms during days after flowering (DAF), extraction efficiency with various solvents, and variations in polypeptide components or amino acid compositions. There was little difference in accumulation profiles between this and previous observations, in which glutelin appeared on 5-7 DAF and prolamin on 7-9 DAF. As for the extraction of prolamin and glutelin, it was most effectively attained by sequential usage of 55% propanol and 21% SDS. As a result of SDS-PAGE, prolamin and glutelin proved to be composed mainly of a single polypeptide with a molecular weight (MW) of 15 kDa and two subunits with MWs of 21 kDa (alpha) and 32 kDa (beta), respectively. A comparison between the ancient and modern rice cultivars revealed the same physicochemical properties in either case of prolamin or glutelin. Additionally, a good relationship was observed on the total protein and glutelin (but not prolamin) contents in the ancient rice cultivar.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

Innate recognition of lipopolysaccharide by Toll-like receptor 4/MD-2 and RP105/MD-1.

The Toll family of receptors has been implicated in innate recognition and subsequent activation of defense programs against pathogens such as bacteria and fungi. TLR4, for example, signals the presence of lipopolysaccharide (LPS), a membrane constituent of Gram-negative bacteria. LPS signaling via TLR4 is greatly enhanced by a molecule referred to as MD-2, which is associated with the extracellular domain of TLR4. The TLR4/MD-2 complex, therefore, recognizes LPS. RP105, another member of the Toll family, has a striking similarity to TLR4 in that it is associated with an MD-2-like molecule MD-1. B-cells lacking RP105 are severely impaired in LPS-induced proliferation and antibody production. Studies employing transfectants showed that RP105/MD-1, like MD-2, enhances the LPS signaling via TLR4. RP105/MD-1 thus constitutes an LPS-signaling complex on B-cells. These results suggest that a variety of cell surface molecules regulate LPS recognition/signaling by TLR4.

Animals↗
PubMed1999

Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase.

BACKGROUND: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). Plasma levels of ADMA are elevated in individuals with hypercholesterolemia or atherosclerosis. We postulated that reduced degradation of ADMA may play a role in the accumulation of ADMA in these individuals. Accordingly, we studied the effects of oxidized LDL (oxLDL) or tumor necrosis factor-alpha (TNF-alpha) on the accumulation of ADMA by transformed human umbilical vein endothelial cells (ECV304) and on the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which degrades ADMA. METHODS AND RESULTS: ECV304 were incubated with or without native LDL (100 micrograms/mL), oxLDL (100 micrograms/mL), or TNF-alpha (250 U/mL) for 48 hours. The concentration of ADMA in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. We assayed DDAH activity by determining L-citrulline formation from ADMA. The addition of oxLDL or TNF-alpha to ECV304 significantly increased the level of ADMA in the conditioned medium. The effect of oxLDL or TNF-alpha was not due to a change in DDAH expression but rather to the reduction of DDAH activity. To determine whether dysregulation of DDAH also occurred in vivo, New Zealand White rabbits were fed normal chow or a high-cholesterol diet. Hypercholesterolemia significantly reduced aortic, renal, and hepatic DDAH activity. CONCLUSIONS: These results suggest that the endothelial vasodilator dysfunction observed in hypercholesterolemia may be due to reduced degradation of ADMA, the endogenous inhibitor of NOS.

Amidohydrolases↗
PubMed1999

The endothelial cell protein C receptor (EPCR) functions as a primary receptor for protein C activation on endothelial cells in arteries, veins, and capillaries.

Plasma protein C functions as an anticoagulant when it is converted to the active form of serine protease. Protein C activation has been found to be mediated by the endothelial cell surface thrombin/thrombomodulin (TM) complex. In addition, we recently identified the endothelial cell protein C/activated protein C receptor (EPCR) which is capable of high-affinity binding for protein C. In this study, we established monoclonal antibodies (mAbs) against EPCR including several function blocking antibodies. Immunohistochemical analysis using these mAbs demonstrated that EPCR is widely expressed in the endothelial cells of arteries, veins, and capillaries in the lung, heart, and skin. Function blocking anti-EPCR mAbs strongly inhibited protein C activation mediated by primary cultured arterial endothelial cells which express abundant EPCR. Anti-EPCR mAbs also prevent protein C activation mediated by microvascular endothelial cells. These results indicate that EPCR functions as an important regulator for the protein C pathway in various types of vessels.

Antibodies, Monoclonal↗