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Biomedical subjects

J Kambayashi

Publications and source records attributed to J Kambayashi.

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

Platelet talin is phosphorylated by calyculin A.

Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin.

Alkalies↗
PubMed1995

Involvement of protein phosphatase-1 in cytoskeletal organization of cultured endothelial cells.

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effects of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vein endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different according to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton.

Actins↗
PubMed1995

Suppression of pseudointimal hyperplasia by a novel prostacyclin analogue: beraprost.

The aim of this study was to evaluate the effect of a novel prostaglandin I2 analogue, beraprost (BPT), on the pseudointimal hyperplasia (PIH) of polytetrafluoroethylene (PTFE) prostheses. A total of 12 rabbits were equally divided into three groups. The control group was given a placebo daily, group 1 was given BPT orally 2 mg/kg per day, and group 2 was given BPT orally 4 mg/kg b.i.d. Exactly 1 cm of the inferior vena cava was resected and replaced by a 3-cm PTFE tube graft. All the grafts were patent when harvested 4 weeks after implantation, but the lumens were narrowed to various extents by PIH. PIH, determined by the dry weight of the intraluminal tissue deposit, was significantly (P < 0.01) suppressed in groups 1 and 2 compared with the control group. High-magnification light microscopy with various staining methods revealed the PIH to be composed mainly of smooth muscle cells (SMCs) and collagen fibrils in all three groups. Transmission electron microscopy revealed that the majority of SMCs in groups 1 and 2 were contractile in form, in contrast with the synthetic form seen in the control group. In conclusion, BPT attenuated the PIH of PTFE grafts by inhibiting the phenotype change in the SMCs.

Animals↗
PubMed1995

PGI2 analogue, sodium beraprost, suppresses superoxide generation in human neutrophils by inhibiting p47phox phosphorylation.

Sodium beraprost, a newly synthesized PGI2 analogue inhibited in a dose-dependent manner formyl-methionyl-leucyl-phenylalanine (fMLP) induced superoxide generation of human neutrophils, but it had no effect on the superoxide synthesis by phorbol myristate acetate (PMA) or A23187. Sodium beraprost inhibited Ca2+ influx in fMLP stimulated neutrophils employing fluorometry and confocal microscopy. These findings suggested that the inhibitory effect of sodium beraprost on fMLP induced superoxide generation was due to suppression of Ca2+ influx. To examine the relationship between the effect of sodium beraprost and phosphorylation of p47phox (the 47kDa cytosolic phagocyte oxidase factor), immuno-precipitation of p47phox and western blotting for phospho-amino acids were performed. Phosphorylation of serine residues of p47phox induced by fMLP was reduced in the presence of sodium beraprost in a dose-dependent manner. The reduction in phosphorylation was accompanied by a reduction in p47phox and p67phox translocation to the plasma membrane and superoxide generation. These findings suggested that p47phox phosphorylation was necessary for translocation and superoxide generation in fMLP activated neutrophils, and that p47phox phosphorylation was regulated by a Ca2+ dependent mechanism. These observations suggested that sodium beraprost inhibited fMLP induced superoxide generation of human neutrophils by the inhibition of p47phox phosphorylation and translocation by a Ca2+ dependent mechanism.

Calcimycin↗
PubMed1995

Dissecting thoracic aorta and fusiform aneurysm of the abdominal aorta.

A 59-year-old woman with dissection of the thoracic aorta and a fusiform aneurysm of the abdominal aorta without evidence of Marfan's syndrome underwent aneurysmorrhaphy with a bifurcated expanded polytetrafluoroethylene graft. Histological specimens of the aneurysmal wall revealed the presence of idiopathic cystic medial necrosis. As typical findings of idiopathic cystic medial necrosis in the aortic wall are very rare except in cases of Marfan's syndrome, the present case is reported and the implications of this condition are discussed.

Aortic Dissection↗
PubMed1995

Contained rupture of infected abdominal aortic aneurysm due to systemic candidiasis.

A case is reported of contained rupture of an infected abdominal aortic aneurysm as a result of vascular catheter-associated candidiasis. Candida was identified in the mural thrombus taken from the aneurysm during surgery. Since Candida has played an important role in nosocomial infection and catheter-associated sepsis during the past decade, it is becoming an increasingly significant pathogen in microbial arteritis. Successful treatment included axillobifemoral bypass grafting followed by a complete excision of the infected aneurysm, retroperitoneal drainage and long-term antifungal therapy. Although infected abdominal aortic aneurysm is not common, without surgical intervention it may lead to serious consequences, such as rupture or uncontrollable sepsis. As the clinical symptoms of this disease are minimal and non-specific during the early stages, a high index of suspicion for this condition is essential for precise diagnosis and successful treatment. Moreover, initial treatment of candidiasis is especially important to prevent this rare but fatal late complication, infected abdominal aortic aneurysm.

Aneurysm, Infected↗
PubMed1995

Clinical evaluation on combined administration of oral prostacyclin analogue beraprost and phosphodiesterase inhibitor cilostazol.

Among various oral antiplatelets, a combination of a novel prostacyclin analogue beraprost (BPT) and a potent phosphodiesterase inhibitor cilostazol (CLZ) may result in untoward clinical effects due to possible synergistic elevation of intracellular cAMP (cyclic adenosine 3',5'-monophosphate). Thereby, a clinical study of the combined administration of the two agents was attempted. Twelve healthy volunteers were assigned to take BPT/CLZ in the following schedule; BPT: 40 micrograms at day 1 and 120 micrograms t.i.d. from day 7 to 14, CLZ: 200 mg t.i.d. from day 3 to 14. At various time intervals, physical examination and blood collection for ex vivo platelet aggregation and determination of intraplatelet cAMP were performed. Throughout the observation period, no significant alteration in vital signs was observed. Seven out of 12 subjects experienced headache of a short duration accompanying facial flush in one and nausea in one, especially after ingestion of CLZ. All of these symptoms, probably caused by the vasodilating effect of the two agents, were of mild degree and no special treatment was required. Intraplatelet cAMP content was gradually but significantly increased to 9.84 +/- 4.59 pmol per 10(9) platelets at day 14 in comparison with the initial value (6.87 +/- 2.25 pmol). The platelet aggregability was significantly suppressed at various time intervals but no additive or synergistic inhibitory effect by the combined administration was noted. In conclusion, the combined administration of BPT/CLZ is safe at doses used in the study, though the beneficial clinical effect of the combined administration has yet to be elucidated.

Adult↗
PubMed1995

An infection-resistant PTFE vascular graft; spiral coiling of the graft with ofloxacin-bonded PTFE thread.

OBJECTIVE: To develop an infection-resistant polytetrafluoroethylene (PTFE) vascular graft for potential clinical use in grafting in sites of bacterial contamination and in replacement of the infected grafts. SETTING: Experimental study in rabbits. MATERIALS AND METHODS: An antibiotic ofloxacin (OFLX) was bonded to a sheet of PTFE by impregnation, which was cut and twisted into fine threads. The in-vitro antibacterial activity of OFLX-PTFE thread was determined by measuring the zone of growth inhibition against Escherichia coli. The thread was spirally coiled around a ridged outerwall PTFE to make the OFLX-PTFE graft. OFLX-PTFE graft or control graft was interposed in the inferior vena cava (IVC) of rabbits and the entire graft was covered with fibrin containing a fixed number of E. coli. Three or 7 days after the grafting, the grafts with perigraft tissue were harvested and subjected to bacteriological studies. RESULTS: In spite of early phase rapid elution of OFLX, a significant antibacterial activity was retained for more than 2 weeks. The antibacterial activity of OFLX-PTFE threads implanted in the subcutaneous space of rabbits decreased to 48% after 24 h and to approximately 1% after a week. The swab culture of all the control grafts was positive, while only one of 13 PTFE-OFLX grafts was positive. The number of viable bacteria in the perigraft tissue of OFLX-PTFE grafts was remarkably low in comparison with that of control grafts. Thus, the OFLX-PTFE grafts exhibited a marked in-vivo antibacterial activity. CONCLUSION: By a unique method, it was possible to furnish PTFE graft with an excellent infection-resistant property, without affecting the original biological behaviour.

Animals↗
PubMed1995

Healing of polytetrafluoroethylene vascular grafts analyzed by anti-smooth muscle myosin heavy chain isoforms.

The purpose of this study was to characterize the cellular contributions to the formation of the pseudointima (PI) using immuno-histochemistry with antibodies to specific smooth muscle myosin isoforms, SM1, SM2, SMemb, and to alpha-smooth muscle actin. The outer wall of polytetrafluoroethylene tube grafts were coated with a 10-microns silicon film except for 5 mm of the midportion and the grafts were implanted into the inferior vena cava of rabbits. The PI in the anastomotic area was mainly formed by dedifferentiation and redifferentiation of medial smooth muscle cells of the host vessel, while PI in the midgraft was formed by differentiation of myofibroblasts into smooth muscle cells in the internodal space.

Animals↗
PubMed1995

Involvement of dysplasminogenemia in occurrence of deep vein thrombosis.

Although various hematological disorders have been considered to be etiologic factors in deep vein thrombosis (DVT), the involvement of dysplasminogenemia (DPG) in DVT has not been studied in detail. In 72 consecutive DVT patients, the presence of DVT was suspected based on a history of lower limb swelling and tenderness with acute onset, and was confirmed by duplex scanning, radioisotope venography, or contrast venography. DPG was identified by the observation of dissociation between the activity and antigenicity of plasminogen. Of the 72 patients, 9 (12.5%) were diagnosed as having DPG, and several antithrombin-III deficiency and protein C and S deficiency were identified. The mean age of the genetically normal and DPG patients was 52 +/- 15 and 40 +/- 15 years, respectively (p < 0.05). Thus, these findings suggest that DPG is deeply related to the development of DVT, and that abnormality of the fibrinolytic system is one of the major etiologic factors in DVT.

Adult↗
PubMed1995

Effect of recombinant human erythropoietin on anaemia after gastrectomy: a pilot study.

OBJECTIVE: To evaluate the role of recombinant human erythropoietin in reducing the need for homologous blood transfusion during operations by studying its effect on the recovery of postoperative anaemia. DESIGN: Randomised controlled trial. SETTING: University hospital, Japan. SUBJECTS: 10 patients with gastric cancer undergoing distal gastrectomy. INTERVENTIONS: 5 Patients were given erythropoietin 200 IU/kg/day together with ferric pyrophosphate 40 mg/day intravenously for seven days before operation and 14 days afterwards, and 5 were given ferric pyrophosphate 40 mg/day alone (control group). MAIN OUTCOME MEASURES: Packed cell volume, haemoglobin concentration, and white and red cell counts. RESULTS: There was no significant change in packed cell volume after the operation in the erythropoietin group, but in the control group it dropped from a mean (SD) of 0.378 (0.074) before operation to 0.329 (0.068) on day 1 (p < 0.05). Haemoglobin concentrations were significantly higher in the erythropoietin group than the control group on day 7 (mean (SD) 137 (14) compared with 110 (19) p < 0.05), and on day 10 (140 (9) compared with 108 (15) p < 0.01) after operation. CONCLUSION: Erythropoietin prevented postoperative anaemia after gastrectomy as judged by packed cell volume, haemoglobin concentration, and red cell count. Erythropoietin given before and after operation therefore has the potential to reduce the need for homologous blood transfusion during and after major operations.

Adult↗
PubMed1994

Suppression by antithrombotic agents of pseudointimal hyperplasia of polytetrafluoroethylene graft implanted into venous system.

The lumen of the polytetrafluoroethylene graft (PTFE) implanted into a rabbit inferior vena cava (IVC) was markedly narrowed by 4 weeks after grafting. This was due to initial thrombosis on the luminal surface of the graft, which was followed by pseudointimal hyperplasia (PIH). To elucidate the role of the initial thrombosis in subsequent PIH, the effect of ticlopidine (T) and/or warfarin (W) on PIH was studied in an animal model. A PTFE tube graft was implanted into a rabbit IVC. Twenty eight rabbits were randomly assigned to the following experimental groups. Eight rabbits without antithrombotic agents were observed for 2 weeks (A, n = 4) and 4 weeks (B, n = 4) after grafting. T (100 mg/kg/day) and W (0.33 mg/kg/day) were orally administered for 4 weeks to group C (n = 4) and group D (n = 4), respectively. A combination of a half dose of T and W ((T + W)/2) was given for 2 weeks (E, n = 4) and 4 weeks (F, n = 4) after grafting. Four rabbits in group G received the combination of T and W for the first 2 weeks and were observed for an additional 2 weeks without medication. All the grafts were patent at time of harvest. The dry weight of the intraluminal deposit (DW) was determined as an indicator of PIH (A:35 +/- 1 mg/graft, B:40 +/- 8, C:22 +/- 3, D:22 +/- 3, E:14 +/- 2, F:15 +/- 3, G:23 +/- 2). Administration of T or W was equally effective in reducing DW and (T+W)/2 was more effective than a single agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The role of protein phosphorylation and cytoskeletal reorganization in microparticle formation from the platelet plasma membrane.

Platelets activated by various agonists produce vesicles (microparticles; MPs) from the plasma membrane. However, the mechanism of this MP formation remains to be elucidated. To investigate the possible involvement of protein phosphorylation and cytoskeletal reorganization in MP formation, the effects of various inhibitors on MP formation were investigated. Flow cytometry was employed to detect the amount of MP formation by using monoclonal antibodies against glycoprotein (GP) IIb-IIIa (NNKY 1-32) or GPIIb (Tab). The relationship between changes in cytoskeletal architecture and MP formation in the platelets activated by thrombin plus collagen was observed by scanning electron microscopy (SEM). MPs were observed in the vicinity of the terminals of pseudopods, suggesting that MPs may be related by budding of the pseudopods. Cytochalasin D (10 microM) inhibited MP formation from the activated platelets almost completely. Moreover, SEM of the cytochalasin D-treated platelets revealed the absence of shape change, pseudopod formation and MPs. These findings suggest that cytoskeletal reorganization is necessary for MP formation. Since cytoskeletal reorganization is considered to be regulated by a dynamic phosphorylation-dephosphorylation process, we investigated the effects of the protein phosphatase inhibitors, calyculin A (CLA) and okadaic acid (OA), on MP formation. Flow cytometry showed that these two inhibitors doubled MP formation in activated platelets. SEM of the platelets treated with CLA or OA demonstrated more prominent shape change and pseudopod formation in these platelets than in those without inhibitor. From these results, we conclude that cytoskeletal reorganization, which is controlled by phosphorylation, is involved in MP formation.

Adult↗
PubMed1994

Potential etiologic role of PAF in two major septic complications; disseminated intravascular coagulation and multiple organ failure.

A possible role of platelet-activating factor (PAF) in the occurrence of the two septic complications, i.e., disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) was investigated, employing a rabbit model and a novel PAF antagonist E5880. By an instillation of fecal suspension into the common bile duct of the rabbit, manifestations of DIC and MOF were observed with high reproducibility by 9 hours after the septic insult. E5880 was intravenously administered to 12 rabbits for 1 hour after the septic insult at dose of 1 mg/kg (n = 6) or 3mg/kg (n = 6). All the rabbits were subjected to observation of vital signs and serial determination of laboratory tests for 9 hours and then lung, liver and kidney were removed for histological examination. Blood endotoxin level increased significantly by 9 hours after the septic insult. Although administration of E5880 did not affect the endotoxemia, the antagonist attenuated in a dose related manner laboratory manifestation of DIC such as thrombocytopenia and prolonged prothrombin time as well as that of MOF such as increase in serum bilirubin and creatinine level. The beneficial effect of E5880 on MOF was also confirmed by the histological evaluation. These observations indicated that PAF is deeply involved in the occurrence of DIC and MOF due to sepsis and E5880 may be one of the modalities to treat or prevent these two major septic complications.

Alanine Transaminase↗
PubMed1994

A new method of purification and sensitive bioassay of platelet-activating factor (PAF) in human whole blood.

There is no satisfactory assay procedure of PAF in human whole blood in terms of sensitivity, reproducibility and simplicity. This is due to coexisting lipids from plasma and cellular membranes which inhibit measurement of PAF in various assay procedures, including bioassay. In the present study, an attempt was made to eliminate these interfering lipid inhibitors from blood samples. Lipids in human whole blood were extracted according to the method of Bligh & Dyer and the organic layer was dried under a stream of nitrogen. Then, the dried organic layer was dissolved in diethyl-ether and the solution was kept at -20 degrees C which was then centrifuged. The resulting supernatant was then applied to an anion-exchange column and the PAF fraction was obtained by step-wise gradient elution. The fraction was further purified by normal phase HPLC. Then PAF in the final sample was determined by sensitive bioassay using rabbit platelets containing fibrinogen and epinephrine. The recovery rate of PAF throughout this procedure was constant and satisfactory (37.4 +/- 9.7%), which was confirmed using [3H]-PAF. The lower limit of the present assay was estimated to be 5pg in 1 ml of blood and it was sensitive enough to detect PAF in blood samples from healthy volunteers and patients with sepsis or liver cirrhosis. Furthermore, attempts were made to compare the sensitivity and the recovery of our method with these of a commercially available radioimmunoassay (RIA) kit for PAF. However, it was not possible to detect any amount of authentic PAF added to whole blood.

Animals↗
PubMed1994

Quantitative determination of circulating platelet microparticles by flow cytometry.

Platelet microparticles are vesicles derived from the plasma membrane of platelets activated by a variety of agonists. To assess the extent of platelet activation, we recently developed a new and simple method of quantitating circulating microparticles in plasma by flow cytometry using the murine monoclonal antibody against glycoprotein IIb-IIIa (NNKY1-32). Using 2-microns fluorescent calibration beads, we succeeded in evaluating the absolute level of circulating microparticles. We determined the microparticle concentrations in 10 patients with arterial occlusive disease and in nine normal healthy donors, to determine whether the detection of microparticles would be useful in assessing the degree of platelet activation in clinical disorders. Microparticle levels were significantly higher in patients not treated with anti-thrombotic drugs than in normal healthy donors (P < 0.001), but not significantly higher in patients with vascular prostheses who had received anti-thrombotic drugs. These observations suggest the usefulness of microparticle quantitation in assessing the platelet activation in arterial occlusive diseases.

Adult↗
PubMed1994

Subendothelial layer of pseudointima of polytetrafluoroethylene graft is formed by transformation of fibroblasts migrated from extravascular space.

A well organised pseudointima is formed in polytetrafluoroethylene (PTFE) grafts within 4 weeks after implantation into inferior vena cava (IVC) of rabbits. To investigate the process of the subendothelial organisation of pseudointima, an animal experiment was conducted. The outer wall of PTFE graft (30 microns fibril length, 3 mm inner diameter, 3 cm long) was coated with 10 um silicon film in the following ways to prevent cellular ingrowth from the extravascular space: non-coating; full-length coating; half-length coating; and full-length coating excluding 5 mm midportion. These grafts were implanted into rabbit IVC and were harvested 4 weeks later. All the grafts were patent but the lumen of the non-coated area was narrowed by pseudointimal hyperplasia. The degree of the hyperplasia estimated by dried tissue deposit was inversely proportional to the length of the coating. The coverage of the luminal surface with endothelial-like cells was noted at anastomotic areas and also at the surface corresponding to the non-coated area. Light microscopy and immunostaining studies on the non-coated midportion revealed the presence of fibroblasts in the interstices of PTFE and smooth muscle cells and myofibroblasts in the pseudointima. Transmission electron microscopy confirmed the presence of myofibroblasts in the midportion of the non-coated area. No transmural capillary ingrowth was observed in the midportion by histological and immunohistochemical analysis. These observations suggest that the subendothelial layer of pseudointima in PTFE grafts is formed by proliferation and transformation of fibroblasts migrating from the extravascular space and that endothelial-like cells may also be derived from such transformation.

Animals↗