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H H Varner

Publications and source records attributed to H H Varner.

At least 19 recordsLinked to original sources
PubMed1991

Proteoglycans in the mouse interphotoreceptor matrix. IV. Retinal synthesis of chondroitin sulfate proteoglycan.

To determine whether the mouse retina contributes chondroitin sulfate proteoglycan (CS-PG) to the interphotoreceptor matrix (IPM), 35SO4(2-) was used as a tracer for newly synthesized proteoglycan by retinas maintained in vitro in the absence of pigment epithelium. Following incubation with the tracer for 3 hr, the 35S-labeled proteoglycans present in the incubation medium and associated with isolated photoreceptor outer segments were analyzed separately. Proteoglycan was extracted with 4 M guanidine, and then separated on a G-25 column followed by DEAE ion exchange chromatography in the presence of 8 M urea. The proteoglycan fraction was eluted with a linear NaCl gradient of 0.15-1.0 M. Eluted 35S-labeled macromolecules were susceptible to chondroitinase AC and ABC degradation, indicating that virtually all the 35S-labeled proteoglycan synthesized by the mouse retina and secreted into the incubation media is of the chondroitin sulfate type. In parallel autoradiographic analysis of retinas following 35SO4(2-) incubation, silver grains were present over all retinal compartments, with 41-48% associated with the photoreceptor layer. Quantitative autoradiography of retinas following chondroitinase AC digestion of fixed retinas revealed significant (P less than 0.025) reduction in silver grains associated with the photoreceptor outer segment layer as compared to controls. These combined biochemical and autoradiographic studies indicate that the retina, possibly the photoreceptors, synthesize at least a portion of the CS-PG present in the IPM of the mouse.

Animals↗
PubMed1990

Regional variation within the interphotoreceptor matrix from fovea to the retinal periphery.

The interphotoreceptor matrix in the human retina comprises a highly organised extracellular compartment. Using fluorescent labelled peanut agglutinin and the cationic dyes Cupromeronic and Cuprolinic Blue, unique cylindrical domains surrounding cone photoreceptors can be demonstrated. These cone specific domains are relatively insoluble and are closely adherent to cone photoreceptors and to the pigment epithelium, suggesting that these structures may play a role in retinal attachment.

Extracellular Matrix↗
PubMed1989

Proteoglycans in the mouse interphotoreceptor matrix. II. Origin and development of proteoglycans.

The development and cellular origin of chondroitin sulfate-type proteoglycans in the interphotoreceptor matrix (IPM) of the mouse retina were examined with electron microscopy after staining with the cationic dye, Cupromeronic Blue. In the IPM of the developing retina, three structural types of Cupromeronic Blue-positive filaments were observed. Type A filaments measured 45-55 nm in length and around 5 nm in diameter. At birth, a few type A filaments were present in the IPM and they increased in number during subsequent days postpartum. Type A filaments were present in high density in the cytoplasm of the pigment epithelial cells at all stages examined where they were associated with the Golgi apparatus and cytoplasmic vesicles. Type B filaments, measuring 60-80 nm in length and 5-10 nm in diameter, were occasionally observed in the IPM of the newborn mouse. Type B filaments increased in number during subsequent development and by 14 days postpartum, their location was restricted predominantly to the IPM around photoreceptor inner segments and the connecting cilia. Type C filaments, measuring 15-25 nm diameter with extremely long profiles (up to several microns), were first consistently observed in the IPM at 6 days postpartum, the time when photoreceptor outer segments begin to develop. During subsequent days postpartum, type C filaments increased in number. By 18 days postpartum, when photoreceptors have achieved their adult length, type C filaments were interconnected in a net-like meshwork investing the photoreceptor outer segments. All three Cupromeronic Blue-positive filament types were sensitive to chondroitinase AC, but not to Streptomyces hyaluronidase. Cupromeronic Blue-stained filaments were rarely observed in the cytoplasm of the photoreceptor or Müller cells at any of the stages examined. These findings suggest that proteoglycans of the chondroitin sulfate-type present between the retina and pigment epithelium of the adult mouse are secreted principally by the retinal pigment epithelial cells, and that these components begin to be elaborated into the IPM a few days prior to the stage when photoreceptor outer segment development is initiated. Organization of the proteoglycans to form an extracellular meshwork surrounding the photoreceptors takes place during the period when photoreceptor outer segments are elongating to achieve their adult length.

Animals↗
PubMed1989

Retinal attachment to the pigment epithelium. Linkage through an extracellular sheath surrounding cone photoreceptors.

The interphotoreceptor matrix, which occupies the so-called subretinal space in normal human eyes, was examined with electron microscopy after treatment with Cuprolinic Blue, a dye that selectively stains sulfated polyanions. A cylindrically shaped, extracellular ensheathing domain, composed of Cuprolinic Blue-positive particles, was observed surrounding each cone photoreceptor examined. Equivalent structures were not present around rods. Surrounding peripheral cones, the unit particles which comprised the sheath, were punctate in shape whereas in foveal and foveolar cones the particles were more elongate. A matrix domain completely enveloped each cone photoreceptor, beginning at the outer limiting membrane, extending beyond the tip of the cone outer segment, and ending at the apical surface of the pigment epithelium. Proximal to the pigment epithelium, specialized extensions of apical microvilli were present in the lumen of the matrix sheath and shorter microvilli appeared to be embedded in the sheath wall. When retinas were experimentally detached from the pigment epithelium and washed extensively before fixation and staining, the cone matrix domains remained associated with the cones, and broken fragments of pigment epithelial cell processes were present embedded in the distal tip of the cone matrix sheath wall. These observations indicate that the cone matrix sheath is insoluble and is closely associated with both the cone photoreceptor and the apical surface of the pigment epithelium. The implication of these findings is that the cone matrix sheath is a bridge through which the retina and pigment epithelium are physically attached.

Adolescent↗
PubMed1989

Distribution and characterization of sulfated proteoglycans in the human trabecular tissue.

The distribution of proteoglycans in the trabecular tissue of human eyes was evaluated histochemically, using Cupromeronic Blue in combination with a series of enzyme digestions and nitrous acid treatment. Within the extracellular matrix of the trabecular meshwork, many Cupromeronic Blue-positive filaments were observed in association with collagen fibrils, basal lamina, a basal lamina-like material and a fine fibrillar-like material. Pretreatment with chondroitinase AC reduced the staining associated with the collagen fibrils, whereas filament staining in this location was completely eliminated by pretreatment with chondroitinase ABC. Nitrous acid treatment eliminated almost all the filament staining associated with the basal lamina and basal lamina-like material. When the tissue was treated with chondroitinase AC, chondroitinase ABC or nitrous acid, filament staining associated with the fine fibrillar-like material was reduced. After a combined treatment with nitrous acid followed by incubation with chondroitinase ABC, all filament staining in the trabecular meshwork was eliminated. These results are consistent with an interpretation that human trabecular tissue contains three distinct types of sulfated proteoglycans, namely chondroitin sulfate, dermatan sulfate and heparan sulfate-type proteoglycans. Proteoglycans containing chondroitin sulfate and dermatan sulfate are present in association with collagen fibrils. Proteoglycans of heparan sulfate-type are associated with the basal lamina and the basal lamina-like material. Chondroitin sulfate, dermatan sulfate and heparan sulfate-type proteoglycans are present in association with the fine fibrillar-like component. The basal lamina, basal lamina-like material and fine fibrillar-like components associated with these negatively charged proteoglycans may be important contributors to aqueous outflow resistance in the juxtacanalicular connective tissue.

Adult↗
PubMed1988

Proteoglycans in the mouse interphotoreceptor matrix. I. Histochemical studies using cuprolinic blue.

Cuprolinic Blue, when applied at a critical electrolyte concentration, can be utilized for assessing the localization and structural characteristics of proteoglycans with electron microscopy. We have used this cytochemical procedure to evaluate the distribution of proteoglycan in the interphotoreceptor matrix of the mouse retina. Cuprolinic Blue-positive filaments of two distinct morphological types were present surrounding both rod and cone photoreceptors. Large filaments, 115-135 nm long and 15-25 nm in diameter, were distributed in the interphotoreceptor matrix around the outer segment and outer portion of the inner segment. These filaments appeared linked to each other to form a complex meshwork. Smaller filaments, 60-70 nm long and 5-10 nm in diameter, were principally observed around the photoreceptor inner segments. Incubation of retinas with chondroitinase AC and chondroitinase ABC eliminated Cuprolinic Blue staining of both large and small filaments, whereas hyaluronidase treatment reduced the size of the filaments but did not eliminate their staining. When retinas were washed extensively prior to fixation and staining, Cuprolinic Blue-positive filaments remained associated with the photoreceptor cell surface. These results suggest that the interphotoreceptor matrix of the mouse retina contains at least two structural types of proteoglycan, of the chondroitin sulfate-type, which are differentially distributed in this compartment. One of the proteoglycans forms a complex meshwork which surrounds the photoreceptors. Both are insoluble and appear to be firmly attached to the photoreceptor plasma membrane.

Animals↗
PubMed1987

Localization of proteoglycan within the extracellular matrix sheath of cone photoreceptors.

The interphotoreceptor matrix of the human retina was examined histochemically by staining with a cationic copper phthalocyanin dye, Cuprolinic Blue, in a critical electrolyte concentration method which allowed staining of sulfated polyanions. In the presence of Cuprolinic Blue, a dense punctate matrix component was visualized covering the cone from the outer limiting membrane of the retina to the tip of the cone outer segment and extending through the matrix to the apical surface of the pigment epithelium. The size and appearance of the stained material are consistent with its being proteoglycan and its distribution is consistent with its being a component of the cone extracellular matrix sheath. A fine reticular pattern was observed in the presence and absence of Cuprolinic Blue.

Extracellular Matrix↗
PubMed1986

Chondronectin interactions with proteoglycan.

We have investigated whether proteoglycans are involved in the attachment of embryonic chick chondrocytes to type II collagen. Chondroitin sulfate proteoglycan, when added exogenously, promotes the binding of chondronectin, the chondrocyte attachment factor, to type II collagen substrates and thereby stimulates chondrocyte adhesion. Blockage of endogenous proteoglycan synthesis with beta-xylosides prevents chondronectin-mediated chondrocyte attachment, confirming that proteoglycan is required. The intact proteoglycan must be present since chondroitin sulfate glycosaminoglycans did not promote chondronectin-mediated cell attachment but, rather, inhibited it in a dose-dependent manner. This inhibition, however, could be overcome with excess exogenous proteoglycan. Consequently, chondronectin interacts with proteoglycan and then the complex interacts with the collagen substrate and with the cell surface to promote cell adhesion. Further evidence for a direct interaction of chondronectin with the glycosaminoglycan portion of the proteoglycan is the selective binding of chondronectin to dextran-Sepharose, dextran having been shown to inhibit attachment to an extent similar to that of chondroitin sulfate.

Animals↗
PubMed1985

Chondronectin: physical and chemical properties.

Chondronectin, the chondrocyte attachment factor, was purified from chicken serum and characterized as to its physical and chemical properties. From sedimentation equilibrium data it was found to have a native molecular weight of 175,800 +/- 800 and a subunit molecular weight of 55,540 +/- 800 in the presence of guanidinium chloride and cysteine, suggesting a trimeric structure linked by disulfide bonds. As visualized by electron microscopy after rotary shadowing, the protein appears compact and globular. The amino acid and carbohydrate compositions of chondronectin are distinct from fibronectin, the fibroblast attachment factor, and laminin, the epithelial cell attachment factor. The activity of chondronectin in promoting attachment of chondrocytes is stable to digestion by collagenase, elastase, and neuraminidase, but is destroyed by trypsin treatment. The data suggest that chondronectin is structurally and chemically distinct from fibronectin and laminin.

Amino Acids↗
PubMed1985

Isolation and characterization of a factor from calf serum that promotes the pigmentation of embryonic and transformed melanocytes.

A protein (Mr = 63,000) from calf serum that promotes the pigmentation of cultured chick neural crest and mouse melanoma cells has been partially isolated and characterized in this study. The stimulation of melanin synthesis in cultured cells was used to follow its activity during purification. The pigment-promoting factor was isolated by sequential column chromatography on dye-agarose matrices followed by hydroxyapatite and high pressure molecular sieve chromatography. The factor was found to stimulate melanin biosynthesis at 2-4 micrograms/ml and was specific for melanin-producing cells and their precursors. Antibodies raised in rabbits against the factor inhibited its pigment-promoting activity as well as that of whole calf serum. Enzyme-linked immunoadsorbent assays demonstrated that calf and bovine sera contain molecules that cross-react with the pigment-promoting factor. Horse, human, rat, and chicken sera, which lack the biological activity, also lacked immunological cross-reactivity. Extracts of certain tissues, particularly the submaxillary gland, were observed to be rich sources of pigment-promoting activity.

Animals↗
PubMed1985

Endocytosis and degradation of interstitial retinol-binding protein: differential capabilities of cells that border the interphotoreceptor matrix.

Between the pigment epithelium and the outer limiting membrane of the retina is an extracellular compartment filled with the interphotoreceptor matrix (IPM). A prominent component of the IPM is a glycoprotein known as interstitial retinol-binding protein (IRBP). Using in vitro techniques, we compared the ability of the cells that border this compartment to internalize colloidal gold (CG) coated with either IRBP or ovalbumin, a glycoprotein not found in the IPM. Neither IRBP-CG nor ovalbumin-CG was internalized by the Muller's cells. Both rod and cone photoreceptors take up IRBP-CG, which is observed in small vesicles and multivesicular bodies. Neither photoreceptor type takes up ovalbumin-CG. Acid phosphatase cytochemistry indicates that acid phosphatase reaction product in the multivesicular bodies co-localizes with IRBP-CG, which suggests that this molecule is degraded by rod and cone photoreceptors and is not recycled. The pigment epithelium internalizes IRBP-CG and ovalbumin-CG, both of which remain in small cytoplasmic vesicles near the apical plasma membrane. There is no indication that vesicles that contain either IRBP-CG or ovalbumin-CG fuse with the lysosomal system in the pigment epithelial cells during the incubation.

Animals↗
PubMed1985

Participation of photoreceptor cells in retrieval and degradation of components in the interphotoreceptor matrix.

Evidence for endocytosis of components of the interphotoreceptor matrix by photoreceptor inner segments is presented. Photoreceptors cultured in the presence of horseradish peroxidase (HRP) rapidly interiorized this tracer as evidenced by the presence of HRP-reaction product within primary endocytotic vesicles in the inner segment cytoplasm. The tracer was also observed in multivesicular bodies. Retinas were incubated in a suspension of interstitial retinol-binding protein (IRBP) coated colloidal gold (CG). IRBP-CG was interiorized by photoreceptors where it was found in endocytotic vesicles and in multivesicular bodies in both rod and cone inner segments. Acid phosphatase reaction product co-localized with IRBP-CG in multivesicular bodies suggesting that IRBP is degraded following endocytosis and is not recycled.

Culture Techniques↗
PubMed1983

Identification of a bone matrix-derived chemotactic factor.

When demineralized bone matrix powder is implanted subcutaneously in the rat, the early responses involve the appearance and proliferation of mesenchymal cells at the site of implantation, followed by cartilage and bone formation. The ability of cells to migrate to the implant suggests that chemotaxis may be a critical event in this process. Therefore, using the modified Boyden chamber assay, we tested extracts of demineralized bone matrix for chemotactic activity. We have identified and partially purified, on molecular sieve chromatography, a heat labile and trypsin-sensitive protein (Mr = 60,000-70,000) that is a potent chemoattractant for mouse calvaria, osteoblast-like cells (MMB-1), but not for monocytes (putative osteoclast precursors). These findings suggest that chemotactic protein(s) have a significant role in the recruitment of osteoprogenitor cells to a site of bone repair.

Bone Matrix↗
PubMed1982

The isolation and partial characterization of chondronectin, an attachment factor for chondrocytes.

We have previously demonstrated that the adhesion of embryonic chick sternal chondrocytes to a type II-collagen substrate is not mediated by fibronectin but rather by a distinct attachment factor which we have named chondronectin. Here we describe the isolation, properties, and biological activity of chondronectin prepared from chicken serum. Chondronectin is shown to be a glycoprotein with an estimated Mr = 180,000. After reduction, it migrates as subunits of Mr = 70,000. Antibodies directed against chondronectin inhibited the attachment of chondrocytes to type II collagen. Chondronectin is immunologically distinct from either fibronectin or laminin. Immunofluorescence studies on frozen sections of embryonic chick sternal cartilage and of cultured sternal chondrocytes showed that chondronectin is cell-associated rather than a major matrix component.

Animals↗
PubMed1982

The role of chondronectin and cartilage proteoglycan in the attachment of chondrocytes to collagen.

The interaction of cells with collagen has previously been demonstrated to be of importance in the growth and differentiation of various cells (reviewed by Hay 1981; Kleinman, et al 1981). Chondronectin has been demonstrated to be involved in mediating the interaction of chondrocytes with their matrix. Unlike fibronectin, which binds directly to collagen before the cell can interact with this macromolecular complex, chondronectin must interact with cartilage proteoglycan monomer in order to function efficiently as an attachment factor. Figure 4 is a schematic representation of the interaction between chondrocytes and their matrix components. We suggest that if these specific interactions are perturbed by the introduction of some outside factor, the phenotypic expression of the cells could be altered. For example, chondrocytes are not normally associated with fibronectin (Dessau, et al 1978; Stenman, Vaheri 1978). However, if chondrocytes are grown in the presence of fibronectin they will become more fibroblastic and stop making the cartilage-specific molecules of type II collagen and cartilage proteoglycan (Pennypacker, et al 1979; West et al 1979). Consequently, it is possible that the specific interactions seen between chondrocytes, chondronectin, type II collagen, and proteoglycan may be essential for the maintenance of the chondrocyte phenotype.

Animals↗
PubMed1980

Polyethylene glycol gradient solubilization of lysyl hydroxylase.

Polyethylene glycol (PEG) was substituted for ammonium sulfate in the gradient solubilization chromatographic procedure for the purification of proteins. Preliminary studies utilizing this method for fractionation of a crude lysyl hydroxylase preparation yielded an increase both in purity and in recovery of activity when compared with fractional precipitation with PEG. Turbidity, evident in resuspended fractional precipitates, was not found in PEG gradient solubilization fractions containing enzymatic activity.

Animals↗
PubMed1979

Purification and enzymatic properties of lysyl hydroxylase from fetal porcine skin.

Lysyl hydroxylase from fetal porcine skin is shown to bind in a highly specific manner to aminoethyl-Sepharose 4B. When coupled to ammonium sulfate fractionation and DEAE-cellulose chromatography, chromatography of lysyl hydroxylase preparations on aminoethyl-Sepharose 4B has yielded a highly purified (greater than 95%) preparation of lysyl hydroxylase. The enzyme consists of two subunits with molecular weights of 70 000 and 115 000. The overall recovery of activity was 2.5%, yielding approximately to 3.5 mg of purified enzyme from 900 g of fetal porcine skin. The enzyme is more active at 30 degrees C than at 37 degrees C and has a pH optimum near 8.0. Both catalase and bovine serum albumin are required by the enzyme for maximum activity. The sulfhydryl reagents p-(chloromercuri)-benzoate, N-ethylmaleimide, and iodoacetamide are potent inhibitors of the enzyme, whereas dithiothreitol appears to be an activator.

Animals↗