Biomedical subjects

J R Hassell

Publications and source records attributed to J R Hassell.

At least 73 records · Page 4Linked to original sources

PubMed1988

Identification of cDNA clones encoding different domains of the basement membrane heparan sulfate proteoglycan.

We have used antibodies to the basement membrane proteoglycan to screen lambda gt11 expression vector libraries and have isolated two cDNA clones, termed BPG 5 and BPG 7, which encode different portions of the core protein of the heparan sulfate basement membrane proteoglycan. These clones hybridize to a single mRNA species of approximately 12 kilobases. Amino acid sequences obtained on peptides derived from protease digests of the core protein were found in the deduced sequence, confirming the identity of these clones. BPG 5 spanned 1986 base pairs and has an open reading frame of 662 amino acids. The amino acid sequence deduced from BPG 5 contains two cysteine-rich domains and two internally homologous domains lacking cysteine. The cysteine-rich domains show homology to the cysteine-rich domains of the laminin chains. A globule-rod structure, similar to that of the short arms of the laminin chains, is proposed for this region of the proteoglycan. The other clone, BPG 7, is 2193 base pairs long and has an open reading frame of 731 amino acids. The deduced sequence contains eight internal repeats with 2 cysteine residues in each repeat. These repeats show homology to the neural-cell adhesion molecule N-CAM and the plasma alpha 1B-glycoprotein. Looping structures similar to these proteins and to other proteins of the immunoglobulin gene superfamily are proposed for this region of the proteoglycan. The sequence DSGEY was found four times in this domain and could be heparan sulfate attachment sites.

Amino Acid Sequence↗

PubMed1988

Visualization of the large heparan sulfate proteoglycan from basement membrane.

Kleinschmidt spreading, negative staining, and rotary shadowing were used to examine the large form of (basement membrane) heparan sulfate proteoglycan in the electron microscope. Heparan sulfate proteoglycan was visualized as consisting of two parts: the core protein and, emerging from one end of the core protein, the glycosaminoglycan side chains. The core protein usually appeared as an S-shaped rod with about six globules along its length. Similar characteristics were observed in preparations of core protein in which the side chains had been removed by heparitinase treatment ("400-kDa core") as well as in a 200-kDa trypsin fragment ("P200") derived from one end of the core protein. The core protein was sensitive to lyophilization and apparently also to the method of examination, being condensed following Kleinschmidt spreading (length means = 52 nm) and extended following negative staining (length means = 83 nm) or rotary shadowing (length means = 87 nm; 400-kDa core length means = 80 nm; P200 length means = 44 nm). Two or three glycosaminoglycan side chains (length means = 146 +/- 53 nm) were attached to one end of the core protein. The side chains often appeared tangled or to merge together as one. Thus, the large heparan sulfate proteoglycan from basement membrane is an asymmetrical molecule with a core protein containing globular domains and terminally attached side chains. This structure is in keeping with that previously predicted by enzymatic digestions and with the proposed orientation in basement membranes, i.e., the core protein bound in the lamina densa and the heparan sulfate side chains in the lamina lucida arranged along the surface of the basement membranes.

Animals↗

PubMed1988

An established rat cell line expressing chondrocyte properties.

Chondrocytes express a well-characterized set of marker proteins making these cells useful for studies on differentiation and regulation of gene expression. Because of the inherent instability of primary rat chondrocytes in culture, and because several rat chondrocyte genes have been cloned and characterized (including the collagen II promoter and enhancer), a rat chondrocyte cell line would be especially useful. To obtain this line we infected primary fetal rat costal chondrocytes with a recombinant retrovirus (NIH/J-2) carrying the myc and raf oncogenes, which have been shown to have an "immortalizing" function. Following infection, a rapidly proliferating clonal line was isolated that maintained a stable phenotype through 45 passages (11/2 year in culture). This line, termed IRC, grows in suspension culture as multicellular aggregates and in monolayer culture as polygonal cells which accumulate an alcian blue-stainable matrix. IRC cells synthesize high levels of cartilage proteoglycan core protein, and link protein, but show reduced collagen II expression. In addition, the cells express virally derived myc mRNA and protein, but do not express v-raf. Retinoic acid, which is a known modulator of chondrocyte phenotype, down-regulates expression of chondrocyte marker proteins, while stimulating v-myc expression by IRC cells. These data suggest that v-myc expression by chondrocytes results in rapid cell division and maintenance of many aspects of the differentiated phenotype. These "immortalized" cells, however, remain responsive to agents such as retinoic acid which modulate cell phenotype. The potential exists for development of chondrocyte cell lines from diseased cartilage, as well as from human cartilage.

Animals↗

PubMed1988

Detection of extracellular matrix molecules synthesized in vitro by monkey and human retinal pigment epithelium: influence of donor age and multiple passages.

The retinal pigment epithelium of various species, including man, can synthesize extracellular matrix components in vitro. We studied extracellular matrix molecule production by human, rhesus and cynomolgus monkey pigment epithelium in vitro, using a panel of specific antibodies to collagen types I-V, fibronectin, laminin, the basement membrane heparan sulfate proteoglycan, human alpha elastin, glial fibrillary acidic protein (GFAP), and Factor VIII in a modified indirect immunofluorescence reaction. Results were similar between human and both monkey species in early passage cultures with respect to the positive detection of collagen types I, III, IV, V laminin, basement membrane proteoglycan, and fibronectin. The fluorescence was strongest in the center of confluent monolayers and was absent in border or isolated cells. Collagen type II, elastin, GFAP, or Factor VIII were not detected. Based on these findings, human and monkey pigment epithelium in vitro should be useful for the study of the synthesis, deposition and supramolecular interactions of a variety of extracellular matrix molecules.

Aging↗

PubMed1988

Glomerular basement membrane proteoglycans are derived from a large precursor.

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.

Animals↗

PubMed1987

Complete primary structure of the rat cartilage proteoglycan core protein deduced from cDNA clones.

We have obtained overlapping cDNA clones for the entire coding sequence of the rat cartilage proteoglycan core protein from the Swarm rat chondrosarcoma. These cDNAs hybridize to two sizes of RNA transcripts of 8.2 and 8.9 kilobase pairs, which contain large 3'-untranslated sequences. The total contiguous cDNA is 6.55 kilobase pairs in size, and codes for a 2124-residue protein, including a 19-residue signal peptide. The sequence forms a series of eight structural domains including two globules, (Mr = 37,000 and 22,000) at the NH2 terminus of the molecule, one a complete and one a partial copy of the cartilage link protein. The major feature of the deduced protein sequence is a 1,104-residue segment containing 117 Ser-Gly sequences, the presumed chondroitin sulfate attachment sites. These are arranged in three domains of 428, 503, and 173 amino acids. The first domain contains 11 complete or partial repeats of a 40-residue unit, and the second domain is composed of six copies of a 100-residue repeating sequence. The first pattern is the more highly conserved, and may have given rise to the second. The carboxyl-terminal domain is a third globule which has homology with animal lectins.

Aggrecans↗

PubMed1987

Detection of specific extracellular matrix molecules in drusen, Bruch's membrane, and ciliary body.

We used specific antibodies to localize a variety of extracellular matrix components by indirect immunofluorescence reactions on cryostat sections of drusen-bearing human donor eye tissue. These included collagen types I through V, fibronectin, laminin, heparan sulfate-containing basement membrane proteoglycan as well as antibodies to human IgG and IgM. The composition of drusen with respect to these specific molecules varied greatly, even within the same region of an eye. Hard or nodular drusen were more likely to show a discrete organized layer of specific basement membrane molecules over their apical surfaces than were diffuse type drusen. Diffuse but not nodular drusen generally contained fibronectin. Immunohistochemical reactivity of Bruch's membrane showed age-related accumulation of type I collagen and localized changes associated with some drusen.

Adult↗

PubMed1987

Domain structure of the basement membrane heparan sulfate proteoglycan.

We have used proteolytic digestions and immunological reactivity to map regional domains of the 400-kilodalton (kDa) core protein of the heparan sulfate containing basement membrane proteoglycan from the Englebreth-Holm-Swarm tumor. Digestion with V8 protease caused the rapid release of numerous large peptides ranging in size from 80 to 200 kDa and a 44-kDa peptide. The 44-kDa peptide (P44) was stable to further digestion, but the larger peptides were eventually degraded to a 46-kDa peptide (P46). Both the P44 and P46 fragments migrate slower in the presence of a reducing agent, indicating intrachain disulfide bonding, and do not have heparan sulfate side chains. Antisera to the P46 fragment, however, did not react with P44 fragment, and the amino acid compositions of P46 and P44 fragments were different. This suggests that these two fragments were unrelated. Trypsin digestion of the proteoglycan immediately released a 200-kDa peptide (P200) that also lacked heparan sulfate side chains. Digestion of the P200 fragment with V8 protease produced the P44 and P46 fragments in the same temporal sequence seen with V8 protease digestion of the proteoglycan. Antisera to the P200 fragment reacted strongly with the P44 and P46 fragments. These results show that the P44 and P46 domains are contained within the P200 domain. The rapid release of the P44 domain indicates that it is located at one end of the core protein. The large size of these proteolytic fragments suggests the core protein contains considerable conformational structure, and the absence of heparan sulfate on the P200 domain indicates that the side chains are asymmetrically located on the core.

Amino Acids↗

PubMed1987

Retinoic acid rapidly reduces cartilage matrix synthesis by altering gene transcription in chondrocytes.

Retinoic acid can alter the differentiation of a variety of cell types, including chondrocytes. This action may explain the high incidence of craniofacial and limb defects resulting from exposure to retinoic acid during development, and may be the basis for the compound's inhibition of a chondrosarcoma tumor in vivo. In order to understand the mechanism of action of retinoic acid, we studied the expression of chondrocyte-specific proteins as well as other proteins that indicate a shift in the differentiated phenotype of the cell following exposure to retinoic acid. After 48 hr of exposure to retinoic acid chondrocytes stopped synthesizing the chondrocyte-specific pro alpha 1 (II) chain of collagen II and a 370-kDa precursor protein of a cartilage-specific proteoglycan. Instead, the cells synthesized increased amounts of fibronectin and the pro alpha 1 chain of collagen III. These changes could be detected as early as 12 hr after treatment. In addition, the steady-state levels of specific mRNA transcripts coding for these differentiation markers correlated with their protein synthesis levels. Also, nuclear runoff experiments indicated that retinoic acid down regulated transcription of the collagen II gene, while stimulating collagen III gene transcription. These observations suggest that retinoic acid may alter the expression of the chondrocyte phenotype by selectively changing the normal pattern of gene expression.

Animals↗

PubMed1987

Extracellular matrix fibers containing fibronectin and basement membrane heparan sulfate proteoglycan coalign with focal contacts and microfilament bundles in stationary fibroblasts.

Double-label immunofluorescence microscopy and immunoelectron microscopy were performed on stationary cultures of Nil 8 fibroblasts to determine if fibronectin and basement membrane heparan sulfate proteoglycans play coordinated roles in cell-to-substrate adhesion. Relationships between subcellular matrix fibers containing fibronectin plus proteoglycan, and focal contacts associated with microfilament bundles, were studied simultaneously using interference reflection microscopy, differential interference contrast microscopy, and immunofluorescence microscopy. Cells maintained in 0.3% FBS were doubly stained with monospecific anti-fibronectin IgG and antibodies against a basement membrane proteoglycan purified from the EHS (Engelbreth-Holm-Swarm) tumor. Coincident patterns of fibronectin and proteoglycan-containing fibers were found to codistribute with focal contacts and microfilament bundles in both early (6-h) and late (24-h) cultures. The early cells showed doubly-stained fibers colinear with substrate adhesion sites in 43% of the sample, while 100% of the later cells exhibited these coaligned matrix-cytoskeletal attachment complexes. Immunoelectron microscopy showed that both of these antigens were situated in the same type of extracellular matrix fiber that appeared to be loosely associated with the cell surface membrane. We hypothesize that the appearance of proteoglycan in subcellular matrix fibers of these fibroblasts might stabilize fibronectin-containing cell-to-substrate contacts.

Actin Cytoskeleton↗

PubMed1987

Identification of a phenotype-specific enhancer in the first intron of the rat collagen II gene.

The regulation of the collagen II gene was investigated by transfecting plasmids containing potential regulatory sequences of this gene coupled to the gene for chloramphenicol acetyltransferase (CAT) into various cells. The 5' flanking region of this gene functioned as a weak promoter when transfected into chicken chondrocytes or fibroblasts. Inclusion of an 800-base fragment from the first intron, however, increased transcription of CAT approximately 18-fold in chicken chondrocytes and in differentiating limb bud cells but not in fibroblasts, myoblasts, muscle-derived fibroblasts, or teratocarcinoma cells. Furthermore, this fragment was active when placed either upstream or downstream of the CAT gene and when present in either orientation. The activity of this enhancer was examined in relation to differentiation by using "micromass" culture of limb bud mesenchyme cells undergoing chondrogenesis. In this system, no response to the enhancer was observed in undifferentiated limb bud mesenchyme cells. Following differentiation into chondrocytes, a 13-fold enhancer effect was observed in these cells. Finally, all-trans-retinoic acid, a known teratogen, dramatically suppressed enhancer activity in chondrocytes and differentiating limb bud mesenchyme cells. These results suggest that the collagen II gene contains an enhancer element in the first intron that is involved in cell-specific expression.

Animals↗

PubMed1987

Response of diabetic basement membrane--producing cells to glucose and insulin.

The effect of insulin and glucose on the synthesis of basement membrane components was studied in organ cultures of a basement membrane-producing tumor grown in diabetic and normal mice. Tumor tissue grown in diabetic mice produced more protein and basement membrane-specific proteoglycan in response to insulin than tissue grown in normal mice. Addition of high levels of glucose to the culture medium did not alter insulin-stimulated protein synthesis by diabetic or normal tissue but dampened insulin-stimulated production of proteoglycan. These data suggest that basement membrane-producing cells in diabetic hosts may be hypersensitive to insulin and that stimulation of protein production by insulin may play some role in the in situ hypertrophy of basement membranes.

Animals↗

PubMed1986

Absence of normal keratan sulfate in the blood of patients with macular corneal dystrophy.

We measured levels of sulfated keratan sulfate in serum using a monoclonal antibody in an enzyme-linked immunosorbent assay. Sulfated keratan sulfate was not detected in the serum of 16 patients with macular corneal dystrophy, but was present at normal levels in 66 patients with other corneal diseases. There were no differences with respect to age, sex, and other ocular findings. This monoclonal antibody recognizes a sulfated carbohydrate epitope present in both corneal and skeletal keratan sulfate. Since most serum keratan sulfate is derived from the cartilages, the defect in keratan sulfate synthesis in macular corneal dystrophy may not be restricted to corneal cells. This assay should prove useful in the diagnosis of macular corneal dystrophy, particularly in children at risk before the appearance of opacification.

Adolescent↗

PubMed1986

Partial cDNA sequence encoding a globular domain at the C terminus of the rat cartilage proteoglycan.

We have isolated and sequenced a cDNA clone of 872 base pairs from the 3' end of the mRNA for the large cartilage specific proteoglycan from rat. Identification was confirmed by a comparison with published protein sequence. Hybridization analysis shows the presence of an 8-9-kilobase mRNA for this proteoglycan in rat and chick sternal chondrocytes and rat chondrosarcoma cells, but not in RNA from rat fibroblasts, vitamin A-treated chick chondrocytes, chick crop, or bone. The carboxyl portion of the proteoglycan is deduced to terminate in a globular domain, which includes a region homologous to a chick hepatic lectin, and is possibly involved in binding to N-acetylglucosamine. The clone extends into a region where serines are clustered, probably the start of the chondroitin sulfate-rich region.

Amino Acid Sequence↗

PubMed1986

Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen.

Rotary shadowing electron microscopy was used to examine complexes formed by incubating combinations of the basement membrane components: type IV collagen, laminin, large heparan sulfate proteoglycan and fibronectin. Complexes were analyzed by length measurement from the globular (COOH) domain of type IV collagen, and by examination of the four arms of laminin and the two arms of fibronectin. Type IV collagen was found to contain binding sites for laminin, heparan sulfate proteoglycan and fibronectin. With laminin the most frequent site was centered approximately 81 nm from the carboxy end of type IV collagen. Less frequent sites appeared to be present at approximately 216 nm and approximately 291 nm, although this was not apparent when the sites were expressed as a fraction of the length of type IV collagen to which they were bound. For heparan sulfate proteoglycan the most frequent site occurred at approximately 206 nm with a less frequent site at approximately 82 nm. For fibronectin, a single site was present at approximately 205 nm. Laminin bound to type IV collagen through its short arms, particularly through the end of the lateral short arms and to heparan sulfate proteoglycan mainly through the end of its long arm. Fibronectin bound to type IV collagen through the free end region of its arms. Using a computer graphics program, the primary laminin binding sites of two adjacent type IV collagen molecules were found to align in the "polygonal" model of type IV collagen, whereas with the "open network" model, a wide meshed matrix is predicted. It is proposed that basement membrane may consist of a lattice of type IV collagen coated with laminin, heparan sulfate proteoglycan and fibronectin.

Animals↗

PubMed1986

Basement membrane complexes with biological activity.

We have studied the reconstitution of basement membrane molecules from extracts prepared from the basement membrane of the EHS tumor. Under physiological conditions and in the presence of added type IV collagen and heparan sulfate proteoglycan, gellike structures form whose ultrastructure appears as interconnected thin sheets resembling the lamina dense zone of basement membrane. The major components of the reconstituted structures include laminin, type IV collagen, heparan sulfate proteoglycan, entactin, and nidogen. These components polymerize in constant proportions on reconstitution, suggesting that they interact in defined proportions. Molecular sieve studies on the soluble extract demonstrate that laminin, entactin, and nidogen are associated in large but dissociable complexes which may be a necessary intermediate in the deposition of basement membrane. The reconstituted matrix was biologically active and stimulated the growth and differentiation of certain cells.

Animals↗

PubMed1986

beta-D-xyloside-mediated alteration in the synthesis of basement membrane proteoglycan.

The effect of nitrophenyl-beta-D-xyloside (xyloside), a synthetic initiator of glycosaminoglycan synthesis, on proteoglycan and glycosaminoglycan synthesis by a basement membrane producing tumor was studied. While xyloside markedly stimulated the formation of chondroitin sulfate chains, it depressed the formation of a basement membrane heparan sulfate proteoglycan and caused only little formation of free heparan sulfate chains. However, when the synthesis of the core protein of the proteoglycan was inhibited by cycloheximide, heparan sulfate chains were produced by xyloside treatment. These heparan sulfate chains had a sulfate content higher than that of heparan sulfate found on the proteoglycan. The data indicate that xyloside can substitute for the heparan sulfate initiation site on the core protein of the proteoglycan and that this initiation is enhanced in the absence of core protein. This suggests that under normal conditions the formation of heparan sulfate chains may be tightly linked to the production of the core protein.

Animals↗

PubMed1986

Altered synthesis of heparan sulfate proteoglycans at low sulfate concentration.

The structural alterations in heparan sulfate produced by sulfate deprivation were studied in cell cultures of the Engelbreth-Holm-Swarm tumor. Tumor cells were labeled in vitro with [3H]glucosamine and/or [35S]sulfate in media containing either 300 microM MgSO4 or no added carrier sulfate, and the newly synthesized proteoglycans isolated by chromatography on DEAE-Sephacel. The proteoglycans isolated from low sulfate cultures showed a reduced affinity for the column eluting at lower salt concentrations compared with the proteoglycans isolated from cultures containing sulfate, suggesting that the former were undersulfated. Analysis of the isolated heparan sulfate side chains indicated that two pools of heparan sulfate were present which differed in their degree of sulfation. Both pools were synthesized by both high sulfate and low sulfate cultures, but the highly sulfated pool was the predominant form produced in sulfate containing cultures, while the undersulfated pool was the predominant form synthesized in low sulfate cultures. The more sulfated pool contained more N-sulfate than the less sulfated pool. Few if any free amino groups were detected in either pool, suggesting that the initial deacetylation step in the biosynthesis of heparan sulfate is tightly coupled to the N-sulfation step in the cells.

Animals↗