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Biomedical subjects

G J Chader

Publications and source records attributed to G J Chader.

At least 37 records · Page 2Linked to original sources
PubMed1995

Isolation of candidate genes for macular degeneration using an improved solid-phase subtractive cloning technique.

An improved solid-phase subtraction procedure was developed to generate a readily amplifiable library of short cDNA fragments highly enriched in the macula (target) versus the peripheral region (driver) of the monkey neural retina. The generated clones were sequenced and 63 were analyzed by northern blotting using total RNA from the monkey macula and peripheral retina. The results indicate that 32% are highly enriched in macula, 36% are below the limits of detection and 32% are not enriched. No clones were found which were enriched in the peripheral retina. Our technique is therefore successful in identifying novel cDNAs enriched in the macula area of the neural retina that may represent potential candidate genes for hereditary ocular diseases. It should thus be useful in other situations where subtle differences in expression between cell types or tissue areas need to be analyzed.

Animals↗
PubMed1995

Structural organization and expression of the human phosphatidylinositol-specific phospholipase C beta-3 gene.

We have cloned and fully sequenced the phospholipase C beta-3 (PLC beta-3) gene. The gene spans approx. 17 kb and consists of 31 exons and 30 introns. All intron-exon junctions obey the GT/AG rule. The gene is highly expressed in several human tissues including retina, brain and kidney; PLC beta-3 mRNA is detected at a much lower level in liver. Because of its importance in signal transduction, its chromosomal localization and its high expression in CNS and other tissues, the PLC beta-3 gene is a candidate in several human genetic diseases which, with the present genomic sequence, can now be fully examined.

Animals↗
PubMed1995

Induction of heme oxygenase 1 in the retina by intense visible light: suppression by the antioxidant dimethylthiourea.

The effect of intense visible light (light damage) on the expression of heme oxygenase 1 (HO-1), a protein induced by oxidative stress, was investigated in the rat retina. A sensitive reverse transcription-PCR assay demonstrated the expression of mRNA for HO-1 as well as HO-2, the noninducible HO form, in the normal retina. As analyzed by Northern blotting, however, HO-1 mRNA was barely detectable under normal circumstances. After exposure to intense visible light, retinas had markedly higher HO-1 mRNA levels than unexposed controls, with increases up to 52- and 98-fold at 12 and 24 hr of exposure, respectively. Intense light exposure also resulted in an increase in HO-1 protein. In contrast, no appreciable change in HO-2 mRNA or protein was observed. The increase in HO-1 message was more pronounced in rats previously reared in the dark than in those reared in a weak cyclic-light environment. A marked decrease from the high level of HO-1 mRNA induced by light insult was observed when the animals were allowed to recover in the dark for 24 hr after light exposure. Most important, treatment of animals with 1,3-dimethylthiourea, a synthetic antioxidant, prior to light exposure effectively blocked the increase in HO-1 mRNA. Thus, HO-1 is a sensitive marker for assessing light-induced insult in the retina. Since increased expression of HO-1 is thought to be a cellular defense against oxidative damage, its expression may play an important role in protecting the retina against light damage.

Amino Acid Sequence↗
PubMed1995

Identification of pigment epithelium-derived factor in the interphotoreceptor matrix of bovine eyes.

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein and a member of the serine protease inhibitor superfamily. Here we describe the identification of PEDF in bovine eyes and optimization of its purification from this natural source. We have developed a polyclonal antibody to recombinant human PEDF, Ab-rPEDF, that immunoreacts in a specific, sensitive, and linear fashion with PEDF protein, and furthermore, blocks its neurotrophic activity. We show that Ab-rPEDF specifically recognizes a 49,500-M(r) polypeptide on Western transfers of a wash of the extracellular matrix between the retinal pigment epithelium and the neural retina-termed interphotoreceptor matrix (IPM). PEDF is present as approximately 1% of total soluble IPM protein. Starting with an IPM wash, PEDF protein is purified 164-fold to near homogeneity by ammonium sulfate fractionation and cation-exchange chromatography, with a recovery of 47%. The highly purified protein has an apparent M(r) of 49,500 +/- 1,500 as assessed by SDS-polyacrylamide gel electrophoresis, and a native pI of 7.0-7.7. It elutes as a single peak on gel-filtration chromatography with a retention time immediately behind that of ovalbumin (43,000 M(r)). N-glycosidase treatment indicates that each PEDF molecule has a 5% carbohydrate content attached to internal asparagine residue(s). Amino terminal sequence of the purified PEDF reveals removal of an amino-terminal peptide region for the mature protein. Purified PEDF has neurotrophic activity on human retinoblastoma cells, as previously observed for IPM. The neurotrophic activities of both PEDF and IPM are blocked by antiserum Ab-rPEDF. Altogether, PEDF is present in the bovine IPM as a soluble, extracellular, monomeric glycoprotein that by itself confers neurotrophic activity to the IPM. Thus, native PEDF isolated and purified as described here should prove useful for biochemical studies as well as other approaches.

Amino Acid Sequence↗
PubMed1995

Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture.

Pigment epithelium-derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y-79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose-response relationship, with a larger effect in chemically defined medium than in serum-containing medium [ED50 = 30 ng/ml (0.70 nM) in chemically defined medium and 100 ng/ml (2.3 nM) in serum-containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme-linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.

Animals↗
PubMed1995

Differences in arylalkylamine N-acetyltransferase activity between inflammatory disease-susceptible Lewis and -resistant Fischer rats.

Lewis (LEW/N) and Fischer (F344/N) rats are histocompatible inbred strains characterized, respectively, by susceptibility and resistance to inflammatory disease. The susceptibility of LEW/N rats to inflammation has been associated with deficient corticotropin-releasing hormone (CRH), ACTH, and corticosterone responses to inflammatory stimuli, specifically attributed to a global impairment in hypothalamic CRH neuron function. In contrast to the LEW/N rats, F344/N rats demonstrate an intact hypothalamic-pituitary-adrenal (HPA) axis. Melatonin, a neurohormone initially isolated in the pineal gland, has been implicated with inhibition of the HPA axis. To investigate melatonin synthesis and secretion in LEW/N and F344/N rats, we examined the diurnal activity of pineal arylalkylamine N-acetyltransferase (NAT1), the rate-limiting enzyme in melatonin biosynthesis, which demonstrates circadian rhythmicity, as well as the diurnal levels of serum melatonin, in both strains. Arylamine N-acetyltransferase (NAT2), a related enzyme activity, thought not to be regulated in a circadian manner, was examined as a control of NAT1 activity. Pineal NAT1 activity peak was observed later and reached significantly higher levels in LEW/N than in F344/N rats. Serum melatonin levels reflected the circadian pattern of the NAT1 activity, without, however, showing any quantitative differences between the two strains. Time-course of pineal NAT1 activity response to beta-adrenergic stimulation was parallel in the two rat strains, whereas the magnitude of the response as greater in LEW/N than in F344/N rats. No circadian or major quantitative differences in NAT2 activity were found between the two strains. Size-exclusion HPLC chromatograms of NAT1 activity revealed similar patterns in both rat strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Cloning and characterization of a chick embryo cDNA and gene for IGF-binding protein-2.

We have isolated and characterized a cDNA for IGF-binding protein-2 (IGFBP-2) and its gene from the chick embryo. Using primers from a conserved region of the mammalian IGFBP-2 sequence, a cDNA clone (1.6 kb) was isolated from an embryonic day-18 chick retina cDNA library. Although the clone was truncated at the 5' end, the complete coding sequence was obtained from 5' rapid amplification of cDNA ends and genomic sequencing. The open reading frame encoded a 311 amino acid precursor protein which contains a putative 36 residue signal peptide. The mature 275 amino acid protein had a predicted M(r) of 33,500 and exhibited 71, 68, 68 and 66% identity to rat, bovine, ovine and human IGFBP-2 cDNA respectively, with conservation of all 18 cysteines. The cDNA contained an RGD peptide but lacked a putative ATP-binding motif. A single transcript of approximately 2.3 kb was present in embryonic day-15 eye, brain, skeletal muscle, heart and intestine, but was virtually absent from embryonic day-15 liver. The chicken IGFBP-2 gene spanned approximately 38 kb, consisted of four exons, and was similarly organized to that of the rat and human. Southern blot analysis of chicken genomic DNA suggested that it is encoded by a single gene. The sequence information from the avian IGFBP-2 should be of value in examining the role of IGFBP-2 in vertebrate development.

Amino Acid Sequence↗
PubMed1995

Identification of arylamine N-acetyltransferase activity in the bovine lens.

Arylamine N-acetyltransferase (NAT) activity was identified and partially characterized in the bovine lens. According to size-exclusion HPLC, the molecular mass of the arylamine NAT is approximately 30-kDa. Based upon substrate specificity analysis, it is best described as an arylamine NAT which has some ability to N-acetylate arylalkylamines. This arylamine NAT acetylates para-aminobenzoic acid thereby demonstrating a monomorphic pattern of N-acetylation. It demonstrates low sensitivity to methotrexate inhibition as indicated by the relatively high IC50 value (470 microM). NAT could be involved in lenticular detoxification of both endogenous amines and exogenous drugs.

4-Aminobenzoic Acid↗
PubMed1995

Vitreous and aqueous humors contain a latent proteinase activity that abolishes IGF binding to specific IGF binding proteins.

Several distinct insulin-like growth factor binding proteins (IGFBPs) are present in tissues and fluids of the developing and adult eye. However, the mechanism(s) involved in the regulation of ocular IGFBP levels is unknown. We have now identified an endogenous factor in vitreous and aqueous humors that, when activated by sodium dodecyl sulfate (SDS), abolishes the capacity of specific low molecular weight IGFBPs (i.e. 24-30 kDa) to bind IGF as assessed by western ligand blotting. In contrast, IGF binding to the 46 and 32 kDa IGFBPs (IGFBP-3 and IGFBP-2 respectively) is not affected by the SDS-activated inhibitory factor (IF). Maximal activation of the IF occurs at an SDS concentration of approximately 0.015%. Incubations in the presence of the serine-proteinase inhibitor aprotinin result in marked inhibition of IF activity. Preliminary characterization by ultrafiltration suggests that the IF is large (< 100 kDa) and/or that it is present in a complex. The finding of a factor, most likely a serine proteinase, that specifically abolishes IGF binding to low molecular weight IGFBPs suggests a mechanism for regulating the levels of these IGFBPs and thus the functional activities of IGFs in ocular fluids under normal and/or pathological conditions.

Animals↗
PubMed1995

Identification and partial characterization of a proteinase specific for insulin-like growth factor binding protein-3 in aqueous and vitreous humors.

The IGFs (-I and -II) are normally found in serum and other extracellular fluids complexed to specific binding proteins (IGFBPs). While several IGFBPs have been identified in vitreous and aqueous humors, the major serum carrier of IGF, IGFBP-3, is notably absent from these fluids. To determine if this paucity could be due to an IGFBP-3 proteinase (IGFBP-3ase), samples of bovine vitreous or aqueous humor were mixed with serum and incubated at 37 degrees C for 4 h followed by western ligand blotting. In these experiments, a distinct loss of the 46 kDa band representing IGFBP-3 was observed while other bands present at 35, 28 and 25 kDa were unaltered. The IGFBP-3ase activity is temperature sensitive, has a pH optimum of about 8.0 and is inhibited by EDTA. Acid treatment of serum to remove endogenously bound IGF does not affect the specificity or activity of the IGFBP-3 proteinase. Size exclusion chromatography of bovine aqueous indicates an approximate molecular weight of 260 kDa. Incubation of recombinant IGFBP-3 or serum with partially-purified IGFBP-3ase results in the appearance of low molecular weight fragments of approximately 30 kDa. These fragments are undetectable by western ligand blotting but are readily visualized using an IGFBP-3 specific antibody. Comparison of normal and diabetic vitreous humor reveals the presence of an increased amount of IGFBP-3 proteolytic fragments in the diabetic as compared to control. These findings indicate the presence of a IGFBP-3 proteinase in aqueous and vitreous humors that may be important in regulating ocular homeostasis.

Aged↗
PubMed1995

Differential temporal and spatial expression of insulin-like growth factor binding protein-2 in developing chick ocular tissues.

PURPOSE: To determine the developmental expression and localization of mRNA for insulin-like growth factor binding protein-2 (IGFBP-2), a major binding protein of IGF-I and IGF-II, in ocular tissues of the embryonic and early posthatched chick. METHODS: In situ hybridization and northern blot analysis were used to analyze the cellular origin and relative expression of IGFBP-2 mRNA in ocular tissues. RESULTS: Wholemount in situ hybridization reveals that, as early as 3.5 days of embryonic development (E3.5), IGFBP-2 mRNA is already expressed in many areas of the embryo, including surface ectoderm, certain regions of the brain, pharyngeal clefts, somites, and limb buds. In the eye, IGFBP-2 mRNA is expressed only in the presumptive corneal epithelium at this time. By E6, IGFBP-2 mRNA expression is present in both the corneal epithelium and endothelium. By E12, IGFBP-2 mRNA is detected clearly in the corneal stroma as well as in several other ocular structures, such as the sclera, eyelid, and ciliary body. In the neural retina, a low, diffuse expression of IGFBP-2 mRNA is found at E6, which becomes more localized to the nuclear layers by E12. Northern blot analysis confirms that a high level of IGFBP-2 expression is present in the cornea and sclera by E8 to E12. A high level of IGFBP-2 mRNA expression, however, is not observed in the retina until E18. At posthatch day 2 (P2), northern blot analyses of ocular tissues reveal that the cornea contains the highest ocular level of IGFBP-2 mRNA expression, a value equal to that of brain and liver. CONCLUSIONS: The early appearance, along with differential temporal and spatial expression of IGFBP-2 mRNA in developing ocular tissues, suggests a role for IGFBP-2 in the regulation of growth and differentiation of several ocular tissues, including the cornea, sclera, and retina.

Aging↗
PubMed1995

Expression, secretion, and age-related downregulation of pigment epithelium-derived factor, a serpin with neurotrophic activity.

Retinal pigment epithelial (RPE) cells form a functional complex with photoreceptor neurons of the retina, interacting through the interphotoreceptor matrix (IPM). We now provide evidence that the gene for pigment epithelium-derived factor (PEDF), a protein possessing neurotrophic and neuronal-survival activities, is highly expressed by both fetal and young adult RPE cells. PEDF mRNA is present in RPE cells of the human eye at 17 weeks of gestation, demonstrating its potential for action in vivo during early retinal development. The PEDF protein is secreted in vivo where it constitutes a part of the fetal and adult IPM surrounding photoreceptor outer segments. A polyclonal PEDF antibody recognizes at least four isoforms of secreted human and bovine PEDF by two dimensional gel analysis, and detects a similar 50 kDa protein in the IPM of several other vertebrate species. Within soluble extracts of RPE cells, however, where little, if any, of the 50 kDa species can be detected, an immunoreactive 36 kDa protein is observed by Western blot analysis. By immunofluorescence, PEDF is localized intracellularly in association with the nucleus, presumptive secretory granules, and cytoskeletal elements of cultured RPE cells with PEDF and actin antibodies colocalizing to the same cytoskeletal structures. During initial stages of attachment, PEDF and actin also concentrate at the tips of pseudopods extended by the cultured RPE cells. However, with successive passages, synthesis, and secretion of the PEDF protein as well as transcription of its mRNA decrease and are lost by about 10 passages. In parallel, cultured RPE cells lose their proliferative potential and change from an epithelial-like morphology in early passages to a more fibroblast-like appearance by about the 10th passage. PEDF is thus apparently present intracellularly and extracellularly in both fetal and early adult periods where it could be involved in cellular differentiation and survival and with its loss, in the onset of senescence.

Aging↗
PubMed1995

Soluble-binding proteins for docosahexaenoic acid are present in neural retina.

PURPOSE: To determine if soluble binding proteins (BP) for fatty acids (FA), particularly docosahexaenoic acid (DHA), could be identified in the cytosol of rat and bovine retinas under in vitro and in vivo conditions. METHODS: In vitro, cytosol fractions from normal bovine and rat retinas were delipidated and incubated with [14C]-DHA with or without a number of competing fatty acids. After crosslinking bound FA to BP, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioactivity determined in each fraction. For in vivo experiments, rats received [14C]-DHA by gavage. At selected periods after ingestion, retinas were collected and cytosolic fractions were prepared from each. These were crosslinked and subjected to SDS-PAGE, and radioactivity was determined in each fraction. RESULTS: In vitro, several peaks of radioactivity (approximately 13, 20, 32, 43-45, 50, 63, and 94-105 kd) were found that exhibited [14C]-DHA binding. Relative specificity of binding was assessed by blocking of the [14C]-DHA binding with unlabeled DHA and by competition experiments with other FAs. After in vivo ingestion of [14C]-DHA, a large peak of radioactivity was observed at 43 kd by 4 hours. At 6 hours, this peak decreased and, after 24 hours, it approached baseline. CONCLUSIONS: These findings demonstrate that there is a discrete grouping of proteins in retinal cytosol capable of binding DHA and other FA. Although the identities of these proteins have yet to be determined, the group may include a member of the 12- to 15-kd group of small fatty acid binding proteins (FABP). In particular, a unique 43-kd binding peak could play a major role in the uptake, binding, or both of DHA by the retina in vivo.

Animals↗
PubMed1995

Neuron-specific enolase: a neuronal survival factor in the retinal extracellular matrix?

To identify soluble proteins of the retinal interphotoreceptor matrix (IPM), we isolated IPM from the bovine eye by gentle lavage and subjected it to SDS-PAGE. In the resultant gel, a 46 kDa band was particularly prominent and appeared to be a single protein. This protein was electroblotted to nitrocellulose membrane, digested with trypsin, and selected peptides were isolated by HPLC and subjected to Edman microsequencing. The amino acid sequences of the peptides were found to be virtually identical to that of human neuron-specific enolase (NSE). A monoclonal antibody specific for human NSE confirmed the presence of this enzyme in the bovine IPM by both Western blotting and immunocytochemical analysis. Immunofluorescence microscopy demonstrated that NSE is mainly localized to the basal domain of the IPM surrounding photoreceptor cells but is also prominent in the inner segments of the cone photoreceptor neurons. When NSE was added to cultures of human retinoblastoma cells, no effect on morphology was observed. However, a positive effect on cell growth and/or survival was readily apparent. It thus seems that not only is NSE a significant component of the retinal extracellular matrix, but that it could function as a survival (neuronotrophic) factor for photoreceptor neurons.

Amino Acid Sequence↗
PubMed1994

Structural analysis of the human hydroxyindole-O-methyltransferase gene. Presence of two distinct promoters.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the metabolic pathway that synthesizes the hormone melatonin. We have found HIOMT mRNA present in small amounts in human retina and in relatively high abundance in the pineal gland. Two distinct 5' ends were found in human retina using a solid-phase 5'-rapid amplification of cDNA ends technique. The two 5' regions appear to originate from two distinct putative promoters. Although many similarities exist between the two promoters, they contain distinctive elements. Putative promoter A, for example, contains a recently discovered photoreceptor-conserved element (PCE-1, CAATTAAG) at -27 not found in promoter B, while promoter B contains an Ap1 site (ATGAGTCAA) at -166 and an octamer site (ATGCAAT) at -59 not found in promoter A. The HIOMT messages are also alternatively spliced in between exons 6 and 8, generating three distinct messages. One of the alternatively spliced messages contains a line-1 repetitive element that is spliced into the mRNA precisely as exon 6. Importantly, the downstream open reading frame is not altered by any of these splicing combinations. The gene is approximately 35 kilobases long containing either 9 or 10 exons (including the line-1 element) depending on which promoter is active. All of the splice sites follow the GT/AG rule. The dual promoters and opportunities for alternative splicing suggest a variety of mechanisms for control of HIOMT expression and biological activity in different tissues not previously recognized.

Acetylserotonin O-Methyltransferase↗
PubMed1994

Characterization of human B cell proteins binding specifically to uveitopathogenic peptide 1169-1191 of bovine IRBP.

Peptide 1169-1191 is a major uveitopathogenic determinant of bovine Interphotoreceptor Retinoid Binding Protein (IRBP) in Lewis rats. Previously, we identified two proteins with approximate molecular masses of 72 and 74 kDa and one with a molecular mass of 40 kDa from B cells of naive Lewis rats and EBV-transformed B cells from a human patient with ocular Behçet's disease that bind to bovine IRBP peptide 1169-1191. In this study, we have partially characterized these proteins. The two proteins with molecular masses 72 and 74 kDa belong to the HSP 70 family of proteins and the 40-kDa protein is actin.

Adenosine Triphosphate↗
PubMed1994

Retinoid processing in retinal pigment epithelium of toad (Bufo marinus).

The formation of 11-cis-[3H]retinal in the retinal pigment epithelium (RPE) and its release to extracellular medium containing interphotoreceptor retinoid-binding protein (IRBP) were studied in the RPE eyecup of the toad (Bufo marinus). The RPE was labeled with all-trans-[3H]retinol during an initial 1-h incubation. In phase 2 of the incubation (0-2 h), the extracellular medium contained initially ligand-free IRBP (0-26 microM). Retinoids subsequently extracted from the extracellular medium and RPE were analyzed by high performance liquid chromatography and scintillation counting. IRBP increased both the molar amount and specific radioactivity of 11-cis-retinal released by the RPE during phase 2. The molar amount of 11-cis-retinal in the RPE was small relative to that of retinal released with high IRBP. With 21 microM IRBP and phase 2 incubations of > or = 10 min, the specific radioactivity of released 11-cis-retinal exceeded that of all-trans-retinyl ester in the RPE. The specific radioactivity of 11-cis-retinyl ester was less than that of all-trans-ester, independent of IRBP concentration. The results indicate that IRBP promotes the formation (from all-trans-precursor) as well as the release of 11-cis-retinal and suggest the preferred utilization of recently incorporated and esterified all-trans-retinol in 11-cis-retinal synthesis in a "last in/first out" manner.

Animals↗