Biomedical subjects

P Eversole-Cire

Publications and source records attributed to P Eversole-Cire.

12 recordsLinked to original sources

PubMed2000

Synergistic effect of Bcl-2 and BAG-1 on the prevention of photoreceptor cell death.

PURPOSE: Ectopic expression of Bcl-2 in photoreceptors of mice with retinal degenerative disease slows progression of the disease. BAG-1 has previously been shown to augment the inhibitory effect of Bcl-2 on programmed cell death in cultured cell systems. This study was designed to determine whether the coexpression of BAG-1 and Bcl-2 in the photoreceptors of mice with an autosomal dominant form of retinitis pigmentosa (RP) would enhance the protective effect provided by Bcl-2 alone. METHODS: An expression vector using the 5' regulatory region of the murine opsin gene was used to target the expression of BAG-1 specifically to photoreceptor cells of mice. The BAG-1 transgenic mice were crossed to Bcl-2 transgenics to obtain animals that coexpress the two transgenes in photoreceptor cells. BAG-1/Bcl-2 animals were then crossed to an RP mouse model (a transgenic line overexpressing the S334ter rhodopsin mutant) to assess the effect of coexpression of BAG-1 and Bcl-2 on retinal degeneration. Morphologic analysis was performed on retinas isolated at various times after birth to monitor disease progression. RESULTS: High levels of BAG-1 expression resulted in retinal degeneration that was not prevented by Bcl-2 expression. However, coexpression of appropriate levels of BAG-1 and Bcl-2 was found to have a profound inhibitory effect on retinal degeneration caused by overexpression of a mutant rhodopsin transgene. Whereas expression of Bcl-2 alone was previously found to delay degeneration of the retina from 2 weeks to approximately 4 weeks of age, coexpression of BAG-1 and Bcl-2 inhibited photoreceptor cell death for as long as 7 to 9 weeks. CONCLUSIONS: The synergistic effect against photoreceptor cell death produced by the coexpression of Bcl-2 and BAG-1 indicates that these proteins can function in concert to prevent cell death. At the correct dosage, coexpression of Bcl-2 and BAG-1 may serve as a potential means to treat retinal degenerative diseases.

Animals↗

PubMed1998

Enhancer-dependent, locus-wide regulation of the imprinted mouse insulin-like growth factor II gene.

The mouse insulin-like growth factor II (IGF-II) gene is subject to parental imprinting and is predominantly expressed from the paternal chromosome. This allele-specific expression is modified further by cell type, developmental stage, and growth conditions. We show that the ratio of the three major IGF-II mRNAs, each produced from a distinct promoter, is consistent in a variety of tissues and cells representing different modes and phases of the complex regulation. Nuclear run-on assays show that the major changes in total IGF-II mRNA level occur at the level of transcription. Moreover, a targeted disruption of the endoderm-specific enhancers, located 90 kb away from the gene, affects all promoters. The dependency of the promoters on distal enhancers is also shown by transgenesis experiments. Our findings suggest that enhancer-dependent, locus-wide mechanisms play a major role in the coordinate regulation of the multiple IGF-II promoters.

Animals↗

PubMed1997

Constitutively active Galphaq and Galpha13 trigger apoptosis through different pathways.

We investigated the effect of expression of constitutively active Galpha mutants on cell survival. Transfection of constitutively active Galphaq and Galpha13 in two different cell lines caused condensation of genomic DNA and nuclear fragmentation. Endonuclease cleavage of genomic DNA was followed by labeling the DNA fragments and subsequent flow cytometric analysis. The observed cellular phenotype was identical to the phenotype displayed by cells undergoing apoptosis. To distinguish between the apoptosis-inducing ability of the two Galpha-subunits, the signaling pathways involved in this cellular function were investigated. Whereas Galphaq induced apoptosis via a protein kinaseC-dependent pathway, Galpha13 caused programmed cell death through a pathway involving the activation of the small G-protein Rho. Both of the pathways leading to apoptosis were blocked by overexpression of bcl-2. In contrast to other apoptosis-inducing systems, expression of constitutively active Galphaq and Galpha13 triggered apoptosis in high serum as well as in defined medium.

Animals↗

PubMed1997

Imprinted genes are up-regulated by growth arrest in embryonic fibroblasts.

It was previously reported that the imprinted insulin-like growth factor II and H19 genes are up-regulated in embryonic fibroblasts upon growth arrest. We have examined six other imprinted genes and found that all were up-regulated upon confluency and at least four of them by serum starvation. The significance of the results was confirmed by demonstrating that only one-third of randomly selected genes are up-regulated upon confluency. Our findings suggest that the cell cycle should be carefully controlled when imprinted genes are studied in cell cultures. Moreover, the unique property could have significance in the mechanistic or the evolutionary aspect of imprinting or both.

Animals↗

PubMed1995

Coordinate regulation of Igf-2 and H19 in cultured cells.

The insulin-like growth factor 2 (Igf-2) and H19 genes are physically linked on mouse distal chromosome 7 and are reciprocally imprinted. We investigated the molecular basis of the parental imprints in somatic cell cultures derived from normal embryos or from their littermates with maternal uniparental disomy for distal chromosome 7 (MatDi7). In normal cells, the two genes appeared to respond to similar regulatory factor(s), since both genes were coordinately up-regulated upon growth arrest and cell clones which had lost expression of one gene had lost expression of the other. However, in a clone of MatDi7 cells (MatDi7 1-1a), which spontaneously began to express the maternally derived copy of Igf-2, Igf-2 and H19 were not coordinately regulated. MatDi7 1-1a cells showed de novo methylation of sites upstream of Igf-2 and also within the H19 promoter, epigenetic modifications normally seen only on the paternal chromosome. The data provide new experimental evidence for previously hypothesized mechanisms suggesting that Igf-2 and H19 are coordinately regulated.

Animals↗

PubMed1994

Progressive increases in the methylation status and heterochromatinization of the myoD CpG island during oncogenic transformation.

Alterations in DNA methylation patterns are one of the earliest and most common events in tumorigenesis. Overall levels of genomic methylation often decrease during transformation, but localized regions of increased methylation have been observed in the same tumors. We have examined changes in the methylation status of the muscle determination gene myoD, which contains a CpG island, as a function of oncogenic transformation. This CpG island underwent de novo methylation during immortalization of 10T1/2 cells, and progressively more sites became methylated during the subsequent transformation of the cells to oncogenicity. The greatest increase in methylation occurred in the middle of the CpG island in exon 1 during transformation. Interestingly, no methylation was apparent in the putative promoter of myoD in either the 10T1/2 cell line or its transformed derivative. The large number of sites in the CpG island that became methylated during transformation was correlated with heterochromatinization of myoD as evidenced by a decreased sensitivity to cleavage of DNA in nuclei by MspI. A site in the putative promoter also became insensitive to MspI digestion in nuclei, suggesting that the chromatin structural changes extended beyond the areas of de novo methylation. Unlike Lyonized genes on the inactive X chromosome, whose timing of replication is shifted to late S phase, myoD replicated early in S phase in the transformed cell line. Methylation analysis of myoD in DNAs from several human tumors, which presumably do not express the gene, showed that hypermethylation also frequently occurs during carcinogenesis in vivo. Thus, the progressive increase in methylation of myoD during immortalization and transformation coinciding with a change in chromatin structure, as illustrated by the in vitro tumorigenic model, may represent a common mechanism in carcinogenesis for permanently silencing the expression of genes which can influence cell growth and differentiation.

5-Methylcytosine↗

PubMed1993

Activation of an imprinted Igf 2 gene in mouse somatic cell cultures.

The mouse insulin-like growth factor II gene (Igf 2), located on distal chromosome 7, is parentally imprinted such that the paternal allele is expressed while the maternal allele is transcriptionally silent. We derived a cell line from a mouse embryo maternally disomic and paternally deficient for distal chromosome 7 (MatDi7) to determine the stability of gene repression in culture. MatDi7 cells maintained Igf2 in a repressed state even after immortalization, except for one randomly picked clone which spontaneously expressed the gene. Igf 2 was expressed in a cell culture derived from a normal littermate; this expression was growth regulated, with Igf 2 mRNA levels increasing in the stationary phase of growth. Analysis of the methylation status of 28 sites distributed over 10 kb of the gene did not show consistent differences associated with expression level in the normal and MatDi7 cell lines, and the CpG island in the Igf 2 promoter remained unmethylated in all of the cell lines. Only with an oncogenically transformed cell line did the promoter become extensively methylated. We attempted to derepress the imprinted gene in MatDi7 cells by treatments known to alter gene expression. Expression of the Igf 2 allele in MatDi7 cells was increased in a dose-dependent manner by treatment with 5-aza-2'-deoxycytidine or bromodeoxyuridine, agents known to change DNA methylation patterns or chromatin conformation. Treatment of the cells with 1-beta-D-arabinofuranosylcytosine, 2'-deoxycytidine, calcium ionophore, heat shock, cold shock, or sodium butyrate did not result in increases in the levels of Igf 2 expression. It seems likely that the mechanism of the Igf 2 imprint involves subtle changes in the methylation or chromatin conformation of the gene which are affected by 5-aza-2'-deoxycytidine and bromodeoxyuridine.

Animals↗

PubMed1988

Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins.

Animals↗

PubMed1988

Identification of a GTP-binding protein alpha subunit that lacks an apparent ADP-ribosylation site for pertussis toxin.

Recent molecular cloning of cDNA for the alpha subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called Tr and Tc, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, we have identified a G-protein alpha subunit, which we refer to as Gz alpha, by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine Tr alpha-subunit cDNA. The deduced amino acid sequence of Gz alpha is 41-67% identical with those of other known G-protein alpha subunits. However, the 355-residue Gz alpha lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein alpha subunits. We suggest that Gz alpha, which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.

Adenosine Diphosphate Ribose↗

PubMed1987

Differential transcription of Xenopus oocyte and somatic-type 5 S genes in a Xenopus oocyte extract.

We have measured the transcription of Xenopus laevis oocyte and somatic-type 5 S RNA genes in S-150 extracts prepared from ovaries of mature X. laevis females (Glikin, G. C., Ruberti, I., and Worcel, A. (1984) Cell 37, 33-41). We find that somatic-type 5 S genes are transcribed at least 40-fold more efficiently than oocyte-type 5 S genes. Since adenovirus VA, Xenopus OAX, and Xenopus tRNAMet genes are all transcribed at levels similar to that of the somatic-type 5 S gene, this difference reflects a low level of oocyte-type 5 S gene transcription. Somatic-type 5 S transcription is a linear function of somatic-type 5 S DNA concentration and this, together with the efficient transcription of other class III genes, suggests that RNA polymerase III and the general class III transcription factors are not limiting under the conditions used here. Moreover, the 5 S gene-specific transcription factor TFIIIA does not limit transcription under these conditions as preincubation with purified TFIIIA does not alter transcription of either gene, and both genes exhibit characteristic TFIIIA footprints under transcription conditions in the S-150. Somatic-type 5 S DNA incubated in the S-150 and then injected into oocyte nuclei is actively transcribed whereas oocyte-type 5 S DNA treated in the same manner is inactive. We conclude that factors in the S-150 distinguish between somatic and oocyte-type 5 S genes, assembling active complexes preferentially on the former and inactive complexes preferentially on the latter. This process is not explained by binding properties of transcription factors for these genes as currently understood and represents a previously unrecognized mechanism of transcriptional regulation of the Xenopus 5 S genes.

Animals↗

PubMed1987

Transcriptionally inactive oocyte-type 5S RNA genes of Xenopus laevis are complexed with TFIIIA in vitro.

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.

Animals↗

PubMed1987

Nonsense mutations of the ornithine decarboxylase structural gene of Neurospora crassa.

Ornithine decarboxylase (ODC) (EC 4.1.1.17) is an early enzyme of polyamine synthesis, and its activity rises quickly at the onset of growth and differentiation in most eucaryotes. Some have speculated that the enzyme protein may have a role in the synthesis of rRNA in addition to its role in catalyzing the decarboxylation of ornithine (G. D. Kuehn and V. J. Atmar, Fed. Proc. 41:3078-3083, 1982; D. H. Russell, Proc. Natl. Acad. Sci. USA 80:1318-1321, 1983). To test this possibility, we sought mutational evidence for the indispensability of the ODC protein for normal growth of Neurospora crassa. We found three new, ODC-deficient mutants that lacked ODC protein. Among these and by reversion analysis of an earlier set of mutants, we found that two ODC-deficient mutants carried nonsense mutations in the ODC structural gene, spe-1. Allele LV10 imparted a complete deficiency for enzyme activity (less than 0.006% of normal) and had no detectable ODC antigen. Allele PE4 imparted a weak activity to cells (0.1% of derepressed spe+ cultures) and encoded a lower-molecular-weight ODC subunit (Mr = 43,000) in comparison to that of the wild-type strain (Mr = 53,000). Strains carrying either mutation, like other spe-1 mutants, grew at a normal rate in exponential culture if the medium was supplemented with spermidine, the main end product of the polyamine pathway in N. crassa. Unless an antigenically silent, N-terminal fragment with an indispensable role persists in the LV10-bearing mutant, we conclude that the ODC protein has no role in the vegetative growth of this organism other than the synthesis of polyamines. The data extend earlier evidence that spe-1 is the structural gene for ODC in N. crassa. The activity found in mutants bearing allele PE4 suggests that the amino acids nearest the carboxy terminus do not contribute to the active site of the enzyme.

Alleles↗