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D B Farber

Publications and source records attributed to D B Farber.

At least 37 records · Page 2Linked to original sources
PubMed1999

Estrogen receptor in the human eye: influence of gender and age on gene expression.

PURPOSE: Many epidemiologic studies indicate an increased incidence of certain vision threatening conditions in postmenopausal women. These data suggest that changes in sex steroid homeostasis may affect the physiology of the eye. To provide support to this hypothesis, the expression of estrogen receptor alpha (ERalpha) in human eye tissues was investigated. METHODS: Complementary studies including RNA analysis by reverse transcription polymerase chain reaction, western blot analysis, and immunocytochemistry were used to provide evidence of ERalpha expression. Protein detection was carried out using a mouse monoclonal antibody raised against an epitope located in the ligand binding domain of the human receptor. Cellular localization was studied on formalin-fixed paraffin-embedded eye sections using conventional immunohistochemical techniques. RESULTS: Gender and age differences in ERalpha mRNA expression were observed in retina. The 65-kDa ERalpha protein was detected in the retina and retinal pigment epithelium (RPE) of young female eyes but not in eye tissues dissected from men and postmenopausal women. Immunocytochemistry corroborated ERalpha staining of a young female neurosensory retina and RPE. In addition, ERalpha could be detected in the ciliary body, in the iris, and in the epithelium of the lens. CONCLUSIONS: The presence of the ERalpha in the human eye suggests that the sex steroid hormone axis may play a role in the pathogenesis of certain ocular diseases.

Adult↗
PubMed1998

Exon screening of the genes encoding the beta- and gamma-subunits of cone transducin in patients with inherited retinal disease.

PURPOSE: To screen the exons of the genes encoding the beta3-subunit (GNB3) and gammac-subunit (GNGT2) of cone transducin for mutations in a large number of unrelated patients with various forms of inherited retinal disease including cone dystrophy, cone-rod dystrophy and macular dystrophy. METHODS: Exons of the two genes were screened for mutations by denaturing gradient gel electrophoresis (DGGE) and/or single strand conformation polymorphism electrophoresis (SSCP); any variants were sequenced directly. RESULTS: Although many sequence variants were found in both genes, none could be associated with disease. Additionally, the gene structure and sequence of the coding exons of GNB3 were determined and compared with those of the dog homolog. Both human and canine GNB3 have nine coding exons and their two predicted amino acid sequences have 97% identity. CONCLUSIONS: The results indicate that GNB3 and GNGT2 are unlikely sites of mutations responsible for inherited retinal degenerations that predominantly effect cone-mediated function (cone and cone-rod dystrophies) or have a predilection for disease in the macula (macular dystrophies).

Amino Acid Sequence↗
PubMed1998

Cloning of a cyclic GMP phosphodiesterase gamma subunit from the ground squirrel retina.

Recent evidence suggests that the two main classes of cones are not only equipped with different photopigments, but also exhibit differences in their downstream phototransduction cascade. An antibody against the gamma subunit of retinal cGMP phosphodiesterase (PDE) had previously been found to label all photoreceptors in the ground squirrel (Spermophilus tridecemlineatus). This property was utilized for the cloning of a cDNA fragment encoding the corresponding polypeptide. A lambda gt11 cDNA library was constructed and screened with the antibody. Positive clones were isolated, subcloned, and sequenced. Clones were used as probes in RNA blot hybridization, and the obtained sequence information was compared to other available gamma-PDE sequences. Four virtually identical cDNA clones were isolated. Both nucleic acid and amino acid sequence alignment placed this gene in the same group as bovine and human cone gamma-phosphodiesterase subunits. The predicted length of the translated protein was 84 amino acids, and its molecular weight 11 kD. The cDNA hybridized with an 0.6-kb transcript in retinal RNA from ground squirrel, Syrian golden hamster, and mouse, and also with a 1.4-kb transcript in the ground squirrel. We conclude that the isolated cDNA fragment encodes a gamma subunit of cone cGMP-PDE. This subunit is expressed in middle-wavelength sensitive cones (the predominant photoreceptor type in this species). It remains to be determined if this subunit is expressed in short-wavelength sensitive cones as well, or if these photoreceptors are equipped with another, unique gamma-PDE subunit.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration.

First (DeltaE1/E3) and second (DeltaE1+DeltaE2/E3/E4) generation adenovirus (Ad) vectors have been shown previously to be of limited use in the treatment of human genetic diseases due to the induction of a host cytotoxic T-cell mediated immune response against virally expressed genes. In addition, a limited cloning capacity of approximately 8 kb does not cater for the incorporation of large upstream sequences essential for regulated tissue-specific expression or inclusion of multiple gene-expression cassettes. In this study we have exploited our recently developed Ad-based vector, the encapsidated adenovirus mini-chromosome (EAM) from which all of the viral genes have been deleted. EAMs contain only the inverted terminal repeats required for replication and five cis -acting Ad encapsidation signals necessary for packaging. We have shown previously that EAMs can efficiently transduce a variety of cell types in vitro. In this study we demonstrate that EAMs can transduce and rescue cells from the neurosensory retina in vivo. EAM-mediated delivery of the beta subunit of cyclic GMP phosphodiesterase (PDE) cDNA to mice affected with retinal degeneration (rd) allows prolonged transgene expression and rescue of rod photoreceptor cells. RT-PCR analysis from the injected retina indicates that transgene products are present for at least 18 weeks post-injection. Both the alpha and beta subunits of PDE could be detected up to 90 days postnatal in EAM-injected rd retina by western analysis. A maximal PDE activity of 150 nm/min/mg was detected at 33 days postnatal. Examination of outer nuclear thickness showed significant differences up to 12 weeks post-injection. These results demonstrate an improved level of rescue over first-generation adenoviral vectors and suggest the possibility of successful EAM-mediated treatment of some retinal diseases in humans.

Adenoviridae↗
PubMed1998

Defective RNA splicing resulting from a mutation in the cyclic guanosine monophosphate-phosphodiesterase beta-subunit gene.

PURPOSE: The authors have previously reported a 3' splice site mutation in intron 2 of the rod cyclic guanosine monophosphate-phosphodiesterase (cGMP) beta-subunit (beta-PDE) gene in a patient with compound heterozygous autosomal recessive retinitis pigmentosa. The purpose of this study is to determine whether this mutation interferes with RNA splicing, what products it generates, and whether the resultant mRNA is able to support the synthesis of the native protein. METHODS: Two expression constructs were prepared by subcloning genomic DNA fragments (one from the control subject DNA and the other from the patient's DNA) to the pCIS2 expression vector. Recombinant plasmid DNA was introduced into 293 human embryonic kidney cells using the calcium phosphate-mediated transfection procedure. Northern blot hybridization, reverse transcription-polymerase chain reaction, and sequencing were used for RNA analysis. RESULTS: Four major products were present in the RNA pool isolated from cells transfected with the expression construct containing the splice site mutation. One of the transcripts resulted from the activation of a cryptic 3' splice site located in exon 3, 12 nucleotides downstream of the mutated site. The second fragment was longer than the correctly spliced mRNA by approximately 1 kb and contained unspliced intron 2. Two other high molecular weight products corresponded to intermediate lariats. CONCLUSIONS: An acceptor splice site mutation in intron 2 of the beta-PDE gene leads to the accumulation of pre-mRNA and intermediate lariats and a 12-nucleotide shorter beta-PDE transcript produced by the use of a cryptic splice site located in exon 3. In the normal beta-PDE mRNA, these 12 nucleotides code for ValPheLeuLys. These amino acids are highly conserved in the putative noncatalytic cGMP-binding domain I of beta-PDE from several species and, probably, are important for the correct folding and function of the protein.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Canine cone transducin-gamma gene and cone degeneration in the cd dog.

PURPOSE: To characterize the cDNA and the organization of the gene encoding the cone-specific gamma subunit of transducin (Tgamma c) and to examine this gene as a candidate for the recessively inherited cone photoreceptor degeneration in the cd dog. METHODS: Canine Tgamma c cDNA was cloned and sequenced. Polymerase chain reaction (PCR) was used to define the Tgamma c gene structure, northern blot analysis to examine the level of expression of Tgamma c mRNA in control and cd-affected retinas, and immunocytochemistry to determine the presence and localization of Tgamma c in normal and cd retinas. RESULTS: Immunocytochemical results showed Tgamma c localized to cone photoreceptor outer segments in the normal retina, whereas no Tgamma c immunoreactivity was observed in the cd retinas. However, the level of transcription and the primary structure of the cloned cDNA coding for the 69-amino acid protein were identical in retinas from wild-type and affected dogs. CONCLUSIONS: Although Tgamma c immunoreactivity was specifically absent in the cd dog retina, no differences were detected between normal and cd retinas in the nucleotide sequence of Tgamma c mRNA or in its synthesis. These results indicate that a mutation in the Tgamma c gene may not be causally associated with the cd dog disease. These findings suggest that possible abnormalities in posttranslational modification of Tgamma c or defective assembly of the transducin alphabetagamma complex could lead to rapid degradation of Tgamma c.

Amino Acid Sequence↗
PubMed1998

Retinal degeneration in the rd mouse in the absence of c-fos.

PURPOSE: Apoptosis is the final common death pathway of photoreceptors in light-induced retinal degeneration and in several animal models for retinal dystrophy. To date, little is known about gene regulation of apoptosis in the retina. The expression of the immediate early gene c-fos is upregulated concomitant with apoptosis in light-induced photoreceptor degeneration and in the rd mouse, an animal model for inherited retinal degeneration. In a recent study it was shown that c-Fos is essential for light-induced apoptosis of photoreceptors in vivo. To determine whether c-Fos is also involved in the apoptotic pathway of inherited retinal degeneration, rd/rd, c-fos -/- double-mutant mice have been generated. METHODS: Double-mutant mice (rd/rd, c-fos -/-) were crossbred from c-fos+/- mice and rd/rd mice. Their genotype was determined by polymerase chain reaction analysis of genomic DNA. Wild-type control mice and homozygous rd mice were killed at 2-day intervals from postnatal day (P)9 through P21. Double-mutant mice were killed at postnatal days P9, P11, P13, P15, and P21. To determine levels of apoptosis in the retina, eyes were enucleated and processed for light microscopy and in situ nick-end labeling. Total retinal DNA was extracted from isolated retinas for DNA fragmentation analysis. RESULTS: Morphologic, histochemical, and biochemical analyses showed that the time course of apoptosis and the outcome of photoreceptor degeneration in rd/rd, c-fos-/- double-mutant mice was indistinguishable from that in rd mice carrying functional c-fos. CONCLUSIONS: These data suggest that in contrast to its role in light-induced photoreceptor degeneration, c-Fos is not essential for apoptosis in the rd mouse.

Animals↗
PubMed1997

Transcriptional activation of the human rod cGMP-phosphodiesterase beta-subunit gene is mediated by an upstream AP-1 element.

During photoactivation retinal cGMP-phosphodiesterase (PDE) mediates signal transduction in the photoreceptor outer segments. Mutations in the beta-subunit gene of rod-specific PDE (beta-PDE) have been associated with inherited retinal degeneration in a number of species, including human. Here we have investigated the proximal upstream sequences that participate in transcriptional activation of this gene. Transient transfections demonstrated that the sequence from -72 to +53 bp contained sufficient information to direct high levels of gene expression in cells of retinal origin. Deletion or mutagenesis of an AP-1 motif present in this region caused 90-95% reduction in reporter gene expression. By gel mobility shift assay we demonstrated specific interactions between putative nuclear transcription factors and this AP-1 element. These findings indicate that the proximal AP-1 site in the human beta-PDE promoter is functionally relevant and necessary for transcriptional activation of this gene.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Structure and analysis of the transducin beta3-subunit gene, a candidate for inherited cone degeneration (cd) in the dog.

The cDNA for the beta3-subunit of cone-specific transducin (Tbeta3) was cloned and characterized from wild type dogs, and used in linkage studies as a candidate gene for cone degeneration. Sequence analysis of the Tbeta3 cDNA revealed an open reading frame of 1020 bp, potentially coding for a protein of 340 amino acids (aa). The deduced aa sequence of canine Tbeta3 shares 97% identity with the previously identified human Tbeta3, and 82% identity with bovine rod-specific transducin (Tbeta1). RT-PCR and sequencing of the amplified products demonstrated that the retinal canine Tbeta3 gene is expressed in two different transcripts which can be generated by alternative splicing of the intron in the 3'-untranslated region (UTR). The short and the long mRNAs differ in the length of their 3'-UTR by 456 nt. We have also determined the genomic organization of the canine Tbeta3 gene; it consists of ten exons and the first exon is in the 5'-UTR. The cDNA encoding Tbeta3 from cd-affected dogs was also cloned and sequenced. We found no differences at the nucleotide level between the cDNAs isolated from normal and diseased retinas. The level of transcription of Tbeta3 mRNA in the cd dog retina appeared to be normal. Linkage analysis of a crossbred informative pedigree showed five obligate recombinants out of nine informative offspring. These results suggest that Tbeta3 is not a candidate gene for the cone degeneration of the cd mutant.

Amino Acid Sequence↗
PubMed1997

TRPM-2 expression and tunel staining in neurodegenerative diseases: studies in wobbler and rd mice.

Neuronal apoptosis has been described during development but little is known about whether apoptosis plays a role in neurodegenerative disease. Neurodegenerative cell death can be difficult to study because it is often a slow process and it is limited to only a few cells among many nondying cells. We used molecular methods to study cell death in the spinal cords of wobbler mice, a model of motoneuron disease, and compared it to retinas of rd mice, a model of retinitis pigmentosa, where it is known that photoreceptors die by apoptosis. Increased levels of mRNA of testosterone-repressed prostate message 2 (TRPM-2) were found in motoneurons of wobbler mice and the retinas of rd mice. In motoneurons, TRPM-2 mRNA colocalized with increased expression of the message for growth-associated protein (GAP-43). In rd retinas, TRPM-2 mRNA was localized to ganglion cells of the inner retina known to survive the disease. These suggest that TRPM-2 expression is associated with cell membrane remodeling in surviving cells associated with synaptic reorganization or change in afferent input. In situ labeling of fragmented DNA (TUNEL staining) identified dying photoreceptors in the rd mouse. In the wobbler spinal cords dying motoneurons were not labeled. These data suggest that the process of neurodegenerative motoneuron cell death in wobbler mice is different from the apoptotic process of rd photoreceptors.

Animals↗
PubMed1997

Identification of genes causing photoreceptor degenerations leading to blindness.

At least 15 genes with defects responsible for various forms of inherited retinal disease involving photoreceptor loss have been identified over the past eight years. Several of the genes were first considered as candidates for study because of their involvement in murine retinal disease, others because of their chromosomal loci. In two cases, novel genes were uncovered by positional cloning. Based on reports of disease loci for which no gene has yet been found, more than twice as many genes remain to be identified in this genetically heterogeneous group of diseases.

Animals↗
PubMed1996

Exclusion of the beta-subunit of type II calmodulin kinase for the wobbler spinal muscular atrophy gene.

The wobbler mouse (wr) is an attractive model for studying motor neuron disease but the genetic defect is unknown. The beta-subunit of calmodulin kinase II (beta-CaMK II) is a good candidate for the wr mutation because of its chromosomal localization and tissue distribution. In this report, we found normal levels of CaM KII mRNA and enzyme activity making it highly unlikely that a mutation in the beta-CaM KII gene is the cause of the wr phenotype.

Animals↗
PubMed1996

Chromosomal localization of murine and human oligodendrocyte-specific protein genes.

Oligodendrocyte-specific protein (OSP) is a recently described protein present only in myelin of the central nervous system. Several inherited disorders of myelin are caused by mutations in myelin genes but the etiology of many remain unknown. We mapped the location of the mouse OSP gene to the proximal region of chromosome 3 using two sets of multilocus crosses and to human chromosome 3 using somatic cell hybrids. Fine mapping with fluorescence in situ hybridization placed the OSP gene at human chromosome 3q26.2-q26.3. To date, there are no known inherited neurological disorders that localize to these regions.

Animals↗
PubMed1996

Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase.

The retinal cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) is a key regulator of phototransduction in the vertebrate visual system. PDE consists of a catalytic core of alpha and beta subunits associated with two inhibitory gamma subunits. A gene-targeting approach was used to disrupt the mouse PDEgamma gene. This mutation resulted in a rapid retinal degeneration resembling human retinitis pigmentosa. In homozygous mutant mice, reduced rather than increased PDE activity was apparent; the PDEalphabeta dimer was formed but lacked hydrolytic activity. Thus, the inhibitory gamma subunit appears to be necessary for integrity of the photoreceptors and expression of PDE activity in vivo.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1996

Screening of the PDE6B gene in patients with autosomal dominant retinitis pigmentosa.

Each of the 22 exons and 140 bp of the 5' untranslated region of the gene encoding the beta-subunit of cGMP-phosphodiesterase (PDE6B) were screened by denaturing gradient gel electrophoresis for mutations in the DNAs of 54 unrelated individuals with autosomal dominant retinitis pigmentosa. Six different sequence variants were found in seven patients. Four of the sequence variants did not segregate with disease in the families of the respective probands and/or were present in control DNAs. The remaining two sequence variants, a Leu228His missense in exon 3 and a G to A transition in the tenth base of the splice acceptor site of intron 8, were both present in the same proband. One or the other of the two sequence variants was present in each affected member of the proband's small family and neither sequence variant was present in the one unaffected member nor in 75 unrelated controls. However, no effect on splicing of mRNA was observed in expression studies of DNA constructs containing the G to A transition. Therefore, mutations in PDE6B could not be shown to be the cause of adRP in this group of patients.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1996

Selective absence of cone outer segment beta 3-transducin immunoreactivity in hereditary cone degeneration (cd).

We have used immunocytochemistry and in situ hybridization to examine the expression of photoreceptor specific genes in retinas of normal dogs and those affected with hereditary cone degeneration (cd), a rare autosomal recessive disorder that selectively affects cones. In the cd retina, cone disease begins early in life; cones are lost by extrusion of the nucleus into the inner segment, and later by displacement of the nucleus, surrounded by a thin rim of cytoplasm, into the interphotoreceptor space. Two micrometer sections from the superior and inferior retinal meridians, extending from the optic disk to the ora serrata, were used for in situ hybridization with a bovine rod opsin and human red/green cone opsin cRNA probes, or were reacted with antibodies directed against photoreceptor-specific proteins and visualized with appropriate biotinylated antibodies. Antibodies against the following proteins were used: alpha- and beta 3-transducins, phosducin, alpha/beta- and gamma-phosphodiesterases, COS-1, and OS-2, opsin, S-antigen and IRBP. Immunoreactivity or hybridization labeling was evaluated in unstained sections; cone pathology was judged in adjacent Toluidine Blue-stained sections. With these methods it was possible to evaluate immunoreactivity or hybridization labeling and cone pathology at the single cell level. Both middle-(COS-1) and short-(OS-2) wavelength-sensitive cones were present in controls and cd affected retinae at 2.2 months, and distinct transcripts of the red/green cone pigment gene were identified in the majority of cones in both normal and affected retinas at this age. However, beta 3-transducin immunoreactivity was completely absent from cd-affected cone outer segments. Both cone types were present but in reduced numbers in older animals (11.5 and 17 months), and no reactivity to beta 3-transducin was noted. No differences were found with the other antibodies used. The specific absence of beta 3-transducin immunoreactivity from the cone outer segments suggests a potential involvement of the beta 3-transducin gene or gene product in the disease process.

Animals↗
PubMed1996

Isolation and characterization of a novel oligodendrocyte-specific protein.

Myelin plays a critical role in nervous system function and alterations in myelin-specific proteins cause a variety of neurologic disorders. We isolated a novel cDNA from the CNS that shares little nucleotide sequence homology with previously reported genes but appears to encode a protein related to peripheral myelin protein-22 (PMP-22) based on its amino acid sequence, predicted structure, and cellular localization. PMP-22 is important in peripheral myelination and Schwann cell proliferation, and mutations in its gene cause diseases of peripheral nerves. The isolated cDNA is 1.8 kb in length with an open reading frame of 621 bp. Northern blot analysis detected hybridization of labeled cDNA with a single 2.1-kb transcript only in the CNS. In situ hybridization revealed expression of this cDNA in oligodendrocytes of brain and spinal cord as well as in oligodendrocyte-enriched cultures; therefore we have named it oligodendrocyte-specific protein (OSP) cDNA. An OSP-specific polyclonal antibody reacted with a single 22-kd protein present in CNS myelin and oligodendrocytes. Developmental expression of OSP mRNA in the spinal cord was similar to that of the mRNA for a major myelin protein, proteolipid protein (PLP), and similar to PMP-22 in peripheral nerves. Since OSP is localized to oligodendrocytes and myelin, has a similar structure with PMP-22, and has a developmental pattern of expression like other myelin proteins, it probably has an important role in CNS myelinogenesis.

Amino Acid Sequence↗