Cynthia Jaworski

A Temperature-sensitive Mutation of Crygs in the Murine Opj Cataract

In Opj, an inherited cataract in mice, opacity is associated with a mutation in Crygs, the gene for γS-crystallin, the first mutation to be associated with this gene. A single base change causes replacement of Phe-9, a key hydrophobic residue in the core of the N-terminal domain, by serine. Despite this highly non-conservative change, mutant protein folds normally at low temperature. However, it exhibits a marked, concentration-dependent decrease in solubility, associated with loss of secondary structure, at close to physiological temperatures. This is reminiscent of processes thought to occur in human senile cataracts in which normal proteins become altered and aggregate. The Opj cataract is progressive and more severe in Opj/Opj than in Opj/+. Lens histology shows that whereas fiber cell morphology in Opj/+mice is essentially normal, in Opj/Opj, cortical fiber cell morphology and the loss of maturing fiber cell nuclei are both severely disrupted from early stages. This may indicate a loss of function of γS-crystallin which would be consistent with ideas that members of the βγ-crystallin superfamily may have roles associated with maintenance of cytoarchitecture.

Molecular and Biochemical Characterization of a Novel Oxysterol-binding Protein (OSBP2) Highly Expressed in Retina

We are interested in understanding the possible function(s) of the oxysterol-binding proteins in mediating oxysterol cytotoxicity in the retina. In this study we describe the cloning, localization, and biological activity of a novel oxysterol-binding protein (OSBP2), and complete the molecular characterization of the previously known OSBP1. Both OSBP genes contain 14 exons and have similar exon sizes and splice sites suggesting they may have arisen from a gene duplication event. OSBP1 is located in chromosome 11q12.1, and OSBP2 is located in 22q12. At the protein level they share 63% overall similarity and although they have unique N termini, both have similar pleckstrin homology domains within the N terminus region. Northern blot analyses indicate that OSBP1 is broadly expressed in human and monkey tissues. OSBP2 is detected mainly in retina, testis, and fetal liver. Western blot analysis using peptide antibodies specific to OSBP1 and OSBP2 detected the proteins in different subcellular fractions in the retinal monkey tissue. OSBP1 is detected mainly in the soluble or cytosolic fraction and nuclei whereas OSBP2 is detected exclusively in the detergent soluble fraction suggesting association with membranes. Immunohistochemical localization of OSBP1 and OSBP2 in the monkey retina placed these two proteins in similar but distinct areas of the inner retina. OSBP2 was found to bind 7-ketocholesterol but to have very little affinity for cholesterol or 25-hydroxycholesterol.

Identification and characterization of PiORP1, a Petunia oxysterol-binding-protein related protein involved in receptor-kinase mediated signaling in pollen, and analysis of the ORP gene family in Arabidopsis

Oxysterol-binding proteins (OSBPs) and oxysterol-binding-protein related proteins (ORPs) are encoded by most eukaryotic genomes examined to date; however, they have not yet been characterized in plants. Here we report the identification and characterization of PiORP1, an ORP of Petunia inflata that interacts with the cytoplasmic kinase domain of a receptor-like kinase, named PRK1, of P. inflata. PiORP1 is phosphorylated by PRK1 in vitro and therefore may be involved in PRK1 signaling during pollen development and growth. RNA gel blot analysis showed that PiORP1 and PRK1 had very similar expression patterns in developing pollen, mature pollen and pollen tubes. GFP fusion proteins of PiORP1 localized in the plasma membrane of pollen tubes at distinct foci and its PH domain alone was sufficient to mediate this localization. The sequence for the oxysterol-binding domain of PiORP1 was used to search the genome of Arabidopsis; 12 ORPs were identified and phylogenetic analysis revealed that they fell into two distinct clades, consistent with the ORPs of other eukaryotes. RT-PCR analysis showed that all 12 Arabidopsis ORPs were expressed; 10 were expressed in most of the tissues examined under normal growth conditions, but only three were expressed in pollen.

A REASSESSMENT OF MAMMALIAN ALPHA A-CRYSTALLIN SEQUENCES USING DNA SEQUENCING : IMPLICATIONS FOR ANTHROPOID AFFINITIES OF TARSIER

AbstractαA-crystallin, a major structural protein in the ocular lenses of all vertebrates, has been a valuable tool for molecular phylogenetic studies. This paper presents the complete sequence for human αA-crystallin derived from cDNA and genomic clones. The deduced amino acid sequence differs at two phylogenetically informative positions from that previously inferred from peptide composition. This led us to examine the same region of the αA-crystallin gene in 12 other mammalian species using direct sequencing of PCR-amplified genomic DNA. New sequences were added to the database, and corrections were made to all anthropoid sequences, defining clear synapomorphies for anthropoids as a clade distinct from prosimians. Within the anthropoids there are further synapomorphies delineating hominoids, Old World monkeys, and New World monkeys. Significantly, sequence revisions and the addition of new sequence for a prosimian, the sifaka, eliminate the previous support for the proposed anthropoid affinities of the tarsier inferred from αA-crystallin protein sequences. In addition, DNA sequences provide greater resolution of certain relationships. For example, although they are identical in protein sequence, comparison of DNA sequences clearly separates mouse and the common tree shrew, grouping the tree shrew closer to prosimians. These results show that adding DNA sequences to the existing αA-crystallin database can enhance its value in resolving phylogenetic relationships.

Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.

The αA-crystallin gene: Conserved features of the 5′-flanking regions in human, mouse, and chicken

SummaryApproximately 2 kb of 5′-flanking sequences of the lens-specific αA-crystallin genes from human and mouse are presented and compared with similar regions of the chicken gene. A repetitive element was found approximately 1 kb upstream from the coding sequences of the αA-crystallin gene in all three species (Alu in human, B2 in mouse, and CR1 in chicken), suggesting that they may have an important functional or structural role.Despite the ability of αA-crystallin promoters to function across species, dot matrix analyses show only limited similarity among the 600 bp 5′ to the structural genes of these three species. The human 5′-flanking sequence is more similar to that of the mouse and chicken than the mouse and chicken are to each other. Numerous short sequences (8–13 bp) are common to all three genes but are distributed differently in each species. The locations and conservation of these sequence motifs suggest functional roles, possibly ascis-regulatory elements of transcription. One motif is similar to the αA-CRYBP1 binding site implicated earlier in the transcriptional regulation of the mouse αA-crystallin gene, and other motifs correspond to sites previously mapped by methylation interference studies in the mouse αA-crystallin promoter. The modular arrangement of conserved sequence motifs is consistent with evolutionary changes occurring at the level of gene regulation.

Resveratrol Attenuates CXCL11 Expression Induced by Proinflammatory Cytokines in Retinal Pigment Epithelial Cells

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). RPE cells adjacent to drusen deposits in the AMD eye are known to contain CXCL11, a chemokine involved in inflammatory cell recruitment. We investigated the CXCL11 production by the human RPE (ARPE-19) cells under inflammatory conditions and tested its response to resveratrol, a naturally occurring anti-inflammatory antioxidant. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1β and TNF-α highly increased CXCL11 mRNA expression and CXCL11 protein secretion by ARPE-19 cells. Resveratrol substantially inhibited the proinflammatory cytokines-induced CXCL11 production while partially blocking nuclear factor-κB activation. This inhibitory action of resveratrol was also observed for the cytokines-induced expression of chemokines CXCL9, CCL2 and CCL5. Our results indicate that resveratrol could potentially attenuate RPE inflammatory response implicated in the pathogenesis of AMD.

Potentiation of hormone-stimulated accumulation of cyclic AMP in cultured fibroblasts by trypsin

Abstract Evidence is presented that brief trypsin treatment of intact normal rat kidney cells significantly increases the intracellular accumulation of cyclic AMP in response to added prostaglandin E1 (PGE1) and (−)-isoproterenol. Trypsin exhibits a concentration-dependent enhancement of hormone-stimulated accumulation of cyclic AMP in intact normal rat kidney cells, with maximal stimulation (two- to threefold) observed with 25 μg/ml trypsin. The trypsin stimulatory effect is rapid, with optimal enhancement noted within 6 to 8 min after protease addition. The extent of stimulation is not altered with increasing PGE1 concentration. Trypsin, by itself, elicits only a 30 to 50% increase in basal cyclic AMP levels. These results suggest that trypsin might act on the external cell surface to modulate hormonal responsiveness, perhaps by eliminating a GTP inhibitory effect on the normal rat kidney adenylate cyclase system.

Proinflammatory cytokine interferon-γ increases the expression of BANCR, a long non-coding RNA, in retinal pigment epithelial cells

HIGHLIGHTSIFN‐&ggr;, IL‐1&bgr; and TNF‐&agr; altered lncRNA expression in retinal pigment epithelial cells.IFN‐&ggr;, but not IL‐1&bgr; or TNF‐&agr;, increased the expression of the lncRNA BANCR.Inhibition of JAK‐STAT1 signaling pathway blocked IFN‐&ggr;‐induced BANCR expression.BANCR may link inflammatory response and retinal pigment epithelial dysfunction. ABSTRACT The inflammatory response may contribute to retinal pigment epithelial (RPE) dysfunction associated with the pathogenesis of age‐related macular degeneration (AMD). We investigated whether the inflammatory response affects the expression of long coding RNAs (lncRNAs) in human RPE‐derived ARPE‐19 cells. This class of regulatory RNA molecules recently came to prominence due to their involvement in many pathophysiological processes. A proinflammatory cytokine mixture consisting of IFN‐&ggr;, IL‐1&bgr; and TNF‐&agr; altered the expression several lncRNAs including BANCR in these cells. The cytokine responsible for increasing BANCR expression in ARPE‐19 cells was found to be IFN‐&ggr;. BANCR expression induced by IFN‐&ggr; was suppressed when STAT1 phosphorylation was blocked by JAK inhibitor 1. Thus, proinflammatory cytokines could modulate the expression of lncRNAs in RPE cells and IFN‐&ggr; could upregulate the expression of BANCR by activating JAK‐STAT1 signaling pathway.

Transcriptional Control of the α-Crystallin Gene Family

Crystallina constitute 80–90% of the soluble protein of the ocular lens. There are a surprisingly large number of crystallin gene families; some (α- and sγ-crystallins) are present in all vertebrate lenses, whereas others (the enzyme-crystallins) are found only in the lenses of certain species. The enzyme-crystallins are not lens-specific and appear to have a double function, i.e., as structural proteins in the lens and as metabolic enzymes in other tissues (see Wistow and Piatigorsky, 1988; Piatigorsky and Wistow, 1989). In addition to their lens-specific or lens-preferred expression, the synthesis of the crystallin polypeptides is developmentally regulated in a temporal and spatial manner in the lens (see McAvoy, 1981; Piatigorsky, 1981).