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Biomedical subjects

B J McLaughlin

Publications and source records attributed to B J McLaughlin.

At least 55 records · Page 3Linked to original sources
PubMed1987

Freeze-fracture label of functional and dysfunctional human corneal endothelium.

Previous freeze-fracture results from our laboratory have shown a reduction in a population of intramembrane particles in the lateral endothelial membranes from dysfunctional human corneas. The size range of these intramembrane particles corresponds to that which has previously been reported for the glycoprotein enzyme Na, K-ATPase in enzyme enriched freeze-fractured membranes. In order to investigate glycoconjugate changes potentially related to the particle reduction, wheat germ agglutinin (WGA), which has been shown to bind to the sugar residues in the ATPase subunit, was used to label three types of corneas with dysfunctional endothelial cells (Fuchs' endothelial dystrophy, aphakic and pseudophakic bullous keratopathy) and two types of corneas (eye bank and keratoconus) with functional endothelium using the technique of thin section freeze-fracture label. Apical WGA labelling on all types of dysfunctional cells was shown to be drastically reduced in comparison to both types of functional corneal endothelial cells. Lateral membranes of dysfunctional cells, exposed by freeze-fracturing, also showed a great reduction in WGA labelling as compared to the fractured lateral membranes of functional cells. The differences observed in lectin labelling of lateral membranes may be related in part to the decreased intramembrane particle density observed in dysfunctional human corneal endothelial cells.

Cornea↗
PubMed1987

Lectin-affinity isolation of microvillous membranes from the pigmented epithelium of rat retina.

The pigmented epithelium of the vertebrate retina phagocytizes the discarded tips of photoreceptors and it is likely that a specific cellular recognition process is involved in this phenomenon. The apical surface of retinal pigmented epithelium (RPE) contains microvilli which interdigitate with the outer segment regions of photoreceptor cells and it is this apical microvillous surface that is of particular interest with respect to phagocytosis. The present study is a report of a method to isolate a fraction that is enriched in microvilli from the apical surface of this highly polarized epithelial cell. Wheat germ agglutinin (WGA) conjugated sepharose beads are used to remove the microvillous membranes which are then observed with scanning and transmission electron microscopy. The proteins of this RPE-subfraction are separated through use of SDS-polyacrylamide gel electrophoresis. The relative molecular weights (Mr) and lectin binding properties of glycoproteins are examined in Western blots through the use of lectin-peroxidase conjugates as probes for carbohydrate residues. A preliminary comparison of membranes isolated from Long Evans (normal) and Royal College of Surgeons (dystrophic) rat retina RPE shows that the glycoproteins in these two preparations are different with respect to the binding of Concanavalin-A (Con-A) and WGA. In particular a glycoprotein in the normal RPE preparation with a Mr of 175K binds Con-A and WGA, but in the dystrophic RPE preparation binds little or no WGA. A glycoprotein present in the normal RPE preparation with a Mr of 86K binds Con-A and WGA, but both lectins have reduced binding sites in the dystrophic preparation. Limax flavus agglutinin (specific for sialic acid residues) binds to a high molecular weight glycoprotein with a Mr of 195K-196K which is present in both normal and dystrophic RPE membrane preparations and which also binds Con-A and WGA.

Animals↗
PubMed1987

Cytochrome oxidase activity of postsurgical bullous keratopathy endothelium.

Proper corneal hydration is normally maintained by a pump-leak mechanism located on the lateral cell membranes of the endothelium (1,2). When endothelial cells become sufficiently dysfunctional in response to trauma, the cornea becomes irreversibly edematous and opaque. Previous studies in our laboratory have shown that mitochondrial cytochrome oxidase (CO) activity, an enzyme important in respiratory activities, is correlated with endothelial cell functional activity in corneas with Fuchs' endothelial dystrophy. In this study we investigated cytochrome oxidase activity in corneas with postsurgical bullous keratopathy. Corneas with aphakic and pseudophakic bullous keratopathy (ABK, PBK) were incubated in diaminobenzidine-cytochrome C media. Results showed that the staining pattern of ABK and PBK corneas was uniformly low compared to keratoconus corneas, and in contrast to previous studies on Fuchs' dystrophy corneas which demonstrated a regional staining pattern. This suggests that CO staining patterns correlate with functional activity and may be a useful technique to detect dysfunctional cells in various disease categories.

Corneal Diseases↗
PubMed1987

Phagocytic interactions of sialated glycoprotein, sugar, and lectin coated beads with rat retinal pigment epithelium.

Latex beads coated with mucin (a sialic acid containing glycoconjugate), N-acetylglucosamine (GlcNAc), or with the lectins, succinylated wheat germ agglutinin (sWGA) (binds to GlcNAc) or Limax flavus agglutinin (LFA) (binds to sialic acid residues) were used as phagocytic particles. Phagocytosis of these coated beads by retinal pigment epithelial (RPE) explants was determined by bead uptake in normal (Long Evans) and dystrophic (Royal College of Surgeons, RCS/p+) rat retinas. Electron microscopy showed that beads coated with mucin or LFA lectin were not phagocytized by either dystrophic or normal RPE. sWGA-coated beads were phagocytized by both dystrophic and normal RPE, while GlcNAc-coated beads were taken up by dystrophic RPE only. Specificity of uptake for sWGA and GlcNAc bead coatings was shown by the reduction in the number of beads phagocytized in the presence of the appropriate competing sugar or lectin. The lack of phagocytic uptake of beads coated with a sialated glycoprotein or a sialic acid binding lectin suggests that sialic acid residues are not recognized as particulate ligands in this phagocytic assay. The data which show the uptake of beads coated with sWGA (binds only GlcNAc) together with results which showed WGA (binds both GlcNAc and sialic acid)-coated beads were not taken up, further suggest that GlcNAc residues may be involved in bead phagocytosis. The most striking difference between normal and dystrophic RPE engulfment of coated beads is the uptake of GlcNAc-coated beads by dystrophic RPE only. These results suggest that the receptor molecules on dystrophic RPE cell surface membranes may be different from those on normal RPE membranes.

Acetylglucosamine↗
PubMed1987

Immunohistochemical localization of ATPase in human dysfunctional corneal endothelium.

Previous light microscopic autoradiographic results from our laboratory have shown a quantitative decrease in ATPase pump site density in dysfunctional corneal endothelium. In order to develop antibody techniques to correlate these findings with electron microscopic localization of pump sites, three types of corneas with dysfunctional endothelium (Fuchs' endothelial dystrophy, aphakic and pseudophakic bullous keratopathy) and two types of corneas with functional endothelium (age matched eye bank and keratoconus) were obtained at the time of transplant surgery. Corneas were fixed, frozen and cryostat sections were incubated in a rabbit kidney ATPase primary antibody followed by incubation in an HRP-labeled secondary antibody. Functional endothelia showed dense antibody labeling along the lateral cell membranes and there was a substantial reduction in labeling along the lateral membranes of dysfunctional endothelia. Positive tissue controls of rabbit kidney showed HRP reaction product in the convoluted tubules. Control tissue, incubated in either non-immune serum or primary antibody preabsorbed with ATPase, confirmed the specificity by having diminished or no reaction product. These results provide immunohistochemical confirmation of our autoradiographic data showing a quantitative reduction in pump site density on dysfunctional corneal endothelium.

Adenosine Triphosphatases↗
PubMed1987

ATPase pump site density in human dysfunctional corneal endothelium.

Proper corneal hydration is maintained by a Na, K-ATPase pump located in the lateral membranes of the endothelial cells. In dysfunctional corneas this pumping action appears to break down as the corneas become edematous. In order to provide quantitative and qualitative data on the Na, K-ATPase pump site density on dysfunctional and functional human corneal endothelial cells, the present study has employed both autoradiographic and histochemical techniques. Computer-assisted morphometrics and statistical analysis showed that there was a significant reduction (P less than 0.001) in 3H-ouabain binding, and thus ATPase pump sites, in the three types of corneas (Fuchs' endothelial dystrophy, aphakic and pseudophakic bullous keratopathy) with dysfunctional endothelia as compared to both types of corneas (eye bank, keratoconus) with functional endothelial cells. There were no significant differences amongst the dysfunctional types or between the two functional types of corneal endothelial cells in respect to density of silver grains. Histochemical staining for ATPase showed less p-nitro-phenylphosphatase histochemical reaction product present on dysfunctional endothelial lateral membranes than in the functional cells.

Aphakia↗
PubMed1986

Cytochrome oxidase activity of Fuchs' endothelial dystrophy.

The normal human corneal endothelial monolayer maintains stromal water equilibrium and thus, transparency, by means of a pump-leak mechanism. Water leaks into the stroma through non-tight lateral cell junctional complexes and is drawn out by an energy dependent cell membrane ion pump. We investigated the histochemical localization of cytochrome oxidase activity (CO), an important energy-deriving mitochondrial enzyme in dysfunctional corneas with Fuchs' endothelial dystrophy (ED), which is a regionally distributed disease. Keratoconus corneas were used as controls for functional control endothelium. In the central area of the corneal button, decreased CO activity was demonstrated which correlated clinically with central corneal edema. This reflects decreased metabolic activity and/or decreased numbers of mitochondria in the attenuated dysfunctional cells. In the mid-periphery, CO activity was increased in the cellular rosettes surrounding guttata, which may be related to increased synthesis of abnormal Descemet's membrane and guttata. Peripherally, the large polygonal cells resembled functional endothelium in their morphology and CO activity. We have, therefore, demonstrated regional differences in energy metabolism in endothelium from Fuchs' ED patients which may be related to decreased numbers of mitochondria in the dysfunctional cells, and/or to synthesis of abnormal Descemet's membrane material.

Cornea↗
PubMed1986

Wheat germ agglutinin and concanavalin A binding during epithelial wound healing in the cornea.

It has been hypothesized that there are differences between membrane-associated glycoconjugates of wounded (migrating) epithelium and those of nonwounded (stationary) epithelium. To test this hypothesis, wheat germ agglutinin-ferritin (WGA-Fe) and concanaval in A-ferritin (Con A-Fe) binding to apical membranes of wounded and nonwounded rabbit corneal epithelia were compared. Epithelial abrasions of the superior half of the cornea were allowed to heal in vivo for six hours. Fixed corneas were then incubated with lectin-ferritin and prepared for electron microscopy. Measurements (ferritin particles per linear um of membrane) of WGA-Fe indicated that binding to leading cells (40.7 particles/um), to areas 20 to 35 cells behind the leading edge (46.5 particles/um) and to nonwounded epithelium (45.1 particles/um) from contralateral eyes were not significantly different. A competitive inhibitor of WGA, 0.2M N-acetylglucosamine, however, blocked 94 percent of WGA binding on leading cells (2.3 particles/um), while binding persisted in areas behind the leading edge (39.5 particles/um) and on nonwounded epithelium (43.6 particles/um). This indicates that leading cell surfaces have a weak affinity for WGA. Unlike WGA, Con A showed a distinct preference for leading-edge cells (33.9 particles/um) compared to nonwounded epithelium (9 particles/um). In areas 20-35 cells behind the leading edge, Con A binding was intermediate to these two extremes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosaminidase↗
PubMed1986

Decreased anionic sites in Bruch's membrane of spontaneous and drug-induced diabetes.

The basal laminae of capillaries, glomeruli, and nerves are thickened in diabetes. Previous studies have shown that diabetic tissues produce increased levels of basal lamina collagen and reduced levels of proteoglycans. Since heparan sulfate and chondroitin sulfate proteoglycans are thought to act as anionic barriers regulating passage of proteins across Bruch's membrane of the eye, the cationic electron microscope tracers, polyethyleneimine (PEI) and ruthenium red, were used to study the distribution of anionic sites in Bruch's membrane of spontaneously diabetic Bio-Breeding/Worcester (BB-W) rats and streptozotocin-diabetic rats. The distribution of the two tracers is similar. In Bruch's membrane of control rats, electron dense particles are present at regular intervals along both sides of the basal laminae of the retinal pigment epithelium (RPE) and of the choriocapillary endothelium (CE), and along collagen fibers in the zone between the two basal laminae. Within 3-6 months after the onset of hyperglycemia in both diabetic rat models, quantitative analysis shows a significant reduction in binding sites along both RPE and CE basal laminae, while thickness of both basal laminae is significantly increased. Because reductions in anionic binding sites along basal laminae in the renal glomerulus have been found to accompany changes in glomerular filtration, these changes suggest that filtration through Bruch's membrane is altered in diabetes.

Animals↗
PubMed1986

Freeze fracture study of human corneal endothelial dysfunction.

Intramembrane changes occurring in dysfunctional corneal endothelial cell membranes were examined using freeze-fracture and transmission electron microscopy techniques. Three categories of dysfunctional endothelium were examined: aphakic bullous keratopathy, pseudophakic bullous keratopathy and Fuchs' endothelial dystrophy. Keratoconus corneas and a donor eye bank eye were examined as normal controls. Four intramembrane changes were observed on replicas of freeze-fractured membranes in each category of dysfunctional endothelium. These were a marked reduction in intramembrane particle density on lateral membranes, altered apico-lateral junctional complexes, increased vesicle fusion sites on apical, lateral and basal membranes, and abnormal desmosome-like particle aggregates on the lateral endothelial cell membranes. The marked reduction in intramembrane particles on lateral membranes may be due to a change in the macromolecular components associated with pump dysfunction. The increase in membrane vesicle fusion sites and the breakdown in intercellular junctions may be associated with increased permeability during barrier dysfunction.

Cornea↗
PubMed1985

Freeze-fracture study of filipin binding in photoreceptor outer segments and pigment epithelium of dystrophic and normal retinas.

We have studied sterol distribution in the retinal pigment epithelial (RPE) microvillous and outer segment disc membranes of rats with inherited retinal degeneration (RCS; RCS-p/+) and of normal genetic controls (RCS-rdy+, RCS-rdy+-p/+) by using the polyene antibiotic filipin, which binds specifically to 3-B-hydroxy-sterols, and freeze-fracture techniques. Retinas were perfusion-fixed, incubated with filipin in the same fixative, and prepared routinely for freeze-fracture electron microscopy. In the normal retina, the distribution of filipin binding sites on both RPE microvillous and outer segment disc membranes changes during development. Prior to outer segment elongation and the onset of phagocytosis (10 days postnatal), filipin sterol complexes are homogeneously distributed in both microvillous and outer segment membranes. With the onset of phagocytosis (2 weeks postnatal and later) filipin binding in both tissues forms a proximal-to-distal gradient, and binding sites decrease as distance from the cell body increases. In the normal RPE microvillous membranes, binding sites are numerous proximally and sparse on the distal tips. In the normal outer segment disc membranes, binding sites are often present on the basal discs, but are sparse on the intact apical discs prior to shedding. As the discs are cast off and engulfed by the RPE, however, filipin binding increases on both disc and phagosome membranes. In the dystrophic retina, the distribution of filipin binding sites differs from the normal. First, in the microvillous membranes, the proximal-to-distal gradient in filipin binding is rarely present at 2 weeks postnatal and becomes prominent only after the buildup of membranous debris has begun (3-5 weeks postnatal). Second, as the photoreceptors degenerate and the membrane debris disappears (4 months postnatal), filipin binding on the microvillous membranes becomes relatively sparse and homogeneous. Third, filipin binding on the intact disc membranes does not change with outer segment elongation, and numerous filipin binding sites are present on both apical and basal outer segment disc membranes. Fourth, large aggregates of filipin binding sites occupy the vast expanses of particle-free areas of debris membranes which accumulate between the photoreceptors and the RPE. These changes in the amount and distribution of filipin binding sites in the dystrophic retina add to the evidence that the disease process involves outer segment as well as RPE membranes and suggest that alterations in cholesterol distribution could contribute to the phagocytic defect.

Animals↗
PubMed1985

Calmodulin-dependent protein phosphatase: immunocytochemical localization in chick retina.

Calmodulin-dependent protein phosphatase, previously called CaM-BP80 or calcineurin, is present in high concentrations in the central nervous system. The level of the phosphatase has been shown by radioimmunoassay to increase during development in the retinas of embryonic and hatching chicks (Tallant, E.A., and W.Y. Cheung, 1983, Biochemistry, 22:3630-3635). The aims of this study are to immunocytochemically localize the phosphatase in developing and mature retinas and to determine if the phosphatase is present in fractions of retinal synaptic membranes and synaptic junctions. Vibratome slices of fixed chick retina and Western blots of detergent-solubilized retinal fractions are both treated sequentially with rabbit primary antisera and goat anti-rabbit Fab fragments conjugated to peroxidase, and then reacted with hydrogen peroxide and diaminobenzidine. The tissue slices are further processed for electron microscopy. This paper demonstrates the presence of peroxidase reaction product in the retina just before synapse formation. In the outer plexiform layer the product is confined to photoreceptor synaptic terminals, whereas in the inner plexiform layer it is present in synaptic terminals of bipolar cells and in dendrites of ganglion cells. In this latter site the product is present postsynaptically at bipolar and amacrine synapses. The phosphatase is detected in Western blots of both synaptic plasma membrane and synaptic junction fractions.

Animals↗
PubMed1985

Lanthanum and freeze-fracture studies of retinal pigment epithelial cell junctions in the streptozotocin diabetic rat.

The blood-retinal barrier breaks down early in diabetes and previous morphological studies suggest that the retinal pigment epithelium (RPE) is the site of this defect. In the present study the electron microscope lanthanum nitrate tracer technique has been used to study RPE cell permeability in the streptozotocin diabetic rat retina. The freeze-fracture technique has been used to study RPE cell tight junction structure as permeability increases. In the lanthanum experiments, RPE cell permeability is normal in control rats and in diabetic rats 3 weeks after the injections. After 8 or 16 weeks of diabetes, however, the RPE cell layer no longer forms a barrier to the tracer and electron dense material is present in the subretinal space, in the apical and basal regions of the RPE cell junctions and intracellularly within the RPE. Freeze-fracture studies of tight junctions during this period show (1) an increase in the complexity of the tight junction network due to an increase in anastomoses between the tight junctions; (2) a change in membrane fracturing properties such that the tight junctional intra-membrane particles adhering to the E-face grooves are more numerous than in the control junctions; (3) no change in the number or size of the tight and gap junctional elements. These results suggest that the blood-retinal barrier breakdown in the diabetic RPE is due to alteration of plasma membrane permeability rather than to a loss of tight junctions.

Animals↗
PubMed1985

Electron microscopy of corneal surface microdiathermy.

Microdiathermy has recently been shown to be effective in the treatment of persistent corneal epithelial recurrent erosion. In order to determine the mechanism of action of microdiathermy on the anterior surface of the cornea, rabbit eyes were treated with microdiathermy and the course of corneal tissue repair studied by electron microscopy. Shortly after treatment, the epithelium is edematous and necrotic and the lamina densa is thickened. Within 24 hours, the epithelium appears healed and some hemidesmosomes are present, but the lamina densa is still thickened. At two weeks the epithelial surface is undulated and protrudes into the stroma in areas where the lamina densa is disrupted. Hemidesmosomes are absent in these regions. Activated fibroblasts are present in superficial stroma. At four weeks following microdiathermy, there is segmental deposition of new lamina densa and a connective tissue zone between the newly deposited lamina densa and the old lamina densa. Hemidesmosomes are present only in areas of newly deposited lamina densa. Between six weeks and three months the epithelial basal surface becomes more uniform with mature hemidesmosomes and the new lamina densa is complete. The old lamina densa remains below it but is no longer present by six months. The mechanism of action for microdiathermy in recurrent erosion is believed to be as follows: in the treated area, epithelium and activated fibroblasts secrete a new connective tissue layer, which provides a suitable substrate to which the epithelium can adhere until it secretes a new lamina densa and hemidesmosome formation can occur.

Animals↗
PubMed1985

Freeze-fracture quantitative comparison of rabbit corneal epithelial and endothelial membranes.

The intramembrane structure of epithelial apical, lateral and basal membranes was compared with that of the endothelium in normal rabbit corneas. The cytoplasmic membrane leaflet (P-face) of apical epithelial membrane displays randomly scattered intramembrane particles over the apical microvilli. Apical endothelial P-face membranes, by comparison, are covered by densely-packed particles. The lateral membranes of superficial epithelial cells display delicate tight junctions, punctate particle aggregates characteristic of desmosomes, and small gap junctions. Background intramembrane particles are randomly distributed and not densely packed. Large gap junctions and fewer desmosomes are present on deeper epithelial cells, including basal cells. By comparison, the endothelial lateral membranes display densely-packed, background particles and numerous triangular-shaped, gap junctional particle aggregates bordered by strands of particles resembling tight junctions. At the apical border, long anastomosing tight junctional strands are present. The basal E-face membranes of both cell layers adjacent to the basement membrane are similar in that they have sparse particles and vesicle fusion sites. Small particle aggregates are present on basal epithelial membranes at sites of hemidesmosomes. Quantitation of particle size and density on P-face apical and lateral membranes of each cell layer reveals that particle density on endothelial membranes is more than twice that on epithelial membranes. Particle sizes are similar on both cell layers and mean diameter ranges from 8.7 to 9.2 nm. The differences in intramembrane particle density and junctional aggregates on epithelial and endothelial membranes reflect membrane constituents associated with functional differences of these two cell layers.

Animals↗
PubMed1984

The distribution of filipin-sterol complexes in photoreceptor synaptic membranes.

The polyene antibiotic filipin, which binds to membrane sterols, has been used to investigate the relative distribution of cholesterol at photoreceptor synaptic junctions in the chick retina. Following anesthesia and aldehyde perfusion fixation, the retina is removed and immersed in fixative solution containing the filipin for 36-48 hours. The retinas are then processed for freeze-fracture. Electron microscopy of freeze-fracture replicas demonstrates that the filipin-sterol complexes are not evident between intramembrane particles of the presynaptic and postsynaptic particle arrays that are present at ribbon and basal junctions. In contrast, the synaptic vesicle fusion zone of ribbon junctions contains large numbers of filipin-sterol complexes which are observed merging with the free margins of the presynaptic particle array. There is a scarcity of such complexes, however, around the free margins of basal junction presynaptic particle arrays. These latter sites do not contain a vesicle fusion zone. Particle-poor areas of membrane that surround postsynaptic particles arrays of ribbon and basal junctions also do not contain filipin binding sites. The nonsynaptic membrane of photoreceptor terminals contains large numbers of filipin-sterol complexes, less tightly packed than in the synaptic vesicle fusion zone. Coated vesicle fusion sites in the presynaptic membrane contain groups of intramembrane particles but the filipin-sterol complexes are excluded from these sites. The observations suggest that synaptic membrane domains which interact with cytoskeletal components, such as clathrin and pre- and postsynaptic densities contain less cholesterol than other domains such as the synaptic vesicle fusion zones.

Animals↗
PubMed1984

Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II. Bovine chymotrypsin A alpha, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates.

The primary subsite specificities of human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin A alpha, and the protease from strain V-8 of Staphylococcus aureus have been mapped with a series of tripeptide thiobenzyl ester substrates of the general formula Boc-Ala-Ala-AA-SBzl, where AA represents one of 13 amino acids. In addition, the effects of a P2 Pro and P4 methoxysuccinyl and succinyl groups were investigated. In an attempt to introduce specificity and/or reactivity into the substrate Boc-Ala-Ala-Leu-SBzl(X), the 4-chloro-, 4-nitro-, and 4-methoxythiobenzyl ester derivatives were studied. Enzymatic hydrolyses of the substrates were measured in the presence of 4,4'-dithiobis(pyridine) or 5,5'-dithiobis(2-nitrobenzoic acid), which provided a highly sensitive assay method for free thiol. The thio esters were excellent substrates for the enzymes tested, and in many cases, the best substrates reported here have kcat/KM values higher than those reported previously. The best substrate for human leukocyte elastase was Boc-Ala-Pro-Nva-SBzl(Cl), which has a kcat/KM of 130 X 10(6) M-1 s-1. A very reactive rat mast cell protease substrate, Boc-Ala-Ala-Leu-SBzl(NO2), was also found. The S. aureus V-8 protease was the most specific enzyme tested since it hydrolyzed only Boc-Ala-Ala-Glu-SBzl. Substituents on the thiobenzyl ester moiety of Boc-Ala-Ala-Leu-SBzl resulted in decreased KM values with human leukocyte elastase and rat mast cell protease I when compared to the unsubstituted derivative.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗