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Biomedical subjects

K Umezawa

Publications and source records attributed to K Umezawa.

At least 91 records · Page 5Linked to original sources
PubMed1995

Inostamycin, an inhibitor of P-glycoprotein function, interacts specifically with phosphatidylethanolamine.

The mechanism of inostamycin action was further studied. When multidrug-resistant KB-C4 cells were preincubated with inostamycin for 30 min, the accumulation of [3H]vinblastine was increased for as long as 48 h thereafter. Inostamycin inhibited azidopine binding to P-glycoprotein, even after KB plasma membranes had been preincubated with inostamycin and washed. Carbon 14-labeled inostamycin bound to KB plasma membranes irreversibly, but the binding capacity did not parallel the amount of P-glycoprotein in three KB cell lines. Inostamycin was found to interact specifically with purified phosphatidylethanolamine. These results suggest that inostamycin can inhibit P-glycoprotein irreversibly by binding to plasma membranes irreversibly through phosphatidylethanolamine.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Granulation in livers of mice infected with Salmonella typhimurium is caused by superoxide released from host phagocytes.

The pathophysiological roles of superoxide (O2.-) at the site of infection of facultative intracellular bacteria were examined in this study. To evaluate the actual in vivo generation of the superoxide, an ex vivo chemiluminescence assay was newly developed. When ICR mice were infected with a sublethal dose (8 x 10(4) CFU) of Salmonella typhimurium, the number of bacteria in the liver reached its peak at 5 days after infection (10(5.05) CFU/g of liver) and decreased thereafter. At 21 days after infection, the bacteria became undetectable. On the other hand, phorbol myristate 13-acetate-stimulated O2.- generation reached a maximum at 7 days after infection (mean photon count, 1,249 cps versus 28.8 cps before infection; n = 4) and decreased thereafter to a level similar to that before infection at 21 days after infection (28.8 cps). Histological examinations revealed that the total area of the lesions reached a peak at 7 days after infection (7.2 x 10(4) microns 2/10 visual fields). In the early phase, a microabscess with infiltration of polymorphonuclear cells was noted, and then, in the late stage, the lesion was replaced by granulation with mononuclear cell infiltration. When microscopic lesions were measured histologically, a significant correlation between the area of the lesions and phorbol myristate 13-acetate-stimulated O2.- generation was observed, which suggested that superoxide was responsible for the generation of the lesions. Modified superoxide dismutase, i.e., alpha-4-([6-(N-maleimido)hexanoyloxymethyl] cumyl)half-butyl-esterified poly(stylrene-co-malelic acid)-conjugated superoxide dismutase (SM-SOD), was then applied. When SM-SOD was administered to suppress the O2.- generation in vivo, the number of bacteria increased (10(6.1) CFU). However, the lesion formation was inhibited (total lesion area, 0.3 x 10(4) microns 2). These results suggest that the establishment of the microabscess and granuloma formation after S. typhimurium infection is not due to the bacteria per se but rather to the O2.- from the host's phagocytes. Two aspects of the O2.-, i.e., the bactericidal role and the tissue-injurious effect, were clearly demonstrated in this study. Therefore, the information obtained from these results is useful in designing treatment strategy for similar kinds of infection.

Animals↗
PubMed1995

Synthesis and protein tyrosine phosphatase inhibitory activity of dephostatin analogs.

We have synthesized derivatives of dephostatin, a protein tyrosine phosphatase (PTPase) inhibitor, to study the structure-activity relationships of this inhibitor. Inactive analogs revealed some insight into structural requirements or PTPase inhibitory activity of dephostatin. Both a nitroso group and phenolic hydroxyl groups were found to be essential for the inhibitory activity. Among the dephostatin derivative synthesized, one of the regioisomers of dephostatin showed PTPase inhibitory activity equivalent to that of dephostatin, and also had increased stability.

Enzyme Inhibitors↗
PubMed1995

Inhibition of phosphatidylinositol-specific phospholipase C activity by fluvirucin B2.

We isolated fluvirucin B2 from the culture broth of Streptomyces as an inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC). It inhibited PI-PLC of A431 cell cytosol with an IC50 of 1.6 micrograms/ml. Fluvirucin B2 also inhibited PI-PLC in cultured A431 cells, whereas it did not inhibit phosphatidylinositol synthesis and macromolecular synthesis markedly. It also inhibited epidermal growth factor-induced rapid rounding of A431 cells, in which PI turnover is involved.

Cells, Cultured↗
PubMed1995

Isolation from Streptomyces of a novel naphthoquinone compound, naphthablin, that inhibits Abl oncogene functions.

In the course of our screening for inhibitors of abl oncogene function, a culture filtrate of Streptomyces aculeolatus, induced normal flat morphology in v-abl-expressing NIH3T3 cells. The active substance was isolated through ethyl acetate extraction, silica gel column chromatography, and reverse-phase HPLC. Mass and NMR spectroscopy including HMBC revealed that it had a novel naphthoquinone structure with a monoterpene, and we named it "naphthablin". Naphthablin inhibited Abl-induced morphological transformation in v-ablts-NIH3T3 cells at around 30 micrograms/ml, and specifically inhibited RNA synthesis.

Cells, Cultured↗
PubMed1994

Localization of vitronectin- and fibronectin-receptors on cultured human glioma cells.

Utilizing a human astrocyte-derived glioma cell line, we have demonstrated the presence of a vitronectin receptor, alpha v beta 3, and a fibronectin receptor, alpha 5 beta 1, on the surface of the cells spreading on the respective adhesion molecules by immunohistochemical analyses. By phase-contrast microscopy, these receptors were found to be expressed predominantly in the focal contact-like area, suggesting that they were involved in the spreading of the cells upon contact with these adhesion molecules. Interestingly, they appeared to have differential functions and roles as integrins as evidenced by different time-dependent distribution profiles on the cell surface in the serum-containing medium. Furthermore, both vitronectin and fibronectin seem to have chemotactic effects onto the glioma cells as observed in a Boyden chamber study. Although these receptors are not expected to be present on the surface of astrocytes under physiological conditions, they may be expressed thereon and involved in gliosis when the cerebral vasculature is traumatized and, thereby, blood proteins, including vitronectin and fibronectin, come into contact with the astrocytes.

Cell Adhesion↗
PubMed1994

Inhibition by two lavendustins of the tyrosine kinase activity of pp60F527 in vitro and in intact cells.

The mutant pp60F527 protein possesses an activated protein-tyrosine kinase (PTK) activity correlated with a transforming activity. We have studied the inhibition of the pp60F527 PTK activity by two EGF-R tyrosine kinase inhibitors, lavendustin A and one of its derivatives, lavendustin C6. In vitro, both molecules were non-competitive inhibitors for the ATP binding site and uncompetitive inhibitors for the peptide binding site. The determined IC50S of the inhibition of pp60F527 kinase activity were 18 microM for lavendustin A and 5 microM for lavendustin C6, as determined on the exogenous substrate enolase, showing that lavendustin C6 was more potent than lavendustin A. Lavendustin C6, but not lavendustin A, inhibited the tyrosine phosphorylation of pp60F527 cellular substrates (the GAP-associated p190, pp125FAK and cortactin) in intact cells. However, this in situ inhibitory effect did not result in a reversion of the morphological changes induced by pp60F527 in cells. On the other hand, lavendustin C6 and lavendustin A exerted antiproliferative effects on cells, suggesting that inhibition of cellular targets related or not to the kinase was also possible.

3T3 Cells↗
PubMed1994

Induction of c-fos and c-myc oncogene expression in the pyloric mucosa of rat stomach by N-methyl-N'-nitro-N-nitrosoguanidine and taurocholate.

The induction of c-fos and c-myc expression in the pyloric mucosa of 8-week-old F344 male rats after oral administration of the glandular stomach carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or the tumor-promoter, taurocholate, was examined by Northern blotting. MNNG at doses of 5-50 mg/kg body weight dose-dependently induced transient increase of up to 30-fold c-fos expression with a maximum after 30 min, and of 8-fold c-myc expression with a maximum after 3 h. It also induced up to 3-fold increase in S-phase cells in the proliferation zone of the pyloric mucosa after 16 h. Similar effects were observed with sodium taurocholate at doses of 200-800 mg/kg body weight. These results suggest that c-fos and c-myc oncogenes play a role in stomach carcinogenesis.

Animals↗
PubMed1994

Inhibition of DNA topoisomerases I and II and induction of apoptosis by erbstatin and tyrphostin derivatives.

Inhibitors of protein tyrosine kinases (PTK) and DNA topoisomerases are potential antitumour agents. Drugs which bind to the ATP site of PTK, such as genistein, are common inhibitors to both types of enzymes. Eleven erbstatin and tyrphostin derivatives, which inhibit epidermal growth factor receptor PTK activity by competing with both the peptide substrate and ATP were tested for their capacity to inhibit DNA topoisomerases I and II. Erbstatin, two synthetic derivatives with a modified side chain and the tyrphostin AG 786 inhibited both topoisomerases in the same range of concentrations (20-50 microM). The tyrphostin AG 213 inhibited only topoisomerase II. In this series, absence of PTK inhibitory effect was correlated with the absence of DNA topoisomerase inhibition, while the detection of PTK inhibition may or may not be associated with DNA topoisomerase inhibition. In contrast to genistein, none of these molecules induced the stabilization of the topoisomerase-DNA cleavable complex, either in vitro or in vivo. Alcaline elution analysis revealed that erbstatin did not induce the formation of protein associated DNA strand breaks. However, an extensive degradation of the cellular DNA was observed which was shown to result from an internucleosomal fragmentation. Furthermore, typical morphological modifications associated with apoptosis were observed in the erbstatin treated cells by electron microscopy. These data indicate that erbstatin induces an apoptotic cell death.

Amino Acid Sequence↗
PubMed1994

Primary culture of human leptomeningeal cells in serum-free medium.

Human leptomeningeal (LM) cells were grown in serum-free medium supplemented with insulin, transferrin and bovine serum albumin. The cultured cells maintained the polygonal morphology characteristic of leptomeningeal cells. Electronmicroscopic and immunofluorescence examinations revealed interdigitation of cell membranes, invagination of cytoplasm into the nucleus, specialized intercellular junctions and the presence of cytokeratin. These characteristics are consistent with previous reports characterizing LM cells. This serum-free culture system may facilitate the in vitro study of the leptomeninges.

Arachnoid↗
PubMed1994

Enhancement of CDP-DG:inositol transferase activity in src- and erbB2-transformed cells.

Phosphatidylinositol (PI) synthesis was activated in Rous sarcoma virus-infected NIH3T3 or activated erbB2-transformed NIH3T3 cells. The in vitro activity of CDP-DG:inositol transferase prepared from these cells was also higher than that from normal parent NIH3T3 cells, although phospholipase C and PI kinase activities were not significantly different among these cells. A tyrosine kinase inhibitor, erbstatin, inhibited the PI synthesis in cultured cells, suggesting that Src and ErbB2-associated tyrosine kinases are involved in activation of CDP-DG:inositol transferase in these cell lines.

1-Phosphatidylinositol 4-Kinase↗
PubMed1994

Involvement of phosphatidylinositol synthesis in the regulation of S phase induction.

The addition of serum to quiescent normal rat kidney (NRK) cells induced phosphatidylinositol (PI) synthesis after 4 h and DNA synthesis after 16 h. Inostamycin, an inhibitor of CDP-DG:inositol transferase, added within 4 h, inhibited both the serum-induced PI synthesis and S phase entry. By contrast, inostamycin added 8 h after release from quiescence did not inhibit the S phase entry. Inostamycin did not affect the G2/M/G1 transition, and incubation with inostamycin induced cell synchronization at early G1 phase. Thus, PI synthesis is involved in the regulation of S phase induction.

Animals↗
PubMed1994

Differences in wound healing pattern between mature and immature brain behavior of extravasated serum protein.

To clarify the fate of extravasated serum protein and tissue reaction following blood brain-barrier breakdown in mature and immature brain, we produced cryogenic injury in the cortices of adult and post natal day 2 rats, and immunohistochemical examination with GFAP, vimentin and albumin, endogenous tracer of serum proteins, antibodies. Mature and immature brain showed the same histological changes by day 3. However, fibrotic scar, cyst formation and GFAP, vimentin-positive astrocytosis were main features in mature brain, fusion of adjoining cortical plate without scar and astrocytosis was typical in the immature brain. In adult rats, presence of albumin was observed near the lesion on day 1, evidently extending to the contralateral hemisphere on day 7 and localized again around scar and cyst on day 14. In the P2 rats, albumin was present in both hemispheres on day 1, but was localized to molecular layer periventricular region and choroid plexus on day 7. No albumin was detected on day 14. These results suggest that rapid spreading and clearance of extravasted serum proteins may take place in the immature brain and this may be deeply involved in the characteristic wound healing pattern.

Animals↗
PubMed1994

Protein tyrosine kinase inhibitors inhibit chemotaxis of vascular smooth muscle cells.

The effects of protein tyrosine kinase inhibitors on platelet-derived growth factor (PDGF)-induced chemotaxis in cultured rat aortic smooth muscle cells (SMCs) were investigated to elucidate the role of tyrosine phosphorylation in the chemotaxis of vascular SMCs. Two tyrosine kinase inhibitors, methyl 2,5-dihydroxycinnamate and genistein, inhibited PDGF-induced chemotaxis, the IC50 being 5 and 150 mumol/L, respectively. Methyl cinnamate and genistein partly inhibited the adhesion of SMC to collagen-coated dishes. A chemotaxis assay using double-well culture dishes revealed that both agents also inhibited cell migration after adhesion. H-7, a C kinase inhibitor, did not inhibit either chemotaxis or SMC adhesion at 100 mumol/L. Western blot analysis using anti-phosphotyrosine revealed that the tyrosine kinase inhibitors inhibited the tyrosine phosphorylation of at least two proteins of molecular weight 85 and 95 kD under our experimental conditions. An immunocytochemical study revealed that these inhibitors eliminated tyrosine phosphorylation along the cell margins; these agents also inhibited the reorganization of microtubules and stress fibers, both of which are involved in directional cell locomotion. These findings suggest that tyrosine kinases may play an important role in SMC chemotaxis.

Animals↗
PubMed1994

Antitumor effect of piericidin B1 N-oxide.

Piericidin B1 N-oxide was isolated from a culture broth of Streptomyces sp. as a novel inhibitor of phosphatidylinositol (PI) turnover. Piericidin B1 N-oxide specifically inhibited orthophosphate labeling of PI induced by epidermal growth factor (EGF) without affecting the formation of phosphatidic acid (PA). Like piericidins A1 and B1, piericidin B1 N-oxide inhibited ATP synthesis in A431 cells; however, the effect of piericidin B1 N-oxide on PI synthesis was stronger than that of piericidins A1 and B1. At the concentration inhibiting PI synthesis, piericidin B1 N-oxide showed no inhibitory effect on DNA, RNA, or protein synthesis. We also demonstrated that piericidin B1 N-oxide reversibly inhibited the growth of A431 cells in situ and suppressed the growth of Ehrlich carcinoma in vivo when administered to mice by intraperitoneal (ip) injection.

Adenosine Triphosphate↗
PubMed1993

Activation of the respiratory burst and tyrosine phosphorylation of proteins in human neutrophils: no direct relationship and involvement of protein kinase C-dependent and -independent signaling pathways.

Human neutrophils maximally stimulated with the optimal concentration (100 ng/ml) of phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), for 5 min at 37 degrees C did not respond with superoxide (O2-) release to the later addition of PMA itself or the Ca2+ ionophore ionomycin. However, these cells did respond with enhanced release of O2- to the later addition of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A (Con A). In these PMA-pretreated cells, an increase in cytoplasmic free Ca2+ ([Ca2+]i) induced by ionomycin was unaffected, whereas that induced by FMLP was inhibited by 50-60% and that induced by Con A was completely abolished. A 42-kDa protein was predominantly and consistently tyrosine-phosphorylated by FMLP, PMA and ionomycin with the different kinetics according to the stimuli. The dose-response curves showed that tyrosine phosphorylation and O2- release were stimulated in parallel by PMA, whereas tyrosine phosphorylation and an increase in [Ca2+]i, but not O2- release, were stimulated in parallel by FMLP or ionomycin. The potency of inducing tyrosine phosphorylation was ionomycin > FMLP = PMA, whereas the potency of triggering of O2- release was PMA > ionomycin = FMLP. UCN-01, a PKC inhibitor, inhibited O2- release and tyrosine phosphorylation induced by PMA, but not by FMLP or ionomycin. In contrast, pertussis toxin inhibited O2- release and tyrosine phosphorylation induced by FMLP, but not by PMA. Tyrosine kinase inhibitors (erbstatin and genistein) inhibited O2- release induced by FMLP, but not by PMA. However, both tyrosine kinase inhibitors did not impair FMLP- or PMA-induced tyrosine phosphorylation of a 42-kDa protein. Increased tyrosine phosphorylation of a 42-kDa protein was also detected in immature myeloid cells (HL-60 cells) stimulated by PMA, but not by ionomycin. These findings suggest that FMLP and Con A trigger the respiratory burst in human neutrophils by activating the definite pathway which include other signals than activation of PKC and an increase in [Ca2+]i; tyrosine phosphorylation of a 42-kDa protein is induced by the PKC-dependent and independent mechanisms according to the stimuli, and the PKC-independent and ionomycin-sensitive mechanism is inoperative in HL-60 cells; and tyrosine phosphorylation of a 42-kDa protein is unlikely to be causally related to activation of the respiratory burst.

Concanavalin A↗