Hiroko Hirano

Presence of Cortisol-Binding Protein in the Lens

The authors have verified the presence of cortisol-binding protein in the rat lens, using gel filtration and cortisol-4-[14C]. Two methods of isolation of the cortisol-binding protein were

Further Studies on the Cortisol-Binding Protein in the Lens

The cortisol binding capacity was decreased by trypsin digestion of the lens protein. The binding capacity was decreased by the addition of 8.0 m of urea and restored partially by dialysis, suggesting

Study on the Conjugation of Cortisol in the Lens

Sulphate and glucuronide conjugation of cortisol in the normal human, cataractous, bovine, rabbit and rat lens in vitro have been investigated using a cortisol-4–14C. The authors have verif

Riboflavin Metabolism in the Single Lens of Rat

To study the mode of riboflavin metabolism in the lens in vivo, the uptake of 14C-labeled riboflavin and synthesis of ester forms from 14C-riboflavin were assayed in the single whole lens of the rats. 14C-riboflavin uptake increased in proportion to the substrate concentration in the incubation medium, reaching a plateau within 60 min. At 4 degrees C the uptake was lowest, at 30, 35 and 40 degrees C it was similar. Neither uncoupler (2,4-dinitrophenol and monoiodoacetate) nor ouabain inhibited the uptake. Within 15 min, approximately 35% of transported 14C-riboflavin was converted to ester forms of riboflavin; this ratio remained constant until 120 min of incubation. Characteristically, 2-3% of 14C-riboflavin was bound to lens protein. 2,4-Dinitrophenol or monoiodoacetate inhibited the synthesis of ester forms of riboflavin, but ouabain did not. No synthesis of ester forms of riboflavin was observed in the lens capsule with epithelium. These results suggest that two enzymes, flavokinase and flavin adenine dinucleotide (FAD) pyrophosphorylase which convert riboflavin to flavin mononucleotide and FAD, respectively, exist in the lens of the rat. These enzymes may participate in riboflavin uptake and riboflavin may pass through the lens capsule by simple diffusion without undergoing a conformational change and then may be metabolized to ester forms of riboflavin with subsequent partial binding to lens protein.

Changes in rat lens proteins and glutathione reductase activity with advancing age

We examined the changes in the amounts of water-soluble and water-insoluble proteins of rat lenses, and in glutathione reductase activity and glutathione reductase gene expression, with advancing age. The lens total protein increased in 1-month-old rats, 3-month-old rats, and 6-month-old rats, but thereafter decreased in 12-month-old-rats. The water-soluble proteins decreased with advancing age, while the water-insoluble proteins increased. The glutathione reductase activity decreased with advancing age, but the decreased glutathione reductase activity almost recovered by addition of flavin adenine dinucleotide (FAD) in vitro. However, advancing age had no effect on the level of mRNA for glutathione reductase.

Biochemical studies on the pathogenesis of steroid cataracts, with particular reference to the pituitary-adrenal-liver axis

Abstract Factors having an effect on the binding and inactivation of [4- 14 C] cortisol in the rat lens, such as hypophysectomy, adrenalectomy, dexamethasone phosphate administration and liver impairment were investigated. Furthermore, the effects of flavin adenine dinucleotide on the cortisol-binding capacity in the lenses of adrenalectomized rats and of dexamethasone phosphate-treated rats were also investigated. The authors found that cortisol-binding by lens protein and sulphate conjugation of cortisol in the lens are greatly influenced by the pituitary-adrenal-liver axis. A decrease in the cortisol-binding capacity in the lens of adrenalectomized rats was slightly prevented by flavin adenine dinucleotide administration. A similar result was found in the lenses of rats treated with dexamethasone phosphate, suggesting that riboflavin may influence the inactivation of steroid hormone in the lens.

Studies on the Inactivation of Cortisol-4-[14C] in the Lenses of Liver-Impaired Rats

Changes in inactivation mechanisms of cortisol, such as cortisol-binding by protein or sulphate and glucuronide conjugation in the lenses of normal and liver-impaired rats, have been studied in order to determine the role of liver in the pathogenesis of steroid cataracts. The cortisol-binding capacity of the rat lens decreased when the liver was impaired by intraperitoneal administration of carbon tetrachloride. Sulphate conjugation of cortisol-4-[14C] showed similar results, however, no significant difference was found in glucuronide conjugation. These results indicate that there is a close relationship between inactivation of steroid hormone in the lens and liver function, and that liver dysfunction in patients receiving long-term oral corticoid therapy plays an important role in causing the onset of steroid cataracts.

5′‐Upstream Structure of the Gene Coding for Chicken Riboflavin‐Binding Protein and Its Relation to Estrogen Induction

To elucidate the mechanism of the estrogen‐dependent induction of chicken riboflavin‐binding protein (RfBP), we analyzed the 5′‐upstream structure of its gene. A noncoding exon exists there, and around this sequence, 9 widely spaced half‐palindromic estrogen‐response element (ERE) motifs (5‐GGTCA or 5‐TGACC) were found. Furthermore, an imperfect ERE‐like palindromic sequence (5′‐ATGTCANNNTGACAT‐3) was also found at the 2.25 kb upstream region. No consensus palindromic ERE was observed. By luciferase reporter assay, the regions containing the half ERE motifs and the imperfect ERE showed estrogen‐dependent enhancer activities, suggesting that these two characteristic sequences might confer estrogen‐inducibility upon the chicken RfBP gene. However the activities were lower than that of a consensus ERE. It remains uncertain whether these sequences act cooperatively.

Effects of Ultraviolet B Irradiation on Lenticular Riboflavin Metabolism and High-Molecular-Weight-Protein Aggregation

We investigated the effects of ultraviolet B (UVB) irradiation on the formation of ester forms of riboflavin and of high-molecular-weight (HMW) protein aggregates and on lenticular riboflavin-binding capacity (LRBC). Esterification of riboflavin decreased as the duration of UV irradiation increased, suggesting the irradiation-induced denaturation of the apoenzyme of synthetases of ester forms of riboflavin. UVB irradiation of lens homogenate supplemented with riboflavin increased LRBC and the formation of HMW protein aggregates, while gamma-crystallin was decreased. These results are consistent with those of our earlier studies in which we obtained data suggesting that, upon exposure of rat lens homogenate to fluorescent light, photosensitized riboflavin may bring about cross-linking of lens protein. Our data demonstrate that the photosensitivity of lenticular riboflavin is increased by longer periods of UV irradiation.

Effects of pH and Light Exposure on the Riboflavin-Binding Capacity in the Rat Lens

To elucidate the binding mode of riboflavin to rat lens protein, we examined the effects of light exposure, different pH (4.0, 7.0 and 9.0) and dialysis on the lenticular riboflavin-binding capacity (LRBC). LRBC increased remarkably by light exposure, irrespective of pH. By dialysis, LRBC of a light-exposed incubation mixture decreased remarkably at pH 9.0, however, at pH 4.0 and 7.0, dialysis did not affect it significantly. Decrease in LRBC in light-exposed dialyzed samples at the higher pH may result from release of dialyzable bound riboflavin from binding protein. These data suggest that the dialyzed bound riboflavin at the higher pH may be responsible for a photosensitization of lens protein.