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Biomedical subjects

A Yamamoto

Publications and source records attributed to A Yamamoto.

At least 19 recordsLinked to original sources
PubMed1995

p53 and human papillomavirus DNA in renal pelvic and ureteral carcinoma including dysplastic lesions.

Ninety-eight cases of transitional-cell carcinoma (TCC) of the renal pelvis and ureter, including dysplastic lesions, were studied for tumor incorporation of human papillomavirus (HPV) type-16 and type-18 DNA by in situ hybridization (ISH) with DNA probes for each HPV viral type. Immunohistochemical analysis of p53 expression was also performed. Fresh tumor tissues from 26 patients were also studied for p53 mutations in exons 4 through 9 by direct sequencing and for HPV infection by polymerase chain reaction (PCR). Thirty-two tumors were positive for HPV DNAs, including 6 double-positive cases. Among these tumors, adjacent dysplastic lesions in 21 cases (66%) also revealed identical reactivity. Overexpressed p53 was detected in 26 cases. Expression of p53 was also detected in dysplastic lesions in 19 out of these 26 cases (73%). Three cases were positive for both HPV DNA and p53 antibody. p53 point mutation was detected in 7 of 26 cases, 6 of which were also positive for p53. HPV type-16 DNA was detected in 6 cases by PCR, 4 of which were also ISH-positive. Overexpressed p53 was frequently detected in invasive and non-papillary tumors (p < 0.01) and in high-grade tumors (p < 0.05). HPV infection was more common in non-invasive and papillary tumors (p < 0.05). These findings suggest that HPV infection or overexpression (mutation) of p53 may be an early event and be related to phenotypes of tumor-cell growth patterns and progression.

Adult↗
PubMed1995

Vesicular L-glutamate transporter in microvesicles from bovine pineal glands. Driving force, mechanism of chloride anion activation, and substrate specificity.

Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic-like microvesicles (MVs) containing the L-glutamate transporter (Moriyama, Y., and Yamamoto, A. (1995) FEBS Lett., 367, 233-236). In this study, the L-glutamate transporter in MVs isolated from bovine pineal glands was characterized as to its driving force, requirement of anions, and substrate specificity. Upon the addition of ATP, the MVs accumulated L-glutamate. The uptake was significantly dependent on the extravesicular Cl- concentration, being negligible in the absence of Cl- and maximum at 2-5 mM and decreasing gradually at 20-100 mM. The membrane potential (inside positive) was maximum at 0-10 mM Cl- and then decreased gradually depending on the Cl- concentration, whereas a pH gradient was practically absent without Cl- and increased gradually up to 100 mM Cl-. Ammonium acetate or nigericin plus K+, a dissipator of a pH gradient, had little effect on or was slightly stimulatory toward the uptake, whereas valinomycin plus K+ inhibited both formation of the membrane potential and the glutamate uptake to similar extents. The ATP- and Cl(-)-dependent glutamate uptake was inhibited by fluoride, iodide, or thiocyanate, without vacuolar H(+)-ATPase being affected. An anion channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, similarly inhibited the glutamate uptake in a Cl- protectable manner. Furthermore, ATP- and glutamate-dependent acidification of MVs was observed when 4 mM Cl- was present. Among more than 50 kinds of glutamate analogues tested, only a few compounds, including 1-aminocyclohexane-trans-1,3-dicarboxylic acid, caused similar acidification. A good correlation was observed between the acidification and the inhibition of glutamate uptake by glutamate analogues. These results indicated that 1) the major driving force of the glutamate uptake is the membrane potential, 2) Cl- regulates the glutamate uptake, probably via anion-binding site(s) on the transporter, and 3) the transporter shows strict substrate specificity. Hence, the overall properties of the vesicular glutamate transporter in the MVs well matched those of the synaptic vesicle glutamate transporter. We concluded that the vesicular glutamate transporter, being similar if not identical to the neuronal counterpart, operates in endocrine cells.

ATP-Binding Cassette Transporters↗
PubMed1995

A single point mutation in the splice donor site of the low-density-lipoprotein-receptor gene produces intron read-through, exon-skipped and cryptic-site-utilized transcripts.

Familial hypercholesterolemia is a genetic disorder caused by mutations of the low-density-lipoprotein (LDL) receptor gene. We characterized the structures of LDL receptor mRNA transcripts in the fibroblasts of a homozygous patient carrying a single base substitution (T-->C) at the 5' splice donor site of intron 12 of the LDL receptor gene. We identified three aberrant transcripts as a consequence of intron-12 read-through, exon-12 skipping and utilization of a cryptic splice donor site. Only a point mutation at the 5' splice donor site caused the production of three alternatively spliced products. None of these transcripts produced a functional LDL receptor protein in this patient.

Alternative Splicing↗
PubMed1995

Syntaxin 1 (HPC-1) is associated with chromaffin granules.

Syntaxin 1 (HPC-1), a component of the receptor for SNAPs (soluble N-ethylmaleimide-sensitive factor attachment proteins), has been implicated in the docking and fusion of synaptic vesicles with the plasma membrane. It was reported that syntaxin 1 in rat brain and chromaffin cells (PC12) is exclusively located on the plasma membrane (Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255-259; Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409-418). By means of biochemical and morphological analyses, we now show that syntaxin 1 is associated with chromaffin granules in the adrenal medulla. This finding raises the possibility that syntaxin 1 in chromaffin cells is a component of vesicle-SNAP receptor as well as one of target-SNAP receptor on the plasma membrane.

Adrenal Medulla↗
PubMed1995

Microvesicles isolated from bovine pineal gland specifically accumulate L-glutamate.

Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic-like microvesicles (MVs) containing synaptophysin. By monitoring cross-reactivity with anti-synaptophysin antibody, the MVs were highly purified from bovine pineal glands. The purified MVs were morphologically similar to but distinct from neuronal synaptic vesicles by their lack of synapsin I. Immunological study indicated that the MVs contained vacuolar H(+)-ATPase, synaptotagmin and synaptobrevin 2 (VAMP2). The MVs accumulated L-glutamate at the expense of ATP hydrolysis by vacuolar H(+)-ATPase. No uptakes of melatonin, serotonin, noradrenaline, gamma-aminobutyrate or acetylcholine were observed. These results indicated that the MVs are organelles for storage of L-glutamate in pinealocytes and suggested a possibility that pinealocytes transmit glutamate signals by MVs-mediated exocytosis.

ATP-Binding Cassette Transporters↗
PubMed1995

Dimer form of phosphorylated Spo0A, a transcriptional regulator, stimulates the spo0F transcription at the initiation of sporulation in Bacillus subtilis.

The Spo0A protein of Bacillus subtilis is a transcriptional regulator that shows extensive homology to the regulator proteins in bacterial two-component regulatory systems. Phosphorylation of Spo0A is absolutely necessary for the initiation of sporulation. We now show that phospho-Spo0A is a dimer, binds specifically to the spo0F promoter region, and stimulates the transcription from the P2 promoter recognized by sigma H-RNA polymerase. Biochemical and biological analyses suggest that phospho-Spo0A interacts directly with the "0A-like box" sequence (TGTCGTA) located in the spo0F promoter region. Phosphorylation of Spo0A enhanced its affinity to the 0A-like box. Evidence is also presented that the spo0F promoter region contains a static bend having two sets of oligo(dA-dT) tracts. It was demonstrated that the bending region overlaps with the recognition site for the phospho-Spo0A.

Bacillus subtilis↗
PubMed1995

Microvesicles isolated from bovine posterior pituitary accumulate norepinephrine.

Histochemical study indicated that the posterior pituitary possesses numerous microvesicles (MVs) containing synaptophysin, a marker protein specific for brain synaptic vesicles (Navone, F., Di Gioia, G., Jahn, R., Browning, M., Greengard, P., and De Camilli, P. (1989) J. Cell Biol. 109, 3425-2433). By monitoring cross-reactivity with anti-synaptophysin antibody, the MVs were highly purified from bovine posterior pituitaries by a combination of differential and sucrose density gradient centrifugations. The purified MVs had an average diameter of about 60 nm and were associated with synaptophysin as revealed by immunoelectron microscopy. The vesicles contained ATPase activity partially sensitive to bafilomycin A1 and to vanadate. The membrane fraction immunoisolated with anti-synaptophysin antibody also exhibited similar ATPase activity. The two ATPases could be purified separately; the vandate-sensitive enzyme was identified as a 115-kDa polypeptide immunochemically similar to chromaffin granule P-ATPase (forming phosphoenzyme intermediate), and the bafilomycin A1-sensitive ATPase showed essentially the same properties as those of vacuolar type H(+)-ATPases. Upon addition of ATP, the MVs formed an electrochemical gradient of protons and took up norepinephrine in a reserpine-sensitive manner, indicating the presence of secondary monoamine transporter coupled with vacuolar type H(+)-ATPase. No uptake of L-glutamate, gamma-aminobutyrate, glycine, or acetylcholine was observed. The identification of MVs as organelles responsible for storage of monoamines is important for understanding the physiological function of the posterior pituitary.

Acetylcholine↗
PubMed1995

Inhibition of cholesterol synthesis ex vivo and in vivo by fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The inhibitory effect of fluvastatin sodium (fluvastatin), a new type of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A inhibitor, on de novo cholesterol synthesis was investigated and compared with that of pravastatin. Fluvastatin at a concentration of 12.5 mg/kg inhibited sterol synthesis ex vivo from [14C]acetate in rat liver and ileum by 97-99% with respect to the control, while the inhibition in kidney was 55%. The inhibition by fluvastatin in the liver and ileum persisted for approximately 9 h after administration. Significant differences between fluvastatin also had an inhibitory effect on cholesterol synthesis in vivo in various tissues of rats given [14C]acetate intraperitoneally. Sterol synthesis in the liver, ileum and kidney was inhibited by over 95% 3 h after administration of 6.25 mg/kg of fluvastatin. Significant differences between fluvastatin and pravastatin were found in the liver and ileum. Fluvastatin was more potent than pravastatin in inhibiting both ex vivo and in vivo sterol synthesis in the ileum (but not in kidney) and liver.

Acetates↗
PubMed1995

T cell receptor-mediated stimulation of mouse thymocytes induces up-regulation of the GM2/GD2 synthase gene.

cDNA clones of the mouse GM2/GD2 synthase (EC 2.4.1.92) gene were isolated, and their analyses revealed that the protein has a type II transmembrane structure with 533 amino acids, which was very similar to the human homolog except for the mRNA size. The mRNA level in thymocytes dramatically increased after treatment with anti-CD3 monoclonal antibody, whereas it was not elevated when treated with prostaglandin E2. In situ hybridization showed an elevation of mRNA levels in medullar thymocytes, suggesting that T cell receptor-mediated signaling induces up-regulation of the GM2/GD2 synthase gene in mature thymocytes.

Amino Acid Sequence↗
PubMed1995

Absorption characteristics of chemically modified-insulin derivatives with various fatty acids in the small and large intestine.

Absorption characteristics of insulin derivatives chemically modified with various fatty acids in the intestine were determined by in situ loop and in vitro modified Ussing chamber methods. The pharmacological activities of these acyl derivatives, as assessed by their hypoglycemic effects after intravenous administration, were reduced upon increasing the carbon number of the fatty acid(s) chemically attached to native insulin. However, high pharmacological activities were seen when mono-and dicaproyl derivatives were administered intravenously. The absorption of insulin after its small intestinal administration could be hardly improved by acylation. In contrast, its absorption after the large intestinal administration was increased by increasing the number of caproic acid molecules attached to insulin. Furthermore, by an in vitro modified Ussing chamber method, it was revealed that the permeability of insulin across both the duodenal and colonic mucous membranes was also improved by increasing the number of caproic acid molecules. These in situ and in vitro results indicated that the chemical modification of insulin with fatty acids was a useful approach for improving insulin absorption from the large intestine.

Animals↗
PubMed1995

Impaired water barrier function in acne vulgaris.

In acne vulgaris, abnormal follicular keratinization is important for comedo formation, yet the precise mechanisms of comedogenesis are not known. The present study examined the interrelationship between sebum secretion rate (SSR), lipid content and water barrier function (WBF) of the stratum corneum (SC) in 36 acne patients and 29 control subjects. All major SC lipid classes were separated and quantified by thin-layer chromatography/photodensitometry. WBF was evaluated by measuring transepidermal water loss (TEWL), and the hygroscopic properties and waterholding capacity of the SC. The SSR over a period of 3 h was significantly higher in patients with moderate acne than in control subjects, but no significant difference was noticed between patients with mild acne and control subjects. Significant differences between patients with both moderate and mild acne and control subjects were noted in the amount of sphingolipids (ceramides and free sphingosine), but not for any other lipid classes. Furthermore in acne patients, lower amounts of sphingolipids were observed corresponding with a diminished WBF. These results suggest that an impaired WBF caused by decreased amounts of ceramides may be responsible for comedo formation, since barrier dysfunction is accompanied by hyperkeratosis of the follicular epithelium.

Acne Vulgaris↗
PubMed1995

Response of 3-hydroxy-3-methylglutaryl CoA reductase to l-triiodothyronine in cultured fibroblasts from FH homozygotes.

The effect of l-triiodothyronine on 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase activity was measured in cultured skin fibroblasts from patients with homozygous familial hypercholesterolemia (FH). The tested strains were obtained from 13 receptor-negative and 6 receptor-defective type homozygotes and 3 heterozygotes. Eleven out of 19 strains of cultured fibroblasts from FH homozygotes demonstrated high levels of the HMG CoA reductase activity when l-triiodothyronine was present in the culture medium. All the 11 strains that responded to l-triiodothyronine were the receptor-negative type in which the binding of LDL on the cell surface was completely lacking. Two receptor-negative type strains showed no response to the addition of l-triiodothyronine. In these strains, partially active receptors were synthesized and rapidly degraded. The effect of l-triiodothyronine on HMG CoA reductase was abolished by cycloheximide, and not by actinomycin D. Furthermore, the effect was abolished by the pre-loading of the cells with free cholesterol. The results indicate that the effect of l-triiodothyronine on HMG CoA reductase activity was a post-transcriptional event, required de novo protein synthesis, and was successful only when cholesterol was depleted from the cells. The difference in the responsiveness of HMG CoA reductase activity to l-triiodothyronine treatment can be utilized to judge the state of impairment of LDL-receptors in the FH homozygote from the viewpoint of ability to incorporate cholesterol into the cells.

Cells, Cultured↗
PubMed1995

Genetic but not diet-induced hypercholesterolemia causes low apolipoprotein A-IV level in rabbit sera.

The present report describes a competitive enzyme immunoassay for rabbit apolipoprotein A-IV (apo A-IV). This assay was applied to the determination of its concentration and distribution in sera from normolipidemic and hyperlipidemic rabbits. The assay was sufficiently sensitive to study this 42-kDa protein in lipoproteins fractionated from 200 microliters of serum by FPLC gel filtration. In normolipidemic sera (n = 8), apo A-IV concentration was 5.32 +/- 0.76 mg/dl. A diet rich in cholesterol (0.5%), which induced an 18-fold increase in serum cholesterol, did not significantly alter apo A-IV concentration (6.65 +/- 1.52 mg/dl, n = 8). By contrast, genetically induced hypercholesterolemia (Watanabe heritable hyperlipidemia, WHHL mutation) caused a significantly reduced level of apo A-IV (3.8 +/- 1.14 mg/dl, n = 7). In each of the groups studied, apo A-IV was distributed in two distinct pools; a high-density lipoprotein-(HDL) associated pool and a lipoprotein-free pool. However, compared to normal, the distribution of apo A-IV in WHHL rabbit sera was shifted towards the lipoprotein-free pool. Consistent with previously reported observations on apo A-I, these results are compatible with the hypothesis of an impaired reverse transport of cholesterol in WHHL rabbits, an animal model for familial hypercholesterolemia.

Amino Acid Sequence↗
PubMed1995

Exogenous supply of artificial lipoproteins does not decrease susceptibility to atherosclerosis in cholesterol-fed rabbits.

We investigated the effects of reconstituted apo A-I-containing high-density lipoprotein (r-HDL, homologous to small pre-beta-migrating HDL) and reconstituted triglyceride-rich lipoprotein (r-TRL, a mixture of Intralipid and apo E) on atherogenesis in rabbits fed a 0.5% cholesterol diet for 8 weeks. Male Japanese white rabbits (n = 17) were divided into three groups: the control group (n = 7) received a placebo and 2.5 ml of Intralipid 20 h later; the second group (n = 6) received r-HDL containing 18 mg of apo A-I followed by 2.5 ml of Intralipid; and the third group (n = 4) received 18 mg of r-HDL and 2.5 ml of r-TRL containing 4 mg of apo E. Rabbits were injected with the agents weekly and the same interval (20 h) was maintained between the two injections. Three minutes after the injection of r-HDL, a sharp increase in the pre-beta-migrating fraction was observed. The cholesterol-rich diet similarly increased serum lipids in the three groups. No significant changes of the HDL cholesterol and apo A-I concentrations were observed in the three groups. Conversely, there was a 12-fold increase of apo E which correlated positively with the total cholesterol level. Injection of r-HDL and r-TRL caused slight inhibition of fatty streak development and lipid deposition in the aortic wall, but neither change was statistically significant. Lipid accumulation in the liver was similar in all three groups. These results suggest that the physiological properties of artificial and native lipoproteins may differ.

Animals↗
PubMed1995

A subpopulation of large ganglion neurons express IsK protein mRNA: an in situ hybridization analysis in the rat eye.

Rat IsK protein is a 130-amino acid membrane protein and subserves generation of K+ outward current. The expression of this putative K+ ion channel mRNA was explored in the eye with in situ hybridization. The ganglion cell layer of the retina expressed IsK mRNA in a subpopulation of ganglion cells composed of large cell bodies. The hybridization-positive cells were scattered throughout the retina. The hybridization signal was also localized to the epithelial cells throughout the cornea. The results provided evidence for IsK message in neuronal cells. Expression of IsK message in the eye was thus shown to be restricted to particular cell types.

Animals↗
PubMed1995

Immunofluorescence techniques for the identification of immune effector cells in rat heart: applications to the study of the myocarditis induced by interleukin-2.

A detailed description is presented of immunohistochemical methods for identification of various types of immune effector cells in rat heart, involving the use of antibodies conjugated with different fluorochromes for the simultaneous demonstration of 2 or 3 different antigens by means of fluorescence microscopy. The initial results of the application of these techniques to the study of the myocarditis induced by interleukin-2 (IL-2) are also presented. Antibodies used included: OX6 antibody (for MHC class II molecules, mainly expressed by dendritic cells): W3/25 and OX8 antibody, for the demonstration of the rat equivalents of CD5 and CD8, respectively: asialo-GM1 ganglioside antibody for the identification of natural killer (NK) cells and lymphokine activated killer (LAK) cells, and ED2 antibody for labeling of macrophages. Fluorochromes used were: fluorescein isothiocyanate (green), tetramethylrhodamine isothiocyanate (red), Texas red sulfonyl chloride (red), and 7-amino-4-methylcoumarin-3-acetic acid (blue). IL-2-induced myocarditis was characterized histologically by infiltration of the myocardium by mononuclear inflammatory cells, microvascular alteration, interstitial edema, and myocyte damage and necrosis. In the initial stages, NK/LAK cells were the predominant type of infiltrating lymphocytes; however, the numbers of these cells decreased sharply in subsequent stages. Macrophages also were initially abundant, and continued to be prevalent throughout the late stages. CD8+ lymphocytes were more numerous than CD4+ lymphocytes. Dendritic-cells showed a diffuse increase in number and also accumulated around foci of myocyte necrosis. Three phenotypes of dendritic cells were recognized, and the possible implications of these findings are discussed. It is hoped that these techniques will prove useful for the immunohistochemical evaluation of various inflammatory diseases of the heart.

Animals↗
PubMed1995

Diverse expression of beta 1,4-N-acetylgalactosaminyltransferase gene in the adult mouse brain.

Among various tissues of mouse, beta 1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.

Animals↗