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Biomedical subjects

K Fukunaga

Publications and source records attributed to K Fukunaga.

At least 127 records · Page 7Linked to original sources
PubMed1999

[Leukocytapheresis for ulcerative colitis].

Leukocytapheresis(LCAP) and Granulocytapheresis(GCAP) are classified as extracorporeal circulation therapy(ECCT). These therapies are a novel, effective way to treat patients with ulcerative colitis(UC). During 7 weeks of intensive therapy(LCAP weekly, predonisolone(PSL) 30-80 mg/day), UC patients treated with LCAP revealed significant improvements on their subjective and objective symptoms compared to patients treated with PSL intravenous administration. Moreover, LCAP has been recognized as safer than other drugs for UC. In our previous study, only 9.9% out of 1,978 LCAP sessions showed some side effects, and most were mild and temporary. Therefore, LCAP could be the first choice to treat UC patients who resist and/or reveal severe complications against ordinary drug therapies.

Adolescent↗
PubMed1998

Serine/threonine phosphatase activity of calcineurin is inhibited by sodium orthovanadate and dithiothreitol reverses the inhibitory effect.

Orthovanadate is known to be an inhibitor of protein tyrosine phosphatases. However, we found that it inhibited calcineurin which has the activity of a serine/threonine protein phosphatase, using casein phosphorylated by cyclic AMP-dependent protein kinase as a substrate. Orthovanadate inhibits the Mn2+-activated activity of purified calcineurin to 20%; this is not the case without Mn2+. Furthermore, 10 mM dithiothreitol (DTT) reversed the inhibitory effects of orthovanadate. Orthovanadate showed the same inhibitory effect for calcineurin activity in homogenates as for the purified enzyme; the inhibitory effect was reversed by DTT. These results indicate that orthovanadate inhibits not only protein tyrosine phosphatases as reported, but also serine/threonine phosphatase activity of calcineurin.

Animals↗
PubMed1998

Lysophosphatidic acid-induced association of SHP-2 with SHPS-1: roles of RHO, FAK, and a SRC family kinase.

SHPS-1 is an approximately 120 kDa glycosylated receptor like protein that contains three immunoglobulin-like domains in its extracellular region as well as four potential tyrosine phosphorylation and SRC homology 2 (SH2) domain binding sites in its cytoplasmic region. Lysophosphatidic acid (LPA) stimulated the rapid tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2, a protein tyrosine phosphatase containing SH2 domains in Rat-1 fibroblasts. LAP-induced tyrosine phosphorylation of SHPS-1 was inhibited by Clostridium botulinum C3 exoenzyme (which inactivates RHO) but not by pertussis toxin. The protein kinase C activator phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) also stimulated tyrosine phosphorylation of SHPS-1; however, down-regulation of protein kinase C by prolonged exposure of cells to TPA did not affect LAP-induced tyrosine phosphorylation of SHPS-1. LPA-induced tyrosine phosphorylation of SHPS-1 was markedly reduced in either focal adhesion kinase (FAK)-deficient mouse cells or CHO cells overexpressing the tyrosine kinase CSK. Overexpression of a catalytically inactivate SHP-2 markedly inhibited MAP kinase activation in response to low concentrations of LPA in CHO cells, whereas overexpression of a wild-type SHPS-1 did enhance this effect of LPA. Furthermore, MAP kinase activation in response to a low concentration of LPA was inhibited by botulinum C3 exoenzyme. These results indicate that LPA-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2 may be mediated by a RHO-dependent pathway that includes FAK and a SRC family kinase. Thus, in addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, the formation of a complex between SHPS-1 and SHP-2 may, in part, play an important role in the activation of MAP kinase in response to low concentrations of LPA.

ADP Ribose Transferases↗
PubMed1998

Integrin-mediated tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Roles of Fak and Src family kinases.

SHPS-1 is a receptor-like glycoprotein that undergoes tyrosine phosphorylation and binds SHP-2, an Src homology 2 domain containing protein tyrosine phosphatase, in response to various mitogens. Cell adhesion to extracellular matrix proteins such as fibronectin and laminin also induced the tyrosine phosphorylation of SHPS-1 and its association with SHP-2. These responses were markedly reduced in cells overexpressing the Csk kinase or in cells that lack focal adhesion kinase or the Src family kinases Src or Fyn. However, unlike Src, focal adhesion kinase did not catalyze phosphorylation of the cytoplasmic domain of SHPS-1 in vitro. Overexpression of a catalytically inactive SHP-2 markedly inhibited activation of mitogen-activated protein (MAP) kinase in response to fibronectin stimulation without affecting the extent of tyrosine phosphorylation of focal adhesion kinase or its interaction with the docking protein Grb2. Overexpression of wild-type SHPS-1 did not enhance fibronectin-induced activation of MAP kinase. These results indicate that the binding of integrins to the extracellular matrix induces tyrosine phosphorylation of SHPS-1 and its association with SHP-2, and that such phosphorylation of SHPS-1 requires both focal adhesion kinase and an Src family kinase. In addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, SHP-2 may play an important role, partly through its interaction with SHPS-1, in the activation of MAP kinase in response to the engagement of integrins by the extracellular matrix.

Animals↗
PubMed1998

Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation.

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathione S-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.

Amino Acid Sequence↗
PubMed1998

Role of MAP kinase in neurons.

Extracellular stimuli such as neurotransmitters, neurotrophins, and growth factors in the brain regulate critical cellular events, including synaptic transmission, neuronal plasticity, morphological differentiation and survival. Although many such stimuli trigger Ser/Thr-kinase and tyrosine-kinase cascades, the extracellular signal-regulated kinases, ERK1 and ERK2, prototypic members of the mitogen-activated protein (MAP) kinase family, are most attractive candidates among protein kinases that mediate morphological differentiation and promote survival in neurons. ERK1 and ERK2 are abundant in the central nervous system (CNS) and are activated during various physiological and pathological events such as brain ischemia and epilepsy. In cultured hippocampal neurons, simulation of glutamate receptors can activate ERK signaling, for which elevation of intracellular Ca2+ is required. In addition, brain-derived neurotrophic factor and growth factors also induce the ERK signaling and here, receptor-coupled tyrosine kinase activation has an association. We describe herein intracellular cascades of ERK signaling through neurotransmitters and neurotrophic factors. Putative functional implications of ERK and other MAP-kinase family members in the central nervous system are give attention.

Animals↗
PubMed1998

Establishment of monolayer culture of pig pancreatic endocrine cells by use of nicotinamide.

A method for the isolation and primary monolayer culture of adult pig pancreatic endocrine (PE) cells was established. Cells were dissociated from the pancreas by autodigestion without addition of proteolytic enzymes and separated into distinct bands in a single centrifugation step using Histopaque-1077 (a mixture of polysucrose and sodium diatrizoate). The cells collected from an interfacial fraction were suspended in RPMI 1640 containing 11 mmol/l D-glucose with or without nicotinamide (0, 10, 20, 40 mmol/l), and then placed in culture dishes. Pancreatic cells formed a monolayer while fibroblasts became detached from the bottom of the dish when cultured in the presence of nicotinamide. More than 80% of monolayer-forming cells were stained for insulin, using an enzymatic method, and were identified as B-cells. Morphologically, the PE cells extended multiple processes terminating in growth-cone-like structures, as visualized by both light microscopy and scanning electron microscopy. Insulin secretion in response to glucose stimulation occurred for 35 days of incubation in the RPMI 1640 medium, with or without nicotinamide. Exposure of the cells to nicotinamide for 35 days resulted in a 2-3-fold increase in insulin secretion in response to high glucose stimulus (16.7 mmol/l) compared with low glucose (5.5 mmol/l). Glucose-induced Ca2+ responses were examined in individual cells cultured for 35 days in the presence of 10 mmol/l nicotinamide, using Ca2+ imaging with fura-2. These results indicate that it is possible to prepare pig PE cells in monolayer culture with low fibroblast contamination and to maintain functioning B-cells in vitro for relatively long periods. The present method provides useful preparations for further morphological and physiological studies on the differentiation, growth and regenerative capacity of islet cells.

Animals↗
PubMed1998

Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization.

Regional distribution of neurons expressing neuronal nitric oxide synthase mRNA in the rat brain was examined by non-radioisotopic in situ hybridization, using digoxigenin-labeled complementary RNA probes. Clustering of intensely positive neurons was observed in discrete areas including the main and accessory olfactory bulbs, the islands of Calleja, the amygdala, the paraventricular nucleus of the thalamus, several hypothalamic nuclei, the lateral geniculate nucleus, the magnocellular nucleus of the posterior commissure, the superior and inferior colliculi, the laterodorsal and pedunculopontine tegmental nuclei, the nucleus of the trapezoid body, the nucleus of the solitary tract and the cerebellum. Strongly-stained isolated neurons were scattered mainly in the cerebral cortex, the basal ganglia and the brain stem, especially the medulla reticular formation. In the hippocampus, an almost uniform distribution of moderately stained neurons was observed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the Ammon's horn, while more intensely stained isolated neurons were scattered over the entire hippocampal region. These observations can serve as a good basis for studies on function and gene regulation of neuronal nitric oxide synthase.

Animals↗
PubMed1998

Detailed acceptor specificities of human alpha1,3-fucosyltransferases, Fuc-TVII and Fuc-TVI.

To clarify the acceptor specificity of Fuc-TVII, its activity toward various analogs of a 2-(trimethylsilyl)ethyl alpha2,3-sialyl lacto-N-neotetraose, an acceptor for both Fuc-TVII and Fuc-TVI, was examined in comparison with that of Fuc-TVI. Fuc-TVII required three portions of alpha2,3-sialylated type-2 oligosaccharide structures (i.e., the hydroxyl group at C-4 of Gal, the hydroxyl group at C-3 of GlcNAc, and the carbonylamino group at C-2 of GlcNAc) for its acceptor recognition. Fuc-TVI required the carbonylamino group at C-2 of GlcNAc for its acceptor recognition. Fuc-TVII showed higher affinity toward two analogs, in which the hydroxyl group at C-6 of GlcNAc has been deoxygenated and the acetamide group of N-acetylneuraminic acid has been replaced with a glycolylamino group, respectively, than that toward the original compound. On the other hand, Fuc-TVI showed higher affinity toward an analog, in which the acetamide group of GlcNAc has been modified with a lauroylamino group, than that toward the original compound. Analysis involving mass spectrometry confirmed that both Fuc-TVII and Fuc-TVI could fucosylate these three analogs to yield sialyl Lewis x derivatives.

Binding, Competitive↗
PubMed1998

Iodine-123 BMIPP and Ga-67 scintigraphy in liposarcoma.

A patient with recurrent retroperitoneal liposarcoma had multiple suspected metastases. I-123 BMIPP imaging showed areas of increased uptake due to accumulation in the myxoid components of the liposarcoma. Ga-67 showed accumulation in the undifferentiated components. The well-differentiated components showed little accumulation of either I-123 BMIPP or Ga-67. Differences in the accumulation of these radionuclides may reflect differences in cell densities, fatty acid metabolism, and the degree of malignancy. I-123 BMIPP and Ga-67 scintigraphy may be useful in determining the prognosis of liposarcoma.

Abdomen↗
PubMed1998

Current therapeutic apheresis technologies for inflammatory bowel disease.

A large number of physicians have indicated that patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD) respond to current apheresis technology treatment. However, the mechanism of the apheresis procedure is undefined for patients with IBD. IBD appears to be caused by a complex of interactions from the genes, environment, and the immune system; therefore, the immune system plays a crucial role in the inflammatory responses. In this process, lots of interactions occur simultaneously, and they cross relate with each other. This review paper briefly discusses the etiology and pathogenesis of IBD and attempts to elucidate the mechanism that occurs after apheresis treatment.

Blood Component Removal↗
PubMed1998

Susceptibilities of plasma antioxidants and erythrocyte constituents to low levels of ozone.

To evaluate the susceptibilities of human blood constituents to the low levels of ozone used in ozonated autohemotherapy (40 microgO3/ml), we quantified plasma antioxidants and erythrocyte constituents after rapid mixing of human whole blood with ozone at 20, 40, 60, and 100 microg/ml blood. Ascorbic acid, uric acid, and alpha-tocopherol in plasma decreased as ozone increased, but bilirubin was unaffected. The content of thiobarbituric acid-reactive substances in plasma was increased by ozone. However, the content of thiobarbituric acid-reactive substances and alpha-tocopherol in the erythrocyte membrane was not significantly affected. No significant changes occurred in the content of methemoglobin, cytoskeleton proteins or erythrocyte enzymes such as Na+/K+-ATPase, acetylcholinesterase, catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase at all the ozone levels tested. A decrease in reduced glutathione in erythrocytes was the only significant change caused by the ozone level used for autohemotherapy. It may be one of the chemical events responsible for the beneficial effects of ozonated autohemotherapy.

Adult↗
PubMed1998

Prolonged hepatic warm ischemia in non-heart-beating donors: protective effects of FK506 and a platelet activating factor antagonist in porcine liver transplantation.

BACKGROUND: Prolonged warm ischemic injury in non-heart-beating donors (NHBDs) significantly affects hepatic allograft function after liver transplantation (LTx). METHODS: The effects of FK506 and the platelet activating factor antagonist E5880 on postoperative function of hepatic allografts procured from NHBDs were evaluated in porcine orthotopic LTx. In donors, livers were subjected to 90 minutes of warm ischemia and a subsequent 4-hour cold preservation. Group 1 (n = 6) was the untreated control group. In group 2 (n = 4), donors were pretreated with FK506 (0.3 mg/kg). In group 3 (n = 4), donors and recipients were treated with E5880 (0.3 mg/kg). In group 4 (n = 6), pigs were treated with both FK506 and E5880. RESULTS: All of the recipients in group 1 died within 12 hours. In groups 2 and 3, half of the recipients survived more than 12 hours. In group 4, all of the recipients survived more than 2 days (p < 0.01 compared with group 1). The improved survival seen in group 4 was associated with a reduction in the serum concentrations of glutamic oxaloacetic transaminase and lactate, and a restoration of hepatic energy charge. CONCLUSIONS: The present study suggests that FK506 and E5880 can improve the function of hepatic allografts subjected to prolonged warm ischemia in NHBDs, and that the protective effects of the two drugs seem to be synergistic.

Adenosine Triphosphate↗