John H. Rockey

Stimulation of retinal capillary pericyte protein and collagen synthesis in culture by high-glucose concentration

The influence of glucose concentration on cell multiplication and protein synthesis was studied in synchronized, long-term cultures of bovine retinal microvessel pericytes. The cell multiplication rate and the mitotic rate were reduced in media containing 20 mM glucose to 57% and 54%, respectively, of that obtained in media containing 5 mM glucose. Elevated glucose, however, did not change the DNA content of individual cells. Protein and collagen synthesis were measured by the incorporation of radioactive proline and lysine, or the posttranslational production of hydroxyproline and hydroxylysine, respectively. High glucose stimulated protein and collagen synthesis per cell 2.2 ± 0.10 (SD) and 2.1 ± 0.06 times, respectively. Aspirin (0.5 mM), an inhibitor of nonenzymatic glycosylation, did not alter the effect of elevated glucose concentration on protein and collagen synthesis.

Inhibitory effects of pyridoxal phosphate, ascorbate and aminoguanidine on nonenzymatic glycosylation

Nonenzymatic glycosylation of serum albumin was studied in the presence of naturally occurring metabolites, pyridoxal, pyridoxal phosphate and ascorbate/dehydroascorbate, and a hydrazine compound, aminoguanidine. Pyridoxal, pyridoxal phosphate, ascorbate and dehydroascorbate, at concentrations of 0.1 mM or greater, significantly inhibited the nonenzymatic glycosylation of albumin. Aminoguanidine was the most potent inhibitor of nonenzymatic glycosylation and 54% or 85% inhibition occurred when 5 or 50 mM aminoguanidine, respectively, was present in the incubation mixture containing 20 mM glucose. A major effect of aminoguanidine was to lower the free glucose concentration in the incubation mixture by a direct reaction with glucose as judged by thin layer chromatography. The present studies suggest that vital metabolites such as pyridoxal phosphate and ascorbate may be potentially important in controlling glucose-induced nonenzymatic glycosylation of proteins. Pyridoxal phosphate forms a Schiff base with proteins as does glucose and therefore may be a preferable drug, over aminoguanidine which is a hydrazine, for inhibiting the effects of glucose-induced nonenzymatic glycosylation.

Immune-mediated adherence of eosinophils to Toxocara canis infective larvae: the role of excretory-secretory antigens.

Summary The participation of Toxocara canis larval excretory‐secretory antigens in immune‐mediated adherence was determined in vitro. Adsorption of immune sera with excretory‐secretory antigens removed some complement components, removed IgG antibody directed against larval surfaces, and abrogated all adherence observed with untreated immune serum. At least four antigens could be implicated in adherence, by Western blot analysis of adherence mediating sera.

Analysis of Toxocara canis larval excretory-secretory antigens: physicochemical characterization and antibody recognition.

Toxocara canis larval excretory-secretory antigens (TEX) were resolved by gradient pore polyacrylamide gel electrophoresis and analyzed using silver, periodic acid-Schiff, and immunoperoxidase stains. At least 15 bands between 29 and 94 kilodaltons (kDa) were detected by silver stain, all of which were recognized by antibodies in serum of a patient with visceral larva migrans. Immunoperoxidase stain detected an additional band at 92 kDa and 4-6 others above 200 kDa. Periodic acid-Schiff stain also detected the high molecular weight components, but did not detect constituents of approximately 53 and 57 kDa. Immunoperoxidase stain using antibody from the vitreous fluid of an ocular larva migrans patient detected 2 TEX components, approximately 76 and 80 kDa. Antigens were compared with respect to batch of larvae and age of larvae in culture. Qualitative differences that correlated with batch were found in the number of constituents above 200 kDa, and in 1 component of 78 kDa. Qualitative differences were noted in many minor components, some of which appeared to correlate with age of larvae in culture. Major TEX constituents were recognized consistently by antibody, regardless of batch or age of larvae. Total protein production per larva was approximately 8 ng/day, and was consistent over time. There was no evidence of neutral proteases in TEX.

Extracellular matrix production by cat retinal pigment epithelium in vitro: characterization of type IV collagen synthesis.

Feline retinal pigment epithelial cells (RPE) produced an extracellular matrix (ECM) in vitro which was located between the basal surface of the RPE and the culture plate. This ECM had three morphological components: bundle, granular and fibrillar. After 14 days in culture the basal extracellular space contained small amounts of bundle material; granular and fibrillar material were infrequently observed at this time. The amount of ECM material increased with increasing time in culture. The accumulation of the granular component extracellularly was greatest between 60 and 108 days. Fibrillar material, although occasionally observed in the ECM, appeared to be an infrequent component. By 145 days, the ECM filled the extracellular space between the RPE and the culture plate. The time-dependent increase of the ECM indicated continued synthesis and secretion of ECM into the basal extracellular space by the RPE. Confluent RPE cultures, or choroidal/scleral fibroblasts, were incubated for 24 hr with [14C]-proline. Newly synthesized collagen, either in the culture medium or the cell layer, was co-precipitated with added carrier collagen by (NH4)2 SO4. The samples, with or without reduction and alkylation, were digested with pepsin and fractioned by selective salt precipitation and carboxymethyl(CM)-cellulose chromatography. The resulting fractions were further analyzed, or purified for thin layer chromatography (TLC) amino acid analysis, by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Cultured RPE cells, but not choroidal/scleral fibroblasts, produced labelled peptides which were characterized as alpha 1 (IV), and alpha 2 (IV) collagen chains by CM-cellulose chromatography, SDS-PAGE, proline: hydroxyproline ratios and sensitivity to bacterial collagenase. In contrast, choroidal/scleral fibroblasts produced labelled alpha 1 (I), beta 12 (I) and alpha 2 (I) collagen chains. The synthesis of type IV collagen by RPE cells may reflect the production of ECM observed by electron microscopy in cultured feline RPE cells.

The effects of glucose and an aldose reductase inhibitor on the sorbitol content and collagen synthesis of bovine retinal capillary pericytes in culture

The absolute rate of collagen synthesis by cultured bovine retinal capillary pericytes, determined using the specific radioactivity of proline in the cellular amino acid pool, was compared in media containing different concentrations of glucose (5, 10 or 40 mM) and Sorbinil (0.0 or 0.1 mM), an inhibitor of aldose reductase. The absolute rate of collagen synthesis, in proline molar terms, by pericytes in medium with 5 mM glucose was 3.3 +/- 1.9 (S.D.) pmol 10(-7) cells 24 hr-1, and increased significantly to 8.8 +/- 3.7 pmol 10(-7) cells 24 hr-1 when the glucose concentration was increased to 40 mM. Sorbinil (0.1 mM) reduced the elevated sorbitol contents of pericytes induced by high concentrations of glucose, but did not significantly change the absolute rate of collagen synthesis per cell.

Equine antihapten antibody II. The γG(7Sγ) components and their specific interaction

Abstract The γ G(7 Sγ ) components of antibody prepared against the haptenic group p -azophenyl-β-lactoside (Lac) were isolated from equine antiserum. The average association constants ( K A ) for the specific interaction of this antibody with the hapten, p -( p -dimethyl-aminobenzeneazo)-phenyl-β-lactoside (Lac dye), were determined at 25° and 37.2° by equilibrium dialysis. Thermodynamic parameters have been calculated and shown to be very similar to those previously obtained for rabbit anti-Lac antibody. A physical-chemical and immunochemical characterization of the purified γG equine anti-Lac antibody has demonstrated chromatographic, electrophoretic and antigenic heterogeneity. Three antigenically distinct immunoglobulins have been identified in the purified γG anti-Lac antibody each having antibody activity. The antigenic individuality has been related to the heavy chains when compared by immunodiffusion to one another or to γA ( β 2 A) antibody from the same serum. A comparison of the binding by the γG antibody with the binding previously described for the γA antibody shows that the affinity of the latter for the Lac dye is much greater. This difference in the affinity for hapten between different immunoglobulins present in the same serum may partially explain the heterogeneity of hapten binding in purified antibody. Furthermore, variation in the relative amounts of these immunoglobulins with time after immunization could in part account for the temporal changes in the affinity of the antiserum for hapten.

Glycosaminoglycan synthesis and secretion by bovine retinal capillary pericytes in culture.

The synthesis and secretion of glycosaminoglycans (GAGs) was characterized in subcultures of bovine retinal capillary pericytes. The GAGs were metabolically labeled with [3H]glucosamine and 35SO4 for 3 days, and then precipitated from the cell layer or media by cetylpyridinium chloride and ethanol, separated by cellulose acetate electrophoresis and further identified by their susceptibility to degradative procedures. The predominant radioactively labeled GAG associated with the pericyte-cell layer was heparan sulfate (HS). Radioactively labeled chondroitin sulfate (CS) and hyaluronic acid (HA) were also present in the pericyte-cell layer. No radioactively labeled dermatan sulfate (DS) was detected. The profile of radioactively labeled GAGs secreted by pericytes into the media differed considerably from that associated with the cell layer. Equal amounts of radioactivity were incorporated into HS and CS. Small quantities of radioactively labeled HA were also present in the media. Although no radioactively labeled DS was detected in the pericyte-cell layer, it was present in the media. The total pericyte-cell layer GAG profile was determined by scanning densitometry of the three bands resolved after cellulose acetate electrophoresis and Alcian Blue staining. The slowest band was identified as HS, and accounted for 17% of the total GAGs. The middle band was identified as DS, and accounted for 34% of the total GAGs. The fastest band was tentatively identified as either DS or chondroitinase AC-resistant CS, and constituted 49% of the total GAGs. The GAGs associated with the fibroblast-cell layer and secreted into the media by fibroblasts also were characterized and compared with those produced by pericytes. The major differences were in the secretion of large amounts of HA into the media by fibroblasts, and the presence of radioactively labeled DS in the cell layer of fibroblasts.

Actin in cultured bovine retinal capillary pericytes: morphological and functional correlation.

Actin in cultured bovine retinal capillary pericytes was identified and partially characterized biochemically. The filamentous actin was localized in bovine retinal capillary pericytes using a fluorescent mushroom toxin (nitrobenzoxadiazole-phallacidin) specific for actin. One-dimensional SDS-polyacrylamide-gel electrophoresis of urea-extracted proteins from bovine retinal capillary pericytes revealed a 46,000 MW protein band corresponding to an actin standard, which comprised 7.3% of the total urea-soluble proteins. Actin-activated skeletal muscle myosin Mg2+-ATPase assay, using [gamma-32P]-ATP as substrate, demonstrated functional actin in bovine retinal capillary pericyte extracts after DEAE-cellulose anion-exchange chromatography. The actin-containing protein fractions were eluted at ionic strengths between 0.25 and 0.35 M KCl. The presence of functional actin in pericytes indicated the ability to generate contractile force. This contraction-generating ability may allow pericytes to regulate microvessel caliber and to maintain the integrity of the capillary wall. A lack of this function when pericytes are preferentially lost in diabetic retinal microangiopathy could destabilize the microvessel wall and predispose the capillary to further pathologic changes.

Induction and Down-Regulation of Conjunctival Type-I Hypersensitivity Reactions in Guinea Pigs Sensitized Topically with Fluoresceinyl Ovalbumin

Type-I hypersensitivity reactions were induced in guinea pigs by repeated topical/conjunctival application of fluoresceinyl ovalbumin (FL-OA). The ocular reactivity in early responding animals was maximal between 16 and 25 days and decreased exponentially thereafter. Desensitized eyes responded minimally to compound 48/80 but maximally to histamine. Unilateral sensitization and challenge with FL-OA produced desensitization of the immunized eye, but the reactivity of the contralateral eye persisted. A significant reduction in conjunctival stained mast cells was found in repeatedly challenged eyes. The desensitization therefore was due to a loss of reactive mast cells. Systemic infection with Ascaris suum, after repeated topical challenge with FL-OA had led to desensitization, produced a reappearance of type-I hypersensitivity reactions toward both FL-OA and ascarid antigens.