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Biomedical subjects

K Kuwabara

Publications and source records attributed to K Kuwabara.

At least 73 records · Page 4Linked to original sources
PubMed1995

Calreticulin, an antithrombotic agent which binds to vitamin K-dependent coagulation factors, stimulates endothelial nitric oxide production, and limits thrombosis in canine coronary arteries.

Coagulation Factor IX/IXa has been shown to bind to cellular surfaces, and Factor IXa expresses its procoagulant activity by assembling into the intrinsic Factor X activating complex (Factors IXa/VIIIa/X), which also forms on membrane surfaces. This led us to identify cellular proteins which bind Factor IX/IXa; an approximately 55-kDa polypeptide was purified to homogeneity from bovine lung extracts based on its capacity to bind 125I-Factor IX in a dose-dependent and saturable manner. From protein sequence data of the amino terminus and internal peptides, the approximately 55-kDa polypeptide was identified as calreticulin, a previously identified intracellular calcium-binding protein. Recombinant calreticulin bound vitamin K-dependent coagulation factors, 125I-Factor IX, 125I-Factor X, and 125I-prothrombin (Kd values of approximately 2.7, 3.2, and 8.3 nM, respectively), via interaction with its C-domain, although it did not affect the coagulant properties of these proteins. 125I-Calreticulin also bound to endothelial cells in vitro (Kd approximately 7.4 nM), and mouse infusion studies showed an initial rapid phase of clearance in which calreticulin could be localized on the vascular endothelium. Exposure of endothelial cells to calreticulin led to dose-dependent, immediate, and sustained increase in the production of nitric oxide, as measured using a porphyrinic microsensor. In a canine electrically induced thrombosis model, intracoronary infusion of calreticulin (n = 7) prevented occlusion of the left circumflex coronary artery in a dose-dependent manner compared with vehicle-treated controls (n = 5). These results indicate that calreticulin interacts with the endothelium to stimulate release of nitric oxide and inhibit clot formation.

Animals↗
PubMed1995

Induction of karyotype instability in a murine tumor cell line by quercetin, 2-amino-1-methyl-6 phenylimidazo[4,5-b]pyridine, and okadaic acid, as revealed by transmission distortion of the inactive X chromosome.

Quercetin, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and okadaic acid are found in various foods and have been shown to have mutagenic or promoter-like activity. The effects of these three compounds on the transmission of the inactive X chromosome were examined in MST-C6 murine tumor cells, which were derived from hybrid F1 mice from matings between C57BL/6 and MSM mice. Polymerase chain reaction analysis using polymorphic markers on the X chromosome detected transmission distortion of the inactive X chromosome due to nondisjunction as a copy-number imbalance in allelic bands. The cells exposed to all three chemicals (but not untreated cells) exhibited such imbalances at high frequencies under exposure conditions similar to those in previous experiments in which tumor progression and recombination were observed. The cells also showed increased frequencies of tumor formation when subcutaneously injected. These results suggest that the three chemicals are capable of inducing transmission distortion of the inactive X chromosome and that such activity may be a causative factor in promoting the tumorigenicity of MST-C6 cells.

Animals↗
PubMed1994

Hypoxia/reoxygenation-mediated induction of astrocyte interleukin 6: a paracrine mechanism potentially enhancing neuron survival.

To elucidate mechanisms underlying neuroprotective properties of astrocytes in brain ischemia, production of neurotrophic mediators was studied in astrocytes exposed to hypoxia/reoxygenation (H/R). Rat astrocytes subjected to H/R released increased amounts of interleukin (IL) 6 in a time-dependent manner, whereas levels of tumor necrosis factor and IL-1 remained undetectable. IL-6 transcripts were induced in hypoxia and the early phase of reoxygenation, whereas synthesis and release of IL-6 antigen/activity occurred during reoxygenation. Elevated levels of IL-6 mRNA were due, at least in part, to increased transcription, as shown by nuclear runoff analysis. The mechanism stimulating synthesis and release of IL-6 antigen by astrocytes was probably production of reactive oxygen intermediates (ROIs), which occurred within 15-20 minutes after placing hypoxia cultures back into normoxia, as the inhibitor diphenyl iodonium inhibited the burst of ROIs and subsequent IL-6 generation (blockade of nitric oxide formation had no effect on ROI generation or IL-6 production). Enhanced IL-6 generation was also observed in human astrocytoma cultures exposed to H/R. Survival of differentiated PC12 cells exposed to H/R was potentiated by conditioned medium from H/R astrocytes, an effect blocked by neutralizing anti-IL-6 antibody. In a gerbil model of brain ischemia, IL-6 activity was lower in the hippocampus, an area sensitive to ischemia, compared with IL-6 activity in the cortex, an area more resistant to ischemia. IL-6 antigen, demonstrated immunohistochemically, was increased in astrocytes from ischemic regions of gerbil brain. These data suggest that H/R enhances transcription of IL-6, resulting in increased translation and release of IL-6 antigen after the burst of ROI generated early during reoxygenation. Release of IL-6 from astrocytes could exert a paracrine neurotrophic effect in brain ischemia.

Animals↗
PubMed1994

Receptor for advanced glycation end products (AGEs) has a central role in vessel wall interactions and gene activation in response to circulating AGE proteins.

The extended interaction of aldoses with proteins or lipids results in nonenzymatic glycation and oxidation, ultimately forming AGEs, the presence of which in the plasma and vessel wall is associated with diabetic vascular complications. We show here that AGE albumin in the intravascular space interacts with the vessel wall via binding to an integral membrane protein, receptor for AGE (RAGE), a member of the immunoglobulin superfamily, resulting in clearance from the plasma and induction of interleukin 6 mRNA. Intravenously infused 125I-AGE albumin showed a rapid phase of plasma clearance with deposition in several organs. Rapid removal of 125I-AGE albumin from the plasma was prevented by administration of a soluble, truncated form of RAGE, which blocked binding of 125I-labeled AGE albumin to cultured endothelial cells and mononuclear phagocytes, as well as by pretreatment with anti-RAGE IgG. Ultrastructural studies with AGE albumin-colloidal gold conjugates perfused in situ showed that in murine coronary vasculature this probe was taken up by endothelial plasmalemmal vesicles followed by transport either to the abluminal surface or by accumulation in intracellular vesicular structures reminiscent of endosomes and lysosomes. Consequences of AGE-RAGE interaction included induction of interleukin 6 mRNA expression in mice. These data indicate that RAGE mediates the interaction of AGEs with the vessel wall, both for removal of these glycated proteins from the plasma and for changes in gene expression.

Animals↗
PubMed1994

Methylation imprinting was observed of mouse mo-2 macrosatellite on the pseudoautosomal region but not on chromosome 9.

Mouse mo-2 macrosatellites consisting of 31-bp tandem repeat units are mainly located at two loci in the C57BL/6 genome, one being at the centromere-distal telomeric region of chromosome 9 and the other at the pseudoautosomal (PA) region of chromosomes X and Y. The two clusters constitute approximately 300 kb and 150 kb, respectively. Southern analysis of a methylation-sensitive enzyme, HpaII-digested DNA showed that the mo-2 macrosatellites are detected as more than 30 polymorphic bands. Comparison of those bands between reciprocally crossed F1 mice revealed that approximately 20% of the allele-specific fragments exhibit different band intensities depending on the sex of the parent of origin. The differential methylation is observed in the mo-2 macrosatellite on the PA region but not in that on chromosome 9. Several fragments including the 3.4-kb fragment without internal HpaII site are more clearly detected when paternally derived, suggesting that the male-derived macrosatellite is undermethylated. Interestingly, the difference is much more remarkable in inter-subspecific F1 mice between C57BL/6 and MSM than F1 between C57BL/6 and C3H/He. This suggests the presence of a modifier(s) that affect(s) the methylation of mo-2 in the MSM genome.

Animals↗
PubMed1994

Hypoxic induction of interleukin-8 gene expression in human endothelial cells.

Because leukocyte-mediated tissue damage is an important component of the pathologic picture in ischemia/reperfusion, we have sought mechanisms by which PMNs are directed into hypoxic tissue. Incubation of human endothelial cells (ECs) in hypoxia, PO2 approximately 14-18 Torr, led to time-dependent release of IL-8 antigen into the conditioned medium; this was accompanied by increased chemotactic activity for PMNs, blocked by antibody to IL-8. Production of IL-8 by hypoxic ECs occurred concomitantly with both increased levels of IL-8 mRNA, based on polymerase chain reaction analysis, and increased IL-8 transcription, based on nuclear run-on assays. Northern analysis of mRNA from hypoxic ECs also demonstrated increased levels of mRNA for macrophage chemotactic protein-1, another member of the chemokine superfamily of proinflammatory cytokines. IL-8 gene induction was associated with the presence of increased binding activity in nuclear extracts from hypoxic ECs for the NF-kB site. Studies with human umbilical vein segments exposed to hypoxia also demonstrated increased elaboration of IL-8 antigen compared with normoxic controls. In mice exposed to hypoxia (PO2 approximately 30-40 Torr), there was increased pulmonary leukostasis, as evidenced by increased myeloperoxidase activity in tissue homogenates. In parallel, increased levels of transcripts for IP-10, a murine homologue in the chemokine family related to IL-8, were observed in hypoxic lung tissue. Taken together, these data suggest that hypoxia constitutes a stimulus for leukocyte chemotaxis and tissue leukostasis.

Animals↗
PubMed1994

[Compression of medulla oblongata by the dissecting aneurysm of the vertebral artery 7 years after its rupture: case report].

A 56-year-old female, who suffered a subarachnoid hemorrhage (SAH) due to spontaneous dissection of the right vertebral artery 7 years previously, was admitted to our hospital with headache and vertigo. She hadn't had any attacks of SAH for 7 years. A magnetic resonance imaging (MRI) showed a high signal intensity mass and a low signal intensity due to calcification on the right ventrolateral surface of the medulla on both T1 and T2 weighted images. Vertebral angiography showed complete occlusion of the cervical segment of the right vertebral artery (VA). Left vertebral angiography didn't reveal any retrograde filling of the intracranial segment of the right VA through VA union. Thus, the spontaneous entrapment by dissection of the vertebral artery was demonstrated 7 years after SAH with MRI and serial angiography.

Aortic Dissection↗
PubMed1993

Active site-blocked factor Xa prevents thrombus formation in the coronary vasculature in parallel with inhibition of extravascular coagulation in a canine thrombosis model.

Factor Xa is a central procoagulant enzyme, linking the intrinsic and extrinsic activation mechanisms to the final common pathway of coagulation. To assess its contribution to pathologic thrombosis, studies were performed in a canine coronary thrombosis model. Thrombus formation was initiated by the application of electric current via a needle electrode placed in the lumen of the left circumflex coronary artery. When 50% occlusion of the vessel developed, the current was stopped and animals received an intravenous bolus of either saline, bovine glutamyl-glycinyl-arginyl-factor Xa (Xai), a competitive inhibitor of factor Xa assembly into the prothrombinase complex, Factor X, or heparin. Animals infused with saline or factor X (300 micrograms/kg) developed total occlusion of the vessel due to a fibrin/platelet thrombus in 70 +/- 11 minutes (36 of 36 animals) and 74 +/- 13 minutes (8 of 8 animals), respectively. In contrast, infusion of Xai prevented thrombus formation completely at a dose of 300 micrograms/kg (8 of 8 animals). As the dose of Xai was decreased, its antithrombotic effect was diminished, with a patency rate of only 2 of 6 animals at a dose of 90 micrograms/kg. Xai at 300 micrograms/kg prevented the accumulation of 125I-fibrinogen/fibrin at the site of the coronary thrombus by approximately 63% and decreased deposition of 111In-labeled platelets by approximately 57%. Hemostatic parameters of animals infused with Xai demonstrated prolongation of the PT and dose-dependent increased extravascular bleeding tendency. These data indicate that factor Xa has a comparably important role in thrombus formation and extravascular hemostasis, and contrast with previous results in this same animal model in which IXai selectively prevented clotting in the coronary vasculature.

Amino Acid Sequence↗
PubMed1993

A variant family of mouse minor satellite located on the centromeric region of chromosome 2.

A variant sequence of minor satellite that exhibits a polymorphism in strains and a subspecies of the mouse by pulsed-field gel analysis has been cloned. A segregation study using intrasubspecific backcross mice reveals that the variant maps to the centromeric region of chromosome 2. A long-range physical analysis of the region shows that the variant family is probably located between the proximal telomere and the major satellite. The sequence probe provides an important marker of the chromosome 2-specific centromere.

Animals↗
PubMed1993

Two mouse hypervariable minisatellites: chromosomal location and simultaneous mutation.

Pc-1 and Pc-2 hypervariable minisatellites of the mouse show a structural similarity and hence have been compared in terms of chromosomal location and mutability. Polymorphisms of the Pc-2 locus detected by analyses of Southern blotting and polymerase chain reaction were used for typing 61 backcross mice that were obtained by mating (C57BL/6 x MSM)F1 females to MSM males. Segregation patterns indicated that the Pc-2 locus maps to the 60 cM region of chromosome 6, while that of Pc-1 maps to chromosome 4. Simultaneous mutation of the two loci was examined in 101 F1 mice. Pc-1 and Pc-2 loci showed mutation rates of 14.4 and 5.0% per gamete, respectively. Of the nine mice with a Pc-2 mutation, four carried a Pc-1 mutation at the same maternally- or paternally-derived allele. The rate of comutation was 44% (4/9), higher than the 12.2% (24/197) of Pc-1 mutation alone. This suggests that the germline mutation of the two hypervariable minisatellites may result from a common mechanism.

Alleles↗
PubMed1992

Influence of oxidative stress on induced tolerance to ischemia in gerbil hippocampal neurons.

We investigated whether reversible oxidative stress induced by the administration of the superoxide dismutase inhibitor, diethyldithiocarbamate, could induce tolerance to subsequent cerebral ischemia in gerbil hippocampal neurons. Mature male gerbils received intraperitoneal injections of diethyldithiocarbamate (1.0 g/kg), which led to reduced superoxide dismutase activity and increases in thiobarbituric acid-reactive substance in the brain. Cerebral ischemia was produced by occluding the bilateral common carotid arteries for 5 min, either 2 or 4 days after diethyldithiocarbamate injection. One week after ischemia, samples from each brain were stained with hematoxylin-eosin to evaluate ischemic neuronal damage in the hippocampal CA1 sector. Diethyldithiocarbamate treatment 4 days before ischemia had significant protective effects against cerebral ischemia, while diethyldithiocarbamate 2-day pretreatment and vehicle treatment failed to show neuroprotection. Biochemical examinations showed a clear induction of heat shock protein 72 and a significant increase in manganese-containing superoxide dismutase in the hippocampus in animals treated with diethyldithiocarbamate 4 days prior to ischemia. These results suggested that the oxidative stress caused by diethyldithiocarbamate could induced tolerance to ischemia in the gerbil brain, and that the increase in the biosynthesis of manganese-containing superoxide dismutase and heat shock protein 72 could provide a biochemical explanation of the tolerance induced under these conditions.

Animals↗
PubMed1992

Anti-inflammatory properties of zinc protoporphyrin disodium (Zn-PP-2Na).

Anti-inflammatory properties of zinc protoporphyrin disodium (Zn-PP-2Na) were studied. Zn-PP-2Na exhibits anti-allergic action against type III and IV reactions (passive Arthus reaction in rats and tuberculin-induced footpad reaction in mice), but does not affect type I and II reactions (homologous passive cutaneous anaphylaxis in mice and reversed cutaneous anaphylaxis in rats). Zn-PP-2Na also clearly inhibits type II collagen-induced arthritis in mice. The agent inhibits general arthritis symptoms, anti-type-II collagen antibody production and type II collagen-induced delayed type hypersensitivity (DTH) in arthritic mice. Zn-PP-2Na, however, did not affect carrageenin-induced paw edema and histamine- and serotonin-induced skin reactions in rats. Zn-PP-2Na inhibits IL-1-induced mouse lymphocyte proliferation, but does not affect PMA-induced O2- generation from guinea-pig neutrophil. These results indicate that Zn-PP-2Na inhibits type II collagen-induced arthritis in mice due to the antagonism of IL-1 activity and the inhibition of DTH against type II collagen.

Anaphylaxis↗
PubMed1992

Hypoxia-induced increased permeability of endothelial monolayers occurs through lowering of cellular cAMP levels.

Prolonged exposure to hypoxia, as at high altitude, results in increased vascular permeability that may be ameliorated by administration of glucocorticoids. To understand mechanisms underlying these observations, cultured bovine aortic and pulmonary artery endothelial cells (ECs) were subjected to hypoxia, and changes in monolayer permeability and adenosine 3',5'-cyclic monophosphate (cAMP) levels were assessed. Exposure of both types of cultured ECs to hypoxia (PO2 approximately 14 Torr) led to a time- and dose-dependent increase in monolayer permeability, as measured by diffusion of radiolabeled solutes, which was associated with a progressive decrease in EC cAMP levels from 60 to 15 pmol/mg protein, and a decrease in EC adenylate cyclase activity. The change in endothelial barrier function was prevented by addition of cAMP analogues. Pertussis toxin protected EC monolayers from hypoxia-mediated increase in permeability while maintaining cAMP levels and adenylate cyclase activity. Addition of dexamethasone to EC monolayers before or simultaneously with their incubation under hypoxic conditions blocked the hypoxia-mediated increase in monolayer permeability. Dexamethasone pretreatment also prevented the decline in cAMP and adenylate cyclase levels in oxygen-deprived cultures. These data indicate that hypoxia decreases EC barrier function by lowering adenylate cyclase activity and cellular cAMP levels. They suggest that dexamethasone may exert its protective effect, in part, by preventing the hypoxia-induced decline in adenylate cyclase activity, leading to an increase in cellular cAMP and maintenance of EC barrier function.

Animals↗
PubMed1992

Synthesis and release of interleukin 1 by reoxygenated human mononuclear phagocytes.

To examine the possible involvement of cytokines in reperfusion injury, we have studied production of IL-1 by human vascular cells, including smooth muscle and mononuclear phagocytes. Exposure of cells to hypoxia (pO2 approximately 14 torr) followed by reoxygenation led to significant release of IL-1 only from the mononuclear phagocytes. Elaboration of IL-1 was dependent on the oxygen tension and duration of hypoxia (optimal at lower pO2s, approximately 14-20 torr, and after 9 h), as well as the time in reoxygenation (maximal IL-1 release at 6-9 h). Although a period of hypoxia was necessary for subsequent IL-1 production during reoxygenation of either peripheral blood monocytes or cultured monocyte-derived macrophages, no IL-1 release occurred during the hypoxic exposure. IL-1 released during reoxygenation was newly synthesized, and its production was triggered by the generation of oxygen free radicals, as it could be blocked by the addition of either allopurinol or free radical scavengers to cultures and could be stimulated in part by low concentrations of hydrogen peroxide or xanthine/xanthine oxidase. The potential pathophysiological effects of IL-1-containing supernatants from reoxygenated macrophages was shown by their induction of endothelial tissue factor and enhancement of endothelial adhesiveness for neutrophils, both of which could be blocked by anti-IL-1 antibody. The relevance of IL-1 to hypoxia/reoxygenation in vivo was suggested by the presence of circulating nanogram amounts of this cytokine in the plasma of mice during the reoxygenation period following a hypoxia.

Animals↗
PubMed1991

Hypoxia induces endothelial cell synthesis of membrane-associated proteins.

Hypoxemia is associated with a prothrombotic tendency. In this study we report the purification and partial characterization of an activator of a central coagulation component, factor X, induced in endothelium by exposure to hypoxia (hypoxia-induced factor X activator or Xact). Expression of Xact occurred in a reversible manner when endothelial cell cultures were exposed to hypoxia or sodium azide but not in response to a variety of other alterations in the cellular milieu, such as heat shock or glucose deprivation. The activity of Xact, which was not detected in normoxic endothelial cells, was maximal under acidic conditions, pH 6.0-6.8, which often coexist with hypoxia in an ischemic milieu. By sequential isoelectric focusing and preparative SDS/PAGE of endothelial membrane-rich fractions, Xact was purified approximately 19,000-fold and found to be a single-chain, approximately 100-kDa polypeptide with pI approximately 5.0. Activation of factor X by purified Xact was not affected by blocking antibodies to other coagulation proteins or by phenylmethylsulfonyl fluoride or leupeptin but was prevented by mercury chloride or iodoacetamide. In addition to the induction of Xact, two-dimensional gel analysis of membrane fractions from metabolically labeled hypoxic endothelial cultures revealed two groups of approximately 10 additional spots: (i) a group for which expression was maximal after 24 hr and (ii) a group for which expression continued to increase up to 48 hr. The pattern of hypoxia-mediated modulation of protein expression was distinct from that seen with other cellular stimuli but could be duplicated, in part, by sodium azide. These results indicate that hypoxia elicits a specific biosynthetic response, including the expression of endothelial cell-surface molecules that can alter cellular function and may potentially serve as markers of hypoxemic vessel-wall injury.

Animals↗
PubMed1991

'Ischemic tolerance' phenomenon detected in various brain regions.

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the 'ischemic tolerance' phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of 'ischemic tolerance'.

Animals↗