Age and cataract-related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens.

Heavy molecular weight (HMW) proteins were detected in normal and cataractous mouse lenses. The HMW aggregates increased with the age of the lens in normal mouse. Alpha and β-crystallins were detected by immunodiffusion in the HMW fractions from normal and Nakano mice. No γ-crystallin could be detected in these aggregates by immunodiffusion; however, a slight amount of this crystallin was detected using the radioimmunoassay. The polypeptide composition of the HMW proteins was different in the Nakano mouse from the normal. By SDS polyacrylamide gel electrophoresis, a polypeptide of 27 000 mol. wt. was evident in the Nakano HMW material that was not present in the normal HMW protein, but a 15 000 mol. wt. band was absent in the Nakano.Two other differences were seen with the Nakano lens. First, the water insoluble lens protein was extremely high. By 90 days, about two thirds of the protein was insoluble in these lenses. Secondly, the sharp drop in γ-crystallin at the time of complete opacification of the lens was in part a result of the leakage of this protein into the anterior chamber of these mice. By radioimmunoassay, the level of γ-crystallin in the Nakano aqueous humor at the time of the cataract was greater than 100 ng per microliter. These data demonstrate that crystallins are converted to the insoluble proteins and some diffuse out of the lens during cataract formation.     

The development and application of a radioimmunoassay to lens crystallins

A radioimmunoassay was developed for mouse α- and γ-crystallins. A standard inhibition curve against known quantities of the crystallins served as a basis for quantitating unknown samples. The sensitivity of the assay was 2 ng for α-crystallin and 4 ng for γ-crystallin. It was possible to quantitate the amount of γ-crystallin present in the cultures from normal mouse and Nakano mouse lenses. The results were consistent with the previous immunofluorescent observation of γ-crystallin localization in the lentoid bodies. In addition, changes in the lens crystallins in normal and Nakano mouse lenses were followed. A dramatic decrease of γ-crystallin occurred during cataract development, however the level of α-crystallin in the soluble lens protein remained unchanged during this period.     

Investigation of Nakano lens proteins

Changes in the protein chemistry of the Nakano lens with age and developing cataract and comparison with normal mouse lens protein are reported. It was found that significant differences exist between the protein of the normal and the cataractous lens. In Nakano lenses high molecular weight disulfide-linked aggregates, disulfide-linked cytosol polypeptides to the fiber membrane, an apparent increase in the concentration of degraded polypeptides, disulfide crosslinking of low molecular weight species and marked differences in membrane polypeptide profiles were observed. A striking similarity was found between these observations with the Nakano cataract and previous reports of the changes in protein chemistry in the development of senile human cataract. It can be concluded that although the initiating event for induction of the cataract may differ, the sequence of events following such insult may be similar.     

Biosynthesis of proteoglycans by lens epithelial cells of cataractous mouse (Nakano strain)

The capsules (with epithelial cells attached) of lenses from normal and cataractous mice (Nakano strain) were biosynthetically labeled in vitro with radioactive precursors. The labeled macromolecules were chromatographed on a Sepharose CL-4B column and analyzed by specific enzyme digestion. The incorporation of [3H]-proline and [3H]-glucosamine into macromolecules was comparable in the cataract and normal capsules, while that of [35S]-sulfate was reduced by 60% in the cataract capsules, indicating that the proteoglycan synthesis was specifically decreased in the cataract lens. Glycosaminoglycan analyses showed an increased synthesis of hyaluronic acid and decreased synthesis of heparan sulfate in the cataract capsules. It is possible that the alterations in the synthetic level and glycosaminoglycan components of proteoglycan affect the permeabilities of macromolecules to lens capsule and lead to cataract in Nakano mouse lens.     

Properties of a Na-K ATPase inhibitor in cultured lens epithelial cells

A Na-K ATPase inhibitor has been isolated from cultured lens epithelial cells from the Nakano mouse. This inhibitory activity elutes from a CM-Sephadex column in the same fraction as the inhibitor from intact Nakano lens. The inhibitor is a polypeptide sensitive to leucine aminopeptidase as well as car?ypeptidase A. In addition, the inhibitor isolated from the cultured lens cells has the same molecular weight as the one present in the Nakano lens. Thus, the inhibitory activity from the cultured cells appears to be similar to that obtained from whole lens.     

Morphological study on cataractogenesis of the Nakano mouse lens

· Background: Although some histopathological features on the Nakano mouse lens have been pointed out by a few investigators, there seem to have been no detailed studies on the sequential changes that occur. · Methods: We used the following two approaches: (1) Observation of the whole lens by dissection microscopy and (2) light and electron microscopic examination of the sectioned lens specimen. · Results: (1) The Nakano mouse lens showed sustained transparency up to 19 days after birth, fine opacity at the 20th day, and development of a mature cataract around the 30th day. In addition, although the Y-shaped posterior suture was normal at the 15th day, bending of the suture line appeared around the 19th day. (2) The cataractous lens revealed degeneration of the epithelial cells and adjacent anterior cortical fibers at the 10th day. Swelling of the anterior cortical fibers became prominent, and swelling of the posterior cortical fibers occurred by the 15th day. Upon separation of the suture around the 20th day, fine opacity occurred in the perinuclear zone, which extended to the anterior cortex and finally led to the formation of a mature cataract. · Conclusions: These results indicate that epithelial degeneration is a major feature of cataract in the Nakano mouse, and the subsequent lens fiber swelling and posterior sutural separation are the underlying causes of the development of opacity. Received: 2 April 1998 Revised version received: 15 June 1998 Accepted: 23 July 1998     

A possible cataractogenic factor in the Nakano mouse lens.

A factor present in the Nakano mouse lens is an inhibitor of NaK ATPase. This inhibitor is not found in the normal mouse lens. It is not associated with any of the major crystallins but is found with the low molecular weight components. It is resistant to high temperatures and to acid and alkaline pHs. It appears to be a polypeptide since its activity is abolished by treatment with carboxy-peptidase A and leucine amino peptidase. Other properties of the inhibitor indicate that this factor appears quite different from other substances which inhibit NaK ATPase.     

Studies on primary cultures of adult lens cells from normal and hereditary cataractous mice.

Lens epithelial cells from normal and congenital cataractous mice strains were cultured under similar conditions. Both normal and cataractous cells actively propagated and reached confluency on the eleventh day. These cells, thereafter, underwent morphological changes characterized by cell elongation, aggregation and formation of lentoid bodies at about 15 days.Electron microscopy revealed these lentoid bodies to consist of immature lens cells. These structures derived from cataractous cells had numerous vacuoles in the cytoplasm much more so than in the normal lens cells. In addition, some lentoid bodies closely resembled mature fibers of the intact lens. It was also demonstrated that these lentoid bodies showed positive immunofluorescence when reacted with fluorescent antiserum to γ-crystallin.There were certain differences observed between the cultured cells derived from normal lens and Nakano cataract. The disappearance of organelles and denucleation process were delayed in the lentoid bodies found in cultured Nakano cells when compared to normal cell culture. In addition a second type of lentoid body, although present as a minor population, was observed in the Nakano cell culture. Other subtle differences were observed during the course of culturing normal and cataractous lens cells.     

Hereditary cataract of the Nakano mouse

The Nakano mouse is a hereditary cataract model whose most characteristic change is a deficiency in lens Na+,K(+)-ATPase. Consequently, there is a change in lenticular sodium and potassium ion levels just before cataract formation. The amounts of calcium ion also change suddenly in the lens, with accumulated levels higher than any other type of cataract. Other biochemical changes coincide with the development of lens opacity, including decreases in the levels of reduced glutathione, ATP, biosynthetic activity of proteoglycans in epithelial cells, and the permeability of gap junction channels in fiber cells. The decrease in the activity of Na+,K(+)-ATPase results in changes in a number of key metabolic parameters, resulting in the eventual opacification of the Nakano mouse lens at approximately 30 days of age.     

Stimulation of the hexose monophosphate pathway by pyrroline-5-carboxylate reductase in the lens

Addition of pyrroline-5-carboxylate (P5C) or its precursors to rat lenses cultured for 24 hr in TC-199 medium containing 14C-glucose results in an apparent concentration-dependent increase in hexose monophosphate-pentose (HMP) pathway activity. Addition of proline, the reduction product of P5C, did not result in an increase, suggesting that stimulation of the HMP pathway is related to the reduction of P5C to proline by the enzyme P5C reductase. No apparent feedback inhibition on P5C reductase was observed. Stimulation of HMP pathway activity by P5C was also observed in the lenses of Philly and Nakano mouse, two models of congenital osmotic cataracts. Compared with its genetic control, the Swiss--Webster mouse, generally no difference in the lenticular levels of HMP pathway activity was observed in the Philly mouse--even after the onset of cataract. Stimulation of the HMP pathway in the Philly lens by P5C, however, was consistently lower than its control. In the lenses from the Nakano mouse and its genetic control, the Balb/c mouse, no difference in the percentage stimulation of the HMP pathway resulting from the addition of P5C was observed, but HMP pathway activity in the Nakano lens was consistently lower than that of the control.     

Swelling of the lens fibers.

Lens cells of congenital mouse cataract (Nakano and Fraser strains) and galactose-fed rats were studied by scanning electron microscopy. Similarly lens cells of normal young mice and rats were examined as controls. Normal lenses of young rodents consist of lens fibers in all maturation stages which have been demonstrated in humans and in monkeys. Lens cells in the congenitally cataractous lenses are irregular in size and shape in the earlier stage of the cataract formation. Swelling of the lens cell occurs corresponding to the occurrence of early optical opacity. Swelling of the cell occurs segmentally in congential cataractous lenses; in the apical ends (Fraser) and in the posterior ends (Nakano). Similar swelling of the lens cell is observed in the main cell body in the superficial lens cortex of galactose-fed rats. However, numerous intercellular cysts are formed by the accumulation of fluid which may have been pumped out of the cells before the cells became degenerative. These numerous shrunken fibers are present among swollen cells in both congenital and galactose-induced cataractous lenses.     

Age-related changes of calpain II and alpha-crystallin in the lens of hereditary cataract (Nakano) mouse

The age-related changes of calpain II (high-Ca2+-requiring form of Ca2+-dependent cysteine proteinase; EC 3.4.22.17) and alpha-crystallin in the lens of hereditary cataract (Nakano; cac/cac) mouse were studied. Before the onset of the cataract formation, i.e., at the end of the 2nd week after birth, the calpain activity in Nakano mice was as high as that in the control ICR mice, but it decreased rapidly as the cataract progressed to completion during the 4th and the 12th week. Marked degradation of lens proteins ensued between the 2nd and the 4th weeks, and one of these proteins was identified, using monospecific antibodies, as B chain of alpha-crystallin. A chain of alpha-crystallin was not degraded in vivo, in contrast to its known susceptibility to calpain in vitro. The present data suggest that in Nakano mice, calpain may be involved in the onset or early stage of the cataract formation.     

Tissue culture of lens epithelial cells from normal and Nakano mice.

Lens epithelial cells from normal and Nakano adult mice have been cultured for over 1 year, and the cells have retained certain differentiated characteristics. Fluorescent antibody to mouse gamma crystallin reacted with the spherical lentoid bodies which appeared approximately 2 weeks after the start of the culture. The lentoid bodies also contained cells which had few cell organelles and homogenous cytoplasms. Both gamma crystallin production and loss of cellular organelles are characteristics of differentiated fiber cells rather than epithelial cells. An inhibitor of the Na-K ATPase is responsible for the hydration and subsequent cataract formation in the Nakamo mouse. The inhibitor of the Na-K ATPase was demonstrated in the lens in culture from the Nakamo mice, but no inhibitory activity was detected in the cultures from normal mice.     

An improved fixation technique for maintaining the fine structure of the nuclear zone of neonatal mouse lens

The fine structure of the nuclear zone of neonatal mouse lenses can vary considerably according to the fixation used. When normal neonatal mouse lenses are fixed in a commonly used chilled glutaraldehyde solution, the nuclear zone develops a grossly visible opacity, and irregular sized protein granules appear in the subsequent sections. Similar artifacts of aggregated irregular sized protein granules appear when cataractous mouse lens are conventionally processed. These artifacts can be avoided by soaking the lens in 0.15 M reduced glutathione solution for 10-15 min before fixation in a phosphate buffered 2% glutaraldehyde solution (pH 7.4) at 27-35 C. Normal lenses treated in this manner maintain translucency in the nuclear zone throughout the fixation-embedding procedure, and the resulting sections show finely uniform granularity with the cell membrane well preserved. Similarly processed nuclear portions of cataractous lenses of Nakano mice show uniformly aggregated protein granules, measuring about 350A in diameter. The cell membranes in the cataractous zone are also not interrupted.     

Biochemical alterations of the lens capsule in mice with hereditary cataract

Lens capsules from normal and cataractous mice (Nakano strain) were compared histologically and chemically. Histologically, the anterior capsule of the cataract lens was thicker than the normal, while the posterior capsule appeared almost the same. Amino acid analysis revealed some differences between normal and cataractous lens capsules: more glycine, hydroxylysine and arginine and less hydroxyproline and tyrosine were found in cataractous capsules. Hydroxyproline showed the largest difference, cataract lens being about 11% lower than normal.To see if the content of hydroxylysine-linked carbohydrates is different in normal and cataractous lenses, as has been shown for renal basement membranes from normal and diabetic animals, a procedure was developed to separate basic amino acids, including the hydroxylysine-linked carbohydrates, from other amino acids. It was found that the content of hydroxylysine-linked disaccharide (Glc-Gal-Hyl) was slightly but significantly higher in cataractous lenses.The possibility was suggested that the compositional alterations observed for the cataract lens might be correlated with functional changes in the lens capsule of the cataract, e.g. lowered elasticity, decreased permeability etc.     

钠尿肽受体A（NPR-A）在小鼠角膜和晶状体发育过程中的表达

目的　检测钠尿肽受体A（natriuretic peptide receptor A,NPR-A）在不同鼠龄小鼠角膜和晶状体内的表达,探讨其在小鼠眼发育过程中的作用。方法　选用C57BL/6转基因小鼠,收集从 E14到P90小鼠眼球标本120只,采用40 g·L^-1多聚甲醛灌注固定后,石蜡包埋切片,采用免疫组织化学方法对NPR-A在角膜和晶状体中的表达进行免疫荧光检测。结果　在E16,NPR-A高表达于角膜上皮细胞中,并持续至成年;其在角膜基质层和内皮细胞的表达也始于E16,而在P14之后,NPR-A表达随鼠龄的增加逐渐减弱。此外,NPR-A在P0小鼠晶状体上皮细胞膜内被检测到,并持续高表达至成年。在E16,由晶状体后壁上皮细胞生成的初级纤维开始高表达NPR-A,但随着鼠龄的增长和纤维结构的改变而逐渐减弱,直到P90消失。结论　NPR-A可能参与了角膜和晶状体生长和发育,并且对维持角膜上皮细胞的增生和修复以及晶状体的通透性具有重要作用。     

Biochemical evidence for conversion to milder form of hereditary mouse cataract by different genetic background

Congenic hereditary cataract mice, BALB/c-nct/nct, were established by introducing the nct gene from Nakano into BALB/c mice. These mice developed a milder cortical form of cataract which developed sporadically and later in life than in Nakano mice. Combined use of BALB/c and BALB/c-nct/nct mice enables biochemical comparison of normal clear lenses, congenic clear lenses which are destined to be opacified some time later, and opacified lenses in the same genetic and aging statuses. We compared the age-related changes in water content and water-soluble and -insoluble fractions among these three types of lenses. Congenic clear lenses and opaque lenses were more similar to BALB/c normal clear lenses and Nakano opaque ones, respectively, in these parameters. These results suggest, in addition to formation of aggregated crystallins and their accumulation in water-insoluble fractions, that decreased protein synthesis, increased protein degradation and augmented leakage of crystallin might have a significant role in the nct-induced lens opacification.     

Changes in lens proteins induced at the early stage of cataractogenesis in cac (Nakano) mice

Changes in lens proteins induced at the early stages of cataractogenesis in cac (Nakano) mice were investigated by two-dimensional gel electrophoresis (2D-PAGE). The 2D-PAGE profile of lens proteins in 21-day-old cac mice differed from that in 27-day-old normal mice, even though the appearance of 'pin-head' nuclear opacity (26-day-old) had not yet been observed in the lenses. Especially noticeable were great differences in the polypeptides associated with the alpha- and beta- crystallin subfractions, the appearances of which corresponded to an increase in a ratio of the amounts of Na+ to that of K+ in the lenses of defective mice. No dramatic decrease in the gamma-crystallin fraction was observed until the mature cataract stage.     

Retarded and distinct progress of lens opacification in congenic hereditary cataract mice, Balb/c-nct/nct

The Nakano cataract gene, nct, was introduced into Balb/c mice by repeated backcrosses to elucidate the possible effects of background genes on its expression. The resulting congenic Balb/c-nct/nct mice were characterized by retarded and sporadic cataract formation with a tendency of further retardation in males and by the different disease process of cataract as compared with Nakano mice. The age of 50% cataract incidence was 60 days in females and 90 days in males compared with 22 days in Nakano mice, and lens opacification commenced in a diffuse, mild form at the cortex in congenic but in a pin-head, intense form at the core in Nakano mice. Sex hormones seemed to be involved in the difference in cateractogenesis between male and female mice. Microphthalmia was slighter in degree in Balb/c background mice. The results indicated that the nct-dependent cataractogenesis may be influenced by background genes and some non-hereditary factors. Balb/c-nct/nct mice will provide a new type of hereditary cataract model.     

Three distinct stages of lens opacification in transgenic mice expressing the HIV-1 protease

Scanning electron microscopy of the lenses from transgenic mice (TG(72)) containing the HIV-1 protease linked to the lens alphaA-crystallin promoter showed structural changes around postnatal day 16. Frank opacification of the lens was observed at day 24. To relate the biochemical and biophysical changes that occur during the process of cataract development, high-resolution two-dimensional gel electrophoresis (2D), quantitative image analysis and ion measurements were carried out on lenses from postnatal day 10 and on days 15-24. The phase separation temperature (Tc), a measure of molecular interactions between proteins, was also determined for normal and transgenic lenses. A comparison of the transgenic and normal lenses on day 10 revealed no significant differences in any of the measured parameters. However, starting around day 16 or the first stage of observed structural changes, the TG(72)crystallin profiles of the alphaA- alphaB-, betaA3-, betaA4-, betaB3 and one gamma-crystallin began to deviate from the normal. By postnatal day 20, a second stage was initiated with an influx of calcium and sodium ions that was accompanied by modifications of betaB1- and betaB2-crystallin. In the third and final stage of the cataract process, a large increase in the proteolysis of crystallins was accompanied by the appearance of the frank cataract on day 24. The Tc initially increased in all of the mouse lenses until just prior to eyelid opening. After that time, the Tc decreased in all lenses. Although the Tc continued to decrease in the normal lenses with age, for the homozygous transgenic mice it exhibited a dramatic increase that began on day 20. Thus, in the TG(72)transgenic mouse, cataract formation occurs in a three-stage process. Tc and other biophysical parameters previously measured appeared to be insensitive to the modifications that occur during stage 1. However, during the second stage of cataract formation, there was a correspondence between abnormal Tc and the abnormal interactions between cellular constituents apparently resulting from lens hydration, the loss of ion homeostasis and continued proteolysis. The last stage of cataract formation results in a total loss of lens transparency and leakage of lens proteins.