Short-chain basement membrane collagen. Further characterization and its biosynthesis by F-9 embryonal carcinoma cells.

The paper describes further characterization of the 55-kDa short-chain collagen from lens capsule. Lens capsules were extracted with 5.5 M guanidine.HCl and the extracted material was fractionated on agarose A-5M followed by high-pressure liquid chromatography (HPLC). By amino acid composition, the major fraction obtained from HPLC was found to be different than type-IV collagen fragments. The 55-kDa short-chain collagen on pepsin digestion produced a 45-kDa pepsin-resistant fragment. The undifferentiated embryonal carcinoma (F-9) cells were found to synthesize increased amounts of 55-kDa short-chain collagen. The identity of this biosynthesized molecule with 55-kDa short-chain collagen from lens capsules was established by immunoprecipitation experiments. The results indicated a close similarity or identical nature of the short-chain collagens from these two sources.

Bound water content of bovine lens capsules.

The anterior parts of bovine lens capsules have been incubated in media with different glucose contents. The freezable water content of the lens capsule was obtained by differential scanning calorimetry and the total water content by thermogravimetric analysis. The bound (nonfreezable) water content was calculated as the difference between total and freezable water. The bound water as percent of the total water increases with the degree of glycosylation of the bovine lens capsules.

Bendazac lysine in selected types of human senile cataract. A long-term double-masked placebo-controlled clinical trial with multilinear densitometric image analysis of Scheimpflug photographs.

A double-masked placebo-controlled clinical trial with hard data evaluation by image analysis of Scheimpflug photographs taken at baseline and 6, 12 and 18 months after starting treatment was performed to assess the efficacy of bendazac lysine in four different types of senile cataract. The study had a classical split-plot design. For statistical evaluation, the analysis of variance and covariance for repeated measures were used for three different lens sections: anterior capsule and superficial layer, anterior cortex and nucleus. In the entire group of 53 evaluable patients (without separation into cataract-type subgroups), there was a significantly less increase over time in light scattering (i.e. film blackening) of the anterior cortex and nucleus with bendazac lysine than with placebo. There was also a strong trend in favour of the active drug at the anterior capsular level. Patients with water clefts and spokes showed a significantly less light scattering of the anterior capsule and cortex when treated with bendazac lysine. Those with nuclear changes also showed significantly less light scattering of the anterior cortex and nuclear region with the active drug than with placebo. The number of patients with subcapsular and wedge-shaped (cuneiform) cataracts was too small to be adequately assessed by statistical procedures. Nevertheless, there were indications of a beneficial effect of bendazac lysine on all the lens sections in patients with subcapsular cataracts and on the anterior cortical region in those with wedge-shaped cataracts. In conclusion, this study showed that the increase in light scattering over time, i.e. the progression of cataract, is less in bendazac lysine-treated patients than in those treated with placebo.

Isolation and characterization of retinal and choroidal biomatrix.

Retinal and choroidal biomatrices (extracellular materials) and lens capsule were isolated from bovine eyes. Water-insoluble biomatrices were collected by filtration after repeated ultrasonic disruption of the cells and treatment with EDTA. Light and electron microscopic examination showed that the isolated lens capsule, retinal and choroidal biomatrices were free of cellular components. These biomatrices were then solubilized after pulverization in liquid nitrogen and applied to SDS-polyacrylamide gel electrophoresis. Electrophoretic patterns of the biomatrices were quit different from one another, while immunoblotting demonstrated the presence of laminin as a common constituent.

Association of elastin with pseudoexfoliative material: an immunoelectron microscopic study.

Using immunoelectron microscopy, the presence of elastin and tropoelastin was demonstrated in pseudoexfoliative (PSX) material in all its classical sites on the lens capsule, ciliary non-pigment epithelium, iris epithelium and stroma, and conjunctiva. Some variability in binding affinity was seen in different sites, and labelling was more often on the periphery than the center of the PSX fibers. The elastin epitope on PSX material was more sensitive to processing than the remarkably stable epitope on mature elastic fibers. Since neither elastin nor a related component of PSX fibers, elastic microfibrillar protein, is a circulating protein, both are likely to be secreted by local ocular cells. Most of these local cells are not involved in elastogenesis normally, suggesting that an abnormal stimulus or defective regulation of matrix synthesis exists in this disease.

Synthesis of lens capsule and plasma membrane glycoproteins by lens epithelial cells and fibers in the rat.

The lens of the eye possesses a capsule which is a greatly hypertrophied basement membrane. To investigate the synthesis of glycoproteins destined for this capsule, 3H-fucose was injected into the vitreous body of intact rats weighing approximately 200 gm. The animals were killed from 10 min to 14.5 months later, and their lenses were processed for electron microscope radioautography. At 10 min after injection, more than 58% of the silver grains were localized to the Golgi apparatus of the lens epithelial cells. By day 1, the heaviest sites of reaction were the plasma membrane (more than 50% of total label), the basal cytoplasm, and the adjacent lens capsule, where a heavy band of reaction was seen. The remainder of the capsule exhibited a lighter diffuse reaction. In the lens fibers, the label was at first localized to clusters of vesicles but then migrated to the plasma membrane and to the region of the capsule adjacent to the basal surface of these fibers. Light microscope radioautographs of the lens capsule at later time intervals revealed that by 1 month after injection the diffuse reaction had disappeared, and only the strongly labeled band remained. By 14.5 months after injection, this band had migrated partially across the lens capsule, but the capsule itself had increased considerably in thickness. On the other hand, the distance between the labeled band and the free edge of the capsule had decreased from that seen at the time of injection.

Nonenzymatic glycation of basement membranes from human glomeruli and bovine sources. Effect of diabetes and age.

The nonenzymatic glycation of glomerular basement membranes (GBMs) from 14 diabetic and 19 nondiabetic human subjects was determined after boronic acid affinity and high-performance cation-exchange chromatography of their NaB[3H]4-reduced ketoamine adducts. The glucitol-lysine (Glc-Lys) and the glucitol-hydroxylysine (Glc-Hyl) content of diabetic GBM was found to be about twofold higher than that of nondiabetic samples (P less than .001). The content of these glycated amino acids did not correlate with age over the range examined (20-91 yr) or with the length of disease in diabetic subjects (2-16 yr). However, analyses of Glc-Lys and Glc-Hyl in calf and adult bovine GBM and lens capsules indicated that the levels of these glycated amino acids were several times greater in basement membranes from older animals. We also observed that guanidine-insoluble collagen of bovine GBM is more extensively glycated (approximately 4-fold) than primarily noncollagenous proteins that are extracted by this reagent. In all of the basement membranes examined, the percentage of glycation of lysine was greater than of hydroxylysine. Characterization of the components released by alkaline hydrolysis indicated that O-glycosylated hydroxylysine residues are nonenzymatically N-glycated to the same extent as those without an enzymatically attached carbohydrate unit. Our study indicates that more than a hundred times as many hydroxylysine residues are enzymatically glycosylated in human and bovine GBM as those containing the nonenzymatically formed ketoamine adduct.

Immunochemical analysis of extracellular matrix during embryonic lens development of the Cat Fraser mouse.

We have characterized the extracellular matrix present during early mouse-lens morphogenesis in Swiss and Cat Fraser mutant mice which produces a thicker capsule. In the two mouse strains, laminin was first detected when the optic vesicle and the head ectoderm are closely associated. At day 10, staining for laminin and fibronectin is especially concentrated at the border of the lens pit. At this stage, type IV collagen and proteoheparan sulphate have a similar distribution to laminin and fibronectin. In the two mouse strains, no major differences were observed in the intensity and the distribution of fluorescent basement-membrane components. This suggests that the overall increase in capsule thickness of the Cat Fraser mutant is more related to an increased cellular synthesis of capsule than to an abnormal distribution of one or more basement-membrane macromolecules.

Characterization of 26K globular domain of a new basement membrane collagen.

In continuing our earlier studies (Biochem. Biophys. Res. Comm. 130, 1-8, 1985) on a new collagenous component (Type XIII) from lens capsule basement membrane, we report here the isolation and characterization of a 26K protein from the 4.5 M guanidine-HCl extracts of lens capsules. The 26K protein was purified by molecular sieve and DEAE-cellulose chromatography. The protein has been characterized by amino acid composition, NaDodSO4-polyacrylamide gel electrophoresis and by immunochemical analyses. On rotary shadowing, the 26K protein appears as a globule. The available information suggests its location at the terminal end of the collagenous component. The presented data show clearly that the 26K protein is a distinct new protein, different from the earlier reported NC-1 domain of type IV collagen.

Basement membrane collagen--evidence for a novel molecular packing.

Type IV collagen is the major structural protein of basement membranes but very little is known about its molecular organisation in vivo. We have used X-ray diffraction of a thick basement membrane, bovine lens capsule, to provide information. Under constant load, lens capsule gave a collagen diffraction pattern of a similar quality to unstretched rat rail tendon. In addition there were clear meridional reflections which indexed as orders of 10 nm, and equatorial reflections at 2.1 and 3.8 nm. These results suggest the ordering of type IV collagen molecules in fibrils, with a 10 nm periodicity along the length of the fibrils.

Collagens of the retinal microvascular basement membrane and of retinal microvascular cells in vitro.

We have analysed the collagens present in vascular basement membranes isolated from bovine retinal and cerebral microvessels and bovine renal glomeruli, and from the non-vascular basement membrane of bovine lens capsule. These are compared with the collagens produced by cultured bovine retinal microvascular pericytes and lens epithelial cells, and by canine retinal microvascular endothelial cells, in vitro. Biochemical and immunocytochemical analyses indicate that all of the vascular basement membrane preparations have an identical collagenous composition, consisting of the same polypeptides present in lens capsule (primarily type IV collagen), together with other polypeptides that are identified as type I, and a small amount of type III collagen. Identification of the latter is based on two-dimensional gel electrophoresis in the presence and absence of a reducing agent. Immunocytochemical studies, however, demonstrate type I, type IV and some type V collagen in the basement membranes of the isolated microvessels. The cultured microvascular cells produce predominantly type I collagen molecules, but they also produce other collagen peptides that appear to be type IV, and, at least in some experiments, small amounts of type III collagen. The biochemical identification of collagens type I and IV is confirmed by immunocytochemistry. However, results with anti-type I collagen and procollagen antibodies in cultured pericytes vary with antibodies from different sources. The quantities of the type IV peptides produced by the cultured cells also vary in different experiments.

Macromolecular organization of bovine lens capsule.

Rabbit antisera to type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin were used to localize these proteins in cross-sections of bovine anterior lens capsule. The antisera were exposed to (a) 10-micron frozen-thawed sections of formaldehyde-fixed tissue for examination in the light microscope by the indirect immunofluorescence method and (b) formaldehyde-fixed and L. R. White plastic-embedded thin sections for electron microscopic examination by the protein A-gold technique. The intensity of immunofluorescence was both uniform and strong throughout for type IV collagen, laminin and entactin, but patchy and weak for fibronectin. Electron microscopic immunolabeling with protein A-gold showed that all five components were distributed throughout the full thickness of the membrane, albeit the density of gold particles was not identical for all basement membrane proteins. In general, the number of particles per micron2 was greatest for type IV collagen and entactin, moderate for laminin and heparan sulfate proteoglycan and low for fibronectin. The ultrastructure of the lens capsule as examined by the electron microscope revealed a relatively uniform parallel alignment of filaments, thought to be collagenous. Since the distribution of the filaments corresponds well with the observed immunocytochemical pattern it is concluded that type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin co-localize throughout the cross-section of the anterior lens capsule.

Pseudoexfoliative material contains an elastic microfibrillar-associated glycoprotein.

Because of histochemical similarities, there is reason for thinking that some component or components of PSX material are also present in zonular fibers. Since the zonule is a member of the elastic microfibrillar system, PSX material on the lens capsule was tested for immunologic affinity to elastic MFP from three widely divergent sources, using an indirect immunoperoxidase electron microscopic method. Positive staining was obtained with all three antibodies on all components of lens PSX material, including the superficial aggregates, deep fibrogranular zone, and capsular inclusions. The results support our hypothesis that PSX material derives from abnormal polymerization of glycoprotein associated with the zonular-elastic microfibrillar system. Similar staining of the abnormal material within the lens capsule indicates that the lens epithelial cell is involved in processing this protein. It might be suspected that other microfibrillar-secreting cells, even beyond the present range of suspected sources, could produce similar material.

Identification of noncollagenous components of calf lens capsule: evaluation of their adhesion-promoting activity.

Extraction of calf anterior and posterior lens capsules with 5 M guanidine HCI resulted in the solubilization of protein (12% of total) with a noncollagenous amino acid composition leaving behind the collagen matrix. Polyacrylamide gel electrophoresis of the solubilized material revealed a number of components, all of which were susceptible to trypsin but resistant to collagenase digestion. Fractionation of the extracted proteins by Sepharose CL-6B filtration as well as by affinity chromatography was undertaken, and laminin, fibronectin, entactin, and beta-crystallin were identified by electrophoresis and solid-phase radioimmunoassays in both anterior and posterior capsules. An entactin (Mr = 150,000), which constituted the most prominent component on electrophoresis, was purified after Sepharose CL-6B filtration by a two-step lectin affinity chromatography procedure, which was based on the failure of this protein to bind to Bandeiraea simplicifolia I but its positive reactivity with wheat germ lectin. Neither the mixture of proteins extracted from lens capsules by guanidine nor fractions prepared therefrom were able to enhance lens epithelial cell attachment to type I or type IV collagen-coated surfaces or to guanidine-prepared lens capsules; adhesion-stimulating activity could not be demonstrated even when cycloheximide-treated cells were employed. Furthermore, the cells were observed to attach as effectively to guanidine-extracted as to native capsules. These observations indicate that noncollagenous proteins are not essential for the in vitro attachment of epithelial cells to lens capsule; it appears that the collagen component itself provides an optimal surface for cell-basement membrane interaction.

Inhibition of collagenase activity by extracts of bovine ocular tissues.

Bovine eyes were dissected and separate pools of lens, lens capsule, cornea and vitreous were extracted in guanidine, subjected to ultrafiltration, and examined for their effects on collagenolytic activity. Although lens extract was not inhibitory, the cornea and vitreous both contained inhibitors of collagenase. More inhibition was present in the filtrate of the vitreous than in the retentate, whereas the total amount of inhibition in the cornea was distributed almost equally between the two fractions. The inhibition observed was dose dependent. The partially purified inhibitors from cornea and vitreous blocked the activity of human skin and tadpole back skin collagenases, but they failed to inhibit the bacterial (Clostridium histolyticum) collagenase. The inhibitor was stable to heating to 60 degrees for 30 minutes and to trypsinization.

Growth and differentiation of human keratinocytes cultured on eye lens capsules.

The efficiency of the outgrowth of human epidermal and hair-follicle-sheath keratinocytes was studied using three different growth substrates: plastic, type-I collagen and bovine eye lens capsules (the Epicult system). It was shown that the eye lens capsule is the best substrate, since a higher percentage of cultures showed outgrowth, and the outgrowth of epidermal keratinocytes was much more rapid. This effect is related to the faster migration (not proliferation) of cells grown on lens capsules as compared to the two other substrates. The view that lens capsules can replace the basement membrane present in vivo was supported by the finding that two basement-membrane components, i.e., laminin and fibronectin, are present on lens capsules. It was shown that, in cultures grown on lens capsules, bullous-pemphigoid antigen is restricted to the basal layer, indicating that the differentiation of these cells is comparable to that of keratinocytes grown on irradiated, non-viable pig dermis.

[Comparative study of bovine ocular lens proteins in native tissue and an extract using the x-ray diffraction method]. / Sravnitel'noe issledovanie belkov khrustalika glaza byka v nativnoi tkani i v ékstrakte metodom difraktsii rentgenovskikh luchei.

It has been shown that the maxima (Bragg-spacings 4,5-19 A) on the X-ray diffraction patterns of the bovine lens native tissues from nuclear and cortical parts are predominantly due to the water-soluble crystallin intramolecular structure. The structures of water-soluble and water-insoluble fractions from bovine lens nucleus and cortex were qualitatively compared. Reversible dependence of the lens water-soluble protein structure on water content in the system was demonstrated.