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R D Cone

Publications and source records attributed to R D Cone.

71 records · Page 4Linked to original sources
PubMed1992

3',5'-cyclic adenosine monophosphate-regulated enhancer binding (CREB) activity is required for normal growth and differentiated phenotype in the FRTL5 thyroid follicular cell line.

The thyroid follicular cell requires elevated levels of cAMP for normal growth and optimal expression of the differentiated phenotype. The recent discovery of cAMP-regulated enhancer binding (CREB) proteins prompted us to analyze the possible role of these transcription factors in controlling thyroid cell growth and differentiated phenotype using the FRTL5 thyroid cell line as a model system. FRTL5 cells were stably transfected with an expression vector containing either the gene for wild type CREB (WTCREB) or a dominant negative mutant form of CREB, termed KCREB, which dimerizes with and inactivates endogenous CREB. Transfected clones were found to express the transfected KCREB and WTCREB mRNAs at higher levels than the endogenous CREB mRNA. Transient expression of a somatostatin-chloramphenicol acetyltransferase fusion gene in these clones demonstrated a 60% reduction of cAMP-regulated enhancer-dependent transcriptional activity in the KCREB transfected clones and wild type levels of activity in the WTCREB transfected clones. Parameters of growth (DNA synthesis and growth rate) and differentiation (iodide uptake and thyroglobulin mRNA levels) were then analyzed in the transfected clones. Transfection of WTCREB had no effect on any of the parameters examined in comparison to untransfected cells, presumably because CREB is already constitutively expressed at maximal levels in normal FRTL5 cells. However, cells expressing KCREB showed an 18-40% reduction in TSH-stimulated thymidine incorporation, a 31% increase in the length of the cell cycle, and a 4-fold reduction in TSH-stimulated iodide uptake in comparison with wild type cells or cells tranfected with wild type CREB.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1991

The interaction of signal transduction pathways in FRTL5 thyroid follicular cells: studies with stable expression of beta 2-adrenergic receptors.

Multiple signal transduction pathways interact in FRTL5 cells to promote thyroid follicular cell differentiated function and cell proliferation. In these cells, TSH is a tissue-specific mitogen that promotes DNA synthesis primarily through activation of adenylate cyclase. To further test the role of adenylate cyclase in regulating cell growth and differentiated function we have introduced into FRTL5 the human beta 2-adrenergic receptor (BAR) complementary DNA and have studied the ability of isoproterenol, alone and in combination with insulin-like growth factor I (IGF-I), to stimulate cAMP accumulation, iodide transport, [3H]thymidine incorporation into DNA, and cell growth. Wild-type FRTL5 were infected with a PLJ retroviral construct containing the BAR in either a sense (FRTL BAR) or antisense (FRTL RBAR) orientation, and cell populations were selected on the basis of resistance to the antibiotic geneticin. FRTL BAR expressed approximately 1.3 x 10(5) high affinity binding sites per cell for the beta 2-specific ligand, CGP-12177, while neither FRTL5 wild-type nor RBAR cells demonstrated any specific binding. FRTL BAR had significantly higher levels of intracellular cAMP, [3H]thymidine incorporation, and iodide uptake in the absence of added isoproterenol than FRTL RBAR or wild-type cells. In FRTL BAR, but not RBAR cells, isoproterenol stimulated a dose-dependent accumulation of cAMP, iodide uptake, [3H]thymidine incorporation, and cell growth. FRTL BAR and RBAR cells were equally responsive to TSH and to IGF-I. Isoproterenol enhanced the ability of IGF-I to stimulate [3H]thymidine incorporation in BAR but not RBAR cells. Isoproterenol partially inhibited the ability of TSH to stimulate cAMP generation and DNA synthesis. These studies demonstrate that activation of adenylate cyclase through the BAR introduced into FRTL5 cells by retroviral infection reproduces the range of biological effects in these cells stimulated by TSH and suggest that activation of adenylate cyclase is sufficient to stimulate thyroid differentiated function and cell growth. FRTL BAR cells will provide an interesting model system with which to study the heterologous regulation of both TSH and BARs through activation of a common signal transduction pathway, adenylate cyclase.

Adenylyl Cyclases↗
PubMed1990

Isolation of TSH and LH/CG receptor cDNAs from human thyroid: regulation by tissue specific splicing.

A TSH receptor (TSH-R) cDNA has been isolated from a human thyroid lambda GT11 library. Unexpectedly, several cDNAs encoding the human LH/CG receptor (LH/CG-R), previously thought to be expressed solely in gonadal cells, were also isolated from the thyroid library. The receptors are structurally related, consisting of a signal sequence, a large extracellular amino terminal domain, seven membrane spanning domains, and a short carboxyl-terminal portion. The TSH-R is encoded by a single 4.2 kilobase mRNA specific to the thyroid. Introns were not present in any hTSH-R cDNAs examined, however, sequencing of several LH/CG-R cDNAs and RNase protection experiments demonstrated that the majority of hLH/CG-R mRNA in the thyroid is incompletely spliced. Consequently, tissue-specific splicing may be an important step in the regulation of the glycoprotein hormone receptor family.

Amino Acid Sequence↗
PubMed1988

Retroviral-mediated transfer and expression of human beta-globin genes in cultured murine and human erythroid cells.

We have cloned human beta-globin DNA sequences from a genomic library prepared from DNA isolated from the human leukemia cell line K562 and have used the retroviral vector pZip-NeoSV(X)1 to introduce a 3.0-kilobase segment encompassing the globin gene into mouse erythroleukemia cells. Whereas the endogenous K562 beta-globin gene is repressed in K562 cells, when introduced into mouse erythroleukemia cells by retroviral-mediated gene transfer, the beta-globin gene from K562 cells was transcribed and induced 5-20-fold after treatment of the cells with dimethyl sulfoxide. The transcripts were correctly initiated, and expression and regulation of the K562 gene were identical to the expression of a normal human beta-globin gene transferred into mouse erythroleukemia cells in the same way. We have also introduced the normal human beta-globin gene into K562 cells using the same retrovirus vector. SP6 analysis of the RNA isolated from the transduced cells showed that the normal beta-globin gene was transcribed at a moderately high level, before or after treatment with hemin. Based on these data, we suggest that the lack of expression of the endogenous beta-globin gene in K562 cells does not result from an alteration in the gene itself and may not result from a lack of factor(s) necessary for beta-globin gene transcription. Retroviral-mediated transfer of the human beta-globin gene may, however, uniquely influence expression of the gene in K562 cells.

Animals↗
PubMed1988

A retrovirus expressing the 12S adenoviral E1A gene product can immortalize epithelial cells from a broad range of rat tissues.

An epithelial cell-transforming virus could be of great use, both in the culture of epithelial cell lines and in the study of carcinogenesis. Since the adenoviral E1A gene has been shown to partially transform some epithelial cells from primary rat cell cultures, we constructed retrovirus vectors containing either the 12S or 13S E1A cDNA sequences to facilitate the transfer of these genes into a variety of primary cell types. The 12S E1A virus induced proliferation and immortalization of epithelial cells in rat kidney, liver, heart, pancreas, and thyroid primary cultures. In the two cases tested, heart and liver cultures, E1A-immortalized cells were nontumorigenic, but could be completely transformed by subsequent introduction of the ras oncogene. To our surprise, the 13S virus had a greatly reduced immortalization potential. We discuss these data in light of the model of Spindler et al. (K. R. Spindler, C. Y. Eng, and A.-J. Berk, J. Virol. 53:742-750, 1985), in which the 12S E1A protein is required for the complete induction of the cellular DNA replication machinery in the quiescent human epithelial cells in which adenoviruses normally replicate.

Adenovirus Early Proteins↗
PubMed1988

Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.

Animals↗
PubMed1988

HLA-DR gene expression in a proliferating human thyroid cell clone (12S).

We have used a retroviral vector carrying the adenovirus E1A oncogene and the neomycin phosphotransferase gene to establish a human thyroid-derived cell line that exhibits TSH-mediated cAMP generation as well as the differential expression of HLA class II antigens in response to recombinant gamma-interferon. Twenty-two-week gestation, histologically confirmed, human fetal thyroid was collagenase digested, cultured as a monolayer, and infected directly with 12S or 13S E1A-containing retrovirus constructs. Infected clones (n = 30) were selected in a hormone-supplemented medium containing bovine TSH (bTSH; 1 mU/ml), 10% fetal bovine serum, and 0.5 mg/ml G418 antibiotic. A rapidly growing clone (designated 12S) was chosen for detailed analysis over 18 months of continuous culture. The 12S clone was sensitive to less than 10 microU/ml bTSH when assessed by extracellular accumulation of cAMP, but TSH had no influence on 72-h incorporation of [3H]thymidine. Clone 12S responded to recombinant human gamma-interferon (1-10(4) U/ml) by induction of HLA DR alpha-chain-specific mRNA and the surface expression of HLA-DR antigen detected by fluorescein isothiocyanate-labeled monoclonal antibody to nonpolymorphic HLA-DR regions using flow cytometry. These studies indicate the potential for immortalizing human thyroid cells for use as targets of anti-TSH receptor immune responses and for long term studies of human throcyte HLA gene regulation.

Adult↗
PubMed1987

Tissue-specific expression of functionally rearranged lambda 1 Ig gene through a retrovirus vector.

To explore the potential use of retrovirus vectors for the transfer of genomic DNA sequences into mammalian cells, recombinant retroviral genomes were constructed that encode a functionally rearranged murine lambda 1 immunoglobulin gene. Several of these genomes could be transmitted intact to recipient cells by viral infection, although successful transmission depended both on the orientation of the lambda 1 sequences and on their specific placement within vector sequences. The lambda 1 gene transduced by viral infection was expressed in a cell lineage-specific manner, albeit at lower levels than endogenous lambda 1 gene expression in cells from the B-lymphocyte lineage. Vectors yielding integrated proviruses that lacked viral transcriptional enhancer sequences were used to show that neither viral transcription nor the viral transcriptional sequences themselves had any effect on the tissue specificity of lambda 1 gene expression or the absolute amount of lambda 1 transcription. Vector transcription did, however, dramatically decrease the amount of lambda 1 protein that could be detected in tranduced cells. These results suggest that retrovirus vectors may be useful reagents not only for the expression of complementary DNA sequences but also for studies of tissue-specific transcription in mammalian cells.

Animals↗
PubMed1987

Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element.

Animals↗
PubMed1986

Tissue-specific and ectopic expression of genes introduced into transgenic mice by retroviruses.

Recombinant retroviruses containing the complete genomic human beta globin gene (under the control of its own promoter) and the bacterial neomycin phosphotransferase gene (under the control of the normal or enhancerless viral promoter) were used to derive transgenic mouse strains by infection of preimplantation embryos. Expression of the beta globin gene in hematopoietic tissues was observed in all transgenic strains. In addition, one strain showed ectopic expression of beta globin in the same tissues that also expressed high levels of RNA from the viral promoter. It is likely that expression from the long terminal repeat (LTR), in contrast to expression from the internal promoter, is dependent on the site of integration. Thus, retroviral vectors can be used for tissue-specific expression of foreign genes in transgenic mice, as well as for the identification of loci that allow developmental activation of a provirus.

Animals↗
PubMed1986

High efficiency gene transfer into murine T cell clones using a retroviral vector.

To establish a gene transfer and expression system for murine T cell clones, we have introduced the neomycin phosphotransferase gene encoding resistance to the neomycin analogue, G418, into non-neoplastic inducer T cell clones by using a replication-defective retroviral vector. This method allowed highly efficient gene transfer (20 to 40%) into two inducer T cell clones. The level of viral RNA expression in G418r T cells was 0.1% of poly(A)+ RNA. The infected G418r cells retained physiologic responsiveness to specific antigen as judged by antigen-specific proliferation and production of IL 3.

Animals↗
PubMed1984

High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.

We have constructed a chimeric retrovirus genome containing ecotropic gag-pol sequences from Moloney murine leukemia virus and envelope sequences derived from the amphotropic virus 4070A. This reconstructed genome, termed pMAV-psi-, lacks the psi site required for encapsidation of the viral genome. NIH 3T3 cells transfected with pMAV-psi-, called psi-AM lines, are capable of producing high titer stocks of helper-free recombinant retrovirus with amphotropic host range after transfection with recombinant retroviral vectors carrying the neomycin phosphotransferase gene. Most transfected psi-AM cells remain helper-free, even after months in culture. psi-AM virus stocks infect nearly all human and murine cell lines tested thus far, as assayed by resistance to the neomycin analogue G418. Southern and RNA blot analyses of psi-AM-infected human cells show that recombinant murine retroviruses integrate randomly into genomic DNA as normal proviruses and express high levels of the subgenomic and genomic viral messages in the expected stoichiometry of 1:1.

Animals↗
PubMed1982

Small nuclear ribonucleoprotein complexes of Drosophila melanogaster.

We report here on the isolation and characterization of small nuclear ribonucleoproteins (snRNPs) and corresponding small nuclear RNA (snRNA) species from nuclei of Drosophila melanogaster. Velocity sedimentation in sucrose gradients was used to partially fractionate the RNPs; analysis of fractions so obtained suggests that, in general, one snRNP contains one snRNA. At least 11 species of snRNA are present in Drosophila nuclei; among them we identify a potential mammalian U1 homolog based on sequence homology. Autoimmune antiserum designated anti-Sm from patients with systemic lupus erythematosus recognizes nuclear antigens in Drosophila and precipitates seven species of snRNPs. The antigens in HeLa and Drosophila nuclei recognized by anti-Sm antibodies have been identified and compared. Anti-Sm antibodies at least bind to a 26,000-dalton polypeptide in HeLa extracts and to two polypeptides, one of 18,000 daltons and one of 26,000 daltons, in Drosophila extracts. This suggests that the 26,000-dalton polypeptide is an evolutionarily conserved antigenic component of Drosophila and HeLa snRNPs.

Animals↗
PubMed1970

Differentiation of Clostridium botulinum types A, B, and E by pyrolysis-gas-liquid chromatography.

Vegetative cells and spores of 10 strains of Clostridium botulinum representing types A, B, and E were grown in Trypticase-peptone-sucrose-yeast extract (TPSY) medium. Five type E strains were also grown in Multipeptone-sucrose-Nutramino acids (MSN) medium. Lyophilized samples were subjected to pyrolysis-gas-liquid chromatography (PGLC) analysis, and the resulting pyrograms were examined for variations in elution patterns between spores and vegetative cells of types A, B, and E grown in the TPSY medium and spores and vegetative cells of type E grown in the TPSY medium and spores and vegetative cells of type E grown in TPSY and MSN media. Growth and toxin production of all 10 strains of C. botulinum were investigated by using a modified dialysis sac culture technique. The dialysate supernatant fluid (DSF) obtained after centrifugation of the 5-day-old cultures from the dialysate was also subjected to PGLC analysis. Control samples consisting of (i) noninoculated DSF, (ii) noninoculated DSF plus partially purified toxin, and (iii) 1.0 mg of partially purified toxin were also analyzed by PGLC. Differences between pyrograms of cultures were suitable for positive identification at the type level but not at the strain level. Pyrograms permitting differentiation were also obtained between spores and vegetative cells as well as between the same cultures grown in different media. The dialysis sac technique was useful in detecting growth but not toxin production of C. botulinum.

Chromatography, Gas↗
PubMedDate not supplied

The melanocortin receptors: lessons from knockout models.

Identifying the role of the melanocortin system in regulating energy homeostasis has relied on both genetic and pharmacological studies. The key findings included 1) that the coat color phenotype in the lethal yellow (A(Y)/a) mouse is due to antagonism of the melanocortin-1 receptor (MC1R) by the agouti gene product; 2) the MC3R and MC4R are expressed in CNS centers involved in energy homeostasis, and 3) the combined results of pharmacological studies showing that agouti is an antagonist of the MC4R and transgenic studies showing that inhibition or loss of the MC4R recapitulate the lethal yellow phenotype. Pro-opiomelanocortin (POMC), MC3R, and MC4R knockouts are obese and are now being used to further analyze melanocortin receptor function. The obesity phenotype observed in the MC3R and MC4R knockouts (KO) differ markedly. MC4RKO mice are hyperphagic, do not regulate pathways that increase energy expenditure (diet-induced thermogenesis) and physical activity in response to hyperphagia, and can develop type 2 diabetes. In contrast, MC3R deficient mice are not hyperphagic, have a normal metabolic response to increased energy consumption, and do not develop diabetes. The mechanism underlying the increased adiposity in the MC3R knockout remains unclear, but might be related to changes in nutrient partitioning or physical activity.

Animals↗