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Biomedical subjects

E Uchida

Publications and source records attributed to E Uchida.

At least 55 records · Page 3Linked to original sources
PubMed1998

Anti-obesity effects of selective agonists to the beta 3-adrenergic receptor in dogs. I. The presence of canine beta 3-adrenergic receptor and in vivo lipomobilization by its agonists.

It is known that in rodents and humans the beta 3-adrenergic receptor (beta 3-AR) is present primarily in adipocytes and plays a significant role in the adrenergic stimulation of lipolysis. We examined the expression of beta 3-AR mRNA in the dog and the lipomobilizing effects of beta 3-AR-selective agonists in vivo. Reverse transcription polymerase chain reaction of RNA extracted from dog adipose tissue produced a cDNA fragment, the nucleotide sequence of which was highly homologous to the corresponding regions of human (86.4%) and mouse (79.5%) beta 3-AR cDNA. The beta 3-AR mRNA was present at high levels in subcutaneous and visceral adipose tissues, but undetectable in other organs. When a selective beta 3-AR agonist, CL316,243, was infused intravenously into beagle dogs, the plasma level of free fatty acid increased in 30 min and persisted at higher levels for several hours. ICI D7114, another beta 3-AR agonist, also showed a similar lipomobilizing effect, but with lower potency. beta 3-AR agonist infusion also increased the plasma insulin level. These results suggested that functional beta 3-AR is present in adipose tissues of the dog and that it is effective for in vivo lipomobilization.

Adipose Tissue↗
PubMed1998

Anti-obesity effects of selective agonists to the beta 3-adrenergic receptor in dogs. II. Recruitment of thermogenic brown adipocytes and reduction of adiposity after chronic treatment with a beta 3-adrenergic agonist.

The aim of this study was to evaluate the effectiveness of beta 3-adrenergic agonists for the treatment and prevention of obesity in the dog. When a selective beta 3-adrenergic agonist, CL316,243 (0.1 mg/kg), was given orally to adult beagles every day for 5-7 weeks, body weight and girth were decreased compared with control placebo-treated dogs. Gross anatomical examinations revealed no noticeable abnormalities in CL316,243-treated dogs, except an apparent decrease in abdominal fat. Immunohistochemical examination of perirenal adipose tissue showed a remarkable increase in brown adipocytes expressing a thermogenic protein, uncoupling protein (UCP). The increased expression of UCP and its mRNA in CL316,243-treated dogs was also confirmed by Western blot and reverse transcription polymerase chain reaction analyses. It was concluded that treatment with a beta 3-adrenergic agonist stimulates UCP expression, which may lead to an increase in energy expenditure, and thereby is useful for the treatment and prevention of obesity in the dog.

Adipose Tissue, Brown↗
PubMed1998

Uptake of acetyl low density lipoprotein (LDL) by stimulated canine macrophages.

Canine monocytes from peripheral blood were stimulated by acetyl low density lipoprotein (LDL) to detect receptor-mediated uptake of lipoproteins and to clarify the role of monocytes involving in atherosclerotic lesions. Monocytes collected from peripheral blood which were treated with phorbol 12-myristate 13-acetate (PMA) took up acetyl LDL and differentiated into macrophage-like cells (foamy macrophages). Uptake of acetyl LDL was inhibited specifically by maleyl canine serum albumin (MSA) depending on its concentration. Monocytes treated with PMA did not take up native LDL. These results suggest that canine monocytes express the receptor-mediated uptake of acetyl LDL by a specific pathway (the so-called scavenger receptor) and that they play a crucial role at the initial stage of canine atherosclerotic lesions.

Animals↗
PubMed1998

Pharmacodynamics and pharmacokinetics of a single oral dose of nitrazepam in healthy volunteers: an interethnic comparative study between Japanese and European volunteers.

Potential interethnic differences in drug disposition and effects between Japanese and white subjects hamper the registration in Japan of medications already used in Western countries. This double-blind, placebo-controlled, crossover study was conducted to compare the pharmacodynamics and pharmacokinetics of a single oral dose of nitrazepam (5 mg) in age- and sex-matched Japanese (n = 8) and white (n = 8) healthy volunteers. The study was performed in centers in Japan and the Netherlands using the same methods and study design. Subjects were individually matched for gender, age, and body stature. Drug effects were measured by means of saccadic and smooth pursuit eye movements and visual analog lines obtained from the scales of Bond and Lader. There were no pharmacokinetic differences between the Japanese and white subjects. Clearance of nitrazepam was 0.91 +/- 0.165 mL/min/kg and 1.17 +/- 0.492 mL/min/kg, and half-life (t1/2) was 22.1 +/- 4.96 hours and 21.5 +/- 7.51 hours for the Japanese and European groups, respectively. Pharmacokinetic parameters showed no significant correlation with age, height, or weight. The average time-effect curves for the different parameters were comparable between groups. Compared with placebo, both groups showed similar significant reductions in average peak velocity and increases in saccadic inaccuracy and reaction time. Visual analog scores showed clear sedation in the white subjects, but insignificant effects in the Japanese subjects. Smooth pursuit did not change significantly in either group. Slope and intercept of the concentration-effect relationships for saccadic peak velocity showed considerable intersubject variability, but no clear differences between groups. The pharmacokinetics and pharmacodynamics of nitrazepam were similar in matched healthy Japanese and white subjects. Interethnic comparative studies are feasible, and provide meaningful information about potential racial differences in disposition and action of drugs. Such studies can form a rational basis for comparative clinical trials.

Administration, Oral↗
PubMed1997

STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation.

Recent studies have indicated that serine phosphorylation regulates the activities of STAT1 and STAT3. However, the kinase(s) responsible and the role of serine phosphorylation in STAT function remain unresolved. In the present studies, we examined the growth factor-dependent serine phosphorylation of STAT1 and STAT3. We provide in vitro and in vivo evidence that the ERK family of mitogen-activated protein (MAP) kinases, but not JNK or p38, specifically phosphorylate STAT3 at serine 727 in response to growth factors. Evidence for additional mitogen-regulated serine phosphorylation is also provided. STAT1 is a relatively poor substrate for all MAP kinases tested both in vitro and in vivo. STAT3 serine phosphorylation, not its tyrosine phosphorylation, results in retarded mobility of the STAT3 protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Importantly, serine 727 phosphorylation negatively modulates STAT3 tyrosine phosphorylation, which is required for dimer formation, nuclear translocation, and the DNA binding activity of this transcriptional regulator. Interestingly, the cytokine interleukin-6 also stimulates STAT3 serine phosphorylation, but in contrast to growth factors, this occurs by an ERK-independent process.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

[The significance of blood group-related antigen A in a pancreatic cell line (PGHAM-1) in hamsters. With special reference to cell proliferation].

The relationship between blood group-related antigen A and cell proliferation was studied in a pancreatic carcinoma cell line (PGHAM-1) induced by N-nitrosobis (2-oxopropyl) amine (BOP) in hamsters. In vitro, the cell proliferation of PGHAM-1 was inhibited in a culture medium with an added monoclonal antibody (MoAb) against blood group-related antigen A depending on its concentration. In in vivo experiments, immunohistochemical double staining was performed using MoAb against blood group-related antigen A and bromodeoxyuridine (BrdU) in pancreatic carcinomas by intrapancreatic transplantation of PGHAM-1. Antigen A was expressed in the cell membrane and cytoplasm of PGHAM-1 cells. The BrdU labeling index (LI) in lesions with strong expression of MoAb A (> or = 50%) was 27.4 +/- 5.00, whereas the BrdU LI in lesions with weak expression of MoAb A (< or = 10%) was 11.9 +/- 2.10. The mean LIs in strongly expressed lesions of MoAb A were higher than those in weakly expressed lesions of MoAb A (p < 0.01). These results indicated that antigen A might be associated with cell proliferation in pancreatic carcinomas induced in hamsters.

ABO Blood-Group System↗
PubMed1997

Effect of active oxygen radicals on protein and carbohydrate moieties of recombinant human erythropoietin.

Our previous study showed that active oxygen radicals generated from a Fenton system and a xanthine plus xanthine oxidase system caused serious loss of in vivo bioactivity of recombinant human erythropoietin (EPO), a highly glycosylated protein. In the present study, we characterized the oxidative modifications to the protein and carbohydrate moiety of EPO, which lead to a reduction of its bioactivity. In vitro bioactivity was reduced when EPO was treated with oxygen radicals generated from a Fenton system in the presence of 0.016 mM H2O2, and the reduction was directly proportional to the loss of in vivo bioactivity. SDS-PAGE analysis showed that dimer formation and degradation was observed under more severe conditions (Fenton reaction with 0.16 mM H2O2). The tryptophan destruction was detected at 0.016 mM H2O2 and well correlated with the loss of in vitro bioactivity, whereas loss of other amino acids were occurred under more severe conditions. Treatment with the Fenton system did not result in any specific damage on the carbohydrate moiety of EPO, except a reduction of sialic acid content under severe condition. These results suggest that active oxygen radicals mainly react with the protein moiety rather than the carbohydrate moiety of EPO. Destruction of tryptophan residues is the most sensitive marker of oxidative damage to EPO, suggesting the importance of tryptophan in the active EPO structure. Deglycosylation of EPO caused an increased of susceptibility to oxygen radicals compared to intact EPO. The role of oligosaccharides in EPO may be to protect the protein structure from active oxygen radicals.

Amino Acids↗
PubMed1996

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialyltransferase.

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol-1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between the in vivo bioactivity and the sialic acid content of the fractionated samples was observed until 12.1 mol mol-1 of EPO, there was no further increase in their activity over 12.4 mol mol-1 of EPO. On the other hand, an inverse relationship between the in vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that the in vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with alpha 2,6-sialyltransferase, but the in vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.

Animals↗
PubMed1996

Polyacrylamide gel electrophoretic serum protein patterns of acute inflammation induced by intramuscular injection of turpentine in young broiler chickens.

Changes in the serum protein patterns accompanying inflammation in young broilers were examined by polyacrylamide gradient gel electrophoresis. Sera were obtained from blood of 4-week-old male broilers after induction of acute inflammation by an injection of 0.5 ml/kg turpentine. Electrophoretic patterns at 48 hr after injection, showed an increase in transferrin (Tf) and a segment containing albumin (Alb) with multiple peaks. The unbound iron binding capacity (UIBC) increased by 10 times, and the serum iron (SI) concentration in acute inflammation was reduced to one-half its initial value. These are considered to be typical changes in serum proteins and iron after acute inflammation.

Animals↗
PubMed1996

Basement membrane thickening in small intramyocardial vessels in eosinophilic myocarditis.

The case reported in this paper, besides illustrating several aspects of the eosinophilic myocarditis, documents a yet undescribed feature of the cardiac involvement in idiopathic hypereosinophilic syndrome characterized by marked basement membrane thickening of small intramyocardial vessels. We are unaware of previous reports showing the presence of an extensive thickening of the basement membrane of arteriolar and capillary intramyocardial vessels in eosinophilic myocarditis. Considering the findings in the present case, i.e., the focal nature of the lesions, the type of necrosis, and the diffuse microangiopathy, it can be hypothesized that the microvascular disease could play a role in the development of the myocardial changes.

Basement Membrane↗
PubMed1996

Trimethadione N-demethylation by rat liver CYP2E1 in vitro.

Trimethadione (TMO) is a model drug utilized for estimation of hepatic metabolism in clinical studies, and it was reported that TMO N-demethylase activity was inhibited by CYP2E1 inhibitors and substrates in rat in vivo. This study was performed to investigate the involvement of the CYP2E1 subfamily on TMO N-demethylation in vitro and to clarify these inhibitory mechanisms. The effects of acetone (AC), imidazole (IM) and N-nitrosodimethylamine (NDA) on TMO N-demethylation were studied in vitro. Rat hepatic microsomal fractions were employed as the enzyme source of TMO N-demethylase and the activity was determined by the production of dimethadione (DMO). DMO was analyzed by a GC/FTD equipped with a narrow-bore capillary column. TMO N-demethylation was biphasic by the graphic analysis of Eadie-Hofstee plots; this suggests the involvement of at least two enzymes in TMO metabolism in the rat. The kinetic parameters for the formation of DMO were analyzed graphically using double-reciprocal plots. The apparent K(m1), K(m2) and Vmax1, Vmax2 values for DMO formation were 4, 20 mM and 182, 595 pmol/mg protein/min, respectively. AC and IM inhibited TMO N-demethylase activity competetively. However, mixed inhibition kinetics was observed by NDA. Furthermore, TMO N-demethylase activity was inhibited by antiserum to CYP2E1 by 62% and CYP3A2 by 46%. These results indicate that the CYP2E1 subfamily is the major enzyme involved in TMO N-demethylation in rat in vitro although the CYP3A2 is also involved in this transformation.

Animals↗
PubMed1996

[The Somatropin Reference Standard (Control 951) of the National Institute of Health Sciences].

Somatropin material was examined for preparation of the "Somatropin Reference Standard". The candidate material was evaluated by a domestic collaborative study in which eight laboratories participated. The protein content was determined to be 4.5 mg/Vial based on amino acid analysis. Because of the possibility of application as a chemical reference standard for assay by the HPLC method, a physico-chemical evaluation of the candidate material was also performed. By SE-HPLC, the content of polymer, dimer were determined to be 0.54%, 0.98%, respectively. By RP-HPLC, the early peak area ascribed to desamido and sulfoxide form was 1.07% of the total peak area. And for informational data, the potency of the candidate material, being estimated by three different biological methods, weight gain assay, tibia test and adiposeconversion assay is 14.8 IU/vial. Based on the above results, the candidate was authorized as the Somatropin Reference Standard of the National Institute of Health Sciences.

Amino Acids↗
PubMed1995

The activity of lecithin:cholesterol acyltransferase in the serum of cows at parturition or with fatty liver.

The activity of lecithin:cholesterol acyltransferase (LCAT), which is responsible for esterification of plasma cholesterol, was evaluated in bovine serum. It was associated with the high-density lipoprotein fraction that contains apolipoprotein A-I, an activator of LCAT. In lactating cows, the activity was around 1000 U (decrease in nmol of free cholesterol per h per ml of serum), slightly higher than in 1-month-old calves. LCAT activity decreased around parturition, at which the time the serum concentrations of cholesteryl esters and free cholesterol were concomitantly decreased. A reduced LCAT activity was also found in cows with fatty livers induced by the administration of ethionine. In the cows with fatty livers, the serum concentration of cholesteryl esters was markedly decreased, whereas that of free cholesterol was only slightly decreased, thereby increasing the free- to esterified-cholesterol ratio. These results suggest that the decrease in LCAT activity may be involved in the reduction in fertility associated with fatty liver because esterification of cholesterol by LCAT is essential for its transport from the liver to peripheral tissues, such as the corpus luteum, and because cholesterol serves as the source of progesterone synthesis in the latter organ.

Animals↗
PubMed1995

Activity of artificial mutant variants of human growth hormone changes in charged residues around 62-67.

Our previous work has shown that the amino acid residues around 62-67 located in the connecting loop between helix I and II of human growth hormone (hGH) are important in eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes. In this study, we evaluated the role of the charged residues around 62-67 in receptor binding and biological activity. Eight artificial mutant variants of hGH were prepared in Escherichia coli by site-directed mutagenesis. Replacement of Arg64 with Tyr (R64Y variant) resulted in a significant loss of binding to the somatogenic receptors on 3T3-F442A cells, but retained full adipose conversion activity on these cells. Replacement of Arg64 with Glu (R64E) produced a considerable loss in receptor binding and a significant loss in biological activity. hGH variants in which either Glu65 or Glu66 was replaced with Asp (E65D and E66D) and with Gln (E65Q and E66Q) showed a slight loss in binding activity and retained almost a full adipogenic activity. An E65P variant (replacement of Glu65 with Pro) possessed the same binding activity as hGH, although it failed to induce full biological activity. The insertion of Ala between Asn63 and Arg64 (63NAR) caused a marked loss in both activities. These results indicate that the positively charged Arg64 is important for receptor binding and thereby in eliciting the biological activity of hGH, while negatively charged Glu65 and Glu66 are less important. In addition, our findings confirm that the conformation and size of the loop region around Arg64 is important for the adipose conversion activity of hGH.

Adipose Tissue↗
PubMed1995

Changes of hepatic microsomal oxidative drug metabolizing enzymes in chronic renal failure (CRF) rats by partial nephrectomy.

Male SD rats, 7-weeks-old, were used to investigate the changes in the hepatic drug metabolizing system of chronic renal failure (CRF) model rats. Partial nephrectomy (5/6) was performed in a two-stage surgical procedure. After nephrectomy, the rats were housed under regular conditions at least 21 days. After confirming the CRF states, trimethadione (TMO, 100 mg/kg, i.p.) was administered for evaluation of the hepatic drug metabolizing capacity; the ratio of dimethadione (DMO: the only metabolite of TMO) to TMO (DMO/TMO) in the serum and the dialysate from the blood microdialysis method were ascertained. The hepatic drug metabolizing enzyme contents and activities were also determined. In the CRF rats, the DMO/TMO ratios decreased significantly; total cytochrome P450 (CYP) contents, aminopyrine N-demethylase activity and delta-aminolevulinic acid synthetase activity also decreased significantly in the CRF rats. The extent of the alterations of these enzyme contents and activities correlated well with the severity of the CRF states evaluated by the serum blood urea nitrogen and creatinine concentrations. With Western blot analysis, the levels of CYP2C6, CYP2C11 and CYP3A2 decreased considerably in the CRF rats. These results suggest that CRF states induce not only a reduction of renal function but also an alteration of hepatic metabolism.

Animals↗
PubMed1995

The loss of in vivo activity of recombinant human erythropoietin by active oxygen species.

The effects of active oxygen species on the in vivo activity of recombinant human erythropoietin (EPO) treated by Fenton system, xanthine (X) plus xanthine oxidase (XO) system and hydrogen peroxide (H2O2) has been studied by means of counting the increase in number of hemolyser-resistant cells (HRCs) in EPO-injected mice. The results showed that both Fenton and X plus XO systems caused a significant reduction of the activity in proportion to the concentration of generated active oxygen species. Meanwhile, the treatment of EPO with H2O2 alone resulted in a relatively slight reduction of the activity. Electrophoretic studies on the structure of EPO revealed that its main protein band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) disappeared in proportion with the extent of exposure to active oxygen generating systems. Both Fenton and X plus XO systems caused a significant loss of fluorescence in the pyridylamino (PA-) sugar chain in proportion to the concentration of generated active oxygen species, and no degradation products in the sugar chain part of the PA-sugar chain were detected. This showed that aromatic groups in EPO were sensitive to attack by active oxygen species. These results provide evidence that hydroxyl radical and other active oxygen species have a potential to react with EPO, leading to a reduction of its in vivo activity.

Animals↗
PubMed1995

Polyacrylamide gel electrophoretic patterns of chicken serum in acute inflammation induced by intramuscular injection of turpentine.

To develop a method to detect hidden inflammation using serum protein in chickens, changes in serum proteins with acute inflammation were analyzed using a turpentine-induced inflammation model. Inflammation in the pectoral muscle of a 14-wk-old White Leghorn became apparent 3 h after the injection of turpentine and became more severe thereafter. Coincident with the development of inflammation, changes in serum proteins were analyzed by electrophoresis on polyacrylamide gradient gels. The electrophoretic patterns were divided into 21 segments. Two of these segments increased remarkably. These were located near the center of the electrophoretic pattern and were identified as transferrin due to iron staining, correlation of movement against a commercial transferrin sample in SDS-PAGE, and immunoblotting. These results suggest that transferrin may serve as a marker for inflammation in chicken.

Acute-Phase Reaction↗