Metal sensitive protease(s) associated with corneal proteoglycans.
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Amino Acid Sequence↗
Biomedical subjects
S S Twining
Publications and source records attributed to S S Twining.
Alpha-1 proteinase inhibitor levels in keratoconus.
The levels of alpha 1-proteinase inhibitor (alpha 1-antitrypsin) in keratoconus, normal human, and other diseased corneas were examined. Using an immunoperoxidase technique, the presence of this inhibitor was demonstrated in the epithelium, stroma and endothelium of all corneal sections. Compared with normal human controls, the staining intensity in the epithelium and stromal lamellae of keratoconus corneas was markedly reduced. Such a reduction was not seen in either scarred or other diseased corneas. Extracts of keratoconus and normal human corneas were subsequently analyzed for alpha 1-proteinase inhibitor by a dot blot assay using a monoclonal antibody against the inhibitor and a 125I-labelled secondary antibody. In agreement with the immunohistochemical findings, the alpha 1-proteinase inhibitor level found in the epithelium of keratoconus corneas was approximately one-fourth of that found in normal human controls. In addition, the stromal extracts of keratoconus corneas contained about one-sixth the inhibitor level of that in normal human extracts. These results lend further support to the hypothesis that degradation processes may be aberrant in keratoconus.
Adolescent↗
Loss of stromal glycosaminoglycans during corneal edema.
This study tried to determine if glycosaminoglycans (GAGs) are released from the rabbit stroma during corneal edema. The GAGs of rabbit corneas were labeled in situ using anterior-chamber injections of 35S-sulfate and 3H-glucosamine. Labeled corneal pairs were excised and the endothelium perfused in vitro in the specular microscope. Edema was induced in one cornea by perfusion with a calcium-free balanced salt solution; the control cornea was perfused with glutathione bicarbonate Ringer's (GBR). Corneal thickness was measured every 15 minutes during the 3-hour perfusion period, and perfusate fractions were collected from each cornea and analyzed for the presence of GAGs. Edematous corneas swelled from 438 +/- 14.8 microns to 688 +/- 10.6 microns compared with control corneas (427 +/- 4.7 microns to 454 +/- 7.2 microns). Total 3H-glucosamine (4.00 +/- 0.68%) and 35S-sulfate (10.36 +/- 0.92%) released from the edematous corneas during perfusion exceeded that lost by control corneas (1.92 +/- 0.18% for 3H-glucosamine; 3.23 +/- 0.52% for 35S-sulfate). Enzymatic digestion studies showed the presence of keratan sulfate in the edematous perfusates. The results suggest that increased loss of radiolabeled components from edematous corneas represent a loss of stromal GAGs and possibly GAG fragments. Therefore, corneal edema involves loss of GAGs and water uptake.
Animals↗
Characterization of corneal proteoglycans under vitamin A deficiency.
Comparison of in vivo radiolabeled corneal proteoglycans from vitamin A deficient, pair-fed control and normal rabbits by chromatographic and enzymatic methods, reveals subtle but reproducible changes in the proteoglycans present in the vitamin A deficient corneas relative to those of pair-fed and normal control corneas. Although the total amounts of [3H]leucine and [35S]sulfate incorporated in vivo into the proteoglycans per cornea are similar in the three types of cornea, the proteoglycan affinity for DEAE-Sepharose is different, with more of the vitamin A deficient proteoglycans requiring a higher NaCl concentration for elution. Analysis of in vivo labeled rabbit corneal proteoglycans reveals that in vitamin A deficient animals, relative to pair-fed controls, there is (1) an increase in the proteoglycans digested by chondroitinase AC indicative of decreased epimerization of glucuronic acid to iduronic acid; (2) an increase in the amount of keratan sulfate proteoglycan with high affinity for an anion exchange resin, consistent with a greater negative charge; (3) differences in proteoglycan affinities for octyl-Sepharose, reflecting differences in hydrophobicity; (4) increased susceptibility to proteolysis by trypsin; (5) no significant difference in sulfation.
Animals↗
Localization and quantification of alpha-1-proteinase inhibitor in the human cornea.
alpha-1-Proteinase inhibitor, formerly called alpha-1-antitrypsin, was detected in human corneal epithelium, stroma, Descemet's membrane and endothelium using an indirect immunolocalization technique. The average alpha-1-proteinase inhibitor levels detected by an immunodot blot assay in the epithelium, stroma and Descemet's membrane-endothelium extracts per total human cornea were 29.5 micrograms, 54.3 micrograms and 3.5 micrograms, respectively. Immunolocalization on Western blots of SDS polyacrylamide electrophoresis gels revealed that all three layers contained a molecule with a molecular weight equal to the native alpha-1-proteinase inhibitor. Additionally, in the epithelial and stromal extracts minor bands at 75 kD and 110 kD were noted which are possibly due to complexes with proteases. The 110 kD band alternatively may be a dimer of the inhibitor. The epithelial extract contained bands at 40 kd and less than 30 kD indicating the presence of proteolysis products. alpha-1-Proteinase inhibitor probably plays a major role in the protection of the cornea from proteases released by inflammatory cells.
Adult↗
Regional comparisons of cathepsin D activity in bovine retinal pigment epithelium.
Cathepsin D is the lysosomal protease in the retinal pigment epithelium which is presumed to be the major enzyme involved in the degradation of shed discs during the photoreceptor renewal process. In this study, the cathepsin D activity in RPE cells from the posterior area centralis was compared to the activity in cells from the equatorial region of the same bovine eyes. Enzyme activities were measured both in paired fresh RPE isolates from the two retinal regions and in paired regional RPE cultures. Cultures were further analyzed for changes in enzyme activity with time in vitro from 3 to 11 weeks. Analysis of freshly isolated RPE cells from 30 eyes indicated that cells from the area centralis have significantly higher cathepsin D activity than cells from the more peripheral retina. Paired cultures of RPE from the two regions did not express the intraeye topographical differences in enzyme activity which were observed in fresh isolates. There were significant variations in enzyme activity in cultured RPE cells with time in vitro, but activity levels did not show progressive increases or decreases with in vitro aging. After 11 weeks in vitro, but not at earlier times, the enzyme activities in the paired regional cultures from the same eye were highly correlated. The data suggest that the higher levels of cathepsin D activity observed in the freshly isolated RPE from the area centralis result from modulators of enzyme activity which are not present in culture.
Animals↗
The immune system in experimental Pseudomonas keratitis. Model and early effects.
A model to study the immune system in Pseudomonas keratitis was developed using defined flora rats (WAG/RijMCW) that have not been exposed to Pseudomonas aeruginosa. One group of rats was made immunocompetent towards P. aeruginosa by intraperitoneal injection of phenol-killed P. aeruginosa while a second group remained naive to this organism. Corneas of both groups were scratched centrally with a 21-g needle, before inoculation with 2 X 10(8) P. aeruginosa organisms. Corneas of control animals were either only scratched or only inoculated with the bacterium. At 18 hr, the naive animals were killed. In naive rat corneas, light and electron microscopy showed bacteria throughout the cornea, polymorphonuclear leukocytes (PMNs) distributed from the limbus towards the center, and little stromal degradation. In contrast, massive corneal degradation was observed in the immunocompetent rats; PMNs were present, but no bacteria were observed free in the stroma. The total acid protease content was higher in the immunocompetent than in the naive rat corneas, a possible reason for the observed difference in corneal degradation. This difference was not due to increased numbers of PMNs since nearly equal numbers of PMNs were counted after enzymatic disaggregation of both types of corneas. Glycogen-induced peritoneal PMNs from both types of rats migrated equally well towards P. aeruginosa culture media and media of corneas incubated with this bacterium. The authors conclude that immune recognition is (1) involved in the corneal host response to P. aeruginosa and (2) required for efficient phagocytosis by PMNs but not their recruitment.
Animals↗
Use of immunoadsorbents for the study of antibody binding to sperm whale myoglobin and its synthetic antigenic sites.
Conditions for preparing immunoadsorbents of sperm-whale myoglobin and its five synthetic antigenic sites and for desorption of radiolabeled antibodies from the immunoadsorbents were studied. In immunoadsorbent titration studies, the sum of the amounts of antibodies bound in the plateau (maximum binding) by the adsorbents of the five sites accounted quantitatively for the entire (100%) antibody response to sperm-whale myoglobin.
Animals↗
Genetic control of immune response to sperm whale myoglobin in mice. II. T lymphocyte proliferative response to the synthetic antigenic sites.
The genetic control of T lymphocyte proliferative response to the five synthetic antigenic sites of myoglobin, two synthetic nonantigenic control peptides, and one "nonsense" peptide was determined in independent and recombinant strains of mice. In all the strains examined, the nonantigenic control peptides and the "nonsense" peptide did not invoke a response in myoglobin-primed mice. Further, when mice were not primed with whole myoglobin, no response was obtained with any of the antigenic sites. Haplotypes H-2d, H-2f, and H-2s are higher responders to sites 1 and 2, whereas haplotypes H-2d and H-2s are high responders to site 5. Response to site 3 may be controlled by a non-H-2-linked gene. Site 4 can stimulate H-2b and H-2k haplotypes that are nonresponders to the whole myoglobin. Studies with the recombinant strains suggested that Ir genes to sites 1 and 2 map in the I-A subregion and I-C subregion and were designated Ir-Mb-1,2(A) and Ir-Mb-1,2(C). Ir genes to sites 4 and 5 mapped only in the I-A subregion and were designated Ir-Mb-4(A) and Ir-Mb-5(A). These studies suggest that individual antigenic sites in a molecule are controlled by unique Ir genes.
Animals↗
Antibody-combining sites can be mimicked synthetically. Surface-simulation synthesis of the immunoglobulin new combining site to the gamma-hydroxyl derivative of vitamin K1.
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Amino Acid Sequence↗
Large scale separation of protease inhibitors from malignant human breast tissue.
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Antithrombins↗
Identification of alpha1-acid glycoprotein, alpha2-macroglobulin and antithrombin III as components of normal and malignant human tissues.
alpha1-Acid glycoprotein, alpha2-macroglobulin, and antithrombin III have been identified, by immunological means, as components of the 90000 X g supernatant fraction of malignant and adjacent normal human breast, colon, and anal tissues, as well as malignant stomach and ileum. Malignant lung tissue only contained alpha1-acid glycoprotein. These protease inhibitors are immunologically equivalent to those present in human plasma.
Antithrombins↗
Characterization of the interaction between chymotrypsin and heparin.
Heparin forms a complex with chymotrypsin which is active towards glutaryl-L-phenylalanine-p-nitroanilide (GPANA) and glutaryl-L-phenylalanine-beta-naphthylamide (GPNA) at pH 7.6. The activity of chymotrypsin towards GPANA at pH 7.6 is enhanced in the presence of heparin. Heparin does not bind at the active site of the enzyme since proflavin is not displaced from the active site of chymotrypsin upon complex formation. The heparin-chymotrypsin complex migrates under basic polyacrylamide disc gel electrophoresis conditions to a position intermediate between heparin and free chymotrypsin. The complex is dissociable under acidic polyacrylamide gel electrophoresis conditions. It is estimated that one to three molecules of heparin can bind to each chymotrypsin molecule on the basis of electrophoretic and enzymic activity data.
Binding Sites↗
Isolation and identification of alpha1-antitrypsin as a component of normal and malignant human breast and other tissues.
Alpha1-Antitrypsin has been detected using radial immunodiffusion in the 90,000g supernatant fraction of malignant and adjacent normal human breast, colon, and anal tissue, as well as malignant lung, stomach, and ileum. Immobilized chymotrypsin, bound to Sepharose or Affi-Gel 10 has been utilized to separate 11 peaks of antiproteolytic activity by affinity chromatography of normal and malignant human breast tissue extracts. Glycoproteins are associated with eight of the peaks. Peak 1 contains predominantly alpha1-antitrypsin in addition to a minor component. The purification of the inhibitors, as judged by disc gel electrophoresis, is extensive. In some peaks, only one or two protein bands are observed, suggesting that affinity chromatography on Sepharose- or Affi-Gel 10-chymotrypsin might be used for the isolation of alpha1-antitrypsin and other inhibitors in preparative amounts.
Anal Canal↗