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J G Hollyfield

Publications and source records attributed to J G Hollyfield.

At least 19 recordsLinked to original sources
PubMed2001

SPACRCAN in the developing retina and pineal gland of the rat: spatial and temporal pattern of gene expression and protein synthesis.

SPACRCAN is a hyaluronan-binding proteoglycan that is present in the pineal gland and interphotoreceptor matrix of the retina. Here, we evaluate the pattern of SPACRCAN gene expression and protein appearance during retinal and pineal gland development in the rat. In situ hybridization histochemistry with SPACRCAN riboprobes indicates that hybridization signals are first evident in the retina over developing photoreceptor cells at embryonic day 16 (E16) and in the pineal gland at E21. Immunocytochemistry using a SPACRCAN antibody shows localization of SPACRCAN protein in the developing interphotoreceptor matrix by Postnatal day 5 (P5) and in the pineal gland by P6. These studies suggest that SPACRCAN mRNA expression may occur substantially earlier than the time when SPACRCAN protein is detectable in both the retina and the pineal gland. The period of retinal histogenesis when SPACRCAN is detected first is coincident with the time photoreceptors begin to extend from the outer retinal surface, suggesting that SPACRCAN may participate in the maturation and maintenance of the light-sensitive photoreceptor outer segment.

Age Factors↗
PubMed2001

Interphotoreceptor matrix in the fovea and peripheral retina of the primate Macaca mulatta: distribution and glycoforms of SPACR and SPACRCAN.

SPACR and SPACRCAN localization in the interphotoreceptor matrix (IPM) of the fovea and peripheral retina of Macaca mulatta was established with antibodies to these core proteins and the chondroitin sulfate epitopes and lectin binding properties of these molecules were defined. The IPM of both rods and cones labeled with anti-SPACR, anti-SPACRCAN, anti-Delta Di6S antibodies and wheat germ agglutinin (WGA). Whereas anti-SPACR and anti-SPACRCAN antibodies labeled rod and cone matrix compartments with similar intensity, the Delta Di6S chondroitin antibody labeling was more intense around cones than rods. Peanut lectin (PNA) labeling was present only around cones. No IPM labeling was observed with Delta Di0S-chondroitin or Delta Di4S-chondroitin antibodies. Western blots of undigested IPM extracts showed anti-SPACR immunoreactivity at 150 kDa, colocalizing with the position of WGA and PNA binding. In Western blots of the chondroitinase ABC digested sample and samples double digested with chondroitinase ABC and AC II, anti-SPACR immunoreactivity, WGA and PNA labeling intensity were virtually identical to that in the undigested sample, with prominent staining of the 150 kDa SPACR band. In contrast, anti-SPACRCAN immunoreactivity was not present in the undigested sample, but was evident in both the chondroitinase ABC and double digested samples as a broad band at approximately 230 kDa. Delta Di6S, Delta Di4S, WGA and PNA labeling colocalized with the anti-SPACRCAN immunoreactivity in the chondroitinase ABC digested sample. These findings indicate that SPACR and SPACRCAN are present around cones in the fovea and both rods and cones in the peripheral retina, but that the specific glycoforms of these molecules are different depending on whether present in the cone or rod associated IPM.

Animals↗
PubMed2001

Glycosaminoglycan synthesis and secretion by the retinal pigment epithelium: polarized delivery of hyaluronan from the apical surface.

Hyaluronan and chondroitin sulfate glycosaminoglycan secretion from retinal pigment epithelial cells was established in confluent cultures with high transepithelial resistance. Cell cultures were maintained on Millicell-PCF culture plates, which allow separation of culture medium exposed to apical and basal epithelial surfaces. Following various times in culture, apical and basal culture media were sampled at three day intervals and the glycosaminoglycan content was quantified. Samples were digested with proteinase K to free the glycosaminoglycans from their core proteins, the glycosaminoglycans were ethanol precipitated, and subjected to hyaluronidase SD and chondroitinase ABC digestion to release hyaluronan and chondroitin sulfate disaccharides. Disaccharides were fluorotagged with 2-aminoacridone, separated on polyacrylamide gels and the molar fluorescence in each disaccharide band quantitated. Hyaluronan in the apical medium was significantly higher than in the basal medium (5-12 times) at all recovery intervals (P<0.0001). In contrast, the distribution of unsulfated chondroitin, 4-sulfated chondroitin and 6-sulfated chondroitin disaccharides in apical and basal media was non-polar. Confocal microscopy of cultures probed with a hyaluronan-specific fluorotag established that the HA evident in these cultures is restricted to the apical border of the RPE cultures. Collectively, these data indicate that hyaluronan synthesized by the retinal pigment epithelium is secreted preferentially from the apical surface, suggesting that this tissue is an important source of hyaluronan present in the interphotoreceptor matrix.

Carbohydrates↗
PubMed2000

Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium.

Best vitelliform macular dystrophy is a dominantly inherited, early onset, macular degenerative disease that exhibits some histopathologic similarities to age-related macular degeneration. Although the vitelliform lesion is common in the fundus of individuals with Best disease, diagnosis is based on a reduced ratio of the light peak to dark trough in the electrooculogram. Recently, the VMD2 gene on chromosome 11q13, encoding the protein bestrophin, was identified. The function of bestrophin is unknown. To facilitate studies of bestrophin, we produced both rabbit polyclonal and mouse monoclonal antibodies that proved useful for Western blotting, immunoprecipitation, and immunocytochemistry. To characterize bestrophin, we initially probed the retinal pigment epithelium (RPE)-derived cell lines ARPE-19, D407, and RPE-J. All of the cell lines expressed bestrophin mRNA by reverse transcription-PCR, but not on Western blots. Bestrophin in human RPE partitioned in the detergent phase during Triton X-114 extraction and could be modified by biotin in intact cells, indicative of a plasma membrane localization. Immunocytochemical staining of macaque and porcine eyes indicated that bestrophin is localized at the basolateral plasma membrane of RPE cells. When expressed in RPE-J cells by adenovirus-mediated gene transfer, bestrophin again was determined by confocal microscopy and cell surface biotinylation to be a basolateral plasma membrane protein. The basolateral plasma membrane localization of bestrophin suggests the possibility that bestrophin plays a role in generating the altered electrooculogram of individuals with Best disease.

Amino Acid Sequence↗
PubMed2000

SPACRCAN, a novel human interphotoreceptor matrix hyaluronan-binding proteoglycan synthesized by photoreceptors and pinealocytes.

The interphotoreceptor matrix is a unique extracellular complex occupying the interface between photoreceptors and the retinal pigment epithelium in the fundus of the eye. Because of the putative supportive role in photoreceptor maintenance, it is likely that constituent molecules play key roles in photoreceptor function and may be targets for inherited retinal disease. In this study we identify and characterize SPACRCAN, a novel chondroitin proteoglycan in this matrix. SPACRCAN was cloned from a human retinal cDNA library and the gene localized to chromosome 3q11.2. Analysis of SPACRCAN mRNA and protein revealed that SPACRCAN is expressed exclusively by photoreceptors and pinealocytes. SPACRCAN synthesized by photoreceptors is localized to the interphotoreceptor matrix where it surrounds both rods and cones. The functional protein contains 1160 amino acids with a large central mucin domain, three consensus sites for glycosaminoglycan attachment, two epidermal growth factor-like repeats, a putative hyaluronan-binding motif, and a potential transmembrane domain near the C-terminal. Lectin and Western blotting indicate an M(r) around 400,000 before and 230,000 after chondroitinase ABC digestion. Removal of N- and O-linked oligosaccharides reduces the M(r) to approximately 160,000, suggesting that approximately 60% of the mass of SPACRCAN is carbohydrate. Finally, we demonstrate that SPACRCAN binds hyaluronan and propose that associations between SPACRCAN and hyaluronan may be involved in organization of the insoluble interphotoreceptor matrix, particularly as SPACRCAN is the major proteoglycan present in this matrix.

Adolescent↗
PubMed2000

SPACR in the interphotoreceptor matrix of the mouse retina: molecular, biochemical and immunohistochemical characterization.

Mouse SPACR cDNA was cloned by screening a mouse retina cDNA library using a PCR probe derived from human SPACR cDNA. Mouse SPACR cDNA comprises 3675 bp containing an open reading frame coding for 742 amino acids. Multitissue Northern blot analysis and in situ hybridization studies indicate that SPACR expression is restricted to retinal photoreceptors. The SPACR core protein was identified with Western blotting following SDS-PAGE with a SPACR C-terminal peptide polyclonal antibody and a chondroitin-6-sulfate Deltadisaccharide monoclonal antibody. The 150 kD immunopositive band was isolated, digested with trypsin and the peptides analysed by MALDI mass spectroscopy. Peptide mass mapping confirmed the identity of the 150 kD immunopositive band to be mouse SPACR core protein. Alignment comparisons of the deduced amino acid sequence of mouse and human SPACR show 64% homology. Like SPACR in the human interphotoreceptor matrix, the mouse orthologue contains a large central mucin-like domain flanked by consensus sites for N-linked oligosaccharide attachment, one EGF-like domain and four hyaluronan-binding motifs. Unlike human SPACR, which contains no conventional consensus sites for glycosaminoglycan attachment, mouse SPACR contains three. Recent biochemical studies of human and mouse SPACR protein indicate that this novel interphotoreceptor matrix molecule is a glycoprotein in human and a proteoglycan in the mouse. The presence of consensus sites for glycosaminoglycan attachment in the deduced sequence of mouse SPACR and the absence of these sites in human SPACR provide molecular verification of our biochemical results, suggesting that differences in post-translational modifications of SPACR may be important in SPACR function in foveate and non-foveate retinas.

Amino Acid Sequence↗
PubMed1999

Chondroitin sulfate proteoglycan core proteins in the interphotoreceptor matrix: a comparative study using biochemical and immunohistochemical analysis.

This study characterizes the core proteins of chondroitin sulfate-type glycosaminoglycans located in the interphotoreceptor matrix and establishes the tissue distribution of chondroitin immunoreactivity in human, bovine, mouse and rat retinas. Monoclonal antibodies specific to unsulfated (DeltaDiOS), 4-sulfated (DeltaDi4S) and 6-sulfated (DeltaDi6S) chondroitin were employed. Retinal sections and IPM samples were either (a) digested with chondroitinase ABC to expose antibody specific epitopes, (b) double digested with chondroitinase ABC and chondroitinase AC II to remove specific epitopes, or (c) left undigested to evaluate mimotope labeling. In tissue sections from each species studied, positive immunoreactivity to the DeltaDi6S antibody was present in the IPM surrounding both rods and cones. In human and bovine, DeltaDi6S labeling of the cone matrix compartments was more intense than labeling of the matrix surrounding rods. Intense DeltaDi6S immunoreactivity was present surrounding the foveal cones. In mouse and rat, no differences in labeling intensity of IPM surrounding rod and cone photoreceptors were evident, although labeling of the IPM near the apical surface of the retinal pigment epithelium and around the photoreceptor inner segments was more pronounced than that surrounding the outer segments. All DeltaDi6S antibody labeling was eliminated with chondroitinase AC II digestion. No IPM immunoreactivity in tissue sections was observed when the DeltaDi0S or DeltaDi4S antibodies were used. In Western blots of IPM extracts treated with chondroitinase ABC, prominent DeltaDi6S immunoreactive bands were present at approximately 230 kD and 150 kD in each species studied, with the exception of the human, where the 150 kD component is not a chondroitin proteoglycan. Each of the prominent DeltaDi6S immunoreactive bands showed minor immunoreactivity to the DeltaDi4S antibody. No DeltaDi0S immunoreactivity was noted in Western blots of IPM samples from any species. All immunoreactivity was lost following chondroitinase AC II digestion. These observations document similarities in the electrophoretic mobility of IPM proteoglycan core proteins released following chondroitinase ABC digestion in the four species studied, but reveal pronounced differences in the tissue distribution. Bovine and human IPM show greater concentrations of DeltaDi6S immunoreactivity surrounding cones than rods, whereas rodent tissues show higher concentrations near the retinal pigment epithelium and around the photoreceptor inner segments than around the outer segments. The pattern of distribution of these proteoglycan molecules is highly conserved in these species, suggesting a common role in IPM structure and function.

Adult↗
PubMed1999

TIMP-3 in Bruch's membrane: changes during aging and in age-related macular degeneration.

PURPOSE: To assess the distribution, content, and function of tissue inhibitor of metalloproteinases (TIMP)-3 during aging in normal eyes for comparison with the levels observed in eyes with age-related macular degeneration (AMD). METHODS: Donor tissues analyzed included 36 normal eyes (14-96 years old) and 15 AMD eyes (74 -98 years old). A tissue strip including the fovea was used for immunohistochemistry. Western blot analysis was performed on extracts of the retinal pigment epithelium (RPE)- choroid complex from the posterior part of each eye. Immunoreactivity of TIMP-3 bands in each western blot was densitometrically quantitated. The inhibitory function of TIMP-3 was evaluated with reverse zymography. RESULTS: TIMP-3 was present uniformly across Bruch's membrane in the normal samples. In samples from donors more than 50 years of age, immunostaining was intense. TIMP-3 content ranged from 92 to 1061 ng/cm2 and increased with age (r = 0.66). In AMD eyes, TIMP-3 distribution in Bruch's membrane was abundant in areas of continuous soft drusen but absent in areas below RPE atrophy. TIMP-3 levels in AMD eyes were significantly higher than in age-matched normal eyes (577 versus 877 ng/cm2; P = 0.009). Inhibitory activity correlated well with TIMP-3 content (r = 0.82) and was also significantly higher in AMD eyes than in age-matched normal eyes (P < 0.001). CONCLUSIONS: During normal aging, TIMP-3 content in Bruch's membrane of the macula shows a significant increase. TIMP-3 content in AMD eyes was elevated relative to that of age-matched normal eyes. Higher levels of TIMP-3 may contribute to the thickening of Bruch's membrane observed in AMD.

Adolescent↗
PubMed1998

SPACR, a novel interphotoreceptor matrix glycoprotein in human retina that interacts with hyaluronan.

SPACR (sialoprotein associated with cones and rods), is the major 147-150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998) Glycobiology 8, 997-1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147-150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.

Amino Acid Sequence↗
PubMed1998

Hyaluronan in the interphotoreceptor matrix of the eye: species differences in content, distribution, ligand binding and degradation.

Hyaluronan (HA) distribution in the posterior eye wall from the vitreous through the sclera, with special consideration to localization in the retina and interphotoreceptor matrix (IPM), was evaluated in human, bovine, guinea pig, dog, rat and mouse tissues using a specific probe for HA (bHABC, biotinylated hyaluronan binding complex). The sclera, some regions of the choroid and vitreous body was positive for HA, as was the basal lamina of the retina (inner limiting membrane). bHABC binding was detected in the IPM of all species studied except the mouse. Predigestion with Streptomyces hyaluronidase for 3 hr before bHABC application eliminated binding in the vitreous, choroid, sclera and basal lamina of the retina, but did not eliminate bHABC binding in the IPM. In tissues from all species studied, incubation for 6 hr with hyaluronidase eliminated bHABC binding in the IPM, except for two human samples. In these two human samples, HA specific binding in the IPM persisted even after 24 hr enzyme treatment. bHABC failed to bind to any tissue layer when bHABC was preincubated with hyaluronan oligosaccharides before application. The resistance of the IPM HA to hyaluronidase digestion may reflect extensive coverage of HA binding sites by ligands present in this compartment which hinder enzyme access. The absence of bHABC binding to the IPM when the probe is preincubated with HA oligosaccharides indicates that the binding reflects specific interaction with HA. We conclude that, with the exception of the mouse, HA is a prominent constituent of the IPM, where it may serve to organize the matrix by functioning as a basic scaffold to which other macromolecules in the insoluble IPM are attached.

Adolescent↗
PubMed1998

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis.

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O-glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1-3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.

Carbohydrate Sequence↗
PubMed1997

Unscheduled DNA replication precedes apoptosis of photoreceptors expressing SV40 T antigen.

Expression of simian virus 40 T antigen (Tag) in the rod photoreceptors of transgenic mice leads to cell death that is completed by the end of the third week of postnatal development. To understand the mechanistic link between Tag expression and the death of the expressing photoreceptors, cell cycle activity was followed in a transgenic mouse family that expresses Tag directed by the mouse opsin promoter. Tag-expressing photoreceptors also expressed rhodopsin suggesting that these cells were differentiated. The presence of Tag in the photoreceptors induced the expression of both proliferating cell nuclear antigen (PCNA) and thymidine kinase (TK). The abnormally high levels of PCNA and TK continued until the complete disappearance of the cells expressing Tag. Photoreceptor cell death was also associated with continued DNA synthesis that ceased shortly after postnatal day 16. The specific loss of the rod photoreceptors that re-entered the cell cycle accounted entirely for the loss of photoreceptors from the outer nuclear layer. The antiproliferative nature of the mature retina is directly involved in the apoptotic death of photoreceptors expressing Tag.

Animals↗
PubMed1997

Hyaluronan localization in tissues of the mouse posterior eye wall: absence in the interphotoreceptor matrix.

The distribution of hyaluronan (HA) in the posterior eye wall from the vitreous through the sclera, with special consideration to localization in the retina and interphotoreceptor matrix (IPM), was evaluated in mouse tissues using an HA specific probe (bHABC, biotinylated hyaluronan binding complex). The vitreous body was positive for HA, as was Bruch's membrane, expansive areas within the choroid, sclera and perimysial connective tissue of extraocular muscle. No HA-staining was detected in the IPM or in any other retina layer except for the basal lamina (inner limiting membrane of the retina) which abuts the vitreous. Predigestion of sections with trypsin or chondroitinase ABC before bHABC application did not produce additional HA-staining in the retina or IPM.

Animals↗
PubMed1997

Elevated mRNA expression of brain-derived neurotrophic factor in retinal ganglion cell layer after optic nerve injury.

PURPOSE: Recent studies show that exogenous brain-derived neurotrophic factor (BDNF) can promote retinal ganglion cell survival in vivo and in vitro. BDNF is expressed by a subpopulation of cells in the ganglion cell layer (GCL). To investigate whether endogenous BDNF may play a role in neuronal protection after ganglion cell trauma, BDNF expression in the retina was examined after optic nerve (ON) injury. METHODS: The optic nerve in Sprague-Dawley rats was crushed intraorbitally posterior to the optic disc. For controls, the optic nerve on the opposite side in each animal was similarly exposed but was not crushed. After intervals of 6 hours to 6 weeks, eye tissues were processed for in situ hybridization, Northern blot, and RNase protection assay using radiolabeled rat riboprobes. RESULTS: After ON injury, BDNF expression was significantly elevated in cells restricted to the GCL, and more cells demonstrated expression of BDNF than were observed in the controls. Elevated BDNF expression was first observed at 24 hours, peaked at 48 hours, and declined to the basal level 2 weeks after ON injury. Quantitative analysis showed a fivefold to sixfold increase in the number of BDNF-positive cells and a 54% increase in BDNF signal intensity in individual cells in the GCL 48 hours after ON injury. In control retinas without ON injury, BDNF expression was localized to some cells in the GCL, as was observed in normal eyes without surgery. Northern blot and RNase protection assay demonstrated a 38% elevation in BDNF expression above control levels 48 hours after ON injury. CONCLUSIONS: These results indicate that cells in the GCL can upregulate gene expression of BDNF in response to ganglion cell axonal injury and suggest that endogenous BDNF may contribute to a natural neuroprotective process after ON injury.

Animals↗
PubMed1996

Basic fibroblast growth factor: increased gene expression in inherited and light-induced photoreceptor degeneration.

In a recent study, we reported that bFGF content and mRNA expression levels are elevated in degenerating photoreceptors in the rd mouse retina as compared to the levels in photoreceptors in age matched normal retinas (Gao and Hollyfield, 1995). To determine whether bFGF up-regulation is a general feature of degenerating photoreceptors, bFGF expression was examined with in situ hybridization in several rodent models with retinal degeneration: (a) the normal mouse retina in which cGMP metabolism had been disrupted pharmacologically using an inhibitor of phosphodiesterase or an analog of cGMP, (b) the rds mouse with an inherited photoreceptor degeneration that does not involve alterations in cGMP metabolism, (c) light-damaged BALB/c mice subjected to bright constant light, and (d) light-stressed albino rats subjected to bright cyclic light. In each of these models, we observed dramatic up-regulation of bFGF gene expression in photoreceptors, although the relative levels varied among the different models and followed different temporal patterns. We conclude that increased expression of bFGF in photoreceptors during their degeneration is a general phenomenon occurring in rd, rds, light-damaging and light-stressing conditions. These observations indicate that bFGF can be up-regulated by a variety of photoreceptor insults. We speculate that endogenous bFGF may function as a photoreceptor protection/rescue factor that is activated in response to photoreceptor stress.

Animals↗
PubMed1995

Development of the interphotoreceptor matrix in Xenopus laevis.

Xenopus laevis interphotoreceptor matrix (IPM) contains a relatively aqueous insoluble wheat germ agglutinin (WGA)-binding component containing unidentified sialoglycoconjugates (Wood et al [1984] J. Comp. Neurol. 228:299-307). The appearance of WGA-binding macromolecules in the IPM was assessed during late embryonic stages (32-45) and in retinal rudiment cultures, using lectin cytochemistry and Western blotting techniques. Metabolic labeling of the neural retina versus retinal pigment epithelium (RPE)-choroid of juvenile Xenopus with 35S-MET was also evaluated in vivo and in vitro. Lectin cytochemistry of eyes from developmental stages 32-42 demonstrated distinct WGA-ferritin-binding sites on the developing outer segment membranes and in the IPM compartment. At stages 44-46 extensive WGA-binding domains were present as an extracellular network with other randomly scattered domains near the retinal pigment epithelium. Retinal rudiments from stage 32-33 were isolated and allowed to differentiate in hanging drop culture (Hollyfield and Witkowsky [1974] J. Exp. Zool. 189:357-377) with or without an investing pigment epithelium. Cultures developing with RPE exhibited an elaborate IPM with an anastomosing meshwork of WGA-ferritin binding sites. In the absence of RPE only limited amounts of binding restricted to the immediate vicinity of the developing photoreceptor outer segment membranes was observed. When Western blots were probed with WGA-HRP, stage 32-45 retinas demonstrated a major WGA-binding band of 126 kD. Similar amounts of WGA-binding macromolecules were synthesized in preparations cultured in the presence or absence of the investing RPE. During development the major WGA-binding component is a 126-kD protein. Equivalent synthesis of this protein in the presence and absence of RPE suggests that the PE is not required for synthesis of this 126-kD component. These results suggest that the retina is the primary site of synthesis of the WGA-binding components of the Xenopus IPM, whereas the PE plays a principal role in their assembly and organization.

Animals↗
PubMed1995

Basic fibroblast growth factor in retinal development: differential levels of bFGF expression and content in normal and retinal degeneration (rd) mutant mice.

The spatial and temporal patterns of expression and content of bFGF during postnatal development of the retina were established in C57BL/6J mice. Western blot analysis, using an anti-rodent bFGF antibody, shows multiple molecular weights of 18, 20.5, and 22 kDa of bFGF protein isolated from the adult retina. A bioassay indicates that this putative basic fibroblast growth factor (bFGF) stimulates proliferation of BALB/c 3T3 fibroblasts in a dose-dependent manner identical to an authentic bFGF standard. Immunocytochemistry reveals that bFGF immunoreactivity is located primarily in the immature photoreceptors during postnatal development and is associated with the photoreceptor outer segment/interphotoreceptor matrix complex in the adult retina. bFGF mRNA expression pattern and levels were evaluated using mouse bFGF riboprobes with in situ hybridization and quantitative ribonuclease protection assay. bFGF mRNA expression is not detectable in the retina until Postnatal Day 10 (P10), although high levels of bFGF mRNA signals were consistently observed in astrocytes in the optic disc at all postnatal ages examined. From P10 to the adult stage, bFGF mRNA was localized mainly to the photoreceptor inner segments, and the bFGF mRNA levels were approximately the same at P10 and in the adult retina. The patterns of retinal bFGF expression and content during normal development established above were compared to these parameters in the retina of rd (C57BL/6J rd/rd), a spontaneous mouse mutant in which photoreceptors degenerate shortly after birth. More bFGF immunoreactivity was detected in the outer retina during photoreceptor degeneration than was present in normal photoreceptors at equivalent ages. Densitometry measurements indicate that the level of immunoreactivity is 56% to 1.8-fold higher in rd than in the normal retina between P6 and P10, respectively. This is at least partially due to elevated bFGF mRNA expression in rd retinas during photoreceptor degeneration. In situ hybridization showed more intense bFGF mRNA hybridization signals in rd photoreceptors from P10 to P15, and RNase protection assay demonstrated much higher hybridization signals in rd retinas from P6 to P10 than in the normal retinas at these stages. More bFGF mRNA hybridization signals were also present in some cells in the inner nuclear layer following photoreceptor cell death in the rd retina but were only weakly evident in the inner nuclear layer in the normal adult retina. These results provide the first evidence that a naturally occurring neuronal degeneration is accompanied by elevated expression of bFGF in degenerating neurons prior to cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Age Factors↗