Neeraj Agarwal

Production of bovine rhodopsin by mammalian cell lines expressing cloned cDNA: Spectrophotometry and subcellular localization

Cloned cDNA encoding bovine rhodopsin has been recombined into an expression vector and cotransfected with an antibiotic resistance plasmid into cultured human embryonic kidney cells. The resulting cell lines produce 100-200 micrograms of bovine opsin per liter of saturated tissue culture medium (10(9) cells). Incubation in vitro with 11-cis retinal produces a photolabile pigment the absorbance spectrum of which is indistinguishable from that of bona fide bovine rhodopsin. Expressed rhodopsin accumulates in the plasma membrane as determined by immunoelectron microscopy.

Opsin distribution and synthesis in degenerating photoreceptors of rd mutant mice

The distribution of opsin and the capacity of photoreceptors to synthesize opsin was studied in retinas of mice bearing the rd (retinal degeneration) mutation and compared to control normal mice. Opsin was localized by means of pre-embedding and post-embedding immunocytochemistry and electron microscopy. Cones were identified with anti-cone antibodies and peanut agglutinin lectin which labels cone matrix sheaths. Opsin synthesis was measured by incorporation of [35S]methionine into opsin which was detected by immunoblots. Immunocytochemistry revealed that degeneration of rod outer segments was accompanied by accumulation of opsin in the plasma membrane enveloping the inner segment, nuclei and synaptic terminals. Rod photoreceptors degenerated faster than cones. By post-natal day 19 (P19), 87% of the remaining inner segments were cones. Opsin synthesis in rd mice could no longer be measured after P15. However, opsin molecules could be detected both by immunocytochemistry and immunoblotting up to P30. Between P20 and P30 all detectable opsin was localized in the plasma membrane which envelopes nuclei and synaptic terminals. Unlabeled surviving nuclei after P30 are probably cones. The opsin which is detected in rod inner segment plasma membranes might be derived, by back diffusion, from degenerating outer segments and represent molecules synthesized at an earlier time. Alternatively opsin accumulation might be a result of low levels of opsin synthesis, undetectable by [35S]methionine incorporation, which continues in the absence of outer segments.

Diurnal expression of c-fos in the mouse retina.

The diurnal levels of c-fos mRNA were studied in the mouse retinas by means of RNA blot analysis. Mice were kept on a 12/12 h dark/light cycle and gene expression was studied at various time points during the day and night periods. The highest levels of c-fos mRNA were measured during the first half of the night period. The high levels persisted for about 4-5 h. The c-fos mRNA levels declined during the second half of the night period and remained low during the day period. Continuous illumination of mice during the first hours of the night period prevented the increase in c-fos mRNA. If mice were kept in the dark during the day period, they failed to show an increase in c-fos mRNA levels in the subsequent night period. Hence, following activation of c-fos during the night period, a refractory period exists at which illumination is required before c-fos can be induced again by dark. Although light appears to suppress the activation of c-fos in the night period, a short lived burst in c-fos expression of 30-60 min in duration was observed when dark-adapted animals were illuminated, either at the transition between the dark and light period or during the dark period. Thus, it appears that c-fos is activated in at least two different cell types in the retina which respond differently to light and dark stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Opsin gene expression during early and late phases of retinal degeneration in rds mice.

Opsin mRNA levels, opsin synthetic rates and localization of opsin were studied throughout the photoreceptors life span in the rds mice. Mutant mice 11 days to 11 months old were investigated. Opsin mRNA levels were studied by means of northern blot analysis. Opsin synthesis was measured by incorporation of [35S]methionine into newly synthesized opsin in vitro. Distribution of opsin in the retina was determined by immunoelectron microscopy. Opsin mRNA was detected in young as well as old retinas, and opsin synthesis could be detected at early phases of degeneration but not in late phases. The absence of opsin synthesis in older rds mice might be due to translational down-regulation or some other defect in the capacity to synthesize opsin. In young mice, opsin was detected in the subretinal space in opsin-laden vesicular membranes: such membranes were absent from retinas of older mice. This disappearance parallels the cessation of opsin synthesis and the consequent failure to deliver opsin to the subretinal space in retinas from older mice. Immunochemical analysis revealed the presence of small amounts of opsin in all retinas up to 11 months of age. Immunoelectron microscopy localized the residual opsin, mostly to the plasma membrane which envelops the nuclei and synaptic terminals. These opsin molecules might be a consequence of very low levels of opsin synthesis, too low to be detected by our assays, or may have been synthesized at an earlier age and retained in the plasma membrane of the old mutant photoreceptors.

Sequence analysis, expression and chromosomal localization of a gene, isolated from a subtracted human retina cDNA library, that encodes an insulin-like growth factor binding protein (IGFBP2)

The metabolic functions of insulin-like growth factors (IGFs) I and II are modulated by a family of binding proteins which are present in biological fluids and are synthesized by a variety of cell types. A cDNA clone, isolated at random from a subtracted human retina library, has been identified to code for a novel IGF-binding protein (IGFBP2) by its sequence homology to the peptide sequence of IGF binding proteins purified from bovine MDBK and rat BRL-3A cells. The complete nucleotide sequence of the IGFBP2 cDNA is 1406 bp long, contains 66% G-Cs and an open reading frame of 328 amino acids with a putative signal or pro-peptide of 39 residues. The mature polypeptide of 289 amino acids has 18 cysteines, a putative ATP-binding site and an RGD tripeptide. The 1.4 kb IGFBP2 transcript is expressed in several human tissues including fetal eye and fetal brain, but not in the human lymphoblastoid cell line against which the retinal cDNA library was subtracted. In situ hybridization to sections of mouse retina localized the mRNA for IGFBP2 primarily in the outer nuclear layer of photoreceptors. Southern blot analysis of DNA from human x rodent and mouse x rodent somatic cell hybrids assigned the gene for IGFBP2 to human chromosome 2q33-qter and mouse chromosome 1 in a known conserved syntenic region.

Maintenance of opsin and S-antigen gene expression in RCS dystrophic rats following RPE transplantation

The effects of retinal pigment epithelium (RPE) transplantation in rescuing photoreceptor cells (PRC) were studied in RCS rats bearing the rdy mutation. RPE preparations from Long-Evans rats were transplanted into the subretinal space of post-natal (P) 17-19-day-old RCS rats. Age-matched RCS animals in the control group received injections of the transplantation vehicle buffer alone. The animals were killed at various post-natal ages after RPE transplantation. Immunocytochemical studies using antibodies to opsin and S-antigen indicated intact photoreceptor cells in the transplanted retinas. In contrast age-matched sham-injected animals did not show any rescue of photoreceptor cells at P90. Single RPE transplantation of 60,000 cells in each eye resulted in restoration of near normal mRNA levels for opsin, and S-antigen proteins. Comparisons of mRNA levels of two visual proteins using cDNA probes demonstrated a therapeutic effect of RPE transplantation in preventing a progressive decline in mRNA levels due to retinal degeneration. In contrast, P109 sham-injected controls showed no detectable mRNA levels for these proteins. In vitro protein synthesis in RPE-transplanted retinas implied further competence of these retinas. These data suggest that RPE transplantation not only rescues photoreceptors from degeneration, but more importantly, it allows normal transcription and translation in these cells rendering them capable of participating in the visual and signal transduction cascade.

Expression of opsin and IRBP genes in mutant RCS rats.

The retinal pigment epithelium of RCS rats bearing the autosomal recessive rdy mutation fails to ingest shed rod outer segment tips. Accumulation of disk debris in the subretinal space of the maturing mutant retina causes a secondary degeneration of photoreceptor cells. Two hypotheses have been offered as possible explanations of the death of photoreceptor cells in this disorder: (1) photoreceptors are starved for amino acids, retinal, oxygen, etc; and (2) that IRBP levels and synthesis may be decreased and interfere with retinal transport and this deficiency is lethal to these cells. To test these hypotheses, we have studied the effect of this mutation on the levels of expression of opsin and IRBP genes, and gene products and on rates of synthesis at various ages in dystrophic RCS p+ rats and compared the results to those obtained with normal Long Evans rats. The mutant rats and normal controls had comparable amounts of opsin and IRBP mRNA transcripts and rates of synthesis up to post-natal day 45 (P45) but opsin transcripts were barely detectable at P60 and thereafter. IRBP mRNA levels were also very low after P62 although somewhat higher than opsin mRNA. Opsin could be detected immunochemically, albeit at lower levels, at all the ages studied up to P310, but IRBP levels fell below detection after P45. We localized opsin and IRBP in the retina by post-embedding EM immunocytochemical procedures and found that opsin is present in the remnants of rod outer segment debris, even at P390, long after detectable opsin synthesis had ceased. These data suggest that expression of opsin and IRBP genes is not influenced by the shape and state of the outer segments, and that the rdy mutation does not influence the expression of the opsin and IRBP in these retinas until the photoreceptor cells are profoundly damaged. Thus, neither hypothesis about the causes of cell death in this disorder is supported.