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G W Conrad

Publications and source records attributed to G W Conrad.

At least 73 records · Page 4Linked to original sources
PubMed1988

Immunoanalysis of keratan sulfate proteoglycan from corneal scars.

Corneal keratan sulfate proteoglycan (KSPG) from scar tissue of experimental penetrating corneal wounds in rabbits was analyzed 2-8 weeks after injury using three previously characterized antibodies. Keratan sulfate (KS) was identified in 2 week scars and normal corneal tissue by indirect immunofluorescence using a monoclonal antibody against sulfated KS epitopes. KSPG was measured in unfractionated extracts of scar and of normal corneal tissue using a "sandwich" enzyme-linked immunosorbent assay (ELISA). In extracts of 2 week scars, KSPG molecules reacting with two different anti-KS monoclonal antibodies were 55% and 82% as abundant as in normal tissue extracts. Ion exchange high performance liquid chromatography (HPLC) of tissue extracts found qualitatively similar elution profiles of KSPG antigens from both scar and normal tissues. Direct ELISA of the HPLC-purified KSPG showed identical quantitative binding of antibodies against core protein and KS from normal and scar tissue. KS in the HPLC-purified extracts was sensitive to digestion with endo-beta-galactosidase, whereas core protein antigens were not affected by this enzyme, as expected. Alteration of the antigenic characteristics of the KSPG of scars was detected with a competitive immunoassay using immobilized monoclonal antibodies against KS. KS in extracts from 2, 6, and 8 week scars competed only 5-11% as effectively as KS from normal cornea, although core protein antigens in the scar extracts competed 61-80% as well as those of normal cornea.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Distribution of proteoglycans antigenically related to corneal keratan sulfate proteoglycan.

Three antibodies reacting with corneal keratan sulfate proteoglycan were used to detect antigenically related molecules in 11 bovine and 13 embryonic chick tissues. Two monoclonal antibodies recognized sulfated epitopes on the keratan sulfate chain and a polyclonal antibody bound antigenic sites on the core protein of corneal keratan sulfate proteoglycan. Competitive immunoassay detected core protein and keratan sulfate antigens in guanidine HCl extracts of most tissues. Keratan sulfate antigens of most bovine tissues were only partially extracted with guanidine HCl, but the remainder could be solubilized by CNBr treatment of the guanidine-extracted residue. Keratan sulfate and core protein antigens co-eluted with purified corneal keratan sulfate proteoglycan on ion exchange high-performance liquid chromatography (HPLC). Endo-beta-galactosidase digestion of the HPLC-purified keratan sulfate antigens eliminated the binding of monoclonal anti-keratan sulfate antibodies in enzyme-linked immunosorbent assay. Extracts of all 11 bovine tissues, except those from brain and cartilage, could bind both anti-keratan sulfate monoclonal antibodies and anti-core protein polyclonal antibody simultaneously. Binding was sensitive to competition with keratan sulfate and to digestion with endo-beta-galactosidase. These results suggest widespread occurrence of a proteoglycan or sulfated glycoprotein bearing keratan sulfate-like carbohydrate and a core protein resembling that of corneal keratan sulfate proteoglycan.

Animals↗
PubMed1987

Effects of calcium antagonists, calmodulin antagonists, and methylated xanthines on polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta.

We have studied the ability of fertilized eggs of Ilyanassa obsoleta to undergo polar lobe formation and cytokinesis in the presence of Ca2+ antagonists (Ca2+ channel blockers, Ca2+ uptake inhibitors). Earlier work had suggested little need for exogenous Ca2+ during these cellular shape changes. Again it appears that exogenous Ca2+ probably is not required, based on cell ability to undergo the shape changes with no, or only minor, delay in the presence of 50 mM La3+ at pH 6.5, 10 mM concentrations of Ni2+ or Co2+, 1 mM Cd2+, and 100 microM concentrations of Mn2+, papaverine, verapamil, D600, or diltiazem. In nominally Ca2+-free seawater (containing approximately 10 microM Ca2+) (CFSW), there still is no effect of Cd2+ (up to 100 microM), Ni2+, Co2+, Mn2+, or diltiazem; however, papaverine, verapamil, and D600 in CFSW cause longer delays in the shape changes than they do in the presence of normal levels of Ca2+ (SW). In 10-50 microM nifedipine, shape changes are progressively delayed to the same extent in both SW and CFSW, but more so in CFSW at concentrations above 50 microM nifedipine. Among calmodulin antagonists, trifluoperazine up to 100 microM was without effect, but chlorpromazine at 25-100 microM and calmidazolium at 50-100 microM caused substantial, concentration-dependent delays in the starting times for the shape changes. Methylxanthines caused a substantial speed-up in the starting times for both polar lobe formation and cytokinesis. The most effective of these, caffeine, at optimal concentrations of 0.7-10 mM in SW or CFSW caused shape changes to occur 12-15 min earlier than in controls undergoing a normal 50-min cycle. Caffeine is known to cause release of Ca2+ from muscle sarcoplasmic reticulum. A putative antagonist of intracellular Ca2+ mobilization, TMB-8, significantly inhibited the shape changes of the Ilyanassa cells, whereas a variety of inhibitors of exogenous Ca2+ uptake noted above did not inhibit. We conclude that Ca2+ may be necessary for polar lobe formation and cytokinesis in Ilyanassa cells, but that it may be released from intracellular, sequestered stores rather than derived from exogenous sources.

Animals↗
PubMed1987

Acetylcholinesterase activity in the cornea of the developing chick embryo.

Activity of acetylcholinesterase (EC 3.1.1.7) and pseudocholinesterase (EC 3.1.1.8) was measured in extracts from chick corneas, in a developmental series from days 7-20 of incubation and at three ages after hatching. Enzyme activity was measured by the biphasic single-vial radiometric assay of Johnson and Russell using [3H-acetyl]choline as substrate. Pseudocholinesterase was inhibited with tetraisopropylpyrophosphoramide (iso-OMPA). True acetylcholinesterase activity was verified by control assays run in the presence of both iso-OMPA and the true acetylcholinesterase inhibitor, 1:5-bis(4-allyldimethyl-ammonium phenyl)-pentane-3-one diiodide (BW284c51). With both inhibitors present, no cholinesterase activity was detected. Corneal acetylcholinesterase had an average Km of 1.1 +/- 0.3 X 10(-3) M at day 7, 14, and 20 of development and retained 90% activity even after 3 hr at 26 degrees C. At least 90% of the total cholinesterase activity was solubilized by Triton X-100 and sonication treatment. Activity decreased with increasing concentrations of NaCl present in the assay. A 60-fold transient increase in acetylcholinesterase specific activity occurs during the period from days 7-20 of embryonic development. This increase begins on the first day measured (day 7), progresses steadily and rapidly during the subsequent week, reaches a peak at day 15, and then decreases rapidly before hatching to a level maintained into adulthood. A similar pattern of transient appearance of highly sialylated gangliosides seen previously on days 14-17 leads to an hypothesis of a structural linkage between acetylcholinesterase and the plasma membrane lipids of corneal epithelial cells.

Acetylcholinesterase↗
PubMed1986

Keratan sulfate proteoglycan during embryonic development of the chicken cornea.

Antibodies to corneal keratan sulfate proteoglycan (KSPG) were used to characterize the pattern of KSPG accumulation during differentiation of neural crest cells in the stroma of embryonic chick cornea. Immunohistochemistry with monoclonal antibody I22 to keratan sulfate found this KSPG antigen localized inside stromal cells at stage 29 (Day 6), ca. 12 hr after migration into the primary stroma. A 2- to 3-day lag then occurred before appearance of extracellular keratan sulfate, first seen on Day 9 (Stage 35) in the posterior stroma. Keratan sulfate antigen accumulated in a posterior to anterior direction during subsequent development. Uniform staining of the stroma for keratan sulfate did not occur until after Day 16. Among several tissues, only corneal stroma contained an extracellular matrix which stained for keratan sulfate, though intracellular staining of some cartilage cells was observed. Accumulation of KSPG antigens in developing cornea was measured in unfractionated guanidine extracts with a quantitative ELISA using three different antibodies against KSPG. Increases were first detected after Day 9 using monoclonal I22, and somewhat later with the other two antibodies. Assays with all three antibodies detected a sustained, exponential increase of KSPG throughout the 5 days prior to hatching. Keratan sulfate continued to accumulate after hatching, but an antibody with specificity to KSPG core protein, detected no relative increase in antigen after hatching. This suggests a modulation of KSPG primary structure late in development and after hatching. Overt differentiation of individual neural crest cells thus appears to begin ca. 12 hr after their arrival in the primary stroma; a lag of 2-3 days precedes active secretion of KSPG.

Age Factors↗
PubMed1986

Platelet activating factor raises intracellular calcium ion concentration in macrophages.

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.

Animals↗
PubMed1986

Positional specificity of corneal nerves during development.

The developing avian cornea is enclosed within a nerve ring from which numerous neurites extend into the cornea as radially ingrowing, regularly bifurcating fascicles. The authors now assess the degree of positional specificity of developing corneal nerves by photographic comparison and digital computer signal averaging. Such analyses demonstrate that nerves display extensive homology of position within the developing nerve ring, indicating that their growth cones originally followed specific pathways around the cornea. During their extension within the cornea, nerve fascicles bifurcate only within distinct, successive, and concentric zones suggesting that the position of an intra-corneal nerve branch point is designated by the intra-corneal milieu. Since the distance between the branch zones reported here increases with corresponding corneal growth, neurite elongation may occur both at the growth cone and along the axon at inter-branch sites of extending corneal nerves.

Animals↗
PubMed1984

Intracellular chloride activity and pH during polar lobe formation and cytokinesis in eggs and embryos of Ilyanassa obsoleta.

The objectives of this study were to compare membrane potential, intracellular chloride activity, and intracellular pH in unfertilized and fertilized eggs of Ilyanassa obsoleta throughout polar lobe formation and cytokinesis. Membrane potential, psi m, was measured with single- and double-barrel microelectrodes containing 3 M KCl. Chloride activity, (Cl)c, was measured with single-barrel, chloride-sensitive microelectrodes. Intracellular pH, pHc, was measured with double-barrel microelectrodes, utilizing an antimony-filled barrel to monitor pHc. Unfertilized eggs having a psi m of -13.7 mV displayed an average (Cl)c of 29.9 mM, whereas at electrochemical equilibrium, (Cl)ce, it would be 155.7 mM. This suggests that Cl is pumped out of the cell in eggs that are not fertilized. Fertilized eggs had a psi m of -78.2 mV and a (Cl)c of 18.6 mM. At electrochemical equilibrium, (Cl)ce would be 12.1 mM, thus suggesting that Cl is pumped into the cell in fertilized eggs. Double-barrel microelectrodes measured a psi m of -69.0 mV and a pHc of 7.3 in fertilized eggs. The psi m, (Cl)c, and pHc remained stable as fertilized eggs underwent the cellular shape changes involved in forming first, second, and third polar lobes and first cleavage.

Animals↗
PubMed1984

Changes in chick corneal lipids during development.

Chick corneas from days 9 through 18 of embryonic development were analyzed for phospholipid and glycolipid content, and for phospholipid and ganglioside classes using chromatographic techniques. Little change in phospholipid headgroup classes was detected during this time span, with the exception of a steady increase in the content of phosphatidylserine from 12 to 18% of the total. Changes in glycolipid content also were observed, with a maximum in the sphingolipid/phospholipid ratio at day 14. This increase was correlated with the appearance of highly sialylated gangliosides at days 14 and 15; these ganglioside species then declined in abundance until they were no longer detectable at day 18. These results indicate that gangliosides with high sialic acid content undergo stage-specific developmental cycles in the chick cornea. Furthermore, the data suggest that phosphatidylserine content may be a sensitive biochemical measure of corneal innervation.

Animals↗
PubMed1982

Antibodies against the predominant glycosaminoglycan of the mammalian cornea, keratan sulfate-I.

Antibodies have been made in rabbits against bovine corneal keratan sulfate proteoglycan. Antisera were titered by their ability to agglutinate sheep red blood cells that had been coated with the proteoglycan. Immune antisera, but not preimmune sera, agglutinate coated cells. Uncoated cells are not agglutinated by either serum. Immune agglutination is inhibited by prior incubation of antiserum with the intact corneal proteoglycan fraction or with 2-mercaptoethanol. Immune agglutination is also sharply reduced by the glycosaminoglycans, keratan sulfate-I (corneal type), and keratan sulfate-II (cartilage type). Desulfated keratan sulfate-I is somewhat less effective as an inhibitor than keratan sulfate-I. In contrast, chondroitin 4- and 6-sulfates, heparin, and hyaluronic acid do not interfere with immune agglutination. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electroblot transfer of the proteins to nitrocellulose paper, incubation with antisera, and reaction with 125I-protein A suggest that the proteoglycan fraction contains high molecular weight antigenic components (Mr = approximately 300,000) whose mobility is sharply decreased by incubation with keratanase to that corresponding to molecular weights of approximately 55,000 and 40,000. No antigenic component appears sensitive to reduction by 2-mercaptoethanol. Chondroitinase ABC does not affect the mobility of proteins in the proteoglycan fraction. These results suggest that antibodies against corneal keratan sulfate proteoglycan may include some that react with the keratan sulfate chains, as well as those directed against the core protein. Keratan sulfate core proteins of two molecular weights may be present.

Animals↗