Biomedical subjects

J J Plantner

Publications and source records attributed to J J Plantner.

At least 19 recordsLinked to original sources

PubMed1998

Increase in interphotoreceptor matrix gelatinase A (MMP-2) associated with age-related macular degeneration.

Matrix metalloproteinases have increasingly been shown to be associated with diseases involving neovascularization and/or abnormal cellular migration or proliferation. A number of diseases of this type affect the retina. In this study, the activity of gelatinase A (MMP-2), the most abundant matrix metalloproteinase in IPM (interphoto receptor matrix) and vitreous, was measured with respect to age in normal human donor eyes and compared to donors with age-related macular degeneration. IPM and vitreous were obtained from a total of 88 human donors. Samples for electrophoresis were normalized for protein content and subjected to quantitative gelatin zymography. The zymograms were scanned and then digitized and quantitated using the NIH 'Image' program. There was not a statistically significant change in the level of gelatinase A in IPM or vitreous as a function of age, although a slight downward trend was found in the total gelatinase A activity within the normal population. Likewise, when comparing normal and age-related macular degeneration donors, there was not a significant difference in the gelatinase A level in vitreous or in retina-associated IPM. However, the level of gelatinase A was nearly doubled specifically in retinal pigment epithelium-associated IPM from eyes with age-related macular degeneration [0.99 +/- 0.09 U mg-1 (56) vs 1.71 +/- 0.28 U mg-1 (14) (mean +/- S.E.M. (number), P < 0.0021; 1 unit = 1.0 ng gelatin cleaved h-1). Gelatinase A may be associated with the changes that occur in age-related macular degeneration, especially the neovascularization which accompanies the exudative ('wet') form of the disease.

Aged↗

PubMed1998

Matrix metalloproteinases and metalloproteinase inhibitors in human interphotoreceptor matrix and vitreous.

PURPOSE: We wished to establish which matrix metalloproteinases (MMPs) and metalloproteinase inhibitors (TIMPs) were present in human interphotoreceptor matrix (IPM) and vitreous. METHODS: IPM and vitreous were obtained from postmortem human eyebank eyes. Western immunoblots were probed with antibodies against human MMPs and TIMPs. Assays specific for elastase activity were also performed. RESULTS: Immunoblot analysis indicated the presence of MMP-1 (interstitial collagenase), MMP-2 and MMP-9 (gelatinases A and B), MMP-3 (stromelysin-1) and TIMP-1, -2 and -3 in both IPM and vitreous. MMP-7 (matrilysin) and MMP-12 (metalloelastase) were not found in either IPM or vitreous. CONCLUSIONS: This is the first demonstration of the MMPs and TIMPs in human IPM and of the TIMPs in human vitreous. While these enzymes are most likely involved in normal turnover within the extracellular matrices that surround the neural retina, they may also play a role in a number of retinal diseases, particularly proliferative diabetic retinopathy and age-related macular degeneration.

Aged↗

PubMed1997

Association of matrix metalloproteinases with interphotoreceptor retinoid binding protein.

PURPOSE: Interphotoreceptor retinoid binding protein (IRBP) is one of the major components of the interphotoreceptor matrix (IPM) where it may function in retinoid transport between the photoreceptor cells and the retinal pigment epithelium. In the course of studies of the metalloproteinases (MPs) of the IPM, we found that some MPs remain associated with IRBP through the standard purification scheme. We wished to report this finding as a caution to those working with IRBP prepared from fresh tissue. METHODS: IRBP was prepared from bovine IPM by procedures commonly used for its purification, including ion exchange, concanavalin A affinity and gel filtration chromatographies. The MPs were detected by zymography, both gelatin and casein, run with and without preactivation. RESULTS: Through each step of the purification both gelatinase and, especially, caseinase (stromelysin) activities were associated with IRBP. Inclusion of gelatin affinity chromatography did not totally remove the gelatinases. Much of this activity was latent and was only revealed following fractionation or by preactivation before zymography. Whether the fractionation steps remove an inhibitor or simply provide conditions appropriate for the activation of the latent zymogen forms is not known. CONCLUSIONS: The close association of the MPs and IRBP may suggest a functional role for this complex. As a practical consideration, it is likely that many preparations of IRBP may be contaminated with one or more MPs. We found no evidence for any other class of proteinase. Caution should thus be exercised in using an IRBP preparation purified from fresh tissue. It should be monitored for proteinase activity by zymography and/or prepared or stored in the presence of EDTA or dithiothreitol as much as possible.

Animals↗

PubMed1997

Membrane type-1 matrix metalloproteinase in human ocular tissues.

PURPOSE: Membrane type-1 matrix metalloproteinase (MT1-MMP) (MMP-14) (EC 3.4.24.xx) is involved in the activation of progelatinase A (MMP-2) (EC 3.4.24.24). MMP-2 is present at least in the interphotoreceptor matrix and vitreous. The purpose of this study was to determine the distribution of MT1-MMP, and MMP-2, in human ocular tissues. METHODS: The distribution of MT1-MMP and MMP-2 was investigated in vitreous and in membrane extracts from eye tissues obtained from postmortem human eyebank eyes. Western blot analysis was performed using mouse monoclonal anti-MT1-MMP and anti-MMP-2 antibodies. RESULTS: MT1-MMP was found in sclera, cornea, lens, choroid, retinal pigment epithelium (RPE) and retina. MMP-2 was found in sclera, cornea, choroid, vitreous, RPE and retina but was absent from lens. CONCLUSION: We provide the first evidence for the presence and distribution of membrane-type-1 matrix metalloproteinase in human ocular tissues. MT1-MMP may be responsible for the activation of progelatinase A in many ocular extracellular matrices in a manner similar to that exhibited in other systems.

Animals↗

PubMed1994

Polarized distribution of metalloproteinases in the bovine interphotoreceptor matrix.

Previous studies from this laboratory had indicated that metalloproteolytic activity was present in the interphotoreceptor matrix. This report extends those observations by providing evidence for the presence of multiple forms of metalloproteinase and for their polarized distribution within the interphotoreceptor matrix as determined by zymogram analysis on substrate-loaded gels. Retinal pigment epithelium-associated interphotoreceptor matrix, both unfractionated as well as fractions separated by gel filtration, exhibited bands of proteolytic activity on gelatin-loaded gels at about 70-75 kDa and 90 kDa, possibly due to gelatinases A and B (MMP-2 and MMP-9, respectively). In contrast, no neutral proteolytic activity was seen in retina-associated interphotoreceptor matrix unless it was first fractionated by gel filtration, whereupon a diversity of forms was exhibited, including additional major bands of proteolytic activity at about 150 kDa and 100 kDa, on gelatin-loaded gels, and at about 180 kDa, on casein-loaded gels, along with many minor species. In all cases, all proteinase activity was inhibited by chelating agents. Since these enzymes may be involved in the turnover and remodeling of components present in the interphotoreceptor matrix, many of which are distributed in a compartmentalized fashion, this unequal distribution of metalloproteinases may be correlated with substrate specificity.

Animals↗

PubMed1992

High molecular weight mucin-like glycoproteins of the bovine interphotoreceptor matrix.

A very high molecular weight mucin-like glycoprotein was isolated by gel filtration of interphotoreceptor matrix (IPM) from fresh bovine eyes and purified to apparent homogeneity by cesium chloride/guanidine hydrochloride (GuHCl) equilibrium density gradient centrifugation. Although a molecular weight in excess of 10(7) Da is suggested by gel filtration, the presence of SDS or GuHCl did not alter its elution position, indicating that the large size was not simply due to aggregation. Treatment of this material with disulfide reagents, however, led to a decrease in molecular size. On a relative basis, substantially more of this glycoprotein is present in IPM prepared from retina than from retinal pigment epithelium. While the carbohydrate and amino acid composition are not those of a true 'mucin', the large size and many other properties are quite 'mucin-like'. The carbohydrate composition suggests the presence of both N- and O-glycosidically linked sugar chains. The presence of a mucin-type O-glycosidic linkage is indicated by its susceptibility to alkaline cleavage, with concomitant loss of serine and threonine and increase in 240 nm absorbance; production of a fluorescent product upon reaction with cyanoacetamide; lectin binding properties; and production of N-acetylgalactosaminitol upon alkaline borohydride elimination. This glycoprotein was digested by pronase and trypsin, confirming its protein nature, but was resistant to digestion with chondroitin ABC lyase, hyaluronidase and heparinase, as well as RNAase, indicating that these components were not present to any appreciable extent. ELISA for cartilage keratan sulfate was also negative. Centrifugation in CsCl/GuHCl gradients indicated a density much lower than that of a proteoglycan or nucleic acid as well. In vitro biosynthetic studies suggest that both retina and retinal pigment epithelium may be major sources of material in the IPM. The elution patterns of radioactivity were strikingly similar to the UV elution patterns of IPM. The medium from retinal incubations contained very high molecular weight material which was resistant to enzymes which hydrolyse glycosaminoglycans, suggesting that retina may be the source of this high molecular weight, mucin-like glycoprotein.

Amino Acids↗

PubMed1992

Effect of enzymatic deglycosylation on the regenerability of bovine rhodopsin.

The influence of the carbohydrate groups of rhodopsin on its ability to regenerate upon incubation with 11-cis retinaldehyde after photobleaching was examined. Rhodopsin was deglycosylated enzymatically with peptide-N-glycosidase F (PNGase F). Verification of deglycosylation was established by: (a) SDS-PAGE; (b) carbohydrate compositional analysis using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD); (c) isolation and carbohydrate analysis by HPAEC-PAD and fast atom bombardment-mass spectrometry of the oligosaccharides liberated from rhodopsin; and (d) absence of reactivity with lectins. Deglycosylated rhodopsin, when present either in rod outer segments or after purification, exhibited the same absorption spectrum as the native molecule. After photobleaching, deglycosylated rhodopsin reacted with 11-cis retinaldehyde in a manner similar to the native material, restoring the spectral properties lost after light-exposure. The carbohydrate portion, therefore, was not required for expressing the spectral properties of rhodopsin nor for regeneration of the photobleached visual pigment.

Amidohydrolases↗

PubMed1992

Reductively methylated, tritiated rhodopsin of high specific activity; a convenient sensitive tracer for use in the radioimmunoassay of rhodopsin.

Bovine rhodopsin was subjected to reductive methylation in the dark using formaldehyde and high specific activity sodium borotritide. After purification by gel filtration and affinity chromatography on Concanavalin A-Sepharose, the product retained its immunoreactive properties. [3H]-Reductively methylated rhodopsin (specific activity, 32 Ci/mmole) was suitable for use in radioimmunoassays for rhodopsin, having many advantages over radioiodinated rhodopsin for this purpose. The site of the reductive methylation was shown to be the non-active site lysines with the production of tritiated N-epsilon-dimethyllysine and tritiated N-epsilon-methyllysine in a molar ratio of about 1.3:1, respectively. In terms of stability, ease of preparation, and specificity, tritiated, reductively methylated rhodopsin presents itself as a preferable ligand to radioiodinated rhodopsin in many applications, such as the radioimmunoassay.

Animals↗

PubMed1992

The presence of neutral metalloproteolytic activity and metalloproteinase inhibitors in the interphotoreceptor matrix.

Neutral proteolytic activity, having a pH optimum of about 7, was present in the high molecular weight fraction of bovine interphotoreceptor matrix (IPM) separated by gel filtration on Sephacryl S-500. The enzyme(s) was active toward a number of exogenous substrates, including albumin, Azocoll, and gelatin. However, it was inactive toward a synthetic substrate for bacterial collagenase. Proteolytic activity was proportional to protein; however, the time course of the reaction was nonlinear, suggesting that "activation" of a precursor form might be necessary. Of a number of specific inhibitors tested, those directed toward metalloproteinases (1,10-phenanthroline greater than EDTA greater than EGTA) proved most effective. While activity was also inhibited by sulfhydryl reagents and dithiothreitol, inhibitors specific for cysteine proteinases were ineffective. Higher specific activity was present in IPM obtained from retinal pigment epithelium (RPE) than from retina. An endogenous proteinase inhibitor(s) was also found in IPM from both RPE and retina. It was effective against the endogenous metalloproteolytic activity of IPM and also against thermolysin, but not against trypsin or papain. Fractionation of IPM on Sephacryl S-500 revealed a broad peak of inhibitory activity at molecular weights of less than 10(5) daltons. This is the first report of the presence of neutral proteolytic activity and metalloproteinase inhibitor(s) in bovine IPM. These materials may function in concert to maintain the proper level of various components within this matrix.

Animals↗

PubMed1991

A microassay for proteolytic activity.

A quantitative procedure for measuring proteolytic activity, utilizing azoalbumin as substrate, has been developed for use in microtiter plates. An enzyme-linked immunosorbent assay reader is used to measure absorbance. The procedure is sensitive, as well as being both rapid and economical. It is particularly convenient for measuring large numbers of samples, such as fractions from column chromatography.

Chromatography, Gel↗

PubMed1991

Enzymatic deglycosylation of bovine rhodopsin.

We have investigated the action of three endo N-acetylglucosaminidases on rhodopsin. The oligosaccharide chains of native and denatured opsin and rhodopsin, both solubilized and membrane-bound, were shown to be cleaved by endohexosaminidase H, endohexosaminidase F, and peptide-N-glycosidase F (PNGase F) as revealed by SDS-PAGE. These enzymes were shown to be free of protease activity. Under correct conditions, the endoglycosidases could release one or both carbohydrate chains. Rhodopsin and opsin at concentrations between 2 and 65 nmol ml-1 were cleaved, with more complete deglycosylation occurring at the higher concentrations.

Amidohydrolases↗

PubMed1988

The dolichol pathway in the retina: oligosaccharide-lipid biosynthesis.

The formation of the oligosaccharide-lipid intermediates of the dolichol pathway by the bovine retina was investigated. Intact retinas were incubated in vitro for various periods of time in the presence of a variety of radioactive sugars (2-[3H]mannose, 6-[3H]glucose, 1-[3H]galactose, 1-[14C]glucosamine) using incubation conditions which have been shown previously to support the glycosylation of rhodopsin. The oligosaccharide-lipids were isolated and partially purified by DEAE cellulose chromatography. After mild acid hydrolysis and reduction, the oligosaccharides were analysed by HPLC. Further identification was obtained by chemical means and after digestion of the oligosaccharides with alpha-mannosidase and endohexosaminidase H. The full array of oligosaccharide-lipids which have been observed in other tissues were detected in the bovine retina, although some striking differences were seen in their relative distribution. Although short-term incubations (up to 15 min) indicated that the major species was the fully glucosylated oligosaccharide-lipid (Glc3Man9GlcNAc2), with longer incubation times the non-glucose-containing intermediate, Man9GlcNAc2, became the predominant species. Since glycerol was the carbon source for these incubations, the possibility was investigated that glucose starvation may have been the basis for this phenomenon, as has been reported in other tissues. It was established that this was not the case. Experiments carried out in the presence of castanospermine and bromoconduritol indicated that alpha-glucosidase activity in the retina may have resulted in the accumulation of the unglucosylated oligosaccharide-lipids. The formation of oligosaccharide-lipid intermediates by cells of the retinal pigment epithelium from the embryonic chick, maintained in cell culture, was also examined. In contrast to the bovine retina, the major species present were the glucose-containing intermediates, similar to other tissues.

Animals↗

PubMed1988

The rhodopsin content of the human eye.

The content of rhodopsin in the human retina was determined using a radioimmunoassay which measures both rhodopsin and opsin. A value of 0.5 nmole of rod visual pigment per mg of extractable protein was obtained, corresponding to 3.94 +/- 0.17 (mean +/- SEM, n = 42) nmole per eye. This value is slightly higher than those previously reported which were based on spectral determinations measuring only native rhodopsin. No significant difference was seen in the population studied with respect to age or sex. Human rhodopsin was partially purified by affinity chromatography on concanavalin A, and some of its properties were studied. A comparison of the immunological reactivity of human, bovine, rat and chicken rhodopsin indicated similarity among the three mammalian species, while the chicken was some 50 fold less reactive.

Adolescent↗

PubMed1988

Biogenesis and content of rhodopsin in the retina of the chick during development.

Developmental aspects of the formation of rhodopsin in the chick were investigated. The content of rhodopsin was measured in the retina of the developing chick embryo, newly hatched and adult chickens by both spectral and immunological procedures. Rhodopsin was first detected spectrally at day 18 (stage 43) with the level increasing about 6 fold through hatching. The concentration in the adult retina was about one half that present in the 1 day old chick. By the more sensitive procedure of radioimmunoassay (RIA), rhodopsin was detected as early as day 12 (stage 37), but was undetectable in the 8 day old embryo. The rhodopsin concentration remained low and relatively constant until day 17 (stage 42), at which point it increased rapidly up to hatching. Retina from the newly-hatched chick had a rhodopsin concentration some 30 fold higher than the 16 day old embryo (stage 41). The results of RIA indicate that the biosynthesis of rhodopsin may significantly precede the morphological appearance of outer segments.

Animals↗

PubMed1987

The influence of carbohydrates on the binding of rod outer-segment (ROS) disc membranes and intact ROS by the cells of the retinal pigment epithelium of the embryonic chick.

The role of carbohydrates in mediating the interaction of rhodopsin-containing membranes with retinal pigment epithelium (RPE) cells was investigated by studying the influence of various monosaccharides on their binding by RPE cells of the embryonic chick maintained in cell culture. Rod outer-segment (ROS) disc membranes were selected as a model rhodopsin-containing membrane system for these studies in view of their high concentration of rhodopsin and the relative purity with which they can be isolated. Disc membranes, frozen and thawed in order to expose the carbohydrate groups of rhodopsin which are oriented intraluminally in situ, were incubated with monolayers of RPE cells under various conditions, and the binding of the membranes by the cells was quantitated by radioimmunoassay for rhodopsin. Cell-membrane association was also verified by indirect immunofluorescence microscopy. The surface accessibility of the sugars in frozen-thawed discs was verified by succinyl concanavalin A-binding studies. From 15- to 20-fold increase in carbohydrate-reactive sites was obtained after freezing and thawing the discs. The RPE cell-membrane binding process was saturable, and time- and temperature-dependent. By means of competition studies carried out in the presence of high concentrations of various monosaccharides, and also by comparing the binding of disc membranes whose carbohydrate groups were either exposed (frozen-thawed) on the surface or inaccessible (native), it was concluded that the carbohydrates of rhodopsin, mannose and N-acetylglucosamine, were not involved in the interaction with the RPE. The possibility was also examined that enzymatically galactosylated rhodopsin might serve as a site for recognition by the RPE cell. The binding of ROS disc membranes modified in this manner was not enhanced, indicating that the presence of galactose groups on rhodopsin did not serve as a site for recognition by the RPE. The influence of monosaccharides on the binding of intact ROS by the RPE cells was also investigated. Similar to the results with the disc membranes, the process was not blocked by the presence in the incubation medium of high concentrations (up to 30,000-fold higher than that of rhodopsin) of mannose or GlcNAc, as with the disc membranes, or by glucose or galactose. Thus, from these studies it is concluded that a lectin-like carbohydrate-recognition process may not be involved in the interaction between rhodopsin-containing membranes and the RPE cells.

Animals↗

PubMed1987

Are sugars involved in the binding of rhodopsin-membranes by the retinal pigment epithelium?

The binding by the retinal pigment epithelium (RPE) cells of the embryonic chick, in culture, of recombinant membranes composed of phosphatidylcholine and bovine rhodopsin (rhodopsin-liposomes [RL]) was investigated to explore the influence that the carbohydrate groups of rhodopsin might have on this process. The reaction was quantitated by measuring the amount of RL associated with the cells using a radioimmunoassay for rhodopsin. The process was saturated at about 8 microM RL (calculated as rhodopsin-equivalents), and a two- to threefold greater rate and extent of binding was observed at 37 degrees C as compared with 4 degrees C. The presence of up to 30 X 10(3)-fold molar excess over RL of mannose and N-acetylglucosamine, the sugars present on rhodopsin, as well as other saccharides, had little or no effect on the binding. These observation indicate that lectin-like recognition involving the sugar groups of rhodopsin is not involved in the binding of rhodopsin-containing membranes by the RPE cells. In contrast, exogenously added concanavalin A and wheat germ agglutinin enhanced the binding, an effect which was blocked by appropriate sugars.

Acetylglucosamine↗

PubMed1986

Rod outer segment lipids in vitamin A-adequate and -deficient rats.

Weanling albino rats were fed a vitamin-A-adequate diet or vitamin-A-deficient diet and maintained in a cyclic light or dark environment for up to 14 weeks. One half of the rats were supplemented with additional dietary linolenic acid in the form of linseed oil. The lipid composition and rhodopsin-opsin contents of isolated rod outer segments were determined after 6-7 weeks or 12-14 weeks on diet. This study shows that feeding rats a standard vitamin A-adequate or -deficient diet results in an age-dependent loss of omega three docosahexaenoic acid and a concomitant increase in omega six docosapentanoic acid in the rod outer segments. The loss of docosahexaenoate appears to be caused by insufficient dietary omega three fatty acids. The increase in omega six docosapentanoic acid appears to arise from the high concentration of linoleic acid in standard diets containing either cottonseed, or peanut oil or supplemental corn oil. Feeding rats diets supplemented with linseed oil, however, results in a rod outer-segment lipid profile which is the same as for chow-fed animals. The same effects were seen in the fatty-acid profile of lipids from liver, although the content of polyunsaturates was much lower than in rod outer segments. Vitamin A deficiency, itself, does not lead to changes in the fatty-acid composition of either the rod outer segments or liver. After 6-7 weeks on A+ or A- diet, rhodopsin levels were, as expected, higher in dark-reared rats than in cyclic-light animals. Although the rhodopsin levels in dark-reared vitamin A-adequate rats were significantly higher than in vitamin A-deficient animals, measurements of the lipid to opsin ratio of rod outer segments indicate that the rods of vitamin A-deficient rats are not markedly different than those of vitamin A-adequate rats. It is concluded that these diets may be useful in providing a means for evaluating the role of docosahexaenoic acid in visual cell death from damaging light.

Age Factors↗

PubMed1984

Immunoreactivity of rhodopsin and opsin.

An examination by a radioimmunoassay of the relative affinity of opsin and rhodopsin for rabbit antibody raised against bovine rhodopsin revealed that opsin was the preferred antigen. About 10-fold greater amounts of rhodopsin than opsin were required to achieve 50% inhibition of binding of 125I-labeled ligand in the RIA. Opsin was more reactive when examined in the light or dark, compared to rhodopsin incubated in the dark. Mixtures of opsin and rhodopsin (prepared by partial bleaching of rhodopsin or synthetic mixtures) exhibited increased reactivity with increasing mole fraction of opsin. This response was nonlinear, with small increases in opsin producing relatively large increases in reactivity. A partial fractionation of the antibody into two groups showing differential reactivities toward opsin and rhodopsin was achieved by affinity chromatography on opsin-Sepharose. However, with both groups, opsin was still the preferred antigen. Scatchard analysis of 125I-labeled rhodopsin and opsin produced nonlinear plots, indicating the presence of multiple species of antibody. The affinities and binding capacities were similar for both labeled antigens. In competitive binding studies, the antibody showed a strong preference for either labeled ligand (rhodopsin or opsin) as compared to the unlabeled material. These latter observations indicate that altering rhodopsin either by bleaching or iodination produced changes in the relative immunoreactivity of the molecule.

Animals↗