Effect of calpain on hereditary cataractous rat, ICR/f.

The crystallins in the lenses of ICR/f mutation rat, a known hereditary cataract model, were analyzed during cataractogenesis. Opacification of the mutant lenses was found to be accompanied by changes in crystallin structure and composition, including several deletions of the N-terminals of beta-crystallins and low molecular weight alpha- crystallins. Because similar deletions were observed when the soluble fraction of normal lens protein was incubated with calpain, we considered that calpain could be related to the deletions in mutant lenses. Although measurement of the content of calpain protein by the ELISA method revealed no significant difference between mutant and normal lenses, it was found that the concentrations of Ca2+ and K+ were different between the two lenses and that calpain activity was dependent on both ion concentrations. Endogenous m-calpain in the soluble fraction from normal lenses was activated by addition of 1 mm calcium chloride in the presence of 50 mm KCl (the same concentration as in mutant lenses), and insoluble protein was found in the fraction 1 d after calpain activation. On the other hand, the presence of 120 mm KCl (the concentration in normal lenses) inhibited calpain activity and prevented this insolubilization. These results suggest that calpain in mutant lenses is involved in the proteolysis of crystallins and the progression of cataract formation.     

Inhibitive effects of enhanced lipid peroxidation on Ca(2+)-ATPase in lenses of hereditary cataract ICR/f rats

Our previous studies have demonstrated that the instillation of eye drops containing disulfiram, a radical scavenger and nitric oxide synthase inhibitor, delays cataract development in ICR/f rats, and we have suggested that the production of nitric oxide (NO) and lipid peroxide (LPO) in the lens may relate to the delay in cataract development brought about by disulfiram. However, the involvement of NO and LPO in lenses of ICR/f rats during cataract development has not yet been established. In the present study, we determined changes in NO and LPO levels in lenses of ICR/f rats during cataract development. Opacification of ICR/f rat lenses started at 77 days of age, and the lenses of 91-day-old ICR/f rats were almost entirely opaque. The Ca(2+)-ATPase activity in the lenses of ICR/f rats decreased with increasing age, and an elevation in Ca(2+) content was observed in ICR/f rat lenses with the decrease in Ca(2+)-ATPase activity. NO levels in the lenses of ICR/f rats increased from 63 to 85 days of age, reaching a maximum at 77 days of age. In addition, LPO levels in the lenses of ICR/f rats also increased with increasing age. LPO levels in the lenses of 63- to 91-day-old ICR/f rats were found to be significantly higher compared with those in 22-day-old ICR/f rats. These changes of Ca(2+), Ca(2+)-ATPase, NO and LPO were attenuated by instillation of DSF eye drops. These results suggest that excessive NO may cause enhanced lipid peroxidation resulting in the inhibition of Ca(2+)-ATPase. The decrease in Ca(2+)-ATPase activity may cause the elevation in lens Ca(2+), leading to lens opacification in ICR/f rats.     

Variation in glycosidase activity in soluble fractions in ICR/f rat lenses with the progression of cataract formation

The current study reports active glycosidases in the lens of ICR/f rats, which generate a hereditary cataract approximately 90 d after birth, and the variation in enzyme activity with cataract progression. Seven active glycosidases, beta-D-galactosidase, alpha-D-glucosidase, beta-D-glucosidase, beta-D-glucuronidase, beta-D-galactosaminidase, beta-D-glucosaminidase and alpha-D-mannosidase, were detected in ICR/f rat lenses. Of these, beta-D-glucuronidase and beta-D-galactosidase showed a tendency to increase in activity with the cataract progression. Furthermore, beta-D-glucosidase and alpha-D-mannosidase showed a transitory increase in activity at the time of cataract formation. This result suggests that several glycosidases in the lens may be involved in the hereditary cataract formation. The optimal pH and temperature of the seven active glycosidases in rat lenses were also measured in this study.     

Effect of disulfiram eye drops on lipid peroxide formation via excessive nitric oxide in lenses of hereditary cataract ICR/f rats

The ICR/f rat is a recessive-type hereditary cataractous strain, and opacity in the lens usually becomes evident at around 75 d of age. We previously found that the instillation of eye drops containing a disulfiram and hydroxypropyl-beta-cyclodextrin inclusion complex (DSF eye drops) delays lens opacification in ICR/f rats. In this study, we attempted to clarify the mechanisms of the delaying effect of DSF eye drops on cataract development in ICR/f rats. The calcium ion (Ca2+) content in the lenses of ICR/f rats increases at 77 d of age, and this elevation is preceded by a decrease in Ca2+-ATPase activity. On the other hand, the levels of nitric oxide (NO) and lipid peroxide (LPO) also increase in the lenses of ICR/f rats at 63 d of age, while the lenses are still transparent. The instillation of DSF eye drops reduces the changes in Ca2+ content, Ca2+-ATPase activity, NO and LPO levels in the lenses of ICR/f rats. The present study demonstrates that excessive NO production induces the increase in LPO, which causes the decrease in Ca2+-ATPase activity, and the increase in Ca2+ content in the lenses of ICR/f rat during cataract development. DSF eye drops have the ability to attenuate the increase in the NO and LPO levels, resulting in a delay in cataract development.     

Phosphoproteome analysis of hereditary cataractous rat lens alpha-crystallin

We reported previously that C-terminal truncated alpha-crystallins were found in lenses of hereditary cataractous rat ICR/f. In this study, we examined the phosphorylation of the crystalline lens proteins, alphaB-crystallin and alphaA-crystallin, in cataractous and normal rats of different ages and have found an increase in the phosphorylation of serine residues of truncated alpha-crystallin in cataractous lens. Phosphorylation and C-terminal truncation of alpha-crystallins could, both, reduce their chaperone-like activity and lead to cataract formation.     

C-terminal truncation of alpha-crystallin in hereditary cataractous rat lens

C-Terminal truncated alpha-crystallins have been found in lenses of hereditary cataractous rat ICR/f, including two truncated alphaB-crystallins and several truncated alphaA-crystallins. These truncated crystallins probably resulted from degradation by m-calpain and Lp82. The alphaB-crystallin with five amino acid residues deleted showed decreased chaperone activity. Compared with alpha-crystallins from the normal rat lenses, overall chaperone activity of alpha-crystallins from the mutant lenses, including the above truncated alphaB-crystallin, was remarkably reduced. The decreased chaperone activity accompanying the increase in C-terminal truncated alpha-crystallins may cause the insolubilization of many proteins in the mutant lenses, which it is likely to lead to the progression of cataract formation.     

Studies on protein and taurine in normal, senile and diabetic cataractous human lenses

An attempt has been made to explore the possible role of Taurine in cataractogenesis. Normal lenses were obtained from eye bank donors and cataractous lenses from patients who had undergone surgery for cataract extraction. Lenses were weighed and homogenised. Extraction, isolation and estimation of protein and taurine were carried out. It has been found that the lens wet weight increased progressively with the stage of maturation of cataract, i.e., from mature to hypermature which was significant and also with increase in age. Diabetic cataract group also showed an increase similar to that of senile cataract. Taurine and total protein decreases with different stages of maturation of cataract but not with age. It may be suggested that in the process of development of human senile cataract, there is (a) alteration in the structural integrity and permeability of lens membrane to protein and amino acids including taurine, (b) changes in the lens function including possible inhibition of proteins and amino acids (taurine) synthesis and transport across the cell membrane.     

正常和老年性白内障晶体VE Vc含量的测定

测定正常和老年性白内障晶体ＶＥ、Ｖｃ含量，发现老年性白内障晶体ＶＥ、ＶＣ含量比正常晶体明显减少，并讨论了这些改变的意义。     

Delay in ICR/f rat lens opacification by the instillation of eye drops containing disulfiram and hydroxypropyl-beta-cyclodextrin inclusion complex

In this study, we attempted to enhance disulfiram (DSF) solubility using a 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) and hydroxypropylmethylcellulose (HPMC). We also investigated the effect of an HPbetaCD solution containing DSF and HPMC (DSF eye drops) on cataract development in ICR/f rat. The solubility of DSF increased with increasing HPbetaCD concentration, and the solubility of DSF in HPbetaCD solution containing 0.1% HPMC was approximately 20% greater than that of DSF in HPbetaCD solution without HPMC. In in vivo transcorneal penetration experiments using rabbits, only diethyldithiocarbamate (DDC) was detected (DSF was not detected) in the aqueous humor. This DSF-DDC conversion in the cornea was inhibited by treatment with a sulfhydryl (SH) inhibitor, p-mercuribenzoate and N-ethylmaleimide, in in vitro transcorneal penetration experiments using rabbit corneas. On the other hand, the instillation of 0.25% and 0.5% DSF eye drops delayed cataract development in ICR/f rats, a recessive-type hereditary cataractous strain. The present study demonstrates that DSF in HPbetaCD solution with HPMC is converted to DDC by the catalysis of proteins containing SH residues in the cornea, and this DDC may cause the delay in cataract development in ICR/f rats.     

Pharmacokinetic and pharmacodynamic evaluation of the anti-cataract effect of eye drops containing disulfiram and low-substituted methylcellulose using ICR/f rats as a hereditary cataract model

We attempted to develop anti-cataract eye drops using disulfiram (DSF) and low-substituted methylcellulose (MC), and evaluated their anti-cataract effect in terms of the lens opacification vs. age-profile curves using a one-exponential equation. The eye drops were prepared using 0.5% DSF and 2% MC (DSF eye drops), and ICR/f rats, a recessive-type hereditary cataractous strain, were used as the experimental model. Gelation of DSF eye drops containing MC was first observed at about 35°C, close to body temperature. In in vivo transcorneal penetration experiments using rabbit corneas, only diethyldithiocarbamate (DDC) was detected in the aqueous humor, while DSF was not detected. The DDC penetration level of DSF eye drops containing MC was approximately 1.3-fold higher than that of DSF eye drops. The opacification rate constant (k) of ICR/f rat instilled with DSF eye drops with or without MC was lower, and the initial time of opacification (τ) was longer than those of ICR/f rats instilled with saline. Furthermore, the k of ICR/f rats instilled with DSF eye drops with MC was lower than that of ICR/f rats instilled with DSF eye drops without MC. In conclusion, the analysis of kinetic parameters including k and τ using a one-exponential equation provided useful information for clarifying the anti-cataract effect of eye drops. ICR/f rats instilled with DSF eye drops using a low-substituted MC-based drug delivery system demonstrated a delay in cataract development, probably resulting from an increase in the retention of DSF eye drops on the cornea.     

Transport of trace elements in lenses of normal and hereditary cataract UPL rats

The multitracer technique was applied to the determination of the uptake of trace elements in the lenses of normal and hereditary cataract UPL rats to investigate the transport mechanisms of trace elements during cataract development. Be, Na, Sc, V, Cr, Mn, Fe, Co, Zn, As, Se, Rb, Sr, Y, Zr, Tc, Ru and Rh accumulate in normal and UPL cataract rat lenses. The rates of uptake of trace elements differ among species and also differ between normal and UPL rat lenses. The uptakes of V and Sr are greater in normal rat lenses, while the uptakes of Mn and Co are greater in UPL rat lenses. High concentrations of Zn are transported into normal rat lenses in comparison with other elements. However, the uptake of Se was highest in the lenses of UPL cataract rats. In addition, the difference in Se uptake between the normal and UPL rat lenses was greatest among the tested trace elements. The present study suggests that the transport characteristics of trace elements are different in the lenses of normal and UPL cataract rats. The different transport characteristics of trace elements in the lenses of normal and UPL cataract rats, especially the higher accumulation of Se in UPL rat lenses, may be implicated in cataract development.     

Adverse effects of excessive nitric oxide on cytochrome c oxidase in lenses of hereditary cataract UPL rats

The UPL rat is a newly developed hereditary cataract model. We previously found that the ATP content in UPL rat lenses decreases during cataract development, and the decrease in ATP content causes Ca(2+)-ATPase dysfunction resulting in an elevation in Ca(2+) and cataract development. In addition, we reported that the oral administration of disulfiram and aminoguanidine ameliorates the decrease in ATP content and the elevation in Ca(2+) content in UPL rat lenses. In this study, we demonstrate the effect of nitric oxide (NO) on the expression and activity of cytochrome c oxidase (CCO) in normal and UPL rat lenses during cataract development. We also determined the effects of the oral administration of disulfiram and aminoguanidine on the mRNA expression and activity of CCO and NO production in UPL rat lenses. The expression of CCO-1 mRNA in UPL rat lenses, determined by a quantitative real-time RT-PCR method, decreased during cataract development. CCO activity in UPL rat lenses also decreased with aging. On the other hand, the oral administration of disulfiram and aminoguanidine attenuated the decrease in CCO-1 mRNA expression and CCO activity. These results suggest that excessive NO causes the decrease in CCO-1 mRNA expression and CCO activity, and that the decrease in CCO may cause the decrease in ATP production in UPL rat lenses. Disulfiram and aminoguanidine may attenuate the decrease in ATP production, resulting in a delay in cataract development.     

Effects of N-acetylcysteine and 2,3-dimercaptosuccinic acid on lead induced oxidative stress in rat lenses

Lead (Pb) is known to disrupt the pro-oxidant/anti-oxidant balance of tissues which leads to biochemical and physiological dysfunction. The present study investigated the effects of exposure on the redox status of the lenses of Fisher 344 rats and examined whether antioxidant or chelator administration reversed these changes. Animals were given 5 weeks of 2000 ppm Pb exposure followed by 1 week of either antioxidant, chelator or distilled water administration. Glutathione (GSH) and cysteine (CYS) levels decreased in the Pb-exposed group. N-acetylcysteine or 2,3-dimercaptopsuccinic acid (Succimer) supplementation following Pb intoxication resulted in increases in the GSH and CYS levels. Protein bound glutathione (PSSG) and cysteine (PSSC) increased following Pb exposure. In the Succimer-treated animals, the PSSG decreased significantly. The glutathione disulfide (GSSG) levels remained unchanged. Malondialdehyde (MDA) levels, a major lipid peroxidation byproduct, increased following Pb exposure and decreased following Succimer treatment. Our results suggest that antioxidant supplementation, as well as chelation, following Pb exposure may enhance the reductive status of lenses.     

Anti-inflammatory,expectorant, and antitussive properties of Kyeongok-go in ICR mice

ContextKyeongok-go (KOG) is a traditional mixed herb preparation consisting of Panax ginseng CA Meyer (Araliaceae), Poria cocos Wolf (Polyporaceae), Rehmannia glutinosa (Gaertner) Liboschitz ex Steudel (Orobanchaceae), and honey. Various pharmacological effects of KOG are reported, but the efficacy on respiratory diseases has not been evaluated.ObjectiveThe anti-inflammatory, expectorant, and antitussive properties of KOG were examined using animal models of respiratory diseases.Materials and methodsKOG (100, 200, and 400 mg/kg) was orally administered to ICR mice (n = 8) once a day for 11 days. Anti-inflammatory effects of vehicle, xylene, KOG and DEXA (1 mg/kg) were determined by monitoring edoema and redness of treated ears, and measuring the relative and absolute weight of each ear. Expectorant properties of vehicle, KOG and AM (250 mg/kg) were evaluated by observing body surface redness, and the amount of mucous secreted by the trachea. The antitussive potential of vehicle, NH₄OH, KOG and TB (50 mg/kg) was evaluated by monitoring changes in the number of coughs (for 6 min).ResultsKOG (400 mg/kg) treated mice showed 31.29% and 30.72% (p < 0.01) decreases in the relative and absolute weights of each ear relative to xylene control mice, 39.06% increases (p < 0.01) in TLF OD values relative to intact vehicle control mice, and 59.53% decrease (p < 0.01) in coughing compared to NH₄OH control mice. Dose-dependent changes were observed in all experimental models.ConclusionsKOG may be a potential therapeutic agent for the treatment of various respiratory diseases, particularly those caused by environmental toxins.     

Gamma-hydroxybutyric acid, unlike gamma-aminobutyric acid, does not stimulate Gi/Go proteins in rat brain membranes

gamma-Hydroxybutyric acid is a naturally occurring substance that may act as a neurotransmitter or neuromodulator to elicit several biological effects. Although the existence of a specific gamma-hydroxybutyric acid receptor has been postulated, the receptor protein itself has not been cloned yet. The current study was designed to elucidate whether gamma-hydroxybutyric acid receptors are functionally coupled with heterotrimeric G-proteins, especially Gi/Go family, by means of high-affinity GTPase activity and guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding assays in rat brain membranes. The stimulatory effects of GABAB receptor activation were always determined in parallel as a positive control. The selective GABAB receptor agonist (+/-)-baclofen stimulated the high-affinity GTPase activity in cerebral cortical, hippocampal, and striatal membranes, whereas gamma-hydroxybutyric acid was inactive up to 1 mM in these brain regions. The optimum assay conditions for [35S]GTPgammaS binding to detect a receptor-mediated activation of G-proteins at the greatest signal to noise ratio were then probed as to the concentrations of constituents in the assay mixture (GDP, MgCl2, and NaCl) and incubation period. Even under such an optimized experimental condition, [35S]GTPgammaS binding was not altered by gamma-hydroxybutyric acid in the membranes prepared from cerebral cortex or hippocampus. On the other hand, the specific [35S]GTPgammaS binding was increased by GABAB receptor agonists in a concentration-dependent manner, which was competitively inhibited by CGP54626, a selective GABAB receptor antagonist. These results indicate that gamma-hydroxybutyric acid receptors, if any, are not associated with G-proteins, at least Gi/Go family.     

Biochemical basis of hereditary resistance to warfarin in the rat

The influence of warfarin on prothrombin formation and the regeneration of vitamin K from vitamin K-2,3-epoxide was examined in control and warfarin-resistant rats. The epoxide reductase from liver of resistant rats was inhibited by warfarin less than the reductase from control animals. The concentration of warfarin required to inhibit the reductase in vitro was similar in control and resistant rats to the concentration of warfarin in vivo during inhibition of prothrombin formation. These results are interpreted as evidence of the biochemical basis for hereditary resistance to warfarin in the rat.     

Teratogenicity of thiabendazole in ICR mice

Thiabendazole (TBZ) was tested for teratogenicity using Jcl:ICR mice. TBZ suspended in olive oil was given orally to pregnant mice at different stages of organogenesis. All foetuses were removed from the uterus on day 18 of gestation, and were examined for external and skeletal anomalies. In mice given 700, 1300 or 2400 mg TBZ/kg body weight/day on days 7–15 of gestation, dose-dependent external and skeletal anomalies, especially cleft palate and fusion of vertebrae, were observed. In mice given a single dose of TBZ (2400 mg/kg) on any one of days 6–13, an increased number of malformations was observed. Various types of malformation occurred, especially in the mice treated on day 9. Reduction deformity of limbs was found in mice given TBZ on days 9–12, a change that has not previously been observed to occur spontaneously in normal ICR mice in our laboratory. In order to determine a dose-response relationship, groups of mice were given one of 17 doses of TBZ (30–2400 mg/kg) on day 9 of gestation. The number of litters having foetuses with reduction deformity of limbs and of those having foetuses with skeletal fusion increased in proportion to the dose of TBZ. The regression lines of Y (probit response) on X (log dose) for reduction deformity of limbs and for skeletal fusion were Y = 2.47X ? 3.65 and Y = 1.54X + 0.48, respectively. The effective doses (ED₁) for the two malformations were 362.0 and 26.4 mg/kg, respectively.     

Estrogen-induced proteins in rat hypothalamus

The identification of estrogen-inducible genes in specific regions of the central nervous system (CNS) will give information about the mechanisms by which this steroid modulates nervous activity. Two-dimensional gel analysis of the proteins synthesized in vitro from mRNA isolated from control or estrogen-treated rats indicated that the levels of mRNA were increased in the estrogen-treated animals. The levels of mRNA were elevated only in those brain regions that express estrogen receptors.