Some plasma constituents correlate with human cataract location and nuclear colour.

AIMS: To look for differences in levels of various plasma constituents between pair-matched controls and patients who had cataracts classified by location and appearance of lens opacity and nuclear colour in order to identify systemic risk factors. METHODS: One thousand patients were taken from the cataract waiting list of a specialist eye hospital. For each patient, a matched control of the same sex and half-decade of age but without cataract was taken from the patient-list of the family doctor of the patient; the control was the next alphabetically after the patient on the doctor's list. At an early morning visit to the homes of both patients and controls, fasting, a team of nurses performed venepunctures and collected information for a questionnaire. Eye examinations were performed by a team of ophthalmologists. RESULTS: Predominantly nuclear cataract was significantly associated with raised plasma alanine aminotransferase and bilirubin, posterior subcapsular cataract with increased calcium and urea, cuneiform with reduced potassium, mature/hypermature with raised potassium and reduced total carbon dioxide. The following were consistently significantly associated with all forms of cataract; diabetes and raised plasma glucose (not in non-diabetics), use of steroid medication, raised levels of cortisol (steroid users excluded), albumin, alkaline phosphatase, gamma-glutamyl transpeptidase, sodium and total protein and reduced levels of cholesterol and albumin/(total protein-albumin) ratio (an approximation for the albumin/globulin ratio). The multivariate analysis indicated that the most important non-specific cataractogenic effects were those of increased total protein, diabetes and use of steroid medication. CONCLUSION: This and other studies support, broadly, the conclusions that senile or age-related cataract is not merely caused by increasing age and also that various morphological types have different risk factors. The mechanisms underlying the biochemical associations with different patterns of lens opacification and the identification of the ultimate risk factors remain to be elucidated.

Skin disease and age-related cataract.

Dermatological conditions and treatments were analysed in a study comparing cataract patients and stringently matched controls. One thousand patients were taken from the cataract waiting list of a specialist eye hospital. For each patient a matched control of the same gender, half-decade of age, and family doctor but without cataract was selected. Venepunctures and eye examinations were performed on both patients and controls; in addition, questionnaire information was obtained from each. Age-related cataract is significantly associated with dermatological abnormality and its treatment, the former association being more significant and more pronounced after 69 years of age. The association of hydrocortisone use after 69 years of age and cataract, however, remains significant even after adjustments for dermatological abnormality and steroid use, suggesting that even among steroid medications hydrocortisone is particularly strongly associated with cataract.

Human cataract risk factors: significance of abstention from, and high consumption of, ethanol (U-curve) and non-significance of smoking.

Current ethanol consumption and cigarette smoking were quantified by questionnaire in Edinburgh and suburbs, Scotland, UK. Stringently matched cataract-control pairs (n = 990 and 858, respectively) were included. For ethanol, 'light and infrequent' consumption and 'light and frequent' were associated with a significantly lower risk of cataract than were total abstention and 'occasional' consumption; the prevalence of cataract rose with further increases in consumption, suggesting a U-shaped curve. For nuclear cataract, white in particular, there is a significant trend with amount consumed. Smoking was not found to be a risk factor.

Some blood plasma constituents correlate with human cataract.

AIMS: To look for differences between matched pairs of patients and controls in concentrations of various plasma constituents which might indicate dysfunctions associated with cataract. METHOD: One thousand patients were taken from the cataract waiting list of a specialist eye hospital. For each patient a matched control of the same sex and half-decade of age but without cataract was taken from the patient list of the family doctor of the patient; the control was the next alphabetically after the patient on the doctor's list. The patients and controls were visited in their homes by a team of nurses who performed venepunctures and collected information for a questionnaire. Eye examinations were performed by a team of ophthalmologists. RESULTS: Significant differences were found between the cataract and control groups in 10 of the 18 examined plasma constituents. A constellation of three--bilirubin, alkaline phosphatase, and gamma glutamyl transpeptidase--was significantly higher in the cataract group, suggesting subclinical liver dysfunction as a risk factor. Steroid treatment and diabetes increased cataract risk. Endogenous basal plasma cortisol levels were raised in the cataract group, irrespective of steroid use and diabetic status. Alkaline phosphatase, calcium, glucose, and sodium were all raised in the cataract group. Given the raised total protein and albumin also found in the cataract group, the lower albumin/(total protein-albumin) ratio (an approximation for albumin/globulin ratio) may imply an increase in globulin, suggestive of possible (chronic) infection. Total cholesterol was lower in the cataract group. CONCLUSION: Human cataract in older age groups seems to be due to an accumulation of risk factors, even if individual mean concentrations are well within normal limits but, of course, differing significantly from the corresponding means in the control population.

Beta B2-crystallin in the mammalian retina.

beta-crystallins are abundant lens proteins in most, if not all vertebrate species. We have previously reported the presence of low levels of beta-crystallins in chick non-lens tissues, both ocular and extra-ocular, including the expression of beta B2-crystallin in the retina. Here we report that extralenticular beta-crystallin expression is also found in mammals. beta B2-crystallin is expressed in mouse and cat neural and pigmented retinas and in cat iris. Although present at levels lower than those found in the lens, the appearance and accumulation of beta B2-crystallin in the neural retina coincides with the functional maturation of this tissue.

Changes in crystallin expression during transdifferentiation and subsequent ageing of embryonic chick neural retina in vitro: comparison with lens epithelium.

We have shown that cultured day-old chick lens epithelial cells undergo changes in crystallin expression during lens fibre (lentoid body) differentiation and ageing in serial subculture which are similar to those found in the adult lens in vivo. Here we have maintained neural retina cells, which transdifferentiate in vitro, for 250 days in serial subculture, in order to determine whether the tissue of origin affects the sequence of changes in crystallin expression and capacity for lentoid body formation both during fibre formation in primary culture and ageing in vitro. Alpha and beta-crystallins were detectable before delta-crystallin in primary cultures of both day-old chick lens epithelium and neural retina from 8-day embryos, but while beta 2 (26 kDa) was detected in pre-lentoid lens epithelial cultures it was not detected until after lentoids formed in neural retina cultures. The relative proportions of the alpha- and beta-crystallin polypeptides were similar in lentoid-rich lens epithelium and neural retina cultures, and both cultures underwent similar changes in serial subculture: a loss of lentoid forming capacity, an early preferential loss of delta-crystallin expression followed by a failure to accumulate first alpha- and then beta-crystallins. The order in which the beta-crystallin polypeptides were lost differed between the cultures. There is evidence for rapid turnover of alpha- and beta-crystallins while actin is the major component expressed in both types of aged cultures. Thus lens and retinal cells show some initial differences in the sequence of crystallin expression in primary cultures and in the eventual characteristics of aged cultures, but during the period beginning with lentoid formation and ending with the onset of senescence, lens cells from either source follow a broadly similar programme of ageing changes which are similar to those which occur during lens ageing in vivo.

Localization of delta-crystallin RNA during lens morphogenesis and differentiation in the normal and talpid3 chick embryo.

Embryonic lens fiber cell differentiation in the chick is marked by the accumulation of delta-crystallin protein. The levels of delta-crystallin RNA are shown here to rise dramatically in the cells of the posterior lens pit prior to their elongation and differentiation as lens fibers. This increase correlates with regional proximity to the underlying optic cup (future retina). This accumulation of delta-crystallin RNA during lens induction operates selectively on the delta 1-crystallin transcripts whereas delta 2-crystallin/argininsosuccinate lyase RNA is detectable at lower levels in all developing ocular tissues throughout this period. The talpid3 mutant forms a flat "bridge" of thickened placode-like cells in the head epithelium between the two lens placodes, and this bridge also accumulates delta 1-crystallin RNA, suggesting that the selective increase in delta 1-crystallin RNA levels over those of delta 2-crystallin represents an early event in cellular commitment to lens fiber differentiation in the chick. The significance of the sequence of temporal changes in inductive sources for lens fiber formation is discussed, and we propose that the role of the optic cup is to provide, bound to its extra-cellular material (ECM), a high local concentration of the same growth factors which act as fiber inducers in the older eye.

Long-term effects of aluminium on the fetal mouse brain.

Potentially noxious substances may act as fetal teratogens at levels far lower than those required to produce detectable effects in adults, and behavioural teratogenicity may occur at levels lower than those which produce morphological teratogenesis. Aluminium (Al) is a potential neurotoxin in adults. Since pregnant women may be exposed to untoward levels of Al compounds under certain conditions, we have examined the long-term effects of treating the pregnant mouse with intraperitoneal or oral aluminium sulphate on brain biochemistry and behaviour of the offspring. The cholinergic system, as evaluated by the activity of choline acetyltransferase (ChAT), was affected differentially in different regions of the brain, and still showed significant effects in the adult. Differences between the intraperitoneal and oral series in the magnitude of effect seen in the regions of the brain probably reflect differences in the effective level of exposure. Growth rate and psychomotor maturation in the pre-weaning mouse were affected in the intraperitoneal series only, showing a marked post-natal maternal effect.

Evidence for the extralenticular expression of members of the beta-crystallin gene family in the chick and a comparison with delta-crystallin during differentiation and transdifferentiation.

The beta-crystallins are major water soluble proteins of vertebrate lens fibre cells and have previously been regarded as lens-specific proteins: however beta B2-and beta A3/A1-crystallin RNAs are transcribed and beta-crystallin polypeptides are detectable in the developing chick retina. The beta-crystallin RNA is transcribed in a subpopulation of retina cells and the number of transcribing cells and the level of beta-crystallin polypeptides increase during the differentiation of the retina. Several tissues express beta-crystallin polypeptides, but individual tissues are characterised by qualitative and quantitative differences in the beta- and delta-crystallin polypeptides expressed. The expression of beta-crystallins appears to be non-random as defined by tissue distribution, cellular localisation and ontogeny, implying a function for extralenticular beta-crystallins and a complex mechanism for the regulation of their expression.

Independent regulation of two coexpressed delta-crystallin genes in chick lens and nonlens tissues.

It is known that delta-crystallin is super-abundant in the early chick lens, but it is found at lower levels in certain other tissues. Ninety-nine percent of the lens delta-crystallin poly(A)+ RNA is from the delta 1-crystallin gene. We report here that the delta 1- and delta 2-crystallin genes are both transcribed in the chick lens and retina throughout embryonic development and that both RNAs are found in embryo adenohypophysis and epiphysis and in day-old posthatch chick tibiofemoral chondrocytes and striated muscle. delta 1-crystallin RNA is more abundant in lens tissues, while delta 2-crystallin RNA is more abundant in all nonlens tissues. However, delta 1-crystallin RNA is processed more efficiently than delta 2-crystallin RNA in all early embryonic tissues examined. A comparison of lens epithelium and fibers established that levels of delta 2-crystallin RNA are the same but those of delta 1-crystallin RNA are over 100-fold higher in fibers compared to epithelial cells. The evidence implies independent regulation both of transcription and of post-transcriptional events for these two genes.

Ageing in the chick lens: in vitro studies.

In principle, ageing may be due to the interaction of several factors, including the accumulation of random changes both genomic and non-genomic, secondary changes in a tissue contingent upon the changing function of other tissues, and programmed non-random changes in the tissue-specific expression of various genes. The use of a single tissue comprising one cell type only, in which the major gene products are well defined, in which there is a well attested series of developmental and age-related changes in cell properties and gene expression and which can be studied and compared in vivo and in vitro, offers advantages for investigation of these questions. The vertebrate eye lens possesses these advantages. The crystallins (proteins expressed at super-abundant levels in the lens) are well characterised. The lens epithelial cells (LEC) grow readily and can differentiate into the lens fibre cells in vitro, and, finally, such terminally differentiated cells may also be derived, by a process of transdifferentiation, from neural retina cells (NRC) in vitro. Thus the effect on ageing changes of the tissue of origin may also be studied. This article reviews our previous studies on long-term changes in growth potential, differentiation capacity and crystallin expression of chick lens cells in ageing cultures, their overall similarity to events in vivo and the effect on ageing changes of genotypes affecting the growth rate. It presents new information on these genetic aspects, and on crystallin expression in long-term ageing cultures of transdifferentiated neural retina, and compares the behaviour of ageing chick lens cells with that reported for mammals.

Age-related changes in the response of chick lens cells during long-term culture to insulin, cyclic AMP, retinoic acid and a bovine retinal extract.

We have reported that 1-day-old post-hatch chick lens epithelial cells lose the capacity for lentoid body formation and delta-crystallin expression during long-term serial subculture, although they continue to synthesize, but not to accumulate, alpha- and beta-crystallins, even in cells with a transformed phenotype. Here we present evidence that dedifferentiation may reflect an age-related change in the capacity for response to regulatory signals. We have tested the capacity of these cells in serial subcultures to respond to agencies which affect lens cell growth and differentiation in primary culture: retinoic acid (RA), insulin, cAMP and bovine retinal extract (BRE). Secondary cultures responded only to RA and BRE, by an increase in lentoid formation and by alpha- and beta-accumulation, while RA also restored delta-crystallin expression. Later cultures showed no such responses. The results suggest that the process of lens cell dedifferentiation may, at first, be reversible but later becomes irreversible, despite the continuing persistence of low levels of crystallin expression.

Differential effects of prenatal exposure to phenobarbital on the behaviour and neurochemistry of CBA and C57BL/6J mice.

Pregnant C57BL/6J and CBA mice were administered 60 mg/kg phenobarbital intraperitoneally from days 10 to 16 of gestation. On day 18 of pregnancy half of the control and drug-treated mice were killed and the embryonic brains removed for cell cultures. The remaining mice were allowed to have their litter. After cross-fostering the mice were used for behavioural studies. Pups born to drug-treated CBA mice had birth-weights similar to controls, but their weights had fallen behind controls by day 18 after birth. They were slower at attaining mature responses in tests for sensory motor development and became progressively more hyperactive (three times more active at day 18) compared to controls. Drug-exposed C57 pups also had birth weights similar to controls. After cross-fostering, 19% of control and 31% of drug-exposed pups died, but the remaining drug-exposed pups showed no deficits in weight gain. In contrast to drug-treated CBA pups, drug-exposed C57 pups were slightly quicker in attaining mature responses in some tests. There was no difference in activity between them and their controls. In neurochemical analyses, uptake of neurotransmitters by cerebral cultures from CBA showed that uptake of GABA was increased by 5%, choline by 95%, dopamine 120%, serotonin 165% and noradrenaline by 160% in cultures from drug exposed embryos compared to controls. In cerebral cultures from C57, GABA uptake was reduced by 18%, choline 33%, dopamine 35% and noradrenaline by 25%. Only serotonin uptake was increased by 182% compared to controls. Differences between C57 and CBA were also apparent in the uptake of neurotransmitters by neuronal cultures from the mesencephalon.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of the genotype on the process of ageing of chick lens cells in vitro.

We reported previously that changes in crystallin expression in differentiating long-term primary cultures of lens cells from five different chick genotypes are similar to those which occur in vivo between hatching and the 8-week-old adult. These changes followed a similar program in all genotypes but occurred more rapidly in cells from the fast-growing than from the slow-growing genotypes. The present study examines ageing changes in lens cell populations from the same five genotypes, over a 4-6 month period, using long-term serial subcultures. The capacity for lentoid differentiation was progressively lost, but the rate of loss was inversely related to the intrinsic growth rate of the cells of these genotypes, occurring at the first passage in the slowest-growing strain, while fifth passage cells of the fastest-growing strain still retained some lentoid-forming capacity. The rate of loss of crystallin expression was also inversely related to the genetic growth rate, but the sequence of changes appears to be nonrandom, since it was broadly similar in all genotypes, starting with a preferential loss of delta-crystallin, as occurs in vivo; although alpha- and beta-crystallins were undetectable in late dedifferentiated cultures, the capacity of the cells for their synthesis was still present. Cultures from both fast-growing genotypes eventually showed senescence, but those from all three slow-growing genotypes underwent transformation. The major cell component in late cultures of all genotypes was actin.

Diuretic drugs as risk factors in cataractogenesis.

The use of diuretic drugs, which previously has been found to be associated with the incidence of cataract, is further investigated to elucidate the nature of the association. Diuretic drugs with different modes of action are considered separately. The degree of association of each correlates with the mean plasma urea level which itself is associated with cataract. However, the association is with cataract in general rather than with any specific type of lens opacity.

Patterns of crystallin expression during differentiation in vitro of several chick genotypes with different effects on lens cell growth rate.

We have recently reported that chick lens cells during differentiation in long-term culture show a programme of change in crystallin expression which mimics events during lens development in vivo. The aim of the present work was to examine the stability of the programme by testing the response to genetic influences and exposure to a carcinogen. Five genetically distinct inbred strains of chick, differing in the intrinsic growth rates of lens epithelial cells in vitro, were used to study the effects of the rate of mitosis on crystallin expression, both during lens development and in long-term cell culture. The time of appearance of lentoids, their size and abundance and the rate of change in crystallin expression were all modified in a genotype-specific way, related to the rate of mitosis, but the programme of changes in crystallin expression was the same for all genotypes. Genetic differences were also found in the patterns of response to treatment of cultures during the logarithmic growth phase with a nitrosoguanidine compound known to affect cell differentiation in lens cultures and several other systems. The changes in crystallin expression and fibre differentiation were delayed, but cultures of the faster growing genotypes were least affected. With further culture, crystallin expression tended to recover to control values although levels of fibre differentiation and cell growth remained depressed. The results indicate that genetic differences in intrinsic growth rate moderate but do not change the programme of crystallin expression shown by lens epithelial cells in culture, and that this programme shows resistance to change.

Alterations in crystallin gene expression during subculture of chick lens cells.

We report here on the changes in crystallin gene expression during serial subculture of lens epithelial cells derived from day-old post-hatch chicks. Total cellular RNA from mass cultures were analysed by in vitro cell-free translation and by RNA blot (Northern) hybridization using a cloned delta-crystallin cDNA. Our results indicate that following subculture, lens epithelial cells which still retain the capacity for lens fibre differentiation (lentoid body formation) show a selective loss of delta-crystallin synthesis, and that this is related to the loss of delta-crystallin mRNA. The data suggest that older epithelial-cell populations give rise to lentoid bodies which in terms of crystallin gene expression closely resemble the later-formed cortical fibres of the adult chick lens. Tertiary cultures had an accelerated growth rate, formed no lentoids, contained no translatable alpha- or delta-crystallin mRNAs but still contained translatable beta-crystallin mRNAs.

Developmental changes in membrane protein expression by chick lens cells in vivo and in vitro and the detection of main intrinsic polypeptide (MIP).

We have compared the long-term developmental changes in water-insoluble protein expression by chick lens cells in vitro and in vivo. Crude membrane fractions were prepared by alkali treatment of the urea-insoluble protein fraction, and the proteins analysed by sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gel electrophoresis. The major component present in the urea-insoluble fraction of chick lens fibres, a 25,000 MW polypeptide (MIP-25K) was more abundant in adult (8 weeks) than day-old post-hatch chick lens fibre masses. MIP-25K was detected in differentiated but not predifferentiated lens cell cultures, and indirect immunofluorescence using anti-bovine MIP antiserum indicated that MIP-25K was localized in the lentoid bodies. Our findings indicate that the urea-insoluble protein profiles of long-term well-differentiated chick lens cell cultures are qualitatively very similar to the profiles of the lens fibres. The data also confirm that the expression of MIP-25K, rather than the expression of water-soluble crystallin protein, is a marker for lens cell differentiation, and confirm earlier reports, which have been disputed, that delta-crystallin (but not alpha-or beta-crystallin) is specifically associated with chick lens fibre membranes.

Cellular heterogeneity in the expression of the delta-crystallin gene in non-lens tissue.

RNA transcripts of the delta-crystallin genes, which code for the major chicken lens protein, have been detected at low levels in many non-lens tissues. Here it is demonstrated by in situ hybridisation that these transcripts are concentrated at a high level in small, infrequent clusters of cells in many non-lens tissues. While the nuclei of these cells are very heavily labelled, there is only light labelling of the cytoplasm. The unlabelled cells surrounding the labelled clusters are of similar morphology and staining properties as the labelled cells, and all have the characteristic morphology of cells of the embryonic tissue used. With the exception of neural retina, it is not yet known whether the labelled clusters are found in specific locations in the tissues, or whether they arise at random.