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D J Zack

Publications and source records attributed to D J Zack.

At least 73 records · Page 4Linked to original sources
PubMed1996

The bZIP transcription factor Nrl stimulates rhodopsin promoter activity in primary retinal cell cultures.

In vitro DNA binding assays and transient transfection analysis with monkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression. We have now further explored the role of the NRE and surrounding promoter elements. Using the yeast one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl. Recovery of truncated clones in the screen demonstrated that the carboxyl-terminal half of Nrl, which contains the basic and leucine zipper domains, is sufficient for DNA binding. To functionally dissect the rhodopsin promoter, transient expression studies with primary chick retinal cell cultures were performed. Deletion and mutation analyses identified two positive regulatory sequences: one between -40 and -84 base pairs (bp) and another between -84 and -130 bp. Activity of the -40 to -84 region was shown to be largely due to the NRE. On co-transfection with an NRL expression vector, there were 3-5-fold increases in the activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or deleted NRE. Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity. The -84- to -130-bp region acted synergistically with the NRE to enhance both the level of basal expression and the degree of Nrl-mediated trans-activation. These studies support Nrl as a regulator of rhodopsin expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the cis-acting response elements and trans-acting factors that regulate photoreceptor gene expression.

Amino Acid Sequence↗
PubMed1996

Ret 4, a positive acting rhodopsin regulatory element identified using a bovine retina in vitro transcription system.

Previous transgenic mouse studies demonstrated that the bovine rhodopsin sequence between -222 and +70 base pairs (bp) contains a minimal promoter, which is sufficient to direct photoreceptor cell-specific expression of a lacZ reporter gene. To more fully define the DNA regulatory elements and protein factors involved in regulating rhodopsin transcription, we have developed an in vitro transcription system derived from bovine retinal nuclear extracts. Retinal extracts, as compared to liver, HeLa, and Drosophila embryonic cell extracts, demonstrated preferential activity for the rhodopsin promoter. A template spanning the bovine rhodopsin upstream region from -590 to +15 bp showed significant activation relative to the basal activity seen with a TATA box containing -38 to +15 bp template. Deletion analysis indicated that the region between -85 and -38 bp contained significant positive regulatory activity. This activity was not observed with HeLa extracts, suggesting that it might be retina-specific. Systematic site-directed mutagenesis of the subregion from -64 to -38 bp indicated that sequences between -60 and -58 bp and between -48 and -40 bp harbor critical elements. The former sequence is part of the binding site for the retina-specific transcription factor Nrl, which has been implicated in rhodopsin regulation. Electrophoretic mobility shift assays showed that the latter sequence (-48 to -40 bp), and flanking DNA, designated Ret 4, is bound by both retina-specific and ubiquitously expressed protein factors. Shift assays with mutant oligomers further defined the putative recognition sequences for these protein factors. Together, our results suggest that multiple promoter elements and transcriptional factors are involved in regulating photoreceptor-specific rhodopsin transcription.

Animals↗
PubMed1996

Rin, a neuron-specific and calmodulin-binding small G-protein, and Rit define a novel subfamily of ras proteins.

cDNAs encoding two novel 25 kDa Ras-like proteins, Rit and Rin, were isolated from mouse retina using a degenerate PCR-based cloning strategy. Using the expressed sequence tag database, human orthologs were also obtained and sequenced. The protein sequences of Rit and Rin, which are 64% identical, are more similar to each other than to any known Ras protein. Their closest homologs in the databases are Mucor racemosus Ras2 and Ras3, to which they show approximately 48% identity. Rit and Rin both bind GTP in vitro. An unusual feature of their structure is that they lack a known recognition signal for C-terminal lipidation, a modification that is generally necessary for plasma membrane association among the Ras subfamily of proteins. Nonetheless, transiently expressed Rit and Rin are plasma membrane-localized. Both proteins contain a C-terminal cluster of basic amino acids, which could provide a mechanism for membrane association. Deletion analysis suggested that this region is important for Rit membrane binding but is not necessary for Rin. Rit, like most Ras-related proteins, is ubiquitously expressed. Rin, however, is unusual in that it is expressed only in neurons. In addition, Rin binds calmodulin through a C-terminal binding motif. These results suggest that Rit and Rin define a novel subfamily of Ras-related proteins, perhaps using a new mechanism of membrane association, and that Rin may be involved in calcium-mediated signaling within neurons.

Animals↗
PubMed1996

Mechanisms of cellular penetration and nuclear localization of an anti-double strand DNA autoantibody.

An anti-dsDNA Ab, mAb 3E10, was identified that bound membranes of fixed human renal tubular cells, penetrated live murine renal tubular cells in vivo, and localized in the cell nucleus. mAb 3E10 binds both dsDNA and an extracellular matrix protein, HP8/HEVIN, expressed in high endothelial venules. Previous studies showed both shared and distinct binding determinants of mAb 3E10 VH for DNA and HP8/HEVIN. To independently assess the requirement of DNA and HP8/HEVIN in cellular penetration, site-directed mutants of mAb 3E10 VH and V kappa were studied for penetrating kidney cell lines. The results showed that residues required for binding DNA, but not HP8/HEVIN, were necessary for Ab penetration, indicating that cellular penetration required the presence of DNA or binding of Ab to a membrane determinant precisely resembling DNA. Ab Fab penetrated cells, indicating that neither the Fc nor multivalent Ab binding is necessary for cellular penetration. Ab synthesized in the cytoplasm as a result of deleting heavy and light chain signal peptides was not translocated to the nucleus, indicating a mechanism distinct from the usual protein nuclear localization signals and suggesting the need for a membrane-mediated pathway or for post-translational modification of the Ab.

Animals↗
PubMed1996

Retinal degeneration in transgenic mice with photoreceptor-specific expression of a dominant-negative fibroblast growth factor receptor.

Mutant cDNAs coding for dominant-negative forms of the fibroblast growth factor receptors 1 (FGFR-1) and 2 (FGFR-2) that lack tyrosine kinase activity were ligated to a 2.2 kb DNA fragment containing the bovine rhodopsin promoter and used to generate transgenic mice. Six independent lines were generated with the FGFR-1 construct, and five were generated with the FGFR-2 construct. Five of the six FGFR-1 mutant lines and all five FGFR-2 mutant lines showed transgene expression in the retina by reverse transcription-PCR. By both in situ hybridization and immunohistochemistry, mutant FGFRs were found to be expressed specifically in photoreceptors of transgene-positive FGFR-1 and FGFR-2 mice. Lines expressing the FGFR-2 mutant showed progressive photoreceptor degeneration; the retinas showed minimal or no abnormalities at 1 month, but by 2 months they showed focal areas of thinning of the outer nuclear layer and disruption of photoreceptors. By 2-4 months, areas of complete loss of photoreceptors were seen. These abnormalities were not seen in control littermates not expressing the transgene. Mice from two FGFR-1 mutant lines showed focal areas of thinning of the outer nuclear layer and numerous photoreceptors with fragmented chromatin, whereas the other FGFR-1 lines showed minimal or no abnormalities. These data indicate that perturbation of FGF signaling in photoreceptors is associated with progressive photoreceptor degeneration, suggesting that one or more of the FGFs may act as a survival factor for photoreceptor cells.

Animals↗
PubMed1996

RER, an evolutionarily conserved sequence upstream of the rhodopsin gene, has enhancer activity.

Previous transgenic mouse experiments localized the mammalian rhodopsin gene promoter to a region just upstream of the mRNA start site, and also suggested the existence of a second more distal regulatory region. A highly conserved 100-base pair (bp) sequence which is homologous to the red and green opsin locus control region is located 1.5-2 kilobases upstream of the rhodopsin gene (depending on the species). In order to test the activity of this 100-bp region, transgenic mice were generated with bovine rhodopsin promoter/lacZ constructs which differed only by the presence or absence of the sequence. Of 11 lines generated, all demonstrated photoreceptor-specific expression of the transgene, but the lines with the putative regulatory region showed significantly higher expression. Additional transgenic lines in which the region was fused to a minimal heterologous promoter did not show transgene expression in the retina. Gel mobility shift and DNase I footprint assays demonstrated that bovine retinal nuclear extracts contain retina-specific as well as ubiquitously expressed factors that interact with the putative regulatory region in a sequence-specific manner. These results indicate that the 100-bp sequence can indeed function in vivo as a rhodopsin enhancer region.

Animals↗
PubMed1996

The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression.

The retinal protein Nrl belongs to a distinct subfamily of basic motif-leucine zipper DNA-binding proteins and has been shown to bind extended AP-1-like sequence elements as a homo- or heterodimer. Here, we demonstrate that Nrl can positively regulate the expression of the photoreceptor cell-specific gene rhodopsin. Electrophoretic mobility-shift analysis reveals that a protein(s) in nuclear extracts from bovine retina and the Y79 human retinoblastoma cell line binds to a conserved Nrl response element (NRE) in the upstream promoter region of the rhodopsin gene. Nrl or an antigenically similar protein is shown to be part of the bound protein complex by supershift experiments using Nrl-specific antiserum. Cotransfection studies using an Nrl-expression plasmid and a luciferase reporter gene demonstrate that interaction of the Nrl protein with the -61 to -84 region of the rhodopsin promoter (which includes the NRE) stimulates expression of the reporter gene in CV-1 monkey kidney cells. This Nrl-mediated transactivation is specifically inhibited by coexpression of a naturally occurring truncated form of Nrl (dominant negative effect). Involvement of Nrl in photoreceptor gene regulation and its continued high levels of expression in the adult retina suggest that Nrl plays a significant role in controlling retinal function.

Animals↗
PubMed1996

Apoptosis in ocular disease: a molecular overview.

Apoptosis is a form of genetically programmed cell death that can be induced by a variety of different stimuli. It is often referred to as a form of cellular suicide. Typically, apoptosis is characterized by the condensation and shrinkage of the cellular nucleus and cytoplasm, followed by the complete fragmentation of the cell and subsequent phagocytosis of the debris by surrounding cells. Although important during development, and also for maintaining homeostasis in some adult tissues, apoptosis can also be associated with disease processes. Recent laboratory studies indicate that apoptosis is a mechanism of cell death in several important ocular diseases including glaucoma, retinitis pigmentosa, cataract formation, retinoblastoma, retinal ischemia, and diabetic retinopathy. This review summarizes the results of these studies and provides a brief description of some of the key molecules that are involved in the genetic regulation of apoptosis. It is possible that a complete understanding of how these molecules function may someday lead to new treatment options aimed at blocking the death of cells in a variety of ocular diseases.

Adult↗
PubMed1996

Localization of TIMP-3 mRNA expression to the retinal pigment epithelium.

PURPOSE: To evaluate ocular sites of expression of the tissue inhibitor of metalloproteinases-3 gene (TIMP-3). METHODS: In situ hybridization was performed on frozen sections of albino mouse eyes using riboprobes generated to the 3' untranslated region of TIMP-3. RESULTS: TIMP-3 mRNA expression was detected strongly in the retinal pigment epithelium (RPE) and to a minor extent in the ciliary epithelium, but not at any other site within the eye. CONCLUSIONS: Expression of TIMP-3 in the RPE is consistent with the recent demonstration of TIMP-3 mutations in patients with Sorsby's fundus dystrophy, a condition marked by the early onset of choroidal neovascularization in the macula. Unlike many of the recently described genes that cause human retinal disease, TIMP-3 is preferentially expressed in the RPE of the normal eye, as opposed to the photoreceptors.

Albinism↗
PubMed1995

Localization of an Fc-binding reactivity to the constant region of human IgG4. Implications for the pathogenesis of rheumatoid arthritis.

The majority of plasma cells in rheumatoid arthritis (RA) synovium produce rheumatoid factors (RF). IgG RF predominate in the immune complexes found in RA synovial fluid and have been implicated in the pathogenesis of RA. IgG4 RF are a major component of IgG RF produced in serum and synovium of RA patients, even though this subclass comprises only 4% of the serum IgG. We produced an IgG4 mAb, hRF-1, with RF reactivity from the synovial tissue of a patient with RA. mAb hRF-1 had binding specificity for mammalian IgG similar to Staphylococcus aureus protein A, which is characteristic of RF from patients with RA. To determine the molecular basis of this particular RF reactivity, the heavy and light chain genes of mAb hRF-1 were amplified by PCR, cloned, and ligated into the pSG5 plasmid for expression in COS-7 cells. Chain recombination experiments localized the Fc-binding reactivity to the hRF-1 heavy chain. Using a series of chimeric Ab sequences, the Fc-binding reactivity was mapped to the constant region of IgG4 rather than the variable region involved in classic RF reactivity. Multiple domains, including Hinge, CH2, and CH3 of the IgG4 constant region were required for Fc binding. Our studies demonstrate an example of RF-like Fc-binding reactivity that is conferred by the gamma-4 constant region rather than the classic Ag binding site and suggest that increased production of IgG4 may contribute to the pathogenesis of RA.

Amino Acid Sequence↗
PubMed1995

DNA mimics a self-protein that may be a target for some anti-DNA antibodies in systemic lupus erythematosus.

Recent studies suggest that some anti-DNA Abs in systemic lupus erythematosus may actually be Abs to specific proteins and that binding to dsDNA is a nonspecific cross-reactive event. To identify such proteins that bind to anti-DNA Abs, a cDNA expression library from human placenta was screened with mAb 3E10, a pathogenic anti-dsDNA Ab. MAb 3E10 was shown to bind to a 44-amino-acid fragment of HP8, a newly identified protein with amino acid sequence homology to the family of SPARC extracellular matrix proteins. To determine if Ab binding to both dsDNA and HP8 protein occurs through a common binding site, and therefore represents molecular mimicry, the Ab binding domains for protein and DNA were mapped. Chain recombinations between mAb 3E10 and a non-anti-DNA mAb showed that both the heavy and the light chains of mAb 3E10 were essential for anti-dsDNA and anti-HP8 reactivity. Mutagenesis experiments demonstrated that dsDNA and HP8 shared several critical binding residues located in all three complementarity-determining regions of mAb 3E10 VH. Moreover, Abs to HP8 were demonstrated in the sera of a subset of lupus patients. These results indicate that DNA mimics the HP8 protein in binding a lupus Ab, and that this protein may be a target for a subpopulation of anti-dsDNA Abs in systemic lupus erythematosus.

Animals↗
PubMed1995

Two kappa immunoglobulin light chains are secreted by an anti-DNA hybridoma: implications for isotypic exclusion.

An anti-DNA hybridoma derived from an MRL/lpr mouse secretes two different kappa light chains in combination with a single heavy chain. Multiple single cell clones express and secrete immunoglobulin containing both kappa light chains. The N-terminal protein sequences of the light chains correspond to sequences predicted from functionally rearranged mRNAs subjected to reverse transcription and amplified by polymerase chain reaction (PCR). Karyotype analysis of the hybridoma indicates a clonal line derived from the fusion of two cells. By amino acid sequence comparison and PCR analysis, both functional kappa light chains are derived from the MRL/lpr spleen. The two functional light chain cDNAs were cloned and co-transfected into COS-7 cells with the heavy chain cDNA. Only one of the light chains in combination with mAb 3E10 heavy chain confers anti-DNA reactivity. The presence of two separate kappa light chains and, therefore, two separate antigen receptors on a single B cell may have ramifications for both polyclonal activation and toleration of lupus B cells.

Amino Acid Sequence↗
PubMed1995

Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis.

PURPOSE: To investigate whether retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. METHODS: Chronic elevated eye pressure was produced in 20 monkey eyes, and the optic nerve was transected unilaterally in the orbit of 10 monkeys and 14 rabbits. Sixteen monkey and 14 rabbit eyes were studied as normal controls. Analytic methods included light and electron microscopy, histochemistry for DNA fragmentation (TUNEL method), and DNA electrophoresis in agarose gels. RESULTS: Dying ganglion cells in the experimental retinas exhibited morphologic features of apoptosis, including chromatin condensation and formation of apoptotic bodies. Cells with a positive reaction for DNA fragmentation were observed in eyes subjected to axotomy and experimental glaucoma but were only rarely encountered in control eyes. No evidence of internucleosomal fragmentation was detected electrophoretically, possibly because of the small proportion of cells that were dying at any given time. CONCLUSION: Some retinal ganglion cells injured by glaucoma and by axotomy die by apoptosis.

Animals↗
PubMed1995

Cloning and characterization of rod opsin cDNA from the Old World monkey, Macaca fascicularis.

PURPOSE: Opsin sequences from different phyla have been compared to examine the structural and functional regions of this molecule and to assess the molecular evolution of genes in the animal kingdom. Of all the sequences reported, there is only one primate sequence (human) for rod opsin. To extend our knowledge of the primate family of opsins, the authors cloned and characterized the opsin cDNA for the Old World monkey Macaca fascicularis and determined its position in the phylogenetic tree of the opsin gene family. METHODS: A fragment of the monkey opsin cDNA was generated using specific primers and the polymerase chain reaction. This fragment was then used as a probe to isolate several cDNA clones from a M. fascicularis retinal cDNA library. The nucleotide sequence of monkey opsin was determined and translated into an amino acid sequence. The amino acid sequences of opsins from a variety of organisms ranging from mollusks, arthropods, and vertebrates, including monkey, were then compared to identify highly conserved amino acid residues and to construct a phylogenetic tree of the rod opsins. RESULTS: The monkey has a single major 2.2-kb transcript for opsin and minor transcripts of 1.5, 4.8, and 8.9 kb. The major transcript is highly abundant in the monkey retina and is localized to the inner segments of rod photoreceptor cells, typical of opsin mRNAs in other organisms. The sequence of monkey opsin closely resembles the human sequence at the nucleotide and the amino acid levels, with the latter having only 7 differences out of 348 residues. Comparison of the monkey sequence with the amino acid sequences from diverse organisms shows that all opsins share only 34 conserved amino acids, with the highest concentration of these residues occurring in the first cytoplasmic loop of rhodopsin. A hypothetical phylogenetic tree of the opsins suggests that both human and monkey opsin shared an ancestral gene with the mammalian Order Carnivora. CONCLUSION: The opsins of man and Old World monkeys have diverged little from the time they shared a common ancestor. Comparison of opsins from diverse phyla, however, shows that only 34 residues have been conserved. These conserved residues are likely to be important in the structure and function of rhodopsin.

Amino Acid Sequence↗
PubMed1994

Murine and bovine blue cone pigment genes: cloning and characterization of two new members of the S family of visual pigments.

Two novel visual pigment genes, mouse blue and bovine blue, have been isolated from mouse and bovine genomic libraries, respectively, using a human blue cone pigment cDNA as probe. Corresponding cDNA clones have also been obtained from mouse retinas. The intron-exon boundaries for the mouse gene were determined by comparing the genomic and cDNA sequences. The visual pigments encoded by the mouse and bovine blue pigment genes are highly homologous to each other (89% amino acid identity) and to human blue and chicken violet cone pigments (greater than 80% identity), but are less homologous to chicken or goldfish blue cone pigments (less than 50% identity). These results indicate that phylogenetically both mouse and bovine blue pigments belong to the S branch of visual pigments, rather than to the M branch.

Amino Acid Sequence↗
PubMed1994

Novel structural features of autoantibodies in murine lupus: a possible superantigen binding site?

The stimulus for the production of anti-DNA autoantibodies in lupus remains unknown. Since double-stranded DNA (dsDNA) is a weak immunogen, other stimuli such as B cell superantigens or anti-idiotypic antibodies may provide an alternative mechanism for their production. The presence of regulatory determinants on autoantibodies might be revealed through their structural characterization, but they have eluded detection, perhaps because they may be three-dimensional and require closer analysis. In this report we cloned and sequenced the heavy chain variable region (VH) of a monoclonal anti-dsDNA antibody, mAb 3E10, derived from MRL/lpr mice with lupus nephritis previously shown to express an idiotype associated with nephritis in murine and human lupus. We now show that mAb 3E10 VH contains novel structural features unrelated to DNA binding which are shared only by a subset of autoantibodies expressed in murine lupus. These lupus autoantibodies can be distinguished from antibodies of non-autoimmune strains by the presence of a specific sequence at the junction of the diversity and joining genes combined with the use of variable region genes with conserved sequences in framework 1 (FR1) and FR3. The location of the novel sequences indicates the possibility of a three-dimensional solvent-exposed determinant located distant from the classical antigen binding site that could regulate their production, possibly through binding B cell superantigens or other infectious agents.

Amino Acid Sequence↗
PubMed1994

Long-term photoreceptor transplants in dystrophic and normal mouse retina.

PURPOSE: To determine the long-term status of transgenic photoreceptors transplanted to the subretinal space of both rd mutant (receptorless) and normal mouse retina. METHODS: Microaggregates of neural retina from transgenic mice containing lacZ-labeled photoreceptors were transplanted to the subretinal space of adult rd mutant and normal mice. The transplant site was examined by light and electron microscopy at monthly intervals up to 9 months after transplantation surgery. RESULTS: Photoreceptors develop and survive well if transplanted with the proper orientation to the retinal pigment epithelium (RPE). The status of the photoreceptors, including outer segments and synaptic terminals, appear normal for at least 9 months after transplantation; they continue to express the lacZ reporter gene. Cones survive as well as rods. Transplants to the normal mouse develop normally, whereas the host photoreceptors displaced from the RPE degenerate. A barrier, formed by Müller cell processes, develops after photoreceptor degeneration in both normal and rd mouse retina and demarcates host from transplant tissue. Areas can be found in which neural processes have penetrated this barrier. There is no evidence of host-graft rejection. CONCLUSION: Transplanted progenitor photoreceptors develop and survive well for long periods of time in either the rd mutant or normal retina if they are properly positioned. In the former, they reconstitute a photoreceptor layer; in the latter, they replace the host photoreceptor layer, which degenerates after being displaced from the RPE. Areas of potential contact between donor and host neurons exist in these transplants.

Animals↗