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Biomedical subjects

Y Yamaguchi

Publications and source records attributed to Y Yamaguchi.

At least 181 records · Page 10Linked to original sources
PubMed2000

NO(2)(+) nitration mechanism of aromatic compounds: electrophilic vs charge-transfer process

The nitration of methylnaphthalenes with NO(2)BF(4) and NOBF(4) was examined in order to shed light on the controversial aromatic nitration mechanism, electrophilic vs charge-transfer process. The NO(2)(+) nitration of 1,8-dimethylnaphthalene showed a drastic regioselectivity change depending on the reaction temperature, where ortho-regioselectivity at -78 degrees C and para-regioselectivity at 0 degrees C were considered to reflect the electrophilic and the direct or alternative charge-transfer process, respectively, because the NO(+) nitration through the same reaction intermediates as in the NO(2)(+) nitration via a charge-transfer process resulted in para-regioselectivity regardless of the reaction temperature. The NO(2)(+) nitration of redox potential methylnaphthalenes higher than 1,8-dimethylnaphthalene gave a similar ortho-regioselectivity enhancement to 1,8-dimethylnaphthalene at lower temperature, thus reflecting the electrophilic process. On the other hand, the NO(2)(+) nitration of redox potential methylnaphthalenes lower than 1,8-dimethylnaphthalene showed para-regioselectivity similar to the NO(+) nitration, indicating the direct or alternative charge-transfer process. In the presence of strong acids where the direct charge-transfer process will be suppressed by protonation, the ortho-regioselectivity enhancement was observed in the NO(2)(+) nitration of 1,8-dimethylnaphthalene, suggesting that the direct charge-transfer process could be the main process to show para-regioselectivity. These experimental results imply that the NO(2)(+) nitration proceeds via not only electrophilic but also direct charge-transfer processes, which has been considered to be unlikely because of the high energy demanding process of a bond coordination change between NO(2)(+) and NO(2). Theoretical studies at the MP2/6-31G(d) level predicted ortho- and para-regioselectivity for the NO(2)(+) nitration via electrophilic and charge-transfer processes, respectively, and the preference of the direct charge-transfer process over the alternative one, which support the experimental conclusion

Journal Article↗
PubMed2000

Pharmacokinetics and biodisposition of fluorescein-labeled arabinogalactan in rats.

Fluorescein-labeled arabinogalactan (FA) was prepared by the reaction with FITC in methyl sulphoxide according to the method of deBelder and Granath. A systemic kinetic analysis of FA in rats was carried out by using a specific high-performance size-exclusion chromatography. Intravenously administered FA was rapidly eliminated from the blood circulation followed by an appreciable distribution to the liver and kidney. FA was accumulated in these organs over a long period whereas negligible levels of FA were detected in the other organs. A marked dose-dependency was seen in the hepatic uptake of FA which was markedly reduced by coinjected asialofetuin whereas the renal uptake of FA was not altered. Measurement of the hepatocellular localization demonstrated the overwhelming distribution of FA in the parenchymal liver cell fraction. Furthermore, the microscopic examination revealed FA that was effectively endocytosed by the parenchymal liver cells. These results suggested that FA which is bound to the asialoglycoprotein receptor with a high affinity is subsequently internalized to the hepatocyte via receptor-mediated endocytosis. FA was partially activated by periodate oxidation in order to acquire aldehyde groups to which guest molecules can be bound. A 12.5% oxidized arabinogalactan keeping a hepatocellular targetability showed a good conjugating reactivity to guest molecules via Schiff-base formation or by reductive amination. It was suggested that arabinogalactan can serve as a potential carrier for the delivery of enzymes and drugs to the parenchymal liver cells via the asialoglycoprotein receptor.

Animals↗
PubMed2000

Different membrane targeting of prostaglandin EP3 receptor isoforms dependent on their carboxy-terminal tail structures.

Mouse prostaglandin EP3 receptor consists of three isoforms, EP3alpha, beta and gamma, with different carboxy-terminal tails. To assess the role of their carboxy-terminal tails in membrane targeting, we examined subcellular localization of myc-tagged EP3 isoforms expressed in MDCK cells. Two isoforms, EP3alpha and EP3beta, were localized in the intracellular compartment but not in the plasma membrane, while the EP3gamma isoform was found in the lateral plasma membrane and in part in the intracellular compartment. Mutant EP3 receptor lacking the carboxy-terminal tail was localized in the intracellular compartment but not in the plasma membrane. Thus, EP3 isoforms differ in subcellular targeting, and the carboxy-terminal tails play an important role in determination of the membrane targeting of EP3 receptor.

Animals↗
PubMed2000

Molecular decipherment of Rho effector pathways regulating tight-junction permeability.

We reported recently that the activation of RhoA induced an increase in transepithelial electrical resistance (TER). To clarify effectors of Rho for this RhoA-induced regulation of tight-junction permeability, we introduced two effector-loop mutants of constitutively active RhoA(V14), RhoA(V14/L40) and RhoA(V14/C42), into Mardin-Darby canine kidney cells in an isopropyl beta-D-thiogalactoside-inducible expression system. RhoA(V14) and the two effector-loop mutants interacted in vitro with the Rho-binding domain of Rho-associated kinase, ROKalpha. Next we examined two parameters of Rho functions, stress-fibre formation and TER elevation, induced by RhoA(V14). Stress-fibre formation was induced by RhoA(V14/C42) but not by RhoA(V14/L40). On the other hand, TER elevation was induced by neither RhoA(V14/L40) nor RhoA(V14/C42). RhoA-associated kinase inhibitor, Y-27632, inhibited both stress-fibre formation and TER elevation induced by RhoA(V14). These results demonstrated that RhoA-induced regulation of tight-junction permeability is mediated by Rho-associated kinase and at least one other unidentified effector, the coupling to RhoA being disrupted by mutation at position 40 or 42 in the effector loop.

Animals↗
PubMed2000

The effect of reaction temperature for nephelometric assays for rheumatoid factor.

Even using the same assay parameter, reagent and calibrator (N-latex RF kit II), the results of the assay for serum rheumatoid factors (RFs) with the Behring Nephelometer Analyzer (BNA) were higher than those with the Behring Nephelometer II Analyzer (BNII) ([BNII]=0.76 [BNA]-5.7 kIU/l, r=0.997, Sy/x=60.73, n=99). The mean bias (BNA minus BNII)+/-S.D. was 52.7+/-85.5 using the Bland and Altman plot method, and the bias was not constant. The only difference in the assay condition with the two methods was the reaction temperature with the BNA being performed at room temperature (25+/-1 degrees C) and the BNII being performed at 37 degrees C. The ratio of the results with the BNII to the BNA (BNII/BNA) ranged from 0.23 to 1.18. A significant difference was observed in the BNII/BNA ratio in patients with high levels of C-reactive protein (CRP) over 2.0 mg/l (mean BNII/BNA ratio; 0.78) in comparison to patients with normal CRP levels under 2.0 mg/l (mean BNII/BNA ratio; 0.65) (P<0.01). The RF concentrations with the BNA were reduced by addition of urea, which has been used as a mild protein-denaturing agent, and there was a significant correlation between the values calculated as (1-value treated with urea/original value without urea)x100 and the BNII/BNA ratio (r=0.652, P<0.01). These data suggested that the bias between the RF values obtained by the BNA and BNII might be caused by the variation in the reactivity of autoantibodies, which might be decreased in some inflammatory diseases.

Arthritis, Rheumatoid↗
PubMed2000

Clinical utility of monitoring sialyl Lewis(X) (CD15S) antigen on peripheral lymphocytes for the diagnosis and treatment of rejection after renal transplantation.

BACKGROUND: In organ transplantation, the grafts must be carefully monitored, but it is often difficult to make a quick and accurate diagnosis of unusual changes. Extensive research has failed to identify a useful marker for rejection. We investigated the clinical utility of sialyl Lewis(X) (CD15s) monitoring in 17 renal transplant patients with acute rejection. METHODS: The expression of CD15s on peripheral lymphocytes was examined using flow cytometry in renal transplant recipients with rejection (n=17), without rejection (n=23), recipients infected with cytomegalovirus (n=7), recipients with other diseases (n=7), and healthy volunteers (n=18). CD15s expression was compared with histological findings, and was also examined before and after steroid pulse therapy to investigate the effects of steroids on CD15s antigen expression on the surface of the peripheral lymphocytes. RESULTS: CD15s was strongly expressed in all patients with rejection, but was not expressed in any of the patients without rejection or in any healthy volunteers. Histologically, cell infiltration into the rejected graft was moderate or severe in all patients with strong expression of CD15s. In contrast, no or only mild infiltration was observed in patients with weak expression of CD15s. In addition, 14 of 17 patients (14/17, 82%) with strong CD15s expression improved upon administration of steroid pulse therapy, although there was no benefit from steroids in any of the patients with weak expression of CD15s. CONCLUSIONS: The CD15s antigen is expressed strongly on the peripheral lymphocytes at the time of rejection. It is interesting that the efficacy of steroid therapy in the patients with elevated creatinine could be predicted by CD15s expression on the peripheral lymphocytes before graft biopsy. There have been only few reports showing the relationship between CD markers and the efficacy of the treatment in patients with elevated creatinine. We report that the detection of CD15s on the peripheral lymphocytes by flow cytometry was an easy, helpful, and noninvasive means for the diagnosis and treatment of patients with elevated creatinine after renal transplantation.

Adult↗
PubMed2000

The levels and kinetics of oxygen tension detectable at the surface of human dermal fibroblast cultures.

Low oxygen tension has recently been shown to stimulate cell growth and clonal expansion, as well as synthesis and transcription of certain growth factors and extracellular matrix components. These results have been obtained by exposing cell cultures to a hypoxic environment. Using an oxygen probe, we have now studied how experimental conditions affect the oxygen tension detectable at the cell surface. Dissolved oxygen tension was directly related to the height of the medium above the cell surface (r = 0.8793, P = 0.021), but was constant when no cells were present in the flask (r = -0. 9732, P = 0.001). In both human dermal fibroblasts and NIH/3T3 cultures, oxygen tension decreased linearly as cell density increased (r = -0.835, P < 0.0001; r = -0.916, P < 0.0001, respectively). When human dermal fibroblasts were exposed to 2% O(2), maximum hypoxic levels (0 mmHg) were achieved within approximately 15 min, and the recovery time was within a similar time frame. The addition of rotenone, an inhibitor of cellular respiration, blocked this decrease in oxygen tension at the cell surface, suggesting that cellular consumption of oxygen is responsible for the decline. Finally, we examined the cell-surface oxygen tension in control and acutely wounded human skin equivalents (HSE), consisting of a keratinocyte layer over a type I collagen matrix containing fibroblasts. We found that oxygen tension dropped significantly (P < 0.0001) in acutely wounded areas of HSE as compared to unwounded areas of HSE and that this drop was prevented by the addition of mitomycin C. These results indicate that cell-surface oxygen tension is indirectly related to cell density, and that the amount of detectable oxygen at the cell surface is a function of cell density, the oxygen tension in the incubator, and increased cellular activity, as occurs after injury.

Cell Count↗
PubMed2000

Proteomic approach to the identification of cell membrane proteins.

The expression of plasma membrane proteins in human monocyte-derived U937 cells was examined by cell disruption and isolation of microsomal fractions. Two alternative procedures for cell disruption, Dounce homogenization and nitrogen cavitation, were compared. Cell homogenization and sequential centrifugation resulted in an approximately fivefold enrichment of plasma membrane proteins in the microsomal fraction. However, identification of 30 such apparently enriched proteins by two-dimensional (2-D) electrophoresis, proteolytic digestion, and mass spectrometry revealed that only eight were plasma membrane proteins, the remaining 22 being contaminants. In contrast, nitrogen cavitation followed by sequential centrifugation and solubilization of proteins with sodium dodecyl sulfate (SDS) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) detergent yielded subcellular fractions, including microsomes, that showed little overlap in constituent proteins as revealed by 2-D electrophoresis. These results highlight the importance of obtaining pure plasma membranes and complete solubilization of membrane proteins for proteomic analysis.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2000

Glypican-4 is an FGF2-binding heparan sulfate proteoglycan expressed in neural precursor cells.

FGF2 is a crucial mitogen for neural precursor cells in the developing cerebral cortex. Heparan sulfate proteoglycans (HSPGs) are thought to play a role in cortical neurogenesis by regulating the action of FGF2 on neural precursor cells. In this article, we present data indicating that glypican-4 (K-glypican), a GPI-anchored cell surface HSPG, is involved in these processes. In the developing mouse brain, glypican-4 mRNA is expressed predominantly in the ventricular zone of the telencephalon. Neither the outer layers of the telencephalic wall nor the ventricular zone of other parts of the developing brain express significant levels of glypican-4, with the exception of the ventricular zone of the tectum. In cultures of E13 rat cortical precursor cells, glypican-4 is expressed in cells immunoreactive for nestin and the D1.1 antigen, markers of neural precursor cells. Glypican-4 expression was not detected in early postmitotic or fully differentiated neurons. Recombinant glypican-4 produced in immortalized neural precursor cells binds FGF2 through its heparan sulfate chains and suppressed the mitogenic effect of FGF2 on E13 cortical precursor cells. The spatiotemporal expression pattern of glypican-4 in the developing cerebral wall significantly overlaps with that of FGF2. These results suggest that glypican-4 plays a critical role in the regulation of FGF2 action during cortical neurogenesis.

Amino Acid Sequence↗
PubMed2000

Antioxidant activity of the extracts from fruiting bodies of cultured Cordyceps sinensis.

Cordyceps sinensis is one of the most valued herbs in traditional Chinese medicine. We investigated the antioxidant activities of the cultured fruiting bodies of Cordyceps sinesis. The water and ethanol extracts of Cordyceps sinensis were found to possess a potent antioxidant activity. The scavenging effects of the extracts on superoxide were very weak, but the extracts moderately inhibited malondialdehyde formation via hydroxyl radical induced by SIN-1, a peroxynitrite generator. Of the extracts examined, the hot water extract (70 degrees C for 5 min) showed the greatest oxygen free radical scavenging activity. Also, when low-density lipoprotein (LDL) was incubated with macrophages in the presence of CuCl2 (1 microM), the hot water extract showed a strong inhibitory effect against lipid peroxidation in the medium and consequent accumulation of cholesteryl ester in macrophages. Their activities were comparable to that of authentic Cu/Zn SOD. These results suggest that the extracts of cultured Cordyceps sinensis possess potent antioxidant and anti-lipid peroxidation activities and inhibit accumulation of cholesteryl ester in macrophages via suppression of LDL oxidation.

Animals↗
PubMed2000

Inhibitory effects of water extracts from fruiting bodies of cultured Cordyceps sinensis on raised serum lipid peroxide levels and aortic cholesterol deposition in atherosclerotic mice.

We investigated the effects of the water extracts of the fruiting bodies of cultured Cordyceps sinensis (WECS) on lipid metabolism in mice fed an atherogenic diet. WECS was orally administered at doses of 50, 100 and 200 mg/kg/day for 12 weeks. WECS showed no toxic effects on the growth rate, liver or kidney weights of the mice. Mice fed the atherogenic diet showed marked increases in serum lipid and lipid peroxide levels and also aortic cholesterol levels, particularly cholesteryl ester level, a major lipid constituent in atherosclerotic lesions. WECS significantly suppressed the increased serum lipid peroxide level but not other lipid levels in a dose-dependent manner. WECS also suppressed the increased aortic cholesteryl ester level in a dose-dependent manner. These results suggest that WECS prevents cholesterol deposition in the aorta by inhibition of LDL oxidation mediated by free radicals rather than by reduction in serum lipid level. WECS may exert beneficial effects on the formation of the atherosclerotic lesion induced by oxidative stress with few side effects.

Animals↗
PubMed2000

Thrombin and factor Xa enhance neutrophil chemoattractant production after ischemia/reperfusion in the rat liver.

BACKGROUND: Clotting proteases may affect leukocyte effector function. Activation of the coagulation cascade after ischemia/reperfusion stimulates cytokine production by activated macrophages. Cytokine-induced neutrophil chemoattractant (CINC) may also be important in the pathophysiology of liver ischemia/reperfusion injury. We investigated the effects of a selective factor Xa inhibitor, DX-9065a, on CINC expression after ischemia/reperfusion in the rat liver. METHODS: Liver ischemia was induced in rats by occluding the portal vein for 30 min. DX-9065a (9 mg/kg) was injected intravenously 5 min before vascular clamping. Serum CINC concentrations were measured by enzyme-linked immunosorbent assay. Levels of CINC mRNA in the liver were determined by Northern blot analysis. We also examined in vitro CINC production by peritoneal macrophages in response to alpha-thrombin or factor Xa. RESULTS: Serum CINC concentrations increased and peaked 6 h after reperfusion. However, pretreatment of animals with DX-9065a resulted in significantly smaller increases in CINC after reperfusion. Pretreatment with DX-9065a also significantly reduced CINC mRNA levels in the liver after ischemia/reperfusion. In vitro CINC production by peritoneal macrophages was enhanced by alpha-thrombin, as well as factor Xa. CONCLUSIONS: Thrombin and factor Xa stimulate CINC production by macrophages. A selective inhibitor of factor Xa, DX-9065a, attenuates neutrophil chemoattractant production after ischemia/reperfusion injury of the rat liver.

Animals↗
PubMed2000

Urinary trypsin inhibitor reduces C-X-C chemokine production in rat liver ischemia/reperfusion.

BACKGROUND AND AIM: Protease inhibitors attenuate ischemia/reperfusion injury. However, the underlying mechanisms by which protease inhibitors prevent reperfusion injury remain obscure. Neutrophils play an important role in reperfusion injury. We studied the effects of urinary trypsin inhibitor (UTI) on production of the C-X-C chemokine, cytokine-induced neutrophil chemoattractant (CINC), by Kupffer cells during ischemia/reperfusion of the liver. METHODS: Liver ischemia was induced in rats by occlusion of the portal vein for 30 min. UTI (50,000 U/kg) was injected intravenously 5 min before vascular clamping. Serum CINC concentrations were measured by enzyme-linked immunosorbent assay. Levels of CINC mRNA in the liver were determined by Northern blot analysis. We also examined the inhibitory effects of UTI on in vitro CINC production by peritoneal macrophages in response to neutrophil elastase (NE). RESULTS: Serum CINC concentrations increased and peaked 6 h after reperfusion. However, pretreatment of animals with UTI blunted this increase in CINC and significantly reduced CINC mRNA levels in the liver after ischemia/reperfusion. UTI also decreased neutrophil accumulation in the liver 24 h after reperfusion. In vitro CINC production by Kupffer cells from rats pretreated with UTI 3 h after ischemia/reperfusion was significantly decreased compared to those from untreated animals. UTI reduced NE activity in vitro in a dose-dependent manner, and UTI significantly reduced in vitro CINC production by peritoneal macrophages stimulated with NE. CONCLUSION: UTI reduces the production of CINC by Kupffer cells stimulated with NE, attenuating ischemia/reperfusion injury of the liver.

Alanine Transaminase↗
PubMed2000

Lecticans: organizers of the brain extracellular matrix.

Lecticans are a family of chondroitin sulfate proteoglycans, encompassing aggrecan, versican, neurocan and brevican. These proteoglycans are characterized by the presence of a hyaluronan-binding domain and a C-type lectin domain in their core proteins. Through these domains, lecticans interact with carbohydrate and protein ligands in the extracellular matrix and act as linkers of these extracellular matrix molecules. In adult brain, lecticans are thought to interact with hyaluronan and tenascin-R to form a ternary complex. We propose that the hyaluronan-lectican-tenascin-R complex constitutes the core assembly of the adult brain extracellular matrix, which is found mainly in pericellular spaces of neurons as 'perineuronal nets'.

Alternative Splicing↗
PubMed2000

Anti-insulin receptor autoantibodies in a patient with type B insulin resistance and fasting hypoglycemia.

We studied a patient with systemic lupus erythematosus and type B insulin resistance who showed almost complete normalization of postprandial plasma glucose in 3 months and a transient occurrence of fasting hypoglycemia from day 35 (i.e. the 35th day of hospitalization) to day 77. To determine the clinical relevance of the biological ability of anti-insulin receptor antibodies (anti-IRAb), we made multiple preparations of the patient's dialyzed serum and IgG. Dialyzed serum prepared on day 1 showed 95% inhibition of insulin binding. The binding inhibition was, however, decreased parallel to the normalization of insulin sensitivity. For 2DG uptake, 6.2 microM IgG purified on 3 different days (days 7, 35 and 78, designated IgG-NOV, -JAN, and -FEB, respectively) stimulated 2DG uptake into CHO-hIR at 3.4-, 3.1-, and 1.5-fold, respectively. Phosphotyrosine immunoblotting revealed that apparent insulin receptor autophosphorylation was visible only with IgG-NOV, not with the IgG-JAN or -FEB. Mutation of tyrosine-960 or lysine-1018 of the insulin receptor failed to transduce the IgG's stimulatory effect. IgG-NOV was not able to stimulate the autophosphorylation of the human IGF-I receptor. In the present study, the insulin binding inhibitory activities of the dialyzed sera prepared at different time points were shown to be altered parallel to insulin sensitivity in vivo. Stimulatory activities of the patient's IgG were, however, discordant for the occurrence of fasting hypoglycemia observed in vivo. Other pathogenic factors or mechanisms in addition to the insulin-like action of the anti-IRAb may be also required to fully understand the development of fasting hypoglycemia in type B insulin resistance.

Animals↗
PubMed2000

Endoscopic biliary stenting for treatment of persistent biliary fistula after blunt hepatic injury.

BACKGROUND: Surgical treatment for persistent biliary fistula after blunt hepatic injury is often technically difficult. Endoscopic treatment for such fistulas has been described only infrequently. METHODS: We reviewed 6 patients who underwent endoscopic biliary stent placement with (n = 1) or without (n = 5) sphincterotomy for persistent (12 to 138 days; mean 48 days) biliary fistula after blunt hepatic injury. RESULTS: ERCP showed bile leakage from a second-order or more peripheral branch of the intrahepatic bile ducts in 5 patients but failed to reveal the fistula in 1. Stent placement was successful without complications in all patients. Bile leakage resolved within 1 to 3 days in 5 patients. After 36 to 86 days, the stent was removed and ERCP confirmed disappearance of the fistula. These patients have remained asymptomatic for a mean of 2.6 years since stent removal. In the patient in whom ERCP had not shown a fistula, bile leakage continued despite successful stent placement. CONCLUSIONS: Endoscopic biliary stent placement is a rapid, safe and effective treatment for persistent post-traumatic biliary fistula demonstrated by ERCP.

Adult↗