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Biomedical subjects

Y Hashimoto

Publications and source records attributed to Y Hashimoto.

At least 217 records · Page 12Linked to original sources
PubMed2000

Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria.

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.

Amino Acid Sequence↗
PubMed2000

Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade.

Rap2 is a member of the Ras family of GTPases and exhibits 60% identity to Rap1, but the function and regulation of Rap2 remain obscure. We found that, unlike the other Ras family proteins, the GTP-bound active form exceeded 50% of total Rap2 protein in adherent cells. Guanine nucleotide exchange factors (GEFs) for Rap1, C3G, Epac (or cyclic AMP [cAMP]-GEF), CalDAG-GEFI, PDZ-GEF1, and GFR efficiently increased the level of GTP-Rap2 both in 293T cells and in vitro. GTPase-activating proteins (GAPs) for Rap1, rap1GAPII and SPA-1, stimulated Rap2 GTPase, but with low efficiency. The half-life of GTP-Rap2 was significantly longer than that of GTP-Rap1 in 293T cells, indicating that low sensitivity to GAPs caused a high GTP/GDP ratio on Rap2. Rap2 bound to the Ras-binding domain of Raf and inhibited Ras-dependent activation of Elk1 transcription factor, as did Rap1. The level of GTP-Rap2 in rat 3Y1 fibroblasts was decreased by the expression of v-Src, and expression of a GTPase-deficient Rap2 mutant inhibited v-Src-dependent transformation of 3Y1 cells. Altogether, Rap2 is regulated by a similar set of GEFs and GAPs as Rap1 and functions as a slowly responding molecular switch in the Rap1 signaling cascade.

3T3 Cells↗
PubMed2000

Diphenhydramine transport by pH-dependent tertiary amine transport system in Caco-2 cells.

Substrate specificity and pH dependence of the transport system for diphenhydramine were investigated in Caco-2 cell monolayers. Diphenhydramine uptake was not affected by any typical substrate for the renal organic cation transport system except procainamide. Along with procainamide, tertiary amine compounds with N-dimethyl or N-diethyl moieties in their structures inhibited the diphenhydramine uptake. Moreover, accumulation of diphenhydramine was stimulated by preloading the Caco-2 cells with these tertiary amines (trans-stimulation effect), indicating the existence of the specific transport system for tertiary amines with N-dimethyl or N-diethyl moieties. Efflux of diphenhydramine from monolayers was enhanced by medium acidification. In addition, intracellular acidification resulted in marked stimulation of diphenhydramine accumulation. ATP depletion of the cells caused an enhancement of diphenhydramine accumulation, suggesting the involvement of an active secretory pathway. However, P-glycoprotein did not mediate the diphenhydramine transport. These findings indicate that a novel pH-dependent tertiary amine transport system that recognizes N-dimethyl or N-diethyl moieties is involved in diphenhydramine transport in Caco-2 cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Transport of procainamide via H(+)/tertiary amine antiport system in rabbit intestinal brush-border membrane.

Transport characteristics of procainamide in the brush-border membrane isolated from rabbit small intestine were studied by a rapid-filtration technique. Procainamide uptake by brush-border membrane vesicles was stimulated by an outward H(+) gradient (pH(in) = 6.0, pH(out) = 7.5) against a concentration gradient (overshoot phenomenon), and this stimulation was reduced when the H(+) gradient was subjected to rapid dissipation by the presence of a protonophore, FCCP. An outward H(+) gradient-dependent procainamide uptake was not caused by H(+) diffusion potential. The initial uptake of procainamide was inhibited by other tertiary amines with N-dimethyl or N-diethyl moieties in their structures, such as triethylamine, dimethylaminoethyl chloride, and diphenhydramine, but not by tetraethylammonium and thiamine. Furthermore, procainamide uptake was stimulated by preloading the vesicles with these tertiary amines (trans-stimulation effect), indicating the existence of a specific transport system for tertiary amines. These findings indicate that procainamide transport in the intestinal brush-border membrane is mediated by the H(+)/tertiary amine antiport system that recognizes N-dimethyl or N-diethyl moieties in the structures of tertiary amines.

Amines↗
PubMed2000

Functional expression of novel peptide transporter in renal basolateral membranes.

We examined the peptide transport activity in renal basolateral membranes. [(14)C]glycylsarcosine (Gly-Sar) uptake in rat renal cortical slices was saturable and inhibited by excess dipeptide and aminocephalosporin cefadroxil. When several renal cell lines were screened for the basolateral peptide transport activity, Madin-Darby canine kidney (MDCK) cells were demonstrated to have the greatest transport activity. [(14)C]Gly-Sar uptake across the basolateral membranes of MDCK cells was inhibited by di- and tripeptide and decreased with decreases in extracellular pH from 7.4 to 5.0. The Michaelis-Menten constant value of [(14)C]Gly-Sar uptake across the basolateral membranes of MDCK cells was 71 microM. The basolateral peptide transporter in MDCK cells showed several different [(14)C]Gly-Sar transport characteristics in growth dependence, pH profile, substrate affinity, and sensitivities to chemical modifiers from those of the apical H(+)-peptide cotransporter of MDCK cells. The findings of the present investigation indicated that the peptide transporter was expressed in the renal basolateral membranes. In addition, from the functional characteristics, the renal basolateral peptide transporter was suggested to be distinguishable from known peptide transporters, i.e., H(+)-peptide cotransporters (PEPT1 and PEPT2) and the intestinal basolateral peptide transporter.

4-Chloromercuribenzenesulfonate↗
PubMed2000

A case of antiepiligrin cicatricial pemphigoid successfully treated by plasmapheresis.

We report the case of a 73-year-old Japanese woman suffering from antiepiligrin (laminin 5) cicatricial pemphigoid (CP) with typical clinical and immunopathological features. Histologically, the lesional mucous membrane showed a subepidermal blister formation. When indirect immunofluorescence techniques with skin split by 1 M NaCl as the substrate were used, the patient's serum reacted only to the dermal side. Immunoprecipitation studies demonstrated that the patient's serum contained IgG autoantibodies directed against a set of polypeptides that corresponded to epiligrin (laminin 5). After corticosteroids and immunosuppressive agents had been administered systemically, the patient's autoantibody titer decreased and the cutaneous and mucosal blister formations were suppressed. However, the ocular lesions persisted in spite of these therapeutic regimens. After combining these treatments with double-filtration plasmapheresis, the ocular lesions improved and showed almost no progression. Plasmapheresis may thus present a new option for the treatment of CP.

Aged↗
PubMed2000

Effects of octreotide infusion, surgery and estrogen on suppression of height increase and 20K growth hormone ratio in a girl with gigantism due to a growth hormone-secreting macroadenoma.

We treated an extremely tall 13-year-old girl with a growth hormone (GH)-secreting macroadenoma and GH levels of 120-495 ng/ml with a combination of preoperative octreotide infusion, surgery and postoperative octreotide infusion plus estrogen, which resulted in reduced tumor size prior to surgery, reduced GH levels and completely suppressed growth after surgery. 20K GH is produced by alternative splicing of 22K GH mRNA and the ratio of 20K GH to 22K GH is within a small range in the normal population and high in a GH-secreting tumor. The 20K/22K GH ratio in this patient was persistently elevated during each phase of the treatment and may serve as a sensitive index of tumor-derived GH secretion.

Adenoma↗
PubMed2000

Relationship between the serum level of leptin and life-style habits in Japanese men.

AIMS/METHODS: To investigate whether smoking affects the serum level of leptin, 708 male workers aged 25-65 years old were cross-sectionally surveyed. RESULTS: Multiple regression analysis indicated that among the various parameters examined, the level of leptin was positively associated with the body mass index and the levels of insulin, total cholesterol and uric acid, and was inversely associated with physical activity and the level of creatinine. The partial correlation coefficient of leptin was highest against the body mass index (r = 0.40), followed by insulin (0.29) and physical activity (-0.14), after adjustment for other leptin-related variables. However, no association was observed between the level of leptin and smoking (0.05), alcohol consumption (0.09) or age (0.09). CONCLUSIONS: The findings suggest that among life-style habits, physical activity, but not smoking or alcohol consumption, significantly affects the serum level of leptin in Japanese men.

Adult↗
PubMed2000

QT dispersion in patients with Takayasu arteritis.

This study was designed to test the hypothesis that myocardial involvement exists in patients with Takayasu arteritis and is associated with increased QT dispersion, which is a marker of repolarization inhomogeneity. Twenty-one consecutive patients with Takayasu arteritis and no significant coronary artery disease were included. Twelve-lead electrocardiogram and exercise-induced thallium-201 myocardial scintigraphy were performed in all patients. Ten of 21 patients (48%) had abnormal findings on scintigraphy. Patients were divided into two groups by the presence (group P, n = 10) or absence (group N, n = 11) of exercise-induced thallium-201 myocardial scintigraphic perfusion abnormalities, including permanent defects in three, reversible defects in four, and slow washout in three. The QT dispersion at rest was significantly greater in group P than that in group N (54 +/- 12 vs 40 +/- 8 msec, p < 0.005). The QTc dispersion at rest was also significantly greater in group P than in group N (59 +/- 15 vs 43 +/- 11 msec, p < 0.01). In patients with Takayasu arteritis, myocardial involvement suggested by exercise-induced thallium-201 myocardial scintigraphic perfusion abnormalities is not rare, even when no significant coronary stenosis is present on angiography. Increased baseline QT dispersion was associated with scintigraphic abnormalities and may be a useful marker of myocardial involvement in patients with Takayasu arteritis.

Adult↗
PubMed2000

Exogenous 20K growth hormone (GH) suppresses endogenous 22K GH secretion in normal men.

The physiological and pharmacological functions of the 20-kDa human GH (20K-hGH) isoform are unknown. We conducted a pharmacokinetic study of recombinant 20K-hGH in human subjects (Phase I clinical trial). Placebo or 20K-hGH was administered sc to normal men (20-31 yr of age, n = 6-8 per group) at 2100 h. Serum 20K- and 22K-hGH levels were monitored every 30 min for 24 h by specific enzyme-linked immunosorbent assays. Serum free fatty acid, insulin-like growth factor I, insulin, and glucose levels were measured for 24 h. In the placebo group, the secretion profiles of endogenous 20K- and 22K-hGH were pulsatile and similar to each other. The proportion of 20K- to 22K-hGH was fairly constant. In the 20K-hGH-treated groups, serum 20K-hGH levels increased in a dose-dependent manner over the dose range of 0.01-0.1 mg/kg. Maximum serum 20K-hGH levels were reached at 3-4 h and decreased with half-lives of 2-3 h. Marked suppression of endogenous 22K-hGH secretion was observed in a time-dependent manner. Serum free fatty acid and insulin-like growth factor I levels were significantly elevated (P < 0.01) at 4, 8, and 12 h and at 8, 12, and 24 h after 20K-hGH administration, respectively. Serum insulin and glucose levels did not change significantly within 24 h. These results suggested that: 1) regulation of 20K-hGH secretion is physiologically the same as that of 22K-hGH; 2) the pharmacokinetics after sc injection of 20K-hGH are comparable with those of 22K-hGH; 3) 20K-hGH regulates hGH secretion through "GH-induced negative feedback mechanisms"; and 4) administration of 20K-hGH is expected to exert GH actions (growth-promoting activity and lipolytic activity). Monitoring of serum 20K- and 22K-hGH levels may be useful in evaluating the effects of administered GH isoforms on their own release from the pituitary.

Adult↗
PubMed2000

Pharmacokinetics and bioavailability of tacrolimus in rats with experimental renal dysfunction.

The effects of renal failure on the pharmacokinetics and bioavailability of tacrolimus were investigated in rats. Experimental renal dysfunction was induced by intraperitoneal injection of cisplatin (5 mg kg(-1)) into rats. The blood concentration of tacrolimus was measured after intravenous and intra-intestinal administration of the drug. The blood concentration of tacrolimus after intravenous administration (1 mg kg(-1)) was slightly increased (up to 1.3 fold) by induction of renal dysfunction. In contrast, the peak tacrolimus concentration after intra-intestinal administration (1 mg kg(-1) or 3 mg kg(-1)) in rats with renal failure was about 2-fold higher than that in normal controls. The bioavailability was increased by about 35% in rats with impaired renal function as compared with normal controls. These results suggested that the bioavailability of tacrolimus, which is mainly metabolized in the liver and intestine after oral administration, is also influenced by renal function.

Alanine Transaminase↗
PubMed2000

Separation of phosphatidylinositol 4-phosphate from a mixture with phosphatidylserine and phosphatidylinositol by Sep-Pak C18 cartridge.

A simple and efficient method for the separation of phosphatidylinositol 4-phosphate (PI 4-P) from phosphatidylinositol (PI) and phosphatidylserine (PS) is described. A mixture of PI, PI 4-P and PS was injected onto a Sep-Pak C18 cartridge. PI and PS were flushed through the cartridge with solvent 1 [methanol-chloroform (3: 1)] while PI 4-P remained in it. Then the cartridge was inverted, and PI 4-P was eluted backward with solvent 2 [chloroform-methanol-0.5 M aqueous ammonium hydroxide (9:7:2)].

Phosphatidylinositol Phosphates↗
PubMed2000

Identification of protein disulfide isomerase as a phorbol ester-binding protein.

A specific binding protein for 12-O-tetradecanoylphorbol 13-acetate (TPA), different from protein kinase C (PKC) and histone H1, was purified from HeLa cell extract by the use of affinity gel pendanted with phorbol ester (TPA-GEL). The purified binding protein was identified as protein disulfide isomerase (PDI, EC 5.4.3.1) by peptide sequence analysis. The dissociation constants (Kd's) of TPA to PDI, histone H1 and PKCalpha were determined to be 1.03 x 10(-6) M, 5.70 x 10(-7) M, and 4.00 x 10(-7) m, respectively, by the surface plasmon resonance (SPR) method. TPA moderately inhibited PDI activity assessed in terms of reactivation of denatured RNase A.

Amino Acid Sequence↗
PubMed2000

Anti-androgenic activity of substituted azo- and azoxy-benzene derivatives.

Substituted phenylazo and phenylazoxy compounds were systematically prepared and their anti-androgenic activity was measured in terms of (1) the growth-inhibiting effect on an androgen-dependent cell line, SC-3, and (2) the binding affinity to nuclear androgen receptor. Generally, azo/azoxy compounds showed cell toxicity, and the growth-inhibiting effects on SC-3 cells correlated with the toxicity. However, some compounds, including 4,4'-dinitroazobenzene (25), 4,4'-dimethoxyazobenzene (33), and 2,2'-dichloroazoxybenzene (47), possessed potent anti-androgenic activity without apparent cell toxicity.

Androgen Antagonists↗
PubMed2000

Enhancing effect of tumor promoters, phorbol esters and teleocidins on nuclear receptor-mediated transcription.

Interaction between a tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), and ligands of nuclear receptors has been interpreted as the result of crosstalk between the nuclear receptors and oncogenic transcription factor AP-1. We examined the effects of various tumor promoters on transcription mediated by several nuclear receptors (RAR, TR, and ROR) by using thymidine kinase promoter-based reporter systems. TPA-type and other types of tumor promoters (okadaic acid, thapsigargin) enhanced reporter gene transcription independently of the cognate ligands for the receptors. Various kinds of TPA-type tumor promoters, teleocidine and its synthetic derivatives (indolactam, benzolactams) enhanced reporter gene transcription in proportion to their differentiation-inducing activities. Although TPA is known to activate protein kinase C (PKC), some PKC inhibitors did not inhibit the effect of TPA on reporter gene transcription. Interestingly, staurosporin, a strong PKC inhibitor and also a tumor promoter, enhanced the effect of TPA and weakly enhanced the reporter transcription itself. These results suggest this reporter system is useful for the evaluation of effects on the gene expression of various tumor promoters, including non-TPA type.

Animals↗
PubMed2000

Modulators of tumor necrosis factor alpha production bearing dicarba-closo-dodecaborane as a hydrophobic pharmacophore.

Previous studies on the structural development of tumor necrosis factor alpha (TNF-alpha) production regulators derived from thalidomide (N-alpha-phthalimidoglutarimide) revealed that a hydrophobic substituent at the nitrogen atom of the phthalimide ring is critical for potent activity. We have designed and prepared phthalimide derivatives bearing a boron cluster, dicarba-closo-dodecaborane (carborane), which has a hydrophobic character and spherical geometry, as a novel candidate of biological response modifiers. These compounds were shown to regulate TNF-alpha production by HL-60 cells, as expected. The result provides a further example of the application of carborane as a hydrophobic pharmacophore of biologically active molecules.

Enzyme-Linked Immunosorbent Assay↗
PubMed2000

Expression and characterization of human rheumatoid factor single-chain Fv.

The variable region of heavy chain [V(H)] of human rheumatoid factor (hRF) IgM was connected with the variable region of light chain [V(L)] with the peptide-linker (GGGSGGGSGGGS) by genetic engineering method and the single-chain Fv (scFv) was expressed in E. coli. On design, scFv and scFv (tag) were planned; the latter had a detection marker at the carboxyl-terminal. These scFvs were expressed as inclusion bodies in E. coli, purified in the presence of 8 M urea by gel filtration and renatured to the active form in vitro. As a control, the Fv, non-covalently associated V(H) and V(L) fragments, was also constructed. The 3 derivatives showed almost the same binding activity to rabbit-IgG to which hRF is cross-reactive. ScFv (tag) was the most stable against urea among the 3 derivatives.

Amino Acid Sequence↗