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Biomedical subjects

T Shiono

Publications and source records attributed to T Shiono.

At least 91 records · Page 5Linked to original sources
PubMed1986

Purification and characterization of rat lens pyrroline-5-carboxylate reductase.

delta 1-Pyrroline-5-carboxylate reductase (L-proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) has been purified from rat lens and biochemically characterized. Purification steps included ammonium sulfate fractionation, affinity chromatography on Amicon Matrex Orange A, and gel filtration with Sephadex G-200. These steps were carried out at ambient temperature (22 degrees C) in 20 mM sodium phosphate/potassium phosphate buffer (pH 7.5) containing 10% glycerol, 7 mM mercaptoethanol and 0.5 mM EDTA. The enzyme, purified to apparent homogeneity, displayed a molecular weight of 240 000 by gel chromatography and 30 000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme is composed of eight subunits. The purified enzyme displays a pH optimum between 6.5 and 7.1 and is inhibited by heavy metal ions and p-chloromercuribenzoate. Kinetic studies indicated Km values of 0.62 mM and 0.051 mM for DL-pyrroline-5-carboxylate as substrate when NADH and NADPH respectively were employed as cofactors. The Km values for the cofactors NADH and NADPH with DL-pyrroline-5-carboxylate as substrate were 0.37 mM and 0.006 mM, respectively. With L-pyrroline-5-carboxylate as substrate, Km values of 0.21 mM and 0.022 mM were obtained for NADH and NADPH, respectively. Enzyme activity is potentially inhibited by NADP+ and ATP, suggesting that delta 1-pyrroline-5-carboxylate reductase may be regulated by the energy level and redox state of the lens.

Adenosine Triphosphate↗
PubMed1986

Acid hydrolases in the bovine corneal epithelium.

The activities of acid phosphatase, N-acetyl-beta-D-glucosaminidase, alpha-mannosidase, alpha-fucosidase, beta-glucuronidase, arylsulfatase, and cathepsin D were biochemically investigated in the bovine cornea by separating the tissue into two layers, epithelium and stroma-endothelium. Acid phosphatase, alpha-mannosidase, alpha-fucosidase, and arylsulfatase disclosed much higher activities in the epithelial layer than in the stroma-endothelial layer. The other enzymes showed little difference in enzyme activity between the two layers.

Animals↗
PubMed1986

Molecular cloning of human ornithine aminotransferase mRNA.

The isolation and characterization of a cDNA clone for the mRNA of human ornithine aminotransferase (OATase; ornithine-oxo-acid aminotransferase; L-ornithine:2-oxo-acid aminotransferase, EC 2.6.1.13), a nonabundant mitochondrial matrix enzyme that is severely deficient in a hereditary chorioretinal degenerative disease (gyrate atrophy), is described. Human liver, retina, and retinoblastoma (Y79) mRNAs were prepared and tested for the OATase mRNA content by in vitro translation, immunoprecipitation, and NaDodSO4/PAGE. The retinoblastoma cells were found to be expressing this enzyme at a relatively high level. The primary translation product of the OATase mRNA is larger than the pure OATase protein on NaDodSO4/PAGE by approximately equal to 4 kDa, suggesting a precursor protein. lambda gt11 cDNA libraries were prepared from the human mRNAs, and the recombinant clones were immunoscreened as plaques with two different preparations of rabbit anti-human OATase antibodies. A clone (lambda gtRB315) was isolated from the retinoblastoma library that reacts with both of the antibody preparations, and the DNA sequence of its 2.1-kilobase-pair cDNA insert was obtained. An open reading frame consisting of 1371 nucleotides is present in the sequence, and a putative translational initiation methionine codon is identified at position 55. A putative leader sequence consisting of 32 amino acid residues is identified, resulting in a precursor protein of 439 amino acid residues and a molecular mass of 48,534 Da and a mature protein of 407 residues and 45,136 Da. The amino acid sequences of seven tryptic peptides (115 amino acid residues) of the pure human OATase were obtained by microsequencing. When the tryptic peptide and cDNA-derived amino acid sequences were compared, homologies in 111 of 115 residues, including a match of 20 consecutive residues, were observed. An RNA blot hybridization of 32P-labeled OATase cDNA to normal human retina and retinoblastoma mRNAs demonstrated an OATase mRNA species of approximately equal to 2.2 kilobases. The level of OATase mRNA in the normal human retina is approximately equal to 1/100th the level of rhodopsin mRNA and 1/5th to 1/10th the level present in the retinoblastoma cells.

Amino Acid Sequence↗
PubMed1986

Gyrate atrophy of the choroid and retina: 15 Japanese patients.

We examined 15 Japanese patients who had gyrate atrophy of the choroid and retina with hyperornithinaemia. Their visual acuities fell to 0.2 or worse in the second or third decade of life. Myopia developed late in the first decade, and the refractions decreased to -10 or -15 dioptres at age 20. Tunnel vision developed at approximately age 20. Our results suggested that the visual functions of Japanese patients were worse in the third decade or later than similarly affected Finnish patients.

Adolescent↗
PubMed1986

Optic atrophy, diabetes mellitus and diabetes insipidus.

A 19-year-old Japanese woman developed diabetes mellitus, diabetes insipidus and optic atrophy. Other abnormal ocular findings included color blindness, elevated dark adaptation threshold, loss of oscillatory potentials after a single white flash electroretinogram (ERG), reduced cone response in photopic ERG, and reduced pupillary response to light and convergence. Diabetic retinopathy, which has been thought to be very rare in this syndrome, also was found in the fundi of this patient.

Adult↗
PubMed1985

Aminoglycoside antibiotics and lysosomal enzymes of human tears.

We examined biochemically the effect of six aminoglycoside antibiotics on the activity of lysozyme, acid phosphatase, and N-acetyl-beta-D-glucosaminidase in human tears. All six antibiotics strongly inhibited lysozyme activity, the degree of inhibition depending on the dose administered. Except for bekanamycin, antibiotics had little effect on the activity of acid phosphatase or N-acetyl-beta-D-glucosaminidase. The nature of the inhibition was competitive. Other kinds of antibiotics, such as sulbenicillin or erythromycin, had no inhibitory effect on lysozyme, acid phosphatase, or N-acetyl-beta-D-glucosaminidase. These results suggested that the inhibition of lysozyme by aminoglycosides is specific and that the decreased effectiveness of a protective system against bacterial infection in the eye is highly possible when aminoglycoside antibiotics are used without prior sensitivity testing.

Acetylglucosaminidase↗
PubMed1985

Ornithine accumulation and metabolism in rat lens.

The non-protein amino acid ornithine is readily accumulated into rat lenses cultured in TC-199 bicarbonate medium. This accumulation, measured by the lens water/lens medium ratios of radiolabeled ornithine, appears to be the result of an apparent energy-dependent basic amino acid transport system. Moreover, competition studies suggest that increased levels of ornithine can depress the concomitant lenticular accumulation of arginine and lysine. Examination of the trichloroacetic acid protein precipitates of these cultured lenses indicates that the radiolabel from ornithine can also be incorporated into lens proteins as proline. This results from the conversion by ornithine aminotransferase (EC 2.6.1.13) of radiolabeled ornithine to delta1-pyrroline-5-carboxylic acid (P5C), which is subsequently converted to proline by P5C reductase (EC 1.5.1.2). This incorporation of radiolabeled into cultured lens proteins is reduced upon addition of either 1mM proline or P5C to the culture medium or inhibited by the addition of the protein synthesis inhibitor, puromycin.

Animals↗
PubMed1985

Stimulation of the hexose monophosphate pathway by pyrroline-5-carboxylate reductase in the lens.

Addition of pyrroline-5-carboxylate (P5C) or its precursors to rat lenses cultured for 24 hr in TC-199 medium containing 14C-glucose results in an apparent concentration-dependent increase in hexose monophosphate-pentose (HMP) pathway activity. Addition of proline, the reduction product of P5C, did not result in an increase, suggesting that stimulation of the HMP pathway is related to the reduction of P5C to proline by the enzyme P5C reductase. No apparent feedback inhibition on P5C reductase was observed. Stimulation of HMP pathway activity by P5C was also observed in the lenses of Philly and Nakano mouse, two models of congenital osmotic cataracts. Compared with its genetic control, the Swiss--Webster mouse, generally no difference in the lenticular levels of HMP pathway activity was observed in the Philly mouse--even after the onset of cataract. Stimulation of the HMP pathway in the Philly lens by P5C, however, was consistently lower than its control. In the lenses from the Nakano mouse and its genetic control, the Balb/c mouse, no difference in the percentage stimulation of the HMP pathway resulting from the addition of P5C was observed, but HMP pathway activity in the Nakano lens was consistently lower than that of the control.

Aging↗
PubMed1985

Enzymes in ornithine-proline metabolic pathway in bovine lens.

Various enzyme activities related to ornithine metabolism were studied using bovine lenses, ie, ornithine ketoacid transaminase, delta-1-pyrroline-5-carboxylate reductase, and delta-1-pyrroline-5-carboxylate dehydrogenase. Ornithine ketoacid transaminase activity was found in the lens epithelium; delta-1-pyrroline-5-carboxylate reductase activity was high in both lens epithelium and cortex plus nucleus; delta-1-pyrroline-5-carboxylate dehydrogenase activity was negligible in either lens epithelium or cortex plus nucleus. These results suggest that in the bovine lens ornithine is converted to proline by the cooperative action of ornithine ketoacid transaminase and delta-1-pyrroline-5-carboxylate reductase.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1984

In vitro effect of hyaluronate on lysosomal enzyme activities in the bovine ciliary body and iris.

The effect of hyaluronate on lysosomal enzyme activities in bovine ciliary body and iris was studied in vitro. Hyaluronates from both the human umbilical cord and bovine vitreous inhibited the activities of cathepsin B and acid phosphatase. Cathepsin D, beta-N-acetyl-glucosaminidase, beta-D-glucuronidase and alpha-L-fucosidase activities were not affected by the hyaluronate.

Acid Phosphatase↗
PubMed1984

Scanning electron microscopic study of polyvinyliden fluoride degradation by ocular tissue extracts.

A scanning electron microscopic study showed that the surface of the polyvinyliden fluoride thread, used to make loops for intraocular lenses in Japan, became significantly rough after treatment with bovine iris-ciliary body extracts. The polyvinyliden fluoride degrading factor in the extract was nondialyzable, heat-labile and active at an acidic pH, suggesting that a lysosomal system might be involved in this phenomenon. Bovine extracts from the retinal pigment epithelium and choroid degraded the surface of the polyvinyliden fluoride substantially, while extracts from the cornea, iris, ciliary body and neuroretina only moderately digested it. Lenticular extract did not affect the polyvinyliden fluoride surface. It is possible that the polyvinyliden fluoride loop undergoes degradation in the eye after a long time.

Animals↗
PubMed1984

Effect of chlorpromazine in vitro on release of enzymes from lysosomes of the bovine retinal pigment epithelium.

The effects of chlorpromazine on lysosomal enzymes and the release of enzymes from lysosomes of bovine retinal pigment epithelial cells were studied in vitro, using cathepsin D, arylsulfatase, and acid phosphatase as lysosomal marker enzymes. Chlorpromazine had little effect on the enzyme activity of cathepsin D and arylsulfatase and slightly decreased that of acid phosphatase. Chlorpromazine accelerated considerably the release of cathepsin D and arylsulfatase, but only minimally affected the release of acid phosphatase. The release of these enzymes from lysosomes depended on the dose of chlorpromazine.

Acid Phosphatase↗
PubMed1983

Effect of temperature and pH on release of enzymes from lysosomes of the bovine retinal pigment epithelium in vitro.

The effects of buffer concentrations, pH, and temperatures on the release of enzymes from lysosomes of the bovine retinal pigment epithelium were studied in vitro. Cathepsin D, arylsulfatase, and acid phosphatase were used as lysosomal marker enzymes. Elevation of temperature caused a marked increase in the release of cathepsin D and arylsulfatase from lysosomes, but little changes in the release of acid phosphatase. Acidic conditions accelerated the release of arylsulfatase and acid phosphatase. Different buffer concentrations had little effect on the release of these enzymes from lysosomes.

Acid Phosphatase↗