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Biomedical subjects

J Yamauchi

Publications and source records attributed to J Yamauchi.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effect of warm ischemia time and organ perfusion technique on liver microvascular preservation in a non-heart-beating rat model.

BACKGROUND: Current organ shortage has led to a reconsideration of non-heart-beating cadaveric donation. METHODS: We assessed the effectivity of dual, i.e., arterial and portal-venous versus exclusive, arterial gravity perfusion for procurement of rat livers after 30 min and 60 min of cardiac arrest, analyzing the rate and homogeneity of microvascular perfusion by in situ fluorescence microscopy. RESULTS: After 30 min of cardiac arrest, a nearly 100% recovery of acinar perfusion with a sinusoidal density not significantly different from that of normal, nonischemic livers was achieved by dual gravity perfusion. Prolongation of cardiac arrest to 60 min caused an almost 50% deficit of acinar and sinusoidal perfusion (P<0.05) with a concomitant 2-3-fold increase of heterogeneity of hepatic microperfusion. Regardless of the warm ischemic time period, dually perfused livers exhibited significantly (P<0.05) higher rates of both acinar and sinusoidal perfusion with increased homogeneity of microcirculation when compared with exclusive arterial perfusion. CONCLUSION: These data underline the need and benefit of dual perfusion as well as the limitation of warm ischemic tolerance to 30 min for safe liver procurement of non-heart-beating donors.

Animals↗
PubMed2000

Heparin and phentolamine combined, rather than heparin alone, improves hepatic microvascular procurement in a non-heart-beating donor rat-model.

Improvement of organ procurement from non-heart-beating donors (NHBDs) could increase the donor organ pool for liver transplantation. Whether anti-coagulative and anti-vasospastic substances can improve hepatic microvascular preservation from NHBDs is unknown. In donor rats which were pretreated with either heparin (n = 6) or heparin combined with phentolamine (n = 7) 10 min prior to cardiac arrest, the extent and homogeneity of hepatic microvascular reperfusion was assessed at the end of a 60-min period of cardiac arrest using in situ fluorescence microscopy. Non-pretreated animals with cardiac arrest for 60 min served as controls (n = 6). In the non-treated NHBDs, arterial gravity perfusion of 100 cm H2O with HTK-solution led to a hepatic acinar reperfusion of only approximately 22 % with a remarkably diminished sinusoidal density. Application of heparin prior to cardiac arrest resulted in a two-fold, but insignificant increase of acinar perfusion and sinusoidal density with a still considerable heterogeneity of both parameters. Livers of NHBDs that additionally received phentolamine exhibited significantly increased values of both acinar perfusion and sinusoidal density. Phentolamine was found to reduce heterogeneity of organ microperfusion. Thus, our results indicate that the combined application of heparin and phentolamine is a useful additive for optimizing the quality of organs harvested from NHBDs.

Alanine Transaminase↗
PubMed2000

[Simultaneous administration of ethanol emphasizes the effect of methamphetamine on central nervous system in rat with high alcohol preference].

We studied about influence of alcohol preference and of simultaneously administered ethanol (EtOH) on the effect of methamphetamine (MA) on the central nervous system neurochemically and behavioral pharmacologically using two strains of rat with high or low preference for alcohol (HAP and LAP). In the neurochemical study, we determined dopamine (DA) and serotonin (5-HT) level in striatum and nucleus accumbens (N.Acc) by means of brain microdialysis after administration of MA 1 mg/kg alone (LAP-MA group, HAP-MA group) or EtOH 2 g/kg + MA 1 mg/kg (LAP-EtOH/MA group, HAP-EtOH/MA group) every 20 minutes for 10 sessions. In the behavioral pharmacological study, we observed rat's behavior in the open field and performed scoring according to Locomotion-Stereotypy (L-S) rating scale by Ellinwood every five minutes, and counted locomotion and rearing by means of infrared beam cutting every 20 minutes after administration of MA 1 mg/kg alone (LAP-MA group, HAP-MA group) or EtOH 2 g/kg + MA 1 mg/kg (LAP-EtOH/MA group, HAP-EtOH/MA group). When MA was given alone, HAP rat showed no significant difference in locomotion, rearing and L-S score compared to LAP rat, although HAP rat showed significantly lower elevation of DA and 5-HT in both striatum and N.Acc. When EtOH was simultaneously administered with MA, in LAP rat, DA and 5-HT elevation in the striatum and N.Acc showed no significant differences between LAP-EtOH/MA group and LAP-MA group. Locomotion and rearing reduced and L-S score temporarily reduced significantly in LAP-EtOH/MA group compared to LAP-MA group. However in HAP rat, HAP-EtOH/MA group showed significantly higher DA and 5-HT elevation in both striatum and N.Acc, and also showed significantly higher L-S score and higher locomotion count compared to HAP-MA group. Our results indicate that effect of MA may be influenced by alcohol preference, and that simultaneous administration of EtOH emphasizes the effect of MA on central nervous system in rat with high alcohol preference.

Animals↗
PubMed1999

Importance of the G protein gamma subunit in activating G protein-coupled inward rectifier K(+) channels.

The G protein-coupled inward rectifier K(+) channel (GIRK) is activated by direct interaction with the heterotrimeric GTP-binding protein betagamma subunits (Gbetagamma). However, the precise role of Gbeta and Ggamma in GIRK activation remains to be elucidated. Using transient expression of GIRK1, GIRK2, Gbeta1, and Ggamma2 in human embryonic kidney 293 cells, we show that C-terminal mutants of Gbeta1, which do not bind to Ggamma2, are still able to associate with GIRK, but these mutants are unable to induce activation of GIRK channels. In contrast, other C-terminal mutants of Gbeta1 that bind to Ggamma2, are capable of activating the GIRK channel. These results suggest that Ggamma plays a more important role than that of an anchoring device for the Gbetagamma-induced GIRK activation.

Cell Line↗
PubMed1999

Phosphorylation of F-actin-associating G protein gamma12 subunit enhances fibroblast motility.

Eleven isoforms of G protein gamma subunit have been found thus far, but the precise roles of individual gamma subunits are not known. The gamma12 subunit has two unique properties: phosphorylation by protein kinase C and association with F-actin. To elucidate the role of gamma12, we overexpressed gamma12 and other gamma subunits in NIH 3T3 cells together with the beta1 subunit. The overexpressed gamma12 as well as endogenous gamma12, but not gamma2, gamma5, and gamma7 subunits, associated with cytoskeletal components. Expression of gamma12 induced remarkable changes including cell rounding, disruption of stress fibers, and enhancement of cell migration, but expression of other gamma subunits did not induce significant changes. Deletion of the N-terminal region of gamma12 decreased the abilities of gamma12 to associate with cytoskeletal fractions, to induce cell rounding, and to increase cell motility. Replacement by alanine of Ser2 of gamma12 (Ser1 of a mature gamma12 protein), a phosphorylation site for protein kinase C, eliminated these effects of gamma12, whereas a mutant in which Ser2 was replaced with glutamic acid showed effects equivalent to wild-type gamma12. These results indicate that phosphorylation of gamma12 at Ser2 enhances the motility of cells.

3T3 Cells↗
PubMed1999

Differential regulation of mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7) by signaling from G protein beta gamma subunit in human embryonal kidney 293 cells.

Heterotrimeric G protein beta gamma subunit (Gbeta gamma) mediates signals to two types of stress-activated protein kinases, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase, in mammalian cells. To investigate the signaling mechanism whereby Gbeta gamma regulates the activity of JNK, we transfected kinase-deficient mutants of two JNK kinases, mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), into human embryonal kidney 293 cells. Gbeta gamma-induced JNK activation was blocked by kinase-deficient MKK4 and to a lesser extent by kinase-deficient MKK7. Moreover, Gbeta gamma increased MKK4 activity by 6-fold and MKK7 activity by 2-fold. MKK4 activation by Gbeta gamma was blocked by dominant-negative Rho and Cdc42, whereas MKK7 activation was blocked by dominant-negative Rac. In addition, Gbeta gamma-mediated MKK4 activation, but not MKK7 activation, was inhibited completely by specific tyrosine kinase inhibitors PP2 and PP1. These results indicate that Gbeta gamma induces JNK activation mainly through MKK4 activation dependent on Rho, Cdc42, and tyrosine kinase, and to a lesser extent through MKK7 activation dependent on Rac.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Exocrine, but not endocrine, tissue is susceptible to microvascular ischemia/reperfusion injury following pancreas transplantation in the rat.

While post-transplant pancreatitis is still a frequently occurring complication of whole pancreas transplantation, dysfunction of the endocrine tissue is rarely observed. Given that microcirculatory disorders play a major role in the pathogenesis of pancreatitis, we hypothesized a dissociation of endocrine and exocrine microvascular control in pancreas transplantation (cold ischemia-reperfusion) and studied this dissociation quantitatively, analyzing the pancreatic microcirculation after heterotopic isogeneic pancreaticoduodenal transplantation in rats by means of fluorescence microscopy. Functional capillary density (FCD) of both exocrine and endocrine tissue of pancreatic grafts after 1 h of cold storage in HTK solution did not differ when compared to sham-operated, time-matched controls. Intermittent capillary perfusion, which is absent under sham control conditions and which is proposed to be operative as a compensatory mechanism to counteract malperfusion, was observed in 52% of the exocrine, but in only 8% of the endocrine, tissue studied (p < 0.05). In contrast, cold storage of pancreatic grafts for 6 h in HTK resulted in a complete loss of intermittent capillary perfusion in exocrine tissue and, consequently, marked exocrine perfusion failure (decrease in FCD), while FCD of pancreatic endocrine tissue was preserved without any significant change in the incidence of intermittent capillary perfusion. Thus, our results indicate a higher susceptibility of the exocrine pancreas to cold ischemia/reperfusion events that is associated with significant alterations in nutritive perfusion and, thus, with limitations of the oxygen supply to the tissue. This may lead to inflammatory tissue reactions in the clinical setting of pancreas transplantation.

Animals↗
PubMed1999

Activation of c-fos promoter by Gbetagamma-mediated signaling: involvement of Rho and c-Jun N-terminal kinase.

Several extracellular stimuli mediated by G protein-coupled receptors activate c-fos promoter. Recently, we and other groups have demonstrated that signals from G protein-coupled receptors stimulate mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. The activation of these three MAPKs is mediated in part by the G protein betagamma subunit (Gbetagamma). In this study, we characterized the signals from Gbetagamma to c-fos promoter using transient transfection of c-fos luciferase into human embryonal kidney 293 cells. Activation of m2 muscarinic acetylcholine receptor and overexpression of Gbetagamma, but not constitutively active Galphai2, stimulated c-fos promoter activity. The c-fos promoter activation by m2 receptor and Gbetagamma was inhibited by beta-adrenergic receptor kinase C-terminal peptide (betaARKct), which functions as a Gbetagamma antagonist. MEK1 inhibitor PD98059 and kinase-deficient mutant of JNK kinase, but not p38 MAPK inhibitor SB203580, attenuated the m2 receptor- and Gbetagamma-induced c-fos promoter activation. Activated mutants of Ras and Rho stimulated the c-fos promoter activity, and the dominant negative mutants of Ras and Rho inhibited the c-fos promoter activation by m2 receptor and Gbetagamma. Moreover, c-fos promoter activation by m2 receptor, Gbetagamma, and active Rho, but not active Ras, was inhibited by botulinum C3 toxin. These data indicated that both Ras- and Rho-dependent signaling pathways are essential for c-fos promoter activation mediated by Gbetagamma.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

A novel diamino-pyridine derivative prevents excessive leukocyte infiltration in aggravation of acute necrotizing pancreatitis.

Leukocyte infiltration in the pancreas is involved in the aggravation of acute pancreatitis from edematous phase into necrotic change, and mild disease into severe disease; however, the mechanism responsible for leukocyte accumulation is not fully understood. This study was designed to clarify the mechanism underlying leukocyte accumulation into the pancreas and to elucidate the therapeutic efficacy of a novel diamino-pyridine derivative, IS-741 on leukocyte-endothelial cell interaction using rat necrotizing pancreatitis model. The number of adherent leukocytes to pancreatic collecting venules assessed by in vivo fluorescence microscopy increased significantly in necrotizing pancreatitis animals in a time-dependent manner. The expression of CD11b on circulating neutrophils determined by flow cytometric analysis was enhanced to approximately 500% after 2 h. IS-741 attenuated the leukocyte adherence significantly, accompanied by a lower up-regulation of CD11b. These findings were further supported by the histological examination that the accumulation of leukocytes in the pancreas was remarkably inhibited by IS-741. These results suggest that the leukocyte accumulation in the early phase of acute necrotizing pancreatitis may be mediated by leukocyte-endothelial cell interaction via leukocyte integrin CD11b/18. IS-741 attenuated the leukocyte endothelial cell interaction as a consequence of its inhibitory effect on CD11b upregulation.

Animals↗
PubMed1999

[Microcirculatory derangement and ischemia of the pancreas].

We present a review of the microvascular morphology of the pancreas and microstructure of the pancreatic lobule, and introduce our experimental results on pancreatic microcirculation following acute pancreatitis. Impairment of pancreatic microcirculation in the early phase of acute pancreatitis may play a key role in the progression of this disease. Possible contributory mechanisms include increased vascular permeability, reduced blood flow, leukocyte-endothelial cell interaction, and intravascular thrombus formation. We achieved direct-visualization and quantification of changes in microvascular permeability and leukocyte behavior in the pancreas with acute pancreatitis using an in vivo microscope system and off-line computer analysis. Bradykinin and oxygen radicals have been demonstrated to be involved in the increased vascular permeability in the early stage of cerulein pancreatitis. Gabexate mesilate (FOY) prevents the increase in vascular permeability, resulting in a decreased number of rolling leukocytes. Leukocyte adherence to the pancreatic microcirculation is a secondary event following permeability changes in acute pancreatitis. Leukocyte infiltration during aggravation of acute pancreatitis is mediated by leukocyte-endothelial cell interaction via leukocyte integrin CD11b/18. The diamino-pyridine derivative IS-741 inhibits the progression of pancreatic inflammation by down-regulating the expression of CD11b/18.

Acute Disease↗
PubMed1999

Role of microcirculation in hepatic ischemia/reperfusion injury.

There is a large body of evidence that the liver microcirculation has to be considered as a major target in hepatic ischemia/reperfusion injury. The nature of microvascular injury, which precedes manifestation of hepatic parenchymal tissue damage, includes both hypoxia due to lack of microvascular perfusion (i.e. no-reflow), and a reperfusion-associated inflammatory response, which includes the activation and dysfunction of leukocytes and Kupffer cells (the reflow paradox). No-reflow in sinusoids is thought to be caused by endothelial cell swelling and intravascular hemoconcentration, and involves also a deterioration of the balance between ET and NO. The reflow paradox is associated with: (i) the release and action of proinflammatory cytokines (TNF-alpha, IL-1) and oxygen radicals; (ii) the up-regulation of endothelial and leukocytic adhesion molecules (selectins, beta-integrins, ICAM-1); and (iii) the interaction of leukocytes with the endothelial lining of the hepatic microvasculature.

Animals↗
PubMed1998

Involvement of protein kinase C and Src family tyrosine kinase in Galphaq/11-induced activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase.

Mitogen-activated protein kinases (MAPKs) are activated by various extracellular stimuli. The signaling pathways from G protein-coupled receptors to extracellular signal-regulated kinase have been partially elucidated, whereas the mechanisms by which G protein-coupled receptors stimulate c-Jun N-terminal kinase (JNK) and p38 MAPK activities remain largely unknown. We have recently demonstrated that the signal from Gq/11-coupled m1 muscarinic acetylcholine receptor to p38 MAPK is mediated by both Galphaq/11 and Gbeta gamma in HEK-293 cells (Yamauchi, J., Nagao, M., Kaziro, Y., and Itoh, H. (1997) J. Biol. Chem. 272, 27771-27777). In the present study, we report that a constitutively activated mutant of Galpha11 (Galpha11 Q209L) activated not only p38 MAPK, but also JNK, and the activation of JNK and p38 MAPK by Galpha11 Q209L was partially inhibited by prolonged treatment with phorbol 12-myristate 13-acetate and calphostin C. In addition, the Galpha11 Q209L-stimulated activation of both kinases was blocked by a specific inhibitor of protein tyrosine kinases (PP2) and Csk (C-terminal Src kinase). Furthermore, we demonstrated that Galpha11 Q209L stimulated Src family kinase activity and induced tyrosine phosphorylation of several proteins in HEK-293 cells. These results suggest that Galphaq/11 stimulates JNK and p38 MAPK activities through protein kinase C- and Src family kinase-dependent signaling pathways.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

Evidence for axial coordination of 5,6-dimethylbenzimidazole to the cobalt atom of adenosylcobalamin bound to diol dehydratase.

It was demonstrated by electron paramagnetic resonance (EPR) spectroscopy that organic radical intermediates disappeared and cob(II)alamin accumulated upon suicide inactivation of diol dehydratase by 2-methyl-1,2-propanediol. The resulting EPR spectra showed that the eight hyperfine lines due to the divalent cobalt atom of cob(II)alamin further split into triplets by the superhyperfine coupling to the 14N nucleus. Essentially the same superhyperfine splitting of the octet into triplets was observed with [14N]- and [15N]apoenzyme. When the adenosyl form of [14N2]- and [15N2]imidazolyl analogues of the coenzyme [Toraya, T., and Ishida, A. (1991) J. Biol. Chem. 266, 5430-5437] was used with unlabeled apoenzyme, the octet showed superhyperfine splitting into triplets and doublets, respectively. Therefore, it was concluded that cobalamin is bound to this enzyme with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. This conclusion is consistent with the fact that the consensus sequence forming part of a cobalamin-binding motif, conserved in methionine synthase and some of the other cobalamin enzymes, was not found in the deduced amino acid sequences of the subunits of diol dehydratase. Adenosylcobinamide methyl phosphate, a coenzyme analogue lacking the nucleotide moiety, underwent cleavage of the cobalt-carbon bond upon binding to the enzyme in the presence of substrate, forming a cob(II)inamide derivative without nitrogenous base coordination, as judged by EPR and optical spectroscopy. Therefore, this analogue may be a useful probe for determining whether the replacement of the 5, 6-dimethylbenzimidazole ligand by a histidine residue takes place upon binding of cobalamin to proteins.

Benzimidazoles↗
PubMed1998

Pancreatic microcirculation in acute pancreatitis.

We present a review of the microvascular morphology of the pancreas and microstructure of the pancreatic lobule, and report our experimental results of the investigation of pancreatic microcirculation following acute pancreatitis. Impairment of pancreatic microcirculation in the early phase of acute pancreatitis may play a key role in the progression of this disease. Possible contributory mechanisms include increased vascular permeability, reduced blood flow, leukocyte-endothelial cell interaction and intravascular thrombus formation. Using an in-vivo microscope system and off-line computer analysis, we achieved direct visualization and quantification of changes in microvascular permeability and leukocyte behavior in pancreas with acute pancreatitis. Bradykinin and oxygen radicals have been demonstrated to be involved in the increase of vascular permeability in the early stage of caerulein pancreatitis. Leukocyte adherence to the vessels in the pancreatic microcirculation is a secondary event following permeability changes in acute pancreatitis. Leukocyte infiltration during exacerbation of acute pancreatitis is mediated by leukocyte-endothelial cell interaction via leukocyte integrin CD11b/18.

Acute Disease↗