Growth factor receptor gene and protein expressions in the human lens.

In this study the mRNAs encoding epidermal growth factor receptor (EGFR), basic fibroblast growth factor receptor (FGFR-2) and insulin-like growth factor receptor (IGFR-1) genes of the human normal lenses at ages varying from 0.5 to 72 years, were identified by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Regulation of EGFR gene expression in the lens did not change with aging, and of FGFR-2 and IGFR-1 genes also remained unaltered up to age 53 years. However, expressions of FGFR-2 and IGFR-1 genes were decreased at ages above 60 years. EGFR, FGFR-2 and IGFR-1 proteins were detected by immunoblot analysis in the epithelial cell membranes of lens at age varying from 40 to 72 years. There was no detectable amount of EGFR protein in fiber cell membranes of the lens, and the levels of FGFR-2 and IGFR-1 proteins were much lower than those in the epithelial cell membranes. The low levels of these receptor proteins in the fiber cell membranes of lens, suggest their possible role in keeping the differentiated function of these unique transparent cells. The findings of the increased protein levels with age of EGFR with the appearance of some degradation products at age 48 years and higher, and the increased FGFR-2 protein at age 60 years and higher in the epithelial cell membranes of lens, were of interest. It appears that this could be a compensatory protective response of the lens to aging process for lifelong continuation of normal growth by proliferation and differentiation of its epithelial cells into new fiber cells in the germinative zone at the equatorial region. Thus, these results could provide a basis for further studies on growth factor receptor gene and protein regulations in the mechanism of lens aging and progression of age-related human cataract.

Lens-specific regulation of the thioredoxin-1 gene, but not thioredoxin-2, upon in vivo photochemical oxidative stress in the Emory mouse.

Thioredoxin (TRX)-1 and TRX-2 redox-regulatory genes were analyzed in the lens and some other tissues of the Emory mouse, a model for age-related human cataract. The cDNA transcripts of mRNAs encoding TRX-1 and TRX-2 genes were isolated and cloned by RT-PCR from the lens, liver, kidney, and tail, and the cDNA sequences were similar to the reported sequences of murine TRX-1 and TRX-2 genes. In vivo photochemical oxidative stress to the Emory mice resulted in fivefold upregulation of the lens TRX-1 gene at 3 weeks and declined thereafter. Western blot analysis revealed a fourfold increase of TRX-1 protein in the lens at 3 weeks after oxidative stress. The TRX-2 gene in the lens was not changed up to 5 weeks and decreased by 50% thereafter. However, the expressions of these genes in the liver, kidney, and tail were not changed. Fluorescent light or riboflavin alone did not affect the expressions of TRX-1 and TRX-2 genes in the lens. Thus, we show the expressions of TRX-1 and TRX-2 genes in the lens, liver, kidney, and tail and lens-specific upregulation of the TRX-1 gene and protein expressions, possibly as a protective response to the altered redox state of the lens after in vivo oxidative stress to the Emory mouse.

Socio-economic factors influencing child mortality in north-eastern Libya.

PIP: The authors analyze determinants of child mortality in northeastern Libya. "This study...attempts to (i) estimate the average child mortality by selected socio-demographic factors, (ii) discriminate the couples by child mortality level, and (iii) identify the important social factors which influence mortality." (EXCERPT)^ieng

Menadione-induced oxidative stress accelerates onset of Emory mouse cataract in vivo.

PURPOSE: We evaluated the effect of the free-radical generator, menadione, on the time of occurrence of cataract in the Emory mouse, a model for human cataract. Concomitant experiments were done to compare production and level of reactive metabolites of oxygen in the Emory mouse and its cataract resistant (CR) genetic control. METHODS: Test and control mice were fed both a normal diet and a diet supplemented with menadione, and the lenses were evaluated for the time of occurrence of cataract and the level of membrane ATPases. Effects of menadione were determined on incubated lenses of Emory and CR mice, assaying reactive species of oxygen, levels of antioxidant enzymes, and formation of the lipid peroxidation product, malondialdehyde. RESULTS: Systemic administration of menadione markedly accelerated the onset of Emory mouse cataract, and decreased ATPase activities suggested oxidative damage to membrane proteins. Cumulative levels of O2.-, H2O2, .OH and malondialdehyde were significantly higher than controls in the lenses incubated in the presence of menadione, showing that it generates oxidative stress. However, [GSH] in lenses decreased equally in test and control mice. The observed increases in catalase and glutathione peroxidase activities in the test lenses indicated an early protective response to oxidative insult. CONCLUSIONS: Acceleration by menadione of the appearance of cataract in the Emory mouse demonstrates that oxidative stress is a risk factor in late-onset cataract. This quinone caused a greater increase in the production and levels of reactive metabolites of oxygen in Emory mice than in CR mice, indicative of a higher susceptibility of the former to oxidative insult.

Testing regression equality to study the fertility differentials by child mortality in North-Eastern Libya.

PIP: The author studies "the impacts of different socio-demographic variables on fertility of couples experiencing differential child mortality in North-Eastern Libya. The test of equality of regression coefficients of any particular variable is also suggested. Results show that the regression coefficients in the fitted linear regression line of fertility of child loss mothers are significantly different than those in fitted linear regression lines of fertility of mothers without child loss." (EXCERPT)^ieng

Crosslinking of aminophospholipids in cellular membranes of lens by oxidative stress in vitro.

We have previously demonstrated by TLC an additional phospholipid spot between phosphatidylethanolamine (PE) and phosphatidylserine (PS) in human cataract. This was further identified as a fluorescent Schiff-base conjugate resulting from crosslinking of reactive carbonyl groups of malondialdehyde (MDA) with the primary amino groups of membrane phospholipids. Evidence presented here shows that such an adduct could be formed in rabbit lens subjected to oxidative stress in vitro. TLC analysis of a lipid extract of a crude membrane fraction obtained from the lens homogenate incubated with 1 mM H2O2, tert-butyl hydroperoxide (TBHP) or MDA for 1-6 h at 25 degrees C, showed that the oxidants and MDA produced time-dependent crosslinking of aminophospholipids. Under identical conditions of incubation with TBHP or MDA, development of the Schiff-base lipid fluorochrome in lens with peak emission at 470 nm when excited at 360 nm also showed a time-dependent increase. The PE.MDA.PS produced in cellular membranes of the lenses cultured for 3 h in Krebs-Ringer medium was 151 nmol/mumol PE, and addition of 1 mM H2O2, TBHP or MDA, increased it to 881, 610 and 375 nmol/mumol PE, respectively. Adduct was also formed when authentic samples of PE and PS were reacted with pure MDA. From the results it is clear that oxidants viz., H2O2 and TBHP, or MDA were effective in promoting crosslinking of lens membrane aminophospholipids by Schiff-base conjugation of primary amino groups with the carbonyl groups of the aldehyde, a breakdown product of lipid peroxides.

Fertility differentials according to females' education, employment and family planning adoption in rural Bangladesh.

PIP: This study examines differential fertility in 16 contiguous villages of both Savar and Dhamrai upazila in Dhaka district, Bangladesh. The sample includes 890 couples. Levels of female education were positively correlated with family planning use. 50.6% of housewives with a secondary or higher education used contraception. 59.8% of employed women with a secondary or higher education used contraception. 21.6% of illiterate women used contraception. Fertility declined with an increase in educational level. Child mortality increased fertility regardless of educational level or contraceptive usage. The average number of children ever born differed significantly between contraceptive and noncontraceptive users who did not work. Fertility between working and nonworking women did not differ significantly. Fertility was higher throughout the life cycle among adopters and nonadopters who had husbands that were laborers. Multivariate analysis reveals that fertility was highly significantly affected by duration of marriage and desired number of children. Differences in fertility between working and nonworking individuals was insignificantly related to differences in socioeconomic factors. The impact of socioeconomic factors on the fertility of adopter and nonadopter women was significantly different. Regression findings show that duration of marriage, desired number of children, and spouse's occupation had the strongest impact on fertility. Significant changes in impacts were due to changes in female education and contraceptive usage. Duration of marriage and desired number of children did not have the same level of impact among educated women and illiterate women. Income had a negative effect on the fertility of contraceptive users.^ieng

Dietary vitamin E reverses the effects of ultraviolet light irradiation on rat skin glycosaminoglycans.

Chondroitin 4-sulfate (Ch 4-S), three dermatan sulfates (DS18, DS45a, DS45b) and hyaluronic acid (HA) were the major glycosaminoglycans (GAG) isolated from the skin of 4 groups of albino rats. The yields from Group 1 (control) were: Ch 4-S, 0.015%; HA, 0.028% and DS (total), 0.098% (w/w). Traces of heparin were detected only in rats irradiated with ultraviolet (UV) light (Group II), in the GAG pool isolated with 45% ethanol. Yields increased by at least 28% (w/w) in Group II, but decreased, except HA's, also by at least 28%, below the level of the control, in irradiated rats that also ingested vitamin E (Group III). The sulfate composition of these GAG determined by infrared spectroscopy was as follows: approx. 17% (w/w) for DS18, 21-30% for DS45a, 21-35% for DS45b and 26-44% for Ch 4-S. A 60-70% (mol/mol) N-acetylation of hexosamine in DS45 was estimated by Fourier transform 1H-NMR spectroscopy; the IdUA composition of this DS was 30-46% (mol/mol), and the uronic acid/hexosamine ratio ranged from 2.50:1 to 1.6:1. The data show UV light irradiation of rat skin to result in an abnormally elevated production of the major GAG and oversulfation of Ch 4-S and DS. These effects are reversed, except for the sulfation of DS45b, when the irradiated animals also ingest vitamin E.

The effects of topical and oral L-selenomethionine on pigmentation and skin cancer induced by ultraviolet irradiation.

This study was conducted to determine whether oral and/or topical selenium (Se) supplementation can reduce the incidence of acute and/or chronic damage to the skin (i.e., sunburn and pigmentation and/or skin cancer, respectively) induced by ultraviolet (UV) irradiation in mice. Groups of 38 BALB:c female mice or 16 Skh:2 hairless pigmented mice were treated with 1) lotion vehicle, 2) 0.02% L-selenomethionine (SeMet) lotion, or 3) vehicle and 1.5 ppm SeMet in the drinking water. Within each group, 30 BALB:c mice or 12 Skh:2 mice were given UV irradiation (Westinghouse FS 40 bulbs) three times per week in doses of 0.575 and 0.24 J/cm2, respectively. The animals' weights and food intakes and the Se concentrations of skin and liver were measured. Skin biopsies were taken from the backs and abdomens of all animals to evaluate the relative amounts of Se and the damage by UV irradiation. Skin pigmentation was scored, and the total number of clinically detectable skin tumors per animal was counted weekly. Results showed that the skin Se concentrations in areas of application of the lotion containing SeMet were greater than those of animals given comparable oral doses, while the Se concentrations of untreated skin and liver were similar to those of animals receiving oral Se. Mice treated with Se showed no signs of toxicity and had significantly less skin damage by UV irradiation, as indicated by reduced inflammation and pigmentation and by later onset and lesser incidence of skin cancer.

Antioxidant and anticataractogenic effects of topical captopril in diquat-induced cataract in rabbits.

1-[(2s)-3-Mercapto-2-methylpropionyl]-L-proline (captopril), an antihypertensive and free radical scavenger, protected the rabbit lens from peroxidative and oxidative damage induced by 1 mM diquat in vitro. To evaluate the anticataract efficacy of captopril, an experimental group of five rabbits was treated with topical captopril (1% in 0.15 M NaCl, w/v), and 50 microliters was instilled onto both eyes four times a day for a total of 8 weeks. Following the same procedure, the eyes of five rabbits were treated with topical 0.15 M NaCl as a control for captopril treatment. At the end of the first week of treatment, a single intravitreal dose of 120 nmole diquat in 30 microliters of 0.15 M NaCl was injected into the right eye of each rabbit of both the groups. As a control for intravitreal diquat injection, the left eye of all the rabbits were injected with the diluent, 30 microliters per eye. The intravitreal diquat or its diluent injection was only for one time. From slit-lamp biomicroscopic observation of the diquat-injected right eyes, the anticataract effect of captopril in the treatment group was indicated by the finding that in four of five rabbits the cataract did not advance; whereas in four of five rabbits treated with the diluent the cataract progressed to grade 3. The lenses in the diluent-injected control left eyes of the rabbits treated with the captopril or diluent were normal. However, since the number of animals used for the in vivo studies was few, further confirmation of the anticataract effect of captopril is necessary. In diquat-injected right eyes of animals treated with captopril, the integrated rate of O2- production was about 50% less (p less than .001) in the aqueous humor, vitreous humor, and lens, compared with O2-, 33.49 +/- 2.26 microM (mean +/- SEM) in the aqueous humor, 17.12 +/- 0.75 microM in the vitreous humor, and 31.44 +/- 1.29 nmole/g wet weight in the lens of the diquat-injected right eyes treated with the diluent. Similar significant (p less than .01) differences in the production of .OH and H2O2 in eye tissues were also observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Desferal-Mn(III) in the therapy of diquat-induced cataract in rabbit.

In rabbit lenses subjected to oxidative stress, induced by 1 mM diquat in vitro, there were 7- to 10-fold increases (p less than 0.001) in malondialdehyde, conjugated dienes, and carbonyl dienes, indicating extensive peroxidation of cellular membrane lipids, and approximately a 60% decrease in reduced glutathione. In the presence of 0.1-5 mM Desferal-Mn(III) these changes were diminished by 50-70%. In an experimental group of 12 rabbits having diquat-induced cataract, Desferal-Mn(III) (5% w/v) applied topically as a 50-microliters eye drop four times per day and a single intraperitoneal dose of 64 mg/kg body wt daily for 5 weeks (including pretreatment for 1 week) retarded the progression of lens opacities, whereas, in a control group of 6 rabbits treated with the vehicle (0.15 M NaCl) cataract progressed to an advanced grade. Treatment with Desferal-Mn(III) also significantly diminished production of O2.- and OH. in the lens, aqueous humor, and vitreous humor, and of H2O2 in the aqueous humor and vitreous humor. It also suppressed lipid peroxidation and oxidation of protein-SH of the lens and restored lenticular glutathione and ascorbate to normal levels.

Free radical enhancer xenobiotic is an inducer of cataract in rabbit.

Free radical enhancers, diquat, paraquat, plumbagin and juglone were used to study the oxy radical-induced damage to the rabbit lens in vitro and in vivo. Each compound caused a 6-8 fold increase in malondialdehyde (MDA) and a 30-55% decrease in reduced glutathione of the lens in vitro. These peroxidative and oxidative changes were potentiated in the presence of 100% O2, abolished by N2 and prevented by desferal-Mn (III) (DF-Mn) or liposomal superoxide dismutase (LSOD) indicating the involvement of O2-. Diquat injected intravitreally as a single dose (300 nmole in 30 microliters of isotonic saline) in the right eye of a 5-wk-old Dutch belted rabbit, induced early cataract after 24-72 h. The lens of the contralateral control eye injected with isotonic saline had no change. In the right eye, O2-. and OH. productions were significantly (P less than 0.01) higher; O2-. was about 16 fold higher in the aqueous humor and vitreous humor, and 5 fold in the lens and retina, and OH. was 35 fold higher in the aqueous humor, 2 fold in vitreous humor and 5 fold in the lens and retina as compared to the respective tissues of the control eye. Enhanced lipid peroxidation in the lens was apparent from the higher levels of MDA and formation of aminophospholipid.MDA Schiff-base conjugates. We propose that cyclic oxidation-reduction of xenobiotics coupled to the endogenous redox systems in the eye, could generate oxy radicals in excessive amounts, triggering cataractogenesis.

Oxy radicals in the eye tissues of rabbits after diquat in vivo.

It is our hypothesis that oxygen free radicals are the triggering agents in cataractogenesis. However, besides H2O2 there is no direct evidence of generation of oxy radicals in the eye tissues. Due to extremely short life of O2-. and OH. it is not possible to measure their cellular steady state levels. We found that indirect spectrophotometric techniques based on superoxide dismutase (SOD)-inhibitable cytochrome c reduction for estimation of O2-., salicylate hydroxylation for OH. and peroxidase catalysed reoxidation of 2,6-dichlorophenolindophenol for H2O2, were suitable, sensitive and reproducible for measurements of the reactive species of O2 produced in the eye tissues by oxy radical enhancer, diquat in the rabbit eye in vivo. After a single intravitreal injection of 60, 120 or 300 nmole diquat in the right eyes, there was a dose-dependent rise in O2. levels, 106-265 fold in the aqueous humor, 34-87 fold in the vitreous humor, 6-19 fold in the lens, and 43-88 fold in the retina as compared to 0.16 microM, 0.21 microM, 2.47 nmole/g and 5.56 nmole/g in tissues of the normal eyes, respectively. There were similar increases of OH. in the eye tissues, and of H2O2 in the aqueous humor and vitreous humor after diquat injection. We propose that endogenous reductants of the eye tissues univalently reduce diquat to its free radical which spontaneously reacts with O2 generating O2. in excessive amounts, further giving rise to H2O2 and OH. triggering cataractogenesis.

Administration of an aldose reductase inhibitor induces a decrease of collagen fluorescence in diabetic rats.

As a consequence of an increased flux through the sorbitol pathway fructose levels rise in various tissues in diabetes. Also, in vitro nonenzymatic fructosylation of protein induces the generation of fluorescence at a rate 10 times greater than glucosylation. The administration of sorbinil, an aldose reductase inhibitor known to lower tissue fructose concentration, to experimental diabetic rats led to a decrease in the fluorescence related to advanced Maillard products in their skin collagen. This effect is consistent with the in vivo occurrence of nonenzymatic fructosylation of collagen. A potential pathogenetic role for this posttranslational modification in diabetic complications should be considered.

Multiple dosing of prostaglandin F2 alpha or epinephrine on cynomolgus monkey eyes. II. Slit-lamp biomicroscopy, aqueous humor analysis, and fluorescein angiography.

Prostaglandin (PG) F2 alpha (250 micrograms in 50 microliters saline) or epinephrine 2% solution (50 microliters) was topically applied twice daily for 2 weeks to one eye of six cynomolgus monkeys for each agent. Contralateral control eyes received their respective vehicles. A trace aqueous humor flare response occurred in some PGF2 alpha-treated eyes, which reached significance (P less than 0.05) only when observed 4 hr after the first or seventh dose. No significant anterior chamber cellular response was observed in treated as compared to control eyes. Slit-lamp biomicroscopic evaluation of the cornea, iris, and lens showed no differences in treated as compared to control eyes throughout the study. Aqueous humor samples were obtained from all eyes 4 hr after the ninth consecutive dose. Soluble protein concentration was significantly (P less than 0.01) greater in the PGF2 alpha-treated eyes (1.22 +/- 0.30 mg/ml) as compared to control (0.56 +/- 0.17 mg/ml) or to epinephrine-treated eyes (0.59 +/- 0.18 mg/ml). Microscopic examination of sediments obtained after centrifugation of the aqueous humor revealed no cells in experimental or control samples. Both PGF2 alpha and PGE2 levels were significantly (P less than 0.025) greater in PGF2 alpha-treated eyes, and showed a trend towards being greater in epinephrine-treated compared to control eyes. Neither cystoid macular edema nor other retinal abnormalities were evident by fluorescein angiography in any eyes during the second week of treatment. Multiple dosing of PGF2 alpha in monkey eyes does not appear to produce clinically significant adverse effects in either the anterior or posterior segment which would contraindicate its use in a multiple-dose clinical trial in glaucoma patients.

Molecular mechanisms of cataractogenesis: IV. Evidence of phospholipid . malondialdehyde adduct in human senile cataract.

Lipid extracts of the human cataractous and normal lenses were analyzed by thin-layer chromatography (TLC) using a solvent system consisting of CHCl3/CH3OH/CH3COOH/H2O (50:25:7:3 by vol.). A novel phospholipid having a Rf intermediate between phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS) was detected besides the four major phospholipids viz., PE, PS, phosphatidyl choline (PC) and sphingomyelin (SP). The novel phospholipid was found to be molybdenum positive and ninhydrin negative having a characteristic fluorescence of Schiff-base conjugate formed between PE, malondialdehyde (MDA) and PS. It was possible to resolve this adduct into PE and PS after acid hydrolysis using two dimensional TLC with CHCl3/CH3OH/NH3 (7 M) (65:25:4 by vol.) as the second solvent. In cataract PE . MDA . PS adduct increased significantly as did diene conjugates and MDA. In plasma membrane lipid extract of cataractous lenses there was a marked increase in fluorescence at 460 nm when excited at 365 nm showing a characteristic fluorescence of a typical Schiff-base conjugate. The evidence suggests that peroxidation of lenticular plasma membrane lipids is one of the molecular mechanisms involved in cataract in the human.

Lipid peroxidation in cataract of the human.

Lipid peroxidation was investigated as one of the possible mechanisms of cataractogenesis in the human. Malondialdehyde (MDA), a major breakdown product of lipid peroxides, was significantly higher in cataractous lenses as compared to that in normal lenses. 2-Thiobarbituric acid-reactive material, isolated from cortical cataracts and purified by Sephadex G-10 column chromatography, was identified as MDA. In cataractous lenses the enzymic defenses against reactive species of O2 were impaired as evidenced by the significant decrease in activities of superoxide dismutase, catalase and glutathione peroxidase. Hydrogen peroxide in aqueous humor and vitreous humor of human eyes associated with cataract was increased 2-3 fold. It is possible that carbonyl groups of MDA could interact with primary amino groups of proteins and phospholipids of lenticular plasmalemmae by a cross-linking reaction forming Schiff-base conjugates and these mechanisms might be involved in the pathogenesis of cataract.

Molecular mechanism of cataractogenesis: III. Toxic metabolites of oxygen as initiators of lipid peroxidation and cataract.

A free radical mechanism of cataractogenesis involving enzymatic and nonenzymatic reactions, is proposed. Supporting experimental evidence is briefly reviewed. H2O2, which is one of the toxic metabolites of oxygen, was significantly increased 2-3 fold in ocular humors in several experimental cataracts and in human senile cataract. Various cataractogenic agents were also found to increase H2O2 in ocular humors in vivo prior to cataract formation. Enzymatic defenses against O2-. and H2O2 provided by superoxide dismutase, catalase and glutathione peroxidase were impaired in cataracts. In some cataracts, catalase and superoxide dismutase were affected earlier. Malondialdehyde (MDA), a major breakdown product of lipid peroxides was significantly increased by 2-4-fold in human senile cataract, in cataracts induced in rabbit and rat, and in hereditary cataracts in mice. All the reactive species of O2 (O2-., H2O2, OH. and 1 delta gO2) may participate in initiating lipid peroxidation of lens in vitro. Various scavengers of these species were capable of preventing lenticular lipid peroxidation, amongst which OH. scavengers were found to be the most effective. Biological antioxidant, vitamin E afforded 44% prevention of lipid peroxidation in lens. The important observation was that vitamin E was therapeutically effective in about 50% of animals in arresting cataract induced in rabbit by 3-aminotriazole. In these rabbits, H2O2 and ascorbic acid of ocular humors and MDA of lens were close to normal. It is our working hypothesis that the carbonyl groups of MDA and amino groups of amino acids, proteins, nucleic acids and their bases, and phospholipids could interact in a cross-linking reaction producing high molecular weight aggregates by Schiff-base conjugate formation in addition to disulfide cross-linking of proteins, and finally resulting in cataract.