Ceramides of human normal and cataractous lens.

Ceramides were quantitatively isolated from human normal and cataractous lens by solvent extraction, silicic acid chromatography, thin-layer chromatography, and gas-liquid chromatography. Only two species of ceramides with normal fatty acids were detected. In the mature cataracts, there was an increase in palmitate and nervonate at the expense of the other fatty acids. Due to the increase of 24 : 1, the ratio of 24 : 1/24 : 0 increased significantly from normals to cataracts. Sphinganine was the major long-chain base, but 4-sphingenine was also present. The total amount of ceramides in the immature and mature cataracts was 1.8 and 3.0 times higher than the normals of the same age group. Such an increase does not seem to be the result of an age-dependent process.     

Uptake of75Se in cataractous rat lens proteins

The purpose of the present study was to measure the pattern of uptake of⁷⁵Se into proteins in normal rat lenses and into the proteins of lenses with selenite-induced cataract. Ten-day-old suckling rats received a single injection of⁷⁵Se with or without a cataractous dose of cold carrier sodium selenite. Four days after injection, the proteins from excised lenses were counted for⁷⁵Se radioactivity and subjected to gel permeation chromatography, amino acid analyses, and mass spectrometry. All three soluble crystallin lens proteins took up⁷⁵Se in both normal and cataractous lenses. However, cataractous lenses did not take up⁷⁵Se into a soluble protein in which major quantities of⁷⁵Se were taken up in normal rats. Futhermore,⁷⁵Se in the gamma-crystallins was associated with an unusual acidic amino acid. It was concluded that selenium metabolism by lens proteins may be unusual compared to other soft tissues.     

Expression and activity of paraoxonase 1 in human cataractous lens tissue

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that is believed to be involved in the protection against oxidative stress. There is evidence that paraoxonase activity is reduced in patients with diabetes and cataract. In the current study, we analyzed mRNA expression of PON1 as well as other members of the paraoxonase family, PON2 and PON3, in human cataractous lens samples. Our results indicate that only PON1 is expressed at the gene and protein levels in human lens tissues. We quantified MDA levels and measured PON1 (paraoxonase/arylesterase) enzymatic activities in subjects suffering from cataract due to aging and diabetes. Decreased PON1 activity was more pronounced in diabetic patients (p  <  0.001) compared to senile subjects, which may be due to glycation and increased oxidative insult. To examine the structural alterations that occur in response to glycation, we constructed a three-dimensional model of PON1 and its glycated variant. Glycation at Lys70 and Lys75 is predicted to cause hindrance in binding of substrate to the active site of the enzyme.     

Human senile cataractous lens protease. Isolation and some chemical characteristics

A proteolytic enzyme was isolated from human senile cataractous lens by anion-exchange and gel-filtration chromatography. Sedimentation and zone-electrophoretic experiments indicated a high degree of homogeneity for the enzyme. A molecular weight of 27000 was calculated from measurements of sedimentation velocity and diffusion coefficient. Chelating agents decreased activity which could be restored by addition of certain bivalent metal ions. Di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride inhibit the proteolytic activities. Optimum rates of hydrolysis were observed at pH5.2.     

Estimation of structural changes in the cataractous rat lens using Raman spectroscopy.

For quantitative evaluation of cataract-related changes in lens proteins and lens water, the relative contents of water and SH residues and changes in the microenvironments of aromatic amino acid residues were quantitatively examined in cataract of the rat lens which had been induced by sodium selenite. Using Raman spectroscopy, results were compared with those of age-matched control lenses. The relative contents of water and SH residues decreased with increasing age in normal lenses from 3 to 8 weeks of age. In the cataractous lens, the relative water content increased constantly as compared with that of age-matched controls. The relative SH residue content continued to decline in the cataractous lenses of animals at every age. The microenvironments of tyrosine residues in cataractous lenses also changed progressively.     

Renal abnormalities and their developmental origin

Congenital abnormalities of the kidney and urinary tract (CAKUT) occur in 1 out of 500 newborns, and constitute approximately 20-30% of all anomalies identified in the prenatal period. CAKUT has a major role in renal failure, and there is increasing evidence that certain abnormalities predispose to the development of hypertension and cardiovascular disease in adult life. Moreover, defects in nephron formation can predispose to Wilms tumour, the most frequent solid tumour in children. To understand the basis of human renal diseases, it is essential to consider how the kidney develops.     

Cx43, ZO-1, alpha-catenin and beta-catenin in cataractous lens epithelial cells

Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (n?=?52) undergoing cataract surgery. Specimens were divided into three groups based on the type of cataract: nuclear cataract, cortical cataract and posterior subcapsular cataract (PSC). Clear lenses (n?=?11) obtained from donor eyes were used as controls. Expression was studied by immunofluorescence, real-time PCR and Western blot. Statistical analysis was done using the student’s t-test. Immunofluorescence results showed punctate localization of Cx43 at the cell boundaries in controls, nuclear cataract and PSC groups. In the cortical cataract group, cytoplasmic pools of Cx43 without any localization at the cell boundaries were observed. Real-time PCR results showed significant up-regulation of Cx43 in nuclear and cortical cataract groups. Western blot results revealed significant increase in protein levels of Cx43 and significant decrease of ZO-1 in all three cataract groups. Protein levels of alpha-catenin were decreased significantly in nuclear and cortical cataract group. There was no significant change in expression of beta-catenin in the cataractous groups. Our findings suggest that ZO-1 and alpha-catenin are important for gap junctions containing Cx43 in the LECs. Alterations in cell junction proteins may play a role during formation of different types of cataract.     

Pollution and developmental abnormalities of Atlantic fishes

Synopsis Planktonic eggs of Atlantic mackerel,Scomber scombrus, provide evidence that pollution is associated with mortality, malformation, and abnormal chromosome division of fish embryos developing about the surface of the U.S. Atlantic coast. Embryo data are substantiated by the finding that adults of mackerel, windowpane flounder,Scophthalmus acquosus, and winter flounder,Pseudopleuronectes americanus, from more polluted coastal areas also have higher frequencies of mitotic abnormality than those from less polluted regions of the Mid- and North Atlantic. No ontogenetic interval escapes contamination. All are likely to be adversely influenced, resulting in considerable direct and indirect cumulative effect on total early-life survival. Development of genetic and epigenetic resistance to reproductively harmful influences of contaminants may interfere with other modifications in structure and function necessitated by natural environmental fluctuations, changes in climate, and by fishing itself.     

Dynamic and differential regulation in the microRNA expression in the developing and mature cataractous rat lens

Recent evidence supports a role for microRNAs (miRNAs) in regulating gene expression, and alterations in gene expression are known to affect cells involved in the development of ageing disorders. Using developing rat lens epithelial cells (LECs), we profiled the expression of miRNAs by a microarray‐based approach. Few gene expression changes known to be involved in pathogenesis or cytoprotection were uniquely influenced by miRNA expression. Most miRNAs increased or decreased in abundance (let 7b, let 7c, miR29a, miR29c, miR126 and miR551b) in LECs/lenses during late embryonic and post‐natal development and in cataract. Among them, miR29a, miR29c and miR126 were dramatically decreased in cataractous LECs from Shumiya Cataract Rats (SCRs). Specifically, the cytoskeleton remodelling genes tropomyosin (Tm) 1α and 2β, which have been implicated in the initiation of pathophysiology, were targets of miR29c and were over‐stimulated as demonstrated by inhibitor experiments. In transfection experiments, increasing the level of miR29c caused a corresponding decrease in the expression of Tm1α and 2β, suggesting that miR29c may regulate the translation of Tm1α and 2β. 3′UTR luciferase activity of Tm1α, not 2β, was significantly decreased in miR29c‐transfected mouse LECs. These findings demonstrate changes in miRNAs expression, and target molecules have potential as diagnostic indicators of ageing and as a foundation of miR‐based therapeutics for age‐related diseases.