Biomedical subjects

Y Okada

Publications and source records attributed to Y Okada.

At least 433 records · Page 24Linked to original sources

PubMed1998

A stable prostacyclin analog, beraprost sodium, attenuates platelet accumulation and preservation-reperfusion injury of isografts in a rat model of lung transplantation.

BACKGROUND: Recent studies have shown that the extent of platelet accumulation in the vasculature of transplanted organs correlates with the degree of preservation-reperfusion injury. In this study, we examined the effect of a stable prostacyclin analog, beraprost sodium, which possesses potent antiplatelet activity, on parameters of platelet accumulation and preservation-reperfusion injury of isografts in a rat model of lung transplantation. METHODS: The heart-lung blocks of donor rats were flushed with and preserved in modified Euro-Collins solution at 4 degrees C for 6 hr or 24 hr. The left lung was transplanted into recipient rats and reperfused for 1 hr. Lung injury was evaluated by the pulmonary blood flow ratios to the lung isografts, the weight gain of the isografts, and histological examination. Small portions of the lung isografts were excised and stained with an antibody specific for rat platelets. A scoring system was developed to semiquantitate the intensity of antibody staining (score 0-4). The recipient rats received oral administration of beraprost sodium (0.3 mg/kg) before lung transplantation. Control animals received no beraprost sodium. RESULTS: In the 6-hr preservation study, administration of beraprost sodium significantly reduced the score for platelet accumulation (1.8+/-0.4 vs. 3.3+/-0.5, P<0.01). This observation was accompanied by a significantly decreased degree of preservation-reperfusion injury as evidenced by an increased blood flow ratio (13.7+/-2.6% vs. 4.5+/-3.6%, P<0.01) and a reduced weight gain (0.7+/-0.2 g vs. 1.1+/-0.2 g, P<0.01). Histological examination revealed severe capillary congestion in three of six cases in the control group, while no capillary congestion was observed in the beraprost group. In the 24-hr preservation study, no differences were seen in platelet accumulation scores or parameters of lung injury. CONCLUSION: Beraprost sodium, an antiplatelet agent, reduces platelet accumulation and preservation-reperfusion injury of lung transplants at 6 hr in this rat isograft model.

Animals↗

PubMed1998

Activation of cAMP-dependent C1- currents in guinea-pig paneth cells without relevant evidence for CFTR expression.

1. To determine whether Paneth cells exhibit functional expression of cAMP-activated Cl- currents and molecular expression of the cystic fibrosis transmembrane conductance regulator (CFTR), we applied whole-cell patch clamp and single-cell mRNA analysis by reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification to single Paneth cells in crypts isolated from the guinea-pig small intestine. 2. Prominent activation of Cl- currents was consistently observed after stimulation with dibutyryl cAMP and forskolin or with vasoactive intestinal polypeptide (VIP). The cAMP-activated Cl- current was inhibited by removal of intracellular ATP or administration of an inhibitor of protein kinase A. 3. Many of the biophysical and pharmacological properties of the currents were phenotypically similar to those of the CFTR Cl- channel, such as the ohmic current-voltage relationship, the anion selectivity with a Type III sequence (Br- > Cl- > I- >> F- >= gluconate-), I--induced blockage, insensitivity to a stilbene-derivative Cl- channel blocker, and sensitivity to a carboxylate analogue Cl- channel blocker. The sensitivity of the current to glibenclamide was, however, much weaker than that reported for the CFTR Cl- channel current. In contrast to the time independence of CFTR currents, the inward component of the Paneth cell Cl- currents exhibited inactivation kinetics. 4. Expression of CFTR mRNA could not be detected by RT-PCR analysis in almost all single Paneth cells, although its expression was consistently detected at the whole-crypt level. The presence of a small number of CFTR-expressing epithelial cells, which were scattered both in villi and crypts but not at the crypt base where Paneth cells were located, was demonstrated by immunocytochemistry. 5. Taken together, it appears that guinea-pig Paneth cells functionally express cAMP-activated Cl- conductance without relevant evidence for molecular expression of CFTR. Functional expression of VIP receptors in the Paneth cells was also demonstrated.

Animals↗

PubMed1998

Expression of membrane-bound transferrin-like protein p97 on the cell surface of chondrocytes.

A concanavalin-A-binding protein of 76 kDa was purified from the plasma membrane fraction of rabbit chondrocyte cultures. Amino acid sequencing of the N-terminal region and of tryptic peptides of the protein, in addition to sequencing of its cDNA revealed that this protein is highly similar to the tumour-associated antigen p97. Hence, it was concluded that this protein is the rabbit form of p97. Western blotting, Northern blotting and reverse-transcription PCR analyses indicated that rabbit p97 is expressed at high levels in cartilage and chondrocytes, but is barely detectable in the bone, liver, kidney, small intestine, eye, pancreas, heart, testis, skeletal muscle, spleen and fibroblasts. Immunocytochemical and immunohistochemical analyses demonstrated that p97 is expressed in the plasma membrane of chondrocytes. p97 transcript was detected in all zones of the cartilage but the level was relatively low in the hypertrophic zone. These findings suggest that p97 is involved in maintaining the cell surface characteristics of chondrocytes.

Amino Acid Sequence↗

PubMed1998

Suppression of hyperemia after brain ischemia by L-threo-3,4-dihydroxyphenylserine.

Although several studies have shown that L-threo3,4-dihydroxyphenylserine (DOPS) may provide a neuroprotective effect against ischemic brain damage, its protective mechanism is not fully understood. Glutamate release and hippocampal blood flow in ischemia with administration of DOPS were investigated to elucidate the neuroprotective mechanism of DOPS. Pre- (but not post-) ischemic administration of DOPS rescued 73% of hippocampal CA1 neurons (p < 0.001, compared with ischemia only) 1 week after transient global ischemia in gerbils. While glutamate release induced by ischemia was not affected, the increase of hippocampal blood flow during reperfusion was significantly suppressed by DOPS. These results demonstrate that DOPS may prevent reperfusion injury by suppression of hyperemia after ischemia, resulting in neuroprotection.

Animals↗

PubMed1998

Intracellular free calcium concentration in human taste bud cells increases in response to taste stimuli.

We examined changes of intracellular free calcium concentration [Ca2+]i elicited by taste stimuli of sucrose, denatonium and NaCl in the taste buds of seven human fungiform papillae. In one taste bud we observed an increase in [Ca2+]i induced by only NaCl. In another bud an increase of [Ca2+]i in response to both NaCl and sucrose was found. The Ca2+ responses to NaCl and sucrose occurred in differential areas within the one taste bud. In the other five fungiform papillae [Ca2+]i was not changed by the taste stimuli. These results suggest that an increase of [Ca2+]i participates in taste transduction mechanisms for sucrose and NaCl, and that taste cells in one taste bud may respond to differential stimuli.

Adult↗

PubMed1998

Prevention of murine AIDS development by (R)-9-(2-phosphonylmethoxypropyl)adenine.

LP-BM5 murine leukemia virus (MuLV) infection causes severe immunodeficiency termed murine AIDS (MAIDS). The acyclic nucleoside phosphonates, (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) were examined, in comparison with zidovudine (AZT), for their inhibitory effect on the development of MAIDS. Although no significant difference in inhibition of LP-BM5 MuLV replication was identified between PMPA and PMEA in cell cultures, PMPA was obviously less cytotoxic to the host lymphocytes. None of the mice treated in vivo with 5 or 25 mg/kg of PMPA or 25 mg/kg of PMEA developed MAIDS at 5 weeks after viral infection. However at 9 weeks, none of the 25 mg/kg PMPA-treated mice progressed to MAIDS, except for one that developed mild MAIDS, whereas PMEA, even at 100 mg/kg, could not prevent disease progression. MAIDS-associated activation of lymphocytes and viral replication were drastically inhibited by PMPA treatment. These results indicate that PMPA is a highly effective antiretroviral agent in vivo.

Adenine↗

PubMed1998

Membrane-type 1 MMP (MMP-14) cleaves at three sites in the aggrecan interglobular domain.

An aggrecan G1-G2 substrate was used to determine sites within the interglobular domain that were susceptible to cleavage by MT1-MMP. Degradation products were identified by Western blotting with neo-epitope antibodies specific for MMP-derived N- and C-terminal sequences. The results showed that MT1-MMP cleaved at the N341-F342 and D441-L442 bonds, as shown for other MMPs, and also at a site 13 amino acids C-terminal to the N341-F342 site. The G2 product of this additional cleavage was identified by sequence analysis and revealed an N-terminus commencing T355VxxPDVELPLP. The data are consistent with MT1-MMP cleavage at three sites in the aggrecan interglobular domain; one at N342-F342, a second at D441-L442 and a third at Q354-T355.

Aggrecans↗

PubMed1998

Targeted disruption of mouse conventional kinesin heavy chain, kif5B, results in abnormal perinuclear clustering of mitochondria.

Mouse kif5B gene was disrupted by homologous recombination. kif5B-/- mice were embryonic lethal with a severe growth retardation at 9.5-11.5 days postcoitum. To analyze the significance of this conventional kinesin heavy chain in organelle transport, we studied the distribution of major organelles in the extraembryonic cells. The null mutant cells impaired lysosomal dispersion, while brefeldin A could normally induce the breakdown of their Golgi apparatus. More prominently, their mitochondria abnormally clustered in the perinuclear region. This mitochondrial phenotype was reversed by an exogenous expression of KIF5B, and a subcellular fractionation revealed that KIF5B is associated with mitochondria. These data collectively indicate that kinesin is essential for mitochondrial and lysosomal dispersion rather than for the Golgi-to-ER traffic in these cells.

Animals↗

PubMed1998

TIMP-2 promotes activation of progelatinase A by membrane-type 1 matrix metalloproteinase immobilized on agarose beads.

Membrane-type 1 matrix metalloproteinase (MT1-MMP)/MMP-14 is the activator of progelatinase A (proGelA)/proMMP-2 on the cell surface. However, it was a paradox that a tissue inhibitor of metalloproteinase-2 (TIMP-2), which is an inhibitor of MT1-MMP, is required for proGelA activation by the cells expressing MT1-MMP. In this study, a truncated MT1-MMP having a FLAG-tag sequence at the C terminus (MT1-F) was immobilized onto agarose beads (MT1-F/B) and used to analyze the role of TIMP-2. The proteolytic activity of MT1-F/B against a synthetic peptide substrate was inhibited by TIMP-2 in a dose-dependent manner. In contrast, TIMP-2 promoted the processing of proGelA by MT1-F/B at low concentrations and inhibited it at higher concentrations. TIMP-2 promoted the binding of proGelA to the MT1-F on the beads by forming a trimolecular complex, which was followed by processing of proGelA. A stimulatory effect of TIMP-2 was observed under conditions in which unoccupied MT1-F was still available. Thus, the ternary complex is thought to act as a means to concentrate the substrate to the bead surface and to present it to the neighboring free MT1-F.

Animals↗

PubMed1998

Lipid lowering by diet reduces matrix metalloproteinase activity and increases collagen content of rabbit atheroma: a potential mechanism of lesion stabilization.

BACKGROUND: Proteolytic enzyme activity in lipid-rich atheroma may promote plaque rupture and precipitate acute coronary syndromes. This study tested the hypothesis that lipid lowering stabilizes plaques by reducing proteolytic activity. METHODS AND RESULTS: We produced experimental atheroma in 33 rabbits by balloon injury and an atherogenic diet (0.3% cholesterol and 4.7% coconut oil) for 4 months. At that time, 15 rabbits were killed (baseline group). The remaining animals were divided into two groups: a hyperlipemic group continued to consume a cholesterol-enriched diet (0.05% to 0.2%) for 16 more months (n=5) and a lipid-lowering group consumed a purified chow diet with no added cholesterol or fat for 8 (n=3) or 16 months (n=10). Macrophage accumulation and interstitial collagenase (matrix metalloproteinase-1, MMP-1) expression in the lesion were measured by quantitative image analysis of standardized sections of immunostained aortas. Baseline lesions expressed high levels of MMP-1 and contained many macrophages. These features of plaque instability persisted in the hyperlipemic group. However, the lipid-lowering group showed progressive reduction in both macrophage content and MMP- 1 immunoreactivity with time. Aortic rings of the baseline and hyperlipemic groups elaborated gelatinolytic, caseinolytic, and elastinolytic activity attributable to MMP-2, MMP-3, or MMP-9, monitored by SDS-PAGE zymography. Proteolytic activity decreased markedly in the lipid-lowering group. Aortic content of interstitial collagen, determined by sirius red staining, increased in the lipid-lowering group compared with the baseline or continued hyperlipemic groups, indicating that lipid lowering reinforced the fibrous skeleton of the atheroma. CONCLUSIONS: These results establish a mechanism by which lipid lowering may stabilize vulnerable plaques by reduced expression and activity of enzymes that degrade the arterial extracellular matrix and render atheroma less susceptible to disruption and thrombosis by favoring collagen accumulation in the fibrous cap.

Animals↗

PubMed1998

A quantitative study of the progress of myelination in the rat central nervous system, using the immunohistochemical method for proteolipid protein.

The temporal changes in intensity of myelination of the nervous pathways in 0 to 42-day-old Wistar rats were quantitatively analyzed by immunohistochemistry with anti-proteolipid protein and compared with that obtained by immunohistochemistry with anti-myelin basic protein. Immunohistochemistry was performed on paraffin-embedded tissue according to the standard ABC technique. Intensity of myelination was examined by an image analyzing system. We analyzed nine nervous pathways: corpus callosum, optic tract, internal capsule, spinal tract of the trigeminal nerve, inferior cerebellar peduncle, cerebellar white matter, pyramidal tract, medial longitudinal fasciculus, and cuneate fasciculus. The presence of immunoreactive fibers for proteolipid protein (PLP) in the spinal tract of the trigeminal nerve, medial longitudinal fasciculus and cuneate fasciculus was noted on postnatal day 0. Those of the corpus callosum, inferior cerebellar peduncle, cerebellar white matter, pyramidal tract and internal capsule were noted on day 7, and that of optic tract on day 14. The time required to reach the intensity of myelination of day 42 was day 14 for the cuneate fasciculus, day 21 for the spinal tract of the trigeminal nerve, inferior cerebellar peduncle and medial longitudinal fasciculus, day 28 for the optic and pyramidal tracts, day 35 for the corpus callosum and day 42 for the internal capsule and cerebellar white matter. The appearance of immunoreactive fibers for PLP was usually earlier than that for myelin basic protein (MBP) and the pattern of difference between PLP and MBP can be classified into three groups: (1) their time of appearance and progress are almost the same, as in the optic tract; (2) the appearance and progress of PLP occurs earlier than those of MBP, as in the pyramidal tract; (3) the appearance of PLP occurs earlier than that of MBP, but their progress is the same. Our findings revealed that the time of appearance and progress of myelination as measured by PLP are different among the nervous pathways, and that there is also a difference between PLP and MBP. This difference between PLP and MBP may indicate a functional difference between them.

Age Factors↗

PubMed1998

The antithrombotic effect of potent bifunctional thrombin inhibitors based on hirudin sequence, P551 and P532, on He-Ne laser-induced thrombosis in rat mesenteric microvessels.

The antithrombotic effect of potent synthetic bifunctional non-substrate type thrombin inhibitors based on hirudin sequences, P551 and P532, on Helium-Neon laser-induced thrombosis was investigated in rat mesenteric microvessels and compared with other types of thrombin inhibitors. P551 and P532, when given intravenously, inhibited platelet-rich thrombus formation in both arterioles and venules in a dose-dependent manner. The inhibitory effect was maximal immediately after intravenous administration and persisted for 20-30 minutes in both arterioles and venules. The minimal effective doses of P551 and P532 were 1.0 mg/ kg (intravenously) in both. However, the time course of the antithrombotic effect was not in keeping with the inhibitory effect measured by an activated partial thromboplastin time and was similar to other types of inhibitors in spite of different half-lives. The current findings show that P551 and P532 had significant inhibitory effects on platelet-rich thrombus formation and suggest that these bifunctional thrombin inhibitors could be potent antithrombotic agents.

Animals↗

PubMed1998

ATP produces potassium currents via P3 purinoceptor in the follicle cell layer of Xenopus oocytes.

Purinoceptor agonists produced potassium currents with the order of potency: ATP > adenosine = ADP = AMP > beta,gamma-methylene ATP, while a small response or no response was induced by 2-methylthio ATP, UTP, or alpha,beta-methylene ATP. The response induced by beta,gamma-methylene ATP was completely inhibited in the presence of alpha,beta-methylene ATP, suggesting that the relevant receptor for these agonists was a P3 purinoceptor. ATP induced currents with a latency of 24 s and the currents were not induced in defolliculated oocytes. The currents were not affected by either the Gi/o-protein inhibitor, pertussis toxin (PTX), or the selective cAMP-dependent protein kinase inhibitor, H-89, or the phospholipase C (PLC) inhibitor, neomycin, or the phospholipase A2 (PLA2) inhibitor, 4-bromophenacyl bromide. The currents were enhanced by the selective protein kinase C (PKC) inhibitor, GF109203X, but otherwise, they were reduced by the potent PKC activator, 12-O-tetradecanoylphorbol-13-acetate. The results of the present study suggest that a P3 purinoceptor in the follicle cell layer of oocytes is involved in activation of potassium channels and that the evoked currents are regulated by PLC/PLA2-independent PKC activation.

Adenosine Triphosphate↗

PubMed1998

Transformation of epithelial Madin-Darby canine kidney cells with p60(v-src) induces expression of membrane-type 1 matrix metalloproteinase and invasiveness.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed both in carcinoma cells and in surrounding stromal fibroblasts. MT1-MMP localizes to the surface of tumor cells and is thought to play an important role in tumor invasion. To analyze the mechanism of MT1-MMP gene expression in epithelial tumor cells, the dog kidney epithelial cell line Madin-Darby canine kidney (MDCK) was transformed by oncogenes, including v-src, and expression of MT1-MMP was examined. Transformation of MDCK cells with v-src resulted in loss of cell-to-cell contacts and morphological change. Expression of MT1-MMP in v-src-transformed cells was identified by Northern and Western blotting. Gelatin zymography analysis showed that progelatinase A in the culture medium was processed from latent to activated form by MDCK cells transformed with v-src. The MDCK cells transformed by v-src were tumorigenic in the subcutis (ectopic) and kidney (orthotopic) of nude mice and spontaneously metastasized to the lung after orthotopic implantation. These results suggest that MT1-MMP induced by v-src transformation may promote invasiveness of transformed cells.

Animals↗

PubMed1998

New beamline optics for the effective use of a wiggler beam.

A hard X-ray beamline, BL13A, has been constructed for precision diffraction experiments conducted downstream of the multipole wiggler MPW#13 at the Photon Factory. The BL13A line can be used along with the main beamline, BL13B, which accepts the central 4 mrad of the 5 mrad horizontal fan. BL13A uses the remaining horizontal off-axis beam. The beam is diffracted by a double-crystal monochromator which uses asymmetric (422) diffraction with a horizontal diffraction vector. A monochromatic beam with 33.5 keV photon energy is directed to a precision goniometer. Good beam stability and high intensity were obtained.

Journal Article↗

PubMed1998

Expression of the Na+/Ca2+ exchanger emerges in hepatic stellate cells after activation in association with liver fibrosis.

Activation of hepatic stellate (Ito) cells is a final common pathway of liver fibrosis. The findings presented in this paper indicate that expression of Na+/Ca2+ exchanger (NCX) emerges in rat hepatic stellate cells after activation in vitro during primary culture or in vivo in response to intoxication with CCl4. NCX mRNA became detectable by Northern blot analysis in cultured stellate cells on day 3, as was alpha-smooth muscle actin, an indicator not only of smooth muscle differentiation but also of stellate cell activation. Western blot analysis showed expression of the exchanger protein in the activated stellate cells. Functional expression of the exchanger, monitored by Ni2+-sensitive, verapamil-insensitive intracellular free Ca2+ increases in response to reduction of extracellular Na+ concentration, became sizable by using Fura-2 in stellate cells by 7 days in culture. Furthermore, increased expression of the exchanger mRNA was found predominantly in stellate cells freshly isolated from the CCl4 model rat of hepatic fibrosis. Thus, it is concluded that NCX expression is closely associated with activation of hepatic stellate cells in vitro and in vivo. Because, even at the whole liver level, increased expression of NCX mRNA became observable after induction of liver fibrosis, it is suggested that NCX expression serves a useful diagnostic marker of liver fibrosis or cirrhosis.

Actins↗

PubMed1998

Defect in synaptic vesicle precursor transport and neuronal cell death in KIF1A motor protein-deficient mice.

The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron- specific microtubule plus end-directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769-780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.

Animals↗

PubMed1998

Expression and tissue localization of matrix metalloproteinase 7 (matrilysin) in human gastric carcinomas. Implications for vessel invasion and metastasis.

We examined the production and tissue localization of matrix metalloproteinase-7 (MMP-7 = matrilysin) in human gastric carcinomas and analyzed the data in connection with the clinicopathological factors. Sandwich-enzyme immunoassay for the zymogen of MMP-7 (proMMP-7) showed enhanced production of MMP-7 in carcinoma tissues compared with control normal gastric mucosa. Immunohistochemical studies demonstrated that MMP-7 is localized predominantly to the carcinoma cells in 71% of the carcinoma samples (30/42 cases). The percentage of immunoreactive carcinoma cells to total carcinoma cells (positive ratio) was significantly higher in intestinal-type carcinomas (26%, median) than in diffuse-type carcinomas (3%, median) (p < 0.05). The positive ratio was markedly higher in carcinoma groups with vascular invasion (28%) or lymphatic permeation (12%) than in those without invasion (6%) or permeation (0%) (p < 0.05). It was also significantly higher in carcinoma groups with liver (49%) or lymph-node metastases (15%) than in those without metastases (6 and 2% respectively) (p < 0.05). Both proMMP-7 of 28 kDa and active MMP-7 of 19 kDa were detected in the carcinoma tissues by immunoblotting. Reverse-transcription-PCR showed specific amplification in 50% of the carcinoma cases (6/12 cases) and 8% of the normal control specimens (1/12 cases). In situ hybridization demonstrated that the carcinoma cells almost selectively express MMP-7 mRNA. These data suggest that enhanced production of proMMP-7 and its activation are implicated in invasion and metastasis of human gastric carcinomas.

Adult↗