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Biomedical subjects

J R Hassell

Publications and source records attributed to J R Hassell.

At least 109 records · Page 6Linked to original sources
PubMed1983

Purification of keratan sulfate proteoglycan from monkey cornea.

An explant culture of 15 cynomolgus monkey corneas was incubated with [35S]sulfate and [2-3H]mannose as labeling precursors. A 4 M guanidine HCl extract of the corneal stromas was prepared and combined with a 4 M guanidine HCl extract of stromas from 300 unlabeled corneas. The keratan sulfate proteoglycans in the combined extracts were purified by a combination of DEAE-cellulose chromatography, chondroitinase ABC digestion to remove chondroitin-dermatan sulfate proteoglycans, and elution from immobilized concanavalin A. The purified keratan sulfate proteoglycan was digested with papain and the digest was eluted on DEAE-Sephacel. The unbound fraction contained 59% of the 3H activity and consisted of intact oligosaccharide-peptides. The bound fraction, consisting of keratan sulfate chains linked to peptides, eluted during a linear 0-0.75 M NaCl gradient as a peak centered at approximately 0.6 M NaCl and contained 41% of the 3H and all of the 35S activity in the original proteoglycan. The chains were digested with endo-beta-galactosidase, and the digest was eluted on DEAE-Sephacel with a linear 0-0.75 M NaCl gradient. Most of the sulfated digestion fragments from the chains eluted as several distinct peaks during the gradient. All the 3H activity eluted in the unbound volume along with a small proportion of 35S activity. This unbound fraction was eluted on Bio-Gel P-10 to give a 3H peak (Kav = 0.46) well resolved from the remaining 35S activity which eluted near the total volume. This 3H peak contained the oligosaccharide-peptides derived from the linkage region between the keratan sulfate chains and the core protein. Structural analyses of the linkage region oligosaccharides and the intact oligosaccharides (Nilsson, B., Nakazawa, K., Hassell, J.R., Newsome, D.A., and Hascall, V.C. (1983) J. Biol. Chem. 258, 6056-6063) in combination with the 3H-labeling data suggest that the intact keratan sulfate proteoglycans contain an average of about one intact oligosaccharide per keratan sulfate linkage site.

Animals↗
PubMed1983

Structure of oligosaccharides and the linkage region between keratan sulfate and the core protein on proteoglycans from monkey cornea.

Structural analyses were performed on the intact glycopeptides and on the linkage region oligosaccharide-peptides derived from the keratan sulfate proteoglycan from monkey cornea (Nakazawa, K., Newsome, D.A., Nilsson, B., Hascall, V.C., and Hassell, J.R. (1983) J. Biol. Chem. 258, 6051-6055) using trifluoroacetolysis, Smith degradation, chromium trioxide oxidation, and gas-liquid chromatography-mass spectrometry. The following structure was found for the linkage region (formula; see text) The following structures were found for the intact oligosaccharide peptides (formula; see text) and (formula; see text) The structure of the linkage region for keratan sulfate on corneal proteoglycans is clearly derived from a complex type of N-linked glycoprotein oligosaccharide precursor, indicating that only the oligosaccharides that have been processed to the complex type are used as primers for synthesizing keratan sulfate chains. The high mannose oligosaccharide in Formula 3 is an intermediate in the normal pathway for biosynthesis of complex type oligosaccharides. The structure in Formula 2, in which a single Man alpha 1-2 is retained on the Man alpha 1-3 branch while the Man alpha 1-6 branch is unsubstituted, can be an intermediate for an alternate, presumably minor pathway for complex oligosaccharide formation (Kornfeld, S., Gregory, W., and Chapman, A. (1979) J. Biol. Chem. 254, 11649-11654) in certain cases. This structure has not previously been shown to be present on normal glycoproteins.

Animals↗
PubMed1983

Interaction of link protein with collagen.

Link protein (Mr = 42,000) is an integral component of cartilage as well as of some noncartilagenous tissues. In cartilage, it forms a macromolecular complex with cartilage proteoglycan and hyaluronic acid, but its function in other tissues is unknown. We provide evidence here that the link protein of cartilage binds well to native collagen types I and III. The binding occurs only if both link protein and collagen are native. The binding of link protein to collagen type fibrils is higher than to monomeric collagen. Link protein binding to collagen fibrils is saturable and occurs at molar ratio of collagen to link protein of 7-13:1. These data suggest that the link protein binds to collagen and that the binding requires the collagen to be in its native triple helical structure. This interaction may play a role in collagen fibril formation.

Animals↗
PubMed1983

Proteoglycan changes during restoration of transparency in corneal scars.

Corneal scars generated in rabbits by penetrating wounds are initially opaque but become transparent within a year. Previous studies have shown that the corneal stroma consists of proteoglycans and collagen fibrils spaced at regular intervals and that the interfibrillar spaces, the presumed location of proteoglycans, are abnormally large in opaque scars. In the present study, the size and glycosaminoglycan composition of the corneal stromal proteoglycans were determined in corneal scars during the restoration of transparency. The results showed that initially opaque scars which contained the large interfibrillar spaces also contained unusually large chondroitin sulfate proteoglycans with glycosaminoglycan side chains of normal size. These opaque scars also lacked the keratan sulfate proteoglycan but did contain hyaluronic acid. In the 1-year-old scars there was a restoration of normal interfibrillar spacing, and a return to corneal stromal proteoglycans of normal size and composition. These correlations suggest that the corneal stromal proteoglycans may play a fundamental role in regulating corneal collagen fibril spacing.

Animals↗
PubMed1983

Heterogeneity of proteoglycans in monkey corneal stroma.

The proteoglycans of the cynomolgus monkey corneal stroma were isolated and characterized by using a combination of physiochemical and biochemical methods. Proteoglycans were biosynthetically radiolabeled by incubating whole corneas in medium containing [35S]sulfate and either [3H]serine or [3H]mannose as precursors. Macromolecules were extracted from the corneal stromas with 4 M guanidine-HCl. After dialysis into 8 M urea, proteoglycans in the extracts were initially purified by DEAE-cellulose chromatography. A portion of the proteoglycan fraction was digested with chondroitinase ABC, and the keratan sulfate proteoglycans were then isolated by rechromatography of the digest on DEAE-cellulose. Another portion of the proteoglycan fraction was digested with endo-beta-galactosidase and the dermatan sulfate-proteoglycans were then isolated by chromatography of the digest on Sepharose CL-4B. Each proteoglycan population was further fractionated by chromatography on concanavalin A-Sepharose and by CsCl density gradient centrifugation. Four subpopulations for both the keratan sulfate proteoglycans and the dermatan sulfate proteoglycans were isolated. Based on differences in binding to concanavalin A-Sepharose, buoyant densities, and glycosaminoglycan content, subpopulations of each proteoglycan differ by the number and properties of both the glycosaminoglycan chains and the mannose-containing oligosaccharides attached to their protein core.

Animals↗
PubMed1983

Loss of heparan sulfate proteoglycan from glomerular basement membrane of nephrotic rats.

Three basement membrane components, heparan sulfate proteoglycan, laminin, and type IV collagen, were localized in renal glomeruli using light and electron microscopic immunohistochemistry in normal and aminonucleoside-treated nephrotic rats. In normal rats, type IV collagen was found in both laminae rarae and in lamina densa; laminin and heparan sulfate were found in the three layers of the basement membrane with more intense staining in both laminae rarae. In nephrotic rats laminin and type IV collagen had the same localization and distribution as in normal rats. This was in contrast to heparan sulfate proteoglycan which was lost to a considerable extent in nephrotic animals. These results support the concept that heparan sulfate proteoglycan is a major charge determinant in normal glomerular basement membrane and suggest a crucial role for this proteoglycan in the pathogenesis of the nephrotic syndrome.

Animals↗
PubMed1982

Biochemical and histological analysis of "recurrent' macular corneal dystrophy.

A patient with macular corneal dystrophy who had a successful 6-mm corneal transplant 23 years ago underwent a second keratoplasty for marked irregular astigmatism. The excised button, which contained the original graft and a rim of host cornea, was divided into several portions. One portion was examined histologically, another portion was incubated in organ culture with radioactive precursors and the biosynthetically labeled products characterized, and a third portion was used for cell culture and karotype analysis. The results indicated that host stromacytes had not invaded the graft and that graft stromacytes had synthesized normal proteoglycans. Furthermore, although there was excessive synthesis of abnormal proteoglycan by host stromacytes and accumulation of this material in the host cornea, minor amounts of this material actually accumulated in the graft cornea, possible contributing to the astigmatism. A large transpant that would leave a minimum of host corneal tissue may be conductive to a longer-term good result in patients with macular corneal dystrophy.

Chromatography, Agarose↗
PubMed1982

Chondrogenesis: a model developmental system for measuring teratogenic potential of compounds.

A simple test for determining the teratogenic potential of compounds is described using embryonic limb bud cells in culture. These mesenchyme cells multiply and differentiate into chondrocytes during a 6-day culture period. The extent of chondrogenesis is assessed by staining for the cartilage specific proteoglycan with alcian blue. The amount of stain is then measured spectrophotometrically. Compounds which interfere with growth or differentiation reduce the amount of proteoglycan and as a consequence, reduce alcian blue staining. Compounds can be added directly to the media or be activated using several different metabolizing systems. The dose of a compound needed to reduce alcian blue staining by 50% is designated the teratogenic potential (TP50) of that compound. TP50's of proven teratogens compare favorably with in vivo teratogenic doses of the teratogens.

Animals↗
PubMed1982

Mammalian eyes and associated tissues contain molecules that are immunologically related to cartilage proteoglycan and link protein.

Monospecific antibodies to bovine nasal cartilage proteoglycan monomer and link protein were used to demonstrate that immunologically related molecules are present in the bovine eye and associated tissues. With immunofluorescence microscopy, reactions for both proteoglycan and link protein were observed in the sclera, the anterior uveal tract, and the endoneurium of the optic nerve of the central nervous system. Antibody to bovine nasal cartilage proteoglycan also reacted with some connective tissue sheaths of rectus muscle and the perineurium of the optic nerve of the central nervous system. Antibody to proteoglycan purified from rat brain cross-reacted with bovine nasal cartilage proteoglycan, indicating structural similarities between these proteoglycans. ELISA studies and crossed immunoelectrophoresis demonstrated that purified dermatan sulphate proteoglycans isolated from bovine sclera did not react with these antibodies but that the antibody to cartilage proteoglycan reacted with other molecules extracted from sclera. Two molecular species resembling bovine nasal link protein in size and reactivity with antibody were also demonstrated in scleral extracts: the larger molecule was more common. Antibody to link protein reacted with the media of arterial vessels demonstrating the localization of arterial link protein described earlier. Tissues that were unstained for either molecule included the connective tissue stroma of the iris, retina, vitreous body, cornea, and the remainder of the uveal tract. These observations clearly demonstrate that tissues other than cartilage contain molecules that are immunologically related to cartilage-derived proteoglycans and link proteins.

Animals↗
PubMed1982

Alterations in the basement membrane (heparan sulfate) proteoglycan in diabetic mice.

We have grown the EHS (Engelbreth-Holm, Swarm) tumor in normal and genetically diabetic mice (db/db) and measured some components of basement membrane produced in the tumor. These studies showed similar amounts of total protein in control and diabetic tissue and similar patterns of proteins on SDS gel electrophoresis of extracts of the tissue. Laminin, a basement membrane specific glycoprotein utilized as an attachment factor by epithelial cells, was present in increased amounts in diabetic tissue. In contrast, the amount of BM-1 (heparan sulfate) proteoglycan was reduced. Less 35S-sulfate was incorporated into this proteoglycan, and the proteoglycan, but not its component glycosaminoglycans, was heterogeneous in size. The data indicate that either the synthesis of proteoglycan was decreased or its degradation was increased in diabetic tissue. Since the heparan sulfate proteoglycan serves to block the passage of anionic macromolecules through the basement membrane, decreased levels could account for the increased porosity of diabetic basement membrane. Compensatory synthesis of the basement membrane components to restore normal permeability could account for the thickened basement membranes observed in diabetes.

Animals↗
PubMed1982

Human corneal stroma contains three distinct collagens.

Acetic acid-pepsin extracts of normal human corneal stromas were shown by carboxymethyl cellulose-column chromatography, sodium dodecyl sulfate-slab gel electrophoresis, highly purified collagenase, trypsin sensitivity, and cyanogen bromide peptide mapping to contain types I, III, and IV collagen, with type I by far the predominant species. This degree of collagen heterogeneity in normal human corneal stroma has not been reported previously and may be important in the understanding of wound healing and disease states.

Adolescent↗
PubMed1981

Identification of a basement membrane proteoglycan in exfoliation material.

We have used the indirect immunperoxidase technique to examine the exfoliation syndrome and can demonstrate that the fibrils so typically found in this disease certainly contain basement membrane proteoglycans. This finding is interesting for two reasons: 1. For the first time, an electron-microscopical technique is described that is able to identify one protein component of the exfoliation material. 2. The fact that the basement membrane proteoglycans are present in the exfoliation material supports the hypothesis that this disease is caused by a disturbance in the biosynthesis of the basement membrane.

Antigen-Antibody Reactions↗
PubMed1981

Detection of specific collagen types in normal and keratoconus corneas.

Keratoconus is a corneal disease of unknown cause that involves a progressive thinning and scarring of the corneal connective tissue. We examined normal human and keratoconus corneas, including one healed penetrating keratoplasty specimen. Organ cell cultures of normal and keratoconus corneal specimens were labeled with radioactive proline and analyzed by CM-cellulose chromatography and slab gel electrophoresis to determine collagen biosynthesis. Collagen types I and III were synthesized in similar amounts by normal and keratoconus stromacytes in culture. Specifically purified antibodies were used to determine the distribution of collagen types in tissue sections by immunofluorescence. The distribution of collagen types I, III, and IV in keratoconus was also similar to that in normal corneas, except that scarred regions in keratoconus and at the host-graft juncture were largely type III. Immunofluorescent reaction of the anti-type IV collagen antibodies with Bowman's layer, in particular, and Descemet's membrane in keratoconus specimens indicated extensive destruction. Basement membrane destruction may play an important role in this disease.

Adult↗
PubMed1980

Macular corneal dystrophy: failure to synthesize a mature keratan sulfate proteoglycan.

Corneal specimens obtained during surgery from patients with macular corneal dystrophy and obtained at autopsy from control eyes were incubated in a medium containing radioactive precursors of glycoproteins and proteoglycans. Biosynthetically radiolabeled material was extracted and characterized by using molecular sieve chromatography and specific enzymes. Cells in control corneas synthesized both a chondroitin sulfate proteoglycan and a keratan sulfate proteoglycan similar to those present in monkey and bovine corneas. Cells in macular corneas synthesized a normal chondroitin sulfate proteoglycan but did not synthesize either keratan sulfate or a mature keratan sulfate proteoglycan. Instead, macular corneas synthesized a glycoprotein with unusually large oligosaccharide side chains. This glycoprotein was not detected in normal corneas and is slightly smaller than normal keratan sulfate proteoglycan. The failure to synthesize a mature keratan sulfate proteoglycan may produce corneal opacity and result in blindness. Because of evidence indicating that the corneal keratan sulfate proteoglycan is normally synthesized through a glycoprotein intermediate [Hart, G. W. & Lennarz, W. (1978) J. Biol. Chem. 253-5795-5801], macular corneal dystrophy may be a defect in glycoprotein processing.

Chondroitin Sulfate Proteoglycans↗
PubMed1980

Isolation of a heparan sulfate-containing proteoglycan from basement membrane.

We have isolated a unique, basement membrane proteoglycan from the Engelbreth-Holm-Swarm (EHS) sarcoma. This proteoglycan, estimated to be 0.75 X 10(6) daltons, was found to contain about equal amounts of protein and covalently linked heparan sulfate. Antibody prepared against this proteoglycan reacts with the basement membrane matrix in the tumor and with the basement membranes in skin, kidney, and cornea. These studies indicate that the heparan sulfate proteoglycan is a normal constituent of basement membranes that presumably plays an important role in the organization of basement membrane components and that also may determine the permeability of basement membranes to acidic molecules.

Amino Acids↗