Repopulation of different layers of host human Bruch's membrane by retinal pigment epithelial cell grafts.
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PURPOSE: To determine the morphology of human retinal pigment epithelium (RPE) after reattachment to different ultrastructural layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from eyes of 23 human donors (age range, 11-89 years). The basal lamina of the RPE, inner collagenous layer, and elastin layer were removed sequentially by mechanical and enzymatic techniques. First-passage cells of human RPE (15,000 cells/6 mm explant) from three donors (ages, 52, 64, and 80 years) were plated onto different layers of human BM, and the explants were examined by scanning and transmission electron microscopy up to 21 days later. RESULTS: RPE flattened and extended footplates 6 hours after plating onto basal lamina. Cells remained round 6 and 24 hours after plating onto the inner collagenous, elastin, or outer collagenous layer. The RPE cells became confluent 14 days after plating onto basal lamina but did not become confluent up to 21 days after plating onto the inner collagenous or elastin layer. Sparse round cells were observed 21 days after plating onto deeper layers, suggesting extensive loss of RPE. CONCLUSIONS: The morphology and subsequent behavior of the RPE reattached to BM depends on the anatomic layer of BM available for cell reattachment. The results suggest that the ability of transplanted RPE to repopulate BM in age-related macular degeneration and other disorders may depend on the layer of BM available to serve as a substrate for cell reattachment. (+info)
Glycosaminoglycans differentially bind HARP and modulate its biological activity.
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Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a family of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellular matrix defined as extracellular compartments. Using Western blot analysis, we detected HARP bound to heparan sulfate proteoglycans in the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment. Binding analyses with a biosensor showed that HARP bound heparin with fast association and dissociation kinetics (kass = 1.6 x 10(6) M-1 s-1; kdiss = 0.02 s-1), yielding a Kd value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (kass = 0.68 x 10(6) M-1 s-1) and a lower affinity (Kd = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP. (+info)
Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition.
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The chondroitin ABC lyase digestion products of normal human femoral condyle articular cartilage and of purified aggrecan were analyzed for their mono- and nonsulfated disaccharide composition. Changes in the total tissue chemistry were most pronounced during the period from birth to 20 years of age, when the -[GlcAbeta,3GalNAc6]- disaccharide content increased from approximately 50% to 85% of the total disaccharide content and there was a concomitant decrease in the content of the 4-sulfated disaccharide. In general, the disaccharide content of the deeper layers of immature cartilage were richer in the 4-sulfated residue than the upper regions of the tissue. As the tissue aged and decreased in thickness, the disaccharide composition became more evenly 6-sulfated. The newly synthesized chondroitin sulfate chains had a similar composition to the endogenous chains and also underwent the same age and zonal changes. The monoclonal antisera 3B3(+) and 2B6(+) were used to immunolocalize the unsaturated 6- and 4-sulfated residues generated at the reducing termini of the chondroitin sulfate chains by digestion with chondroitin ABC lyase, and these analyses indicated that the sulfation pattern at this position did not necessarily reflect the internal disaccharide composition of the chains. In summary, the sulfation pattern of chondroitin sulfate disaccharides from human normal articular cartilage varies with the age of the specimen, the position (topography) on the joint surface, and the zone of cartilage analyzed. Furthermore, these changes in composition are a consequence of both extracellular, post-translational processing of the core protein of aggrecan and changes in the sulfotransferase activity of the chondrocyte. (+info)
Molecular polymorphism of the syndecans. Identification of a hypo-glycanated murine syndecan-1 splice variant.
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We have identified a cDNA that encodes a variant form of murine syndecan-1. The variant cDNA lacks the sequence corresponding to the first 132 nucleotides of the third exon of the syndecan-1 gene. The corresponding message is rare. The alternative splice respects the reading frame and deletes 44 amino acids from the protein, joining the S45GS47GT sequence to a variant immediate downstream context. This sequence context initiates with alanine instead of glycine as residue 50, reducing the number of SGXG sequence motifs in the protein from two to one. Expression of this variant syndecan-1 in Madin-Darby canine kidney or MOLT-4 cells yielded a recombinant proteoglycan with a reduced number and clustering of the heparan sulfate chains. Both the conversions of Ala50 and of Lys53 into glycine enhanced the heparan sulfate substitution of the variant protein. These findings support the concept that serine-glycine dipeptide signals for glycosaminoglycan/heparan sulfate synthesis depend on sequence context (Zhang, L., David, G., and Esko, J. D. (1995) J. Biol. Chem. 270, 27127-27135) and imply that alternative splicing mechanisms may in part control the molecular polymorphism of syndecan-1 and, therefore, the efficiency and versatility of this protein in its co-receptor functions. (+info)
Quantitative alterations of hyaluronan and dermatan sulfate in the hairless mouse dorsal skin exposed to chronic UV irradiation.
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The quantitative alterations of hyaluronan and dermatan sulfate in the upper dermis (fibrous tissue) and the lower dermis (adipose tissue) of the hairless mouse skin chronically exposed to the UV irradiation as solar-simulating irradiation (lambda(max) 352 nm, UV distribution: 300-310 nm, 0.9%; 310-320 nm, 2.0%; 320-420 nm, 97.1%) were evaluated. Hyaluronan and dermatan sulfate contents in each part of dermis were determined as follows: skin sections on a glass slide prepared by histological technique were processed into the upper dermis and the lower dermis with a small surgical knife, and treated with chondroitinase ABC and ACII in the presence of bacterial collagenase. The resulting unsaturated disaccharides were determined by HPLC method. By applying this method to the UV-irradiated hairless mouse skin, it was found that the chronic UV irradiation increased dermatan sulfate in the upper dermis, whereas an increase of hyaluronan content was not statistically significant. In the lower dermis, on the contrary, both hyaluronan and dermatan sulfate contents remarkably increased as compared with the control mice. Furthermore, the histological study showed the accumulation of the collagen fibers in the lower dermis of the UV-irradiated hairless mouse skin following the disappearance of adipocytes. These findings indicate that the increases of glycosaminoglycan contents in the UV-irradiated skin are related to the accumulation of the extracellular matrix components in the lower dermis. (+info)
Molecular characterization of a novel basement membrane-associated proteoglycan, leprecan.
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A monoclonal antibody was used in early studies to identify a novel chondroitin sulfate proteoglycan, secreted by L-2 cells, the core protein of which was approximately 100 kDa. To characterize this proteoglycan core protein at the molecular level, an L-2 cell cDNA library was probed by expression screening and solution hybridization. Northern blot analysis assigned transcript size to approximately 3.1 kilobases and, after contig assembly, the coding region of the mRNA corresponded to 2.18 kilobases. Immunoassays were performed to confirm the identity of this sequence, using a polyclonal antibody raised against an expressed fusion protein encoded by sequence representing the carboxyl half of the molecule. The antibody recognized the core protein in Western blots after prior digestion of the intact proteoglycan with chondroitinase ABC. Immunostaining tissue sections with the same antibody localized the proteoglycan to basement membranes, and expression of the entire sequence in Chinese hamster ovary K-1 cells showed that the protein encoded by the sequence secreted as a chondroitin sulfate proteoglycan. The core protein not only has motifs permitting glycosylation as a proteoglycan, but also possesses the endoplasmic reticulum retrieval signal, KDEL, which suggests that, in addition to its role as a basement membrane component, it may also participate in the secretory pathway of cells. (+info)
Effects of hyaluronan lyase, hyaluronidase, and chondroitin ABC lyase on mammalian vitreous gel.
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PURPOSE: To determine the effects of enzymes on mammalian vitreous gel and to thus infer the structural roles of hyaluronan and chondroitin sulfate in the gel. METHODS: The wet weights of bovine vitreous gels were compared before and after incubation with Streptomyces hyaluronan lyase, chondroitin ABC lyase, testicular hyaluronidase, or buffer alone. The extent of hyaluronan depolymerization was determined by chromatography and that of chondroitin sulfate depolymerization by western blot analysis. RESULTS: After digestion with Streptomyces hyaluronan lyase (30 U/gel), the gel wet weight was the same as that of controls (incubated with buffer alone) despite 94% of the hyaluronan having been depolymerized; when digested with 100 U/gel, the gel wet weight decreased (to 57% of original wet weight versus 86% for controls, P = < 0.001) and hyaluronan was completely depolymerized. Chondroitin ABC lyase digestion (0.2 U/gel) resulted in a slight reduction in gel wet weight (90% versus 96%, P = < 0.001) and depolymerization of 88% of the hyaluronan; the presence of fully digested chondroitin sulfate chains was established. Digestions with 100 and 500 U/gel of testicular hyaluronidase resulted in a decrease (P = < 0.001, both cases) in gel wet weight (53% versus 82%, 100 U/gel; 57%, versus 86%, 500 U/gel) with 75% and 97% hyaluronan depolymerization, respectively. CONCLUSIONS: Depolymerization of all vitreous hyaluronan and of chondroitin sulfate resulted in gel wet weight reduction but not gel destruction. Digestion with 30 U/gel of Streptomyces hyaluronan lyase revealed a small pool (6%) of relatively enzyme-resistant hyaluronan that specifically contributed toward maintaining gel wet weight. (+info)
Identification of a nervous tissue-specific chondroitin sulfate proteoglycan, neurocan, in developing rat retina.
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PURPOSE: To identify the expression of neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan, in retina and to elucidate its changes during development. METHODS: Expressional changes of neurocan mRNAs in developing rat retinas were investigated by a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The localization and characterization of neurocan core proteins were also investigated with the use of Western blot analysis and immunohistochemistry. RESULTS: Gene expression of neurocan was identified in retinas by RT-PCR. Semiquantitative analysis using Southern blot analysis revealed that mRNA expression for neurocan increased at increasing postnatal stages and that it reached its peak around postnatal day 7 (P7). Immunohistochemical studies demonstrated that in differentiating rat retinal (neuroblast) cells weak neurocan immunoreactivities were observed throughout the retina on embryonal days 14 (E14) and E16. During the early postnatal period, the immunoreactivities became most conspicuous in the inner and outer plexiform layers on P7 through P14. In adult retinas, only faint immunostaining was detected. Immunoblot analysis showed two positive bands of 220- and 150-kDa core glycoproteins after treatment with chondroitinase ABC. Further immunoblot analysis revealed that the expression of these two immunolabeled variants was regulated differently during retinal development. CONCLUSIONS: The temporal and spatial regulation of expression of neurocan and its proteolytic variant during retinal development suggest that it may play a role in differentiation and neural network formation. (+info)