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Biomedical subjects

N Watanabe

Publications and source records attributed to N Watanabe.

At least 487 records · Page 27Linked to original sources
PubMed1996

Functional properties of murine macrophages promoted by nerve growth factor.

The stimulating effect of nerve growth factor (NGF) on phagocytosis, parasite killing, and interleukin-1beta (IL-1beta) production of murine peritoneal macrophages was assessed. In the presence of various doses of NGF, macrophages showed the increased phagocytosis of both nonspecific hydrophilic microspheres and sheep red blood cells (SRBC) opsonized with anti-SRBC antibodies (Ab) or complement in a dose-dependent manner. NGF also enhanced killing of Leishmania donovani promastigotes by macrophages, and its ability was comparable with that of an optimal dose of recombinant granulocyte-macrophage colony-stimulating factor or recombinant interferon-gamma. The addition of NGF to peritoneal macrophages and monocyte-macrophage J774A.1 cells led to a significant release of IL-1beta in a dose-dependent manner and expression of IL-1beta mRNA. Because pretreatment of peritoneal macrophages and J774A.1 cells with K-252a, a tyrosine kinase inhibitor, completely suppressed these NGF-mediated stimulating effects and p140trk phosphorylation and because flow cytometric analysis with specific Ab against two distinct NGF receptors showed the expression of p140trk, unlike p75LNGFR, on the surface of macrophages, the stimulating activity of NGF to murine macrophages may be mediated through p140trk. Thus, NGF may act as an activator for murine macrophages in the process of inflammatory and immune actions.

3T3 Cells↗
PubMed1996

Circadian blood pressure variation related to morbidity and mortality from cerebrovascular and cardiovascular diseases.

The mortality rate of stroke has decreased dramatically over the past 30 years in Japan, but the prevalence of cerebral infarction has increased, mainly because of the higher incidence of nonfatal cerebral infarction in the elderly. Significant advances in the development of antihypertensive drugs over the last 30 years have contributed to the decrease in stroke mortality, but antihypertensive treatment appears to relate to an increase in the prevalence of cerebral infarction in the elderly. We investigated the risks of low BP levels determined by ambulatory BP monitoring and home BP measurements to clarify the previously reported J-shaped relationships between the incidence of ischemic cardiovascular disease and BP levels after treatment. A long-term prospective study of all causes of mortality and cerebrovascular and cardiovascular mortality has been conducted in Ohasama, Japan, since 1987. Cox proportional hazard model after adjusting for age and sex demonstrated that during a 5-year follow-up period involving 893 of the subjects aged 50 years and over in this cohort, those with the lowest quintile of ambulatory BP levels exhibited a significantly high hazard ratio of cardiovascular as well as all causes of mortality. During the same follow-up period in 1,226 subjects aged 50 years and over, those with the lowest and highest quintiles of home BP levels demonstrated a significantly high hazard ratio of cardiovascular as well as all causes of mortality (i.e., J-shaped relationship). The amplitude of the fall in nocturnal BP obtained by ambulatory BP monitoring correlated positively with the severity of silent cerebrovascular lesions in elderly women, but not in elderly men. These results suggest the significantly high risk associated with low BP levels, which can be determined only by ambulatory and home BP measurements, but not by casual BP measurements.

Age Factors↗
PubMed1996

Characterization of peripheral blood stem cells in mice.

Peripheral blood stem cells (PBSCs) were mobilized in mice by treatment with cytosine-arabinoside on day 0, followed by the administration by injection of granulocyte colony-stimulating factor for 4 days. There were remarkable increases in the numbers of cells with lineage-negative (Lin-) c-kit+ markers, cells with colony-forming unit-cell (CFU-C) and colony-forming unit-spleen (CFU-S) activities, and cells with marrow-repopulating ability (MRA) in the extramedullary sites (the spleen, peripheral blood, and liver) on day 5, whereas the number of these immature hematopoietic cells decreased in the bone marrow (BM) on day 5. This finding suggests the mobilization of immature hematopoietic cells from the BM to the extramedullary sites. Three-color flow cytometric analyses showed that CD4 antigen was not expressed on the Lin-Sca-1+ cells in the mobilized PB cells (PBCs), although CD4lo cells were found in those of normal BM cells. Lin-c-kit+ cells in the mobilized PBCs contained more cells with immature phenotypes (Sca-1+, Thy1.2lo, CD71-, and Rh123dull) than in normal BMCs, indicating an alteration of the hierarchical composition of the Lin-c-kit+ cells. The Lin-c-kit+Sca-1+ cells in the mobilized PBCs had similar CFU-C and CFU-S activities to those in normal BMCs. Electron microscopic studies of these cells in the mobilized PBCs showed that only 10% to 20% of these cells had a thin rim of cytoplasm with poorly developed organelles. Allogeneic transplantation [B6 --> C3H] of PBSCs showed long-term reconstituting activity across the major histocompatibility complex barrier 24 weeks after transplantation, although longer observation is necessary.

Animals↗
PubMed1996

Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain.

Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho. One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese "teki," meaning target). The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35S-labeled guanosine 5'-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays. In addition, this peptide inhibited both endogenous and GTPase-activating protein-stimulated Rho GTPase activity. The amino acid sequence of this region shares approximately 30% identity with the Rho-binding domains of rhophilin and a serine/threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). Thus, not only is rhotekin a novel partner for Rho, but it also belongs to a wide family of proteins that bear a consensus Rho-binding sequence at the N terminus. To our knowledge, this is the first conserved sequence for Rho effectors, and we have termed this region Rho effector motif class 1.

Adaptor Proteins, Signal Transducing↗
PubMed1996

The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1996

Interleukin 1 (IL-1) production is not essential for acquired resistance of human A375 melanoma cells to anti-proliferative effect of IL-1.

The proliferation of human melanoma cell line A375-6 is inhibited by interleukin l (IL-l). However, the cells acquired resistance to IL-l after a long period of culture. We have reported that 2 resistant subclones, A375-R8 and -R19, produced IL-l alpha constitutively and that IL-l induced IL-6 production in an autocrine manner. Therefore, we supposed that IL-l alpha production renders the cells resistant to IL-l. To investigate the relationship between IL-l alpha production and IL-l resistance, we transfected the IL-l alpha expression plasmid to the IL-l-sensitive clone, A375-6, and the anti-sense mRNA expression plasmid to IL-l-resistant cells, A375-R8 and -R19. A375-6MS, a transfectant of mature IL-l alpha expression plasmid, expressed IL-l alpha mRNA and produced IL-l activity at a level comparable to the resistant cells. The transfectant also produced IL-6 and exhibited augmented expression of Mn-SOD mRNA. However, IL-l sensitivity of this transfectant was not affected. With respect to sensitivity to anti-proliferative effects of other cytokines, such as IL-6 and TNF alpha, there was no difference between the transfectant and parent cells. Although A375-R8PH10 and -R19PH10, transfectants of IL-l alpha anti-sense mRNA expression plasmid, exhibited a decrease in the level of IL-l production, their IL-l sensitivity did not differ from parent cells. These results, therefore, suggest that IL-l alpha production is not essential or sufficient for the acquisition of resistance to the anti-proliferative effect of IL-l.

Antineoplastic Agents↗
PubMed1996

Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies.

Nucleic acid specificity was tested for two monoclonal anti-double-stranded DNA autoantibodies, 2C10 and H241, derived from two lupus-prone MRL/Mp-lpr/lpr mice. Antibody 2C10 bound double-stranded oligonucleotides with a preference for dA-dT over dG-dC base pairs and did not bind single-stranded oligonucleotides. Distamycin A, an antibiotic that binds to the minor groove, inhibited 2C10 binding of double-stranded DNA, suggesting that this antibody interacts with dA-dT base pairs in the minor groove. Antibody H241 binding was previously shown to have a dG-dC preference and to involve both major and minor grooves. In attempted footprinting assays, both 2C10 and H241 markedly en- hanced rather than protected against cleavage of DNA by hydroxyl radical-generating systems. With 2C10, this enhancement effect was observed only when hydroxyl radical generation was associated with oxidation of Fe(II). In contrast, H241 enhancement occurred in the presence of H2O2 and ascorbate or UV light irradiation and did not depend on added metal ion. The enhancement sites were related to the antibody binding specificities. The oligonucleotide 5'-AAAATATATATTT-3' was a much more effective inhibitor of the 2C10 enhancement than of the H241 effect, whereas the oligonucleotide 5'-GGGGCGCGCGCCC-3' was a much more effective inhibitor of the H241 enhancement. In addition, the enhanced cleavage occurred preferentially at dA-dT-rich regions with 2C10 and at dG-dC-rich regions with H241. These findings raise the possibility that anti-DNA autoantibodies could enhance DNA damage in inflammatory lesions in which hydroxyl radicals are generated.

Animals↗
PubMed1996

Dependence of cyclin E-CDK2 kinase activity on cell anchorage.

Most nonmalignant cells are anchorage-dependent; they require substrate attachment for growth and, in some instances, survival. This requirement is lost on oncogenic transformation. The cyclin E-CDK2 complex, which is required for the G1-S transition of the cell cycle, was activated in late G1 phase in attached human fibroblasts, but not in fibroblasts maintained in suspension. In transformed fibroblasts the complex was active regardless of attachment. The lack of cyclin E-CDK2 activity in suspended cells appeared to result from increased expression of CDK2 inhibitors and a concomitant decrease in phosphorylation of CDK2 on threonine-160. Suppression of cyclin E-CDK2 activity may thus underlie the anchorage dependence of cell growth.

CDC2-CDC28 Kinases↗
PubMed1996

Increased nitric oxide synthase activity as a cause of mitochondrial dysfunction in rat hepatocytes: roles for tumor necrosis factor alpha.

Kupffer cells have been implicated in playing an important role in the pathogenesis of endotoxemia-associated liver injury. The present study was designed to investigate whether Kupffer cell-derived mediators alter the mitochondrial oxidative phosphorylation of hepatocytes in the endotoxemic condition. Liver cells were isolated from male Wistar rats. Oxidative phosphorylation was monitored as the fluorescence of rhodamine 123 (Rh123), which is the fluorescent cationic dye used to indicate mitochondrial energy synthesis. Two hours after coculture of hepatocytes with lipopolysaccharide (LPS)-pretreated Kupffer cells, a marked decrease in hepatocyte rhodamine 123 fluorescence was observed. The hepatocyte mitochondrial dysfunction was attenuated by the addition of either N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) synthesis, or aminoguanidine, an inducible-type of NO synthase inhibitor, to the culture medium of cocultures, to the pretreatment of LPS-activated Kupffer cells with antisense oligodeoxynucleotides against iNOS messenger RNA (mRNA), or to tumor necrosis factor alpha (TNF-alpha) mRNA. Four hours after the coculture, hepatocyte Rh123 fluorescence further decreased, and an iNOS induction as well as an increased NO production were observed in hepatocytes that were cocultured with LPS-pretreated Kupffer cells. The membrane barrier dysfunction of hepatocytes, indicated by propidium iodide staining, was also induced by a 4-hour coculture with LPS-pretreated Kupffer cells. These late-phase changes were inhibited either by the pretreatment of hepatocytes with antisense oligodeoxynucleotides against iNOS mRNA or by treatments that are effective in the early phase (within 2 hours). Incubation with recombinant rat TNF-alpha decreased hepatocyte Rh123 fluorescence within 2 hours. Thus, the present study suggests that NO and TNF-alpha released from LPS-pretreated Kupffer cells directly inhibit the hepatocyte mitochondrial function in the early phase, and then NO synthesized by TNF-alpha-induced hepatocyte iNOS causes lethal hepatocyte injury, characterized by diminished mitochondrial energization and membrane barrier function in the late phase.

Animals↗
PubMed1996

Cytokine production by SV40-transformed adherent synovial cells from rheumatoid arthritis patients.

We examined the mRNA levels for various cytokines, including IL-1 alpha, IL-1 beta, TNF alpha, TGF-beta 1, GM-CSF, IL-6, IL-8, bFGF, PDGF-A, PDGF-B and IL-1ra, and IL-1 beta converting enzyme, and the protein levels of some of these cytokines in 19 SV40-transformed synovial cell clones. Among those tested, the mRNA levels for IL-6, bFGF and PDGF-A in rheumatoid arthritis (RA) cell clones were greater than those in non-RA cell clones. Moreover, except for one osteoarthritis (OA) cell clone, the mRNA levels for IL-8 in RA cell clones were also greater than those in non-RA cell clones. Although the protein levels were not always correlated with the mRNA levels, the exception being the same OA cell clone, the protein levels of cytokines, such as IL-1 alpha, IL-1 beta, IL-6 and IL-8, in RA cell clones were greater than those in non-RA cell clones. TNF-a was not detected in any cells tested at either the mRNA or the protein level. TNF-alpha upregulated the expression of GM-CSF mRNA in both RA cell clones and one OA cell clone, but not in the other OA cell clone or the normal cell clone. Taken together, these SV-40 transformed synovial cell clones retained many of the original characteristics in terms of cytokine production.

Adult↗
PubMed1996

Inhalation of diesel engine exhaust increases bone mineral concentrations in growing rats.

Experiments were conducted to determine whether diesel engine exhaust affects bone metabolism in growing rats. The rats were assigned to three groups: those exposed to total diesel engine exhaust with 5.63 mg/m3 particulate matter, 4.10 ppm nitrogen dioxide, 8.10 ppm nitrogen monoxide; those exposed to filtered exhaust without particulate matter; and those exposed to clean air. Dosing experiments were performed for 3 months beginning at birth (6 h/day for 5 days/week). Bone mineral content (BMC) values in lumbar vertebral bone were significantly increased in both groups exposed to diesel exhaust (P < 0.01) compared to that of rats exposed to clean air. Bone mineral density (BMD) values were also significantly increased in both exposed groups, total exhaust (P < 0.01) and filtered exhaust (P < 0.001), compared to that of rats exposed to clean air. BMD values in the mid-femur were also significantly greater in animals exposed to diesel exhaust, total exhaust (P < 0.05), and filtered exhaust (P < 0.01), compared to that of those exposed to clean air. Urinary excretion of deoxypyridinolines, a biochemical marker for bone resorption, was significantly reduced in animals exposed to total diesel exhaust and filtered exhaust (P < 0.001, P < 0.01) compared to control. There was also a significant difference between the two exposure groups of diesel exhaust (P < 0.05). Since these effects were not inhibited by filtration, the gaseous phase of the exhaust was considered more responsible than particulate matter for reducing bone resorption.

Administration, Inhalation↗
PubMed1996

IL-1 is an important mediator for microcirculatory changes in endotoxin-induced intestinal mucosal damage.

Although small intestine is frequently injured in endotoxin shock, the exact pathological sequence has not been fully understood. The major objective of this study is to elucidate the role of interleukin (IL)-1 in endotoxin-induced microcirculatory disturbance of rat small intestine. Mucosal and submucosal microvessels of the rat ileum were observed by intravital microscope with a high speed video camera system and the attenuating effect of E5090, an inhibitor of IL-1 generation, on endotoxin-induced intestinal microcirculatory disturbances was investigated. Endotoxin infusion produced significant mucosal damage, but before these morphological changes became significant, microvascular stasis in villi, decreased red blood cell velocity, and increased leukocyte adherence to venular walls were observed in intestinal microcirculatory beds 30 min after endotoxin administration. Intestinal IL-1alpha levels were also significantly increased at that time. Endotoxin treatment enhanced chemiluminescence activity from neurophils and rapidly mobilized CD18 on leukocytes. E5090, which suppressed the IL-1 production in intestinal mucosa, attenuated the microcirculatory disturbances induced by endotoxin, and significantly reduced the subsequent mucosal damage. E5090 also attenuated the increased chemiluminescence activity and CD18 expression on leukocytes. In conclusion, the production of IL-1alpha is enhanced in the intestinal mucosa during endotoxin infusion. IL-1 may be an important mediator of microcirculatory changes, including decreased red blood cell velocity and increased leukocyte sticking and its activation, leading to the mucosal damage.

Acrylates↗
PubMed1996

Determining depth of invasion of advanced colorectal cancer using MRI short inversion time inversion recovery sequences.

To examine the usefulness of magnetic resonance imaging (MRI) in the preoperative determination of cancerous invasion, we examined 39 patients with advanced colorectal cancer with 0.5T MRI. We employed short inversion time inversion recovery (STIR) sequences, in addition to ordinary spin echo sequences for T1- and T2-weighted images. Preoperatively, the estimated depth of tumor invasion was classified into three grades according to MRI findings, and confirmed on the basis of surgical and histopathologic results. The depth of tumor invasion estimated preoperatively using STIR sequences corresponded well with the surgical and histopathologic results in 85% of the cases. In contrast, assessments based on T1-weighted images corresponded well in only 62% of the cases and T2-weighted images corresponded well in only 64%.

Colon, Sigmoid↗
PubMed1996

Laparoscopically assisted ileocecal resection for Crohn's disease associated with intestinal stenosis and ileovesical fistula.

We describe a 22-year-old man with Crohn's ileocolitis accompanied by intestinal stenosis and ileovesical fistula in whom laparoscopically-assisted surgery was successfully performed after thorough nutritional therapy. Laparoscopic procedures are characterized by minimal access and minimal invasion, features which can contribute to the early recovery of patients who undergo surgery. It is suggested that laparoscopic (or laparoscopically-assisted) surgery after strict nutritional therapy can be effective in the treatment of patients with Crohn's disease who have intestinal complications.

Adult↗
PubMed1996

Bacterial adhesion on hydrophilic heparinized catheters, with compared with adhesion on silicone catheters, in patients with malignant obstructive jaundice.

To study the inhibitory effects on bacterial adhesion of a newly devised, hydrophilic heparinized catheter to be used in patients with malignant obstructive jaundice, a randomized controlled study of indwelling endoprostheses was performed, using implantable port-connected heparinized catheters (n = 25) and silicone catheters (n = 21). Catheters withdrawn from patients were cultured for bacteria and examined by electron microscopy for the presence of adherent organisms. In vitro examination of the two type of catheters exposed to suspensions of Eschericia coli and Staphylococcus aureus was performed using electron microscopy and a luminometer. The formation of a biofilm coated with glycocalyces was found in silicone catheters, but not in the heparinized catheters. In vitro experiments demonstrated little bacterial adhesion to the heparinized surface, but significant formation of biofilm on the silicone surface. Anionically charged heparinized catheters have inhibitory effects on bacterial adhesion, and the surface charge of the catheter may be a factor in inhibiting this adhesion.

Aged↗
PubMed1996

Binding of the V proteins to the nucleocapsid proteins of human parainfluenza type 2 virus.

Interaction of the nucleocapsid (NP) and V proteins of human parainfluenza type 2 virus (HPIV-2) was investigated using a transient expression system. When the NP proteins were co-expressed with the V proteins, some of the NP proteins were translocated into the nuclei. These findings suggest that the NP protein interact with the V proteins. We examined the interaction of the NP proteins and the P, V proteins or deletion mutants of V protein using immunofluorescence and co-immunoprecipitation plus Western blotting analyses, and showed that the V proteins of HPIV-2 bind to the NP proteins and that the N-terminal domain of V protein interacts directly with the NP proteins. When the NP proteins were co-expressed with the V proteins or the N-terminal fragments (aa 1-46), the NP proteins were detected diffusely in the nuclei of the transfected cells, and were also detected in cytoplasmic inclusions. The NP and V proteins were co-localized in the nuclei or cytoplasm. Furthermore, the NP proteins were co-precipitated with the P, V, and V (1-164) proteins by a specific antibody. The P proteins interact more closely with the NP proteins than do the V proteins. These findings indicate that the V proteins have the ability to bind the NP proteins.

Binding Sites↗
PubMed1996

Eosinophil chemotactic activity in Leishmania amazonensis promastigotes.

Tissue eosinophilia was observed in the subcutaneous tissue of mice shortly after their inoculation not only with living but also with lysed promastigotes of Leishmania amazonensis. Intraperitoneal inoculation of lysed promastigotes from five different Leishmania species (L. donovani, L. chagasi, L. tropica, L. amazonensis, and L. braziliensis) induced eosinophil accumulation in the mouse peritoneum. This eosinophil infiltration was also detected in C5-deficient AKR mice, indicating complement independent eosinophil chemotaxis by the parasite. The induced eosinophils were hypodense, suggesting activation of the cells. Finally, we demonstrated in vitro eosinophil chemotactic activity in the promastigote lysates using purified eosinophils and blind well chambers. These results suggest the presence of an eosinophil chemotactic factor in Leishmania, a protozoan parasite.

Animals↗