Subject(s)
Cell Differentiation , Cinnamates , Electroporation/methods , Mice, Transgenic/genetics , Stem Cells/cytology , Stem Cells/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Blastocyst , Clone Cells , DNA/genetics , DNA/isolation & purification , Drug Resistance/genetics , Embryo, Mammalian , Female , Genetic Markers , Hygromycin B/analogs & derivatives , Hygromycin B/pharmacology , In Vitro Techniques , Male , Mice , Mice, Knockout/genetics , Mitomycin/pharmacology , Neomycin/pharmacology , Stem Cells/drug effects , TransfectionABSTRACT
Serotonin exerts its diverse physiological effects by interacting with multiple distinct receptor subtypes. We have isolated a rat brain 5HT2 serotonin receptor cDNA by virtue of its homology with the 5HT1c receptor. The 5HT2 receptor is a member of the family of receptors that are linked to guanine nucleotide-binding proteins and are predicted to span the lipid bilayer seven times. Overall sequence identity between the 5HT2 and 5HT1c receptors is 49%, but identity within the transmembrane domains is 80%. Expression of both the 5HT2 and 5HT1c receptors in transfected mouse fibroblasts activates phospholipase C signaling pathways and promotes cellular transformation. However, RNA blotting shows that these two receptor subtypes are differentially expressed in the central nervous system. In this manner, structurally and functionally homologous receptor subtypes may elicit distinct physiologic actions.
Subject(s)
Receptors, Serotonin/genetics , Amino Acid Sequence , Animals , Antiparkinson Agents/pharmacology , Brain/metabolism , DNA/genetics , DNA/isolation & purification , Ergolines/pharmacology , Fibroblasts/metabolism , Gene Library , Genes , Mice , Molecular Sequence Data , Organ Specificity , RNA, Messenger/analysis , RNA, Messenger/genetics , Rats , Receptors, Serotonin/drug effects , Receptors, Serotonin/metabolism , Sequence Homology, Nucleic Acid , Spiperone/pharmacology , Transfection , Type C Phospholipases/metabolismABSTRACT
Neurotransmitter receptors are usually restricted to neuronal cells, but the signaling pathways activated by these receptors are widely distributed in both neural and non-neural cells. The functional consequences of activating a brain-specific neurotransmitter receptor, the serotonin 5HT1c receptor, in the unnatural environment of a fibroblast were examined. Introduction of functional 5HT1c receptors into NIH 3T3 cells results, at high frequency, in the generation of transformed foci. Moreover, the generation and maintenance of transformed foci requires continued activation of the serotonin receptor. In addition, the injection of cells derived from transformed foci into nude mice results in the generation of tumors. The serotonin 5HT1c receptor therefore functions as a protooncogene when expressed in NIH 3T3 fibroblasts.
Subject(s)
Cell Transformation, Neoplastic , Gene Expression Regulation , Receptors, Serotonin/genetics , Animals , Calcium/pharmacology , Cell Division , Cell Line , Cloning, Molecular , Fibroblasts/metabolism , Genetic Vectors , Mice , Mice, Nude , Neoplasm Transplantation , Receptors, Serotonin/physiology , Second Messenger Systems , Serotonin/pharmacology , Serotonin/physiology , TransfectionABSTRACT
A variety of recombinant DNA molecules were constructed in which an avian retroviral long terminal repeat (LTR) was ligated to the bovine growth hormone (bGH) gene. The retroviral LTR was derived from a plasmid clone of a Schmidt Ruppin B strain of Rous sarcoma virus while the bGH gene was subcloned from a lambda bacteriophage genomic library. Using a transient eukaryotic expression assay system, recombinant plasmid constructs were screened for their ability to direct expression and secretion of bGH. One such plasmid DNA construct, termed pBGH-4, was found to be active in the production of bGH. Stable mouse fibroblast cell lines were generated containing pBGH-4 DNA integrated into the mouse cell genome. Many of these mouse cell lines express and secrete bGH. One line, L-Pd lambda-BGH4-13, was found to secrete bGH at a rate of 75 micrograms per 5 X 10(6) cells per 24 hr. Bovine growth hormone derived from this cell line is biologically active.
Subject(s)
Avian Sarcoma Viruses/genetics , Genetic Engineering/methods , Growth Hormone/genetics , Animals , Cells, Cultured , DNA, Recombinant , Gene Amplification , Gene Expression Regulation , Genetic Vectors , L Cells , Leucine/analogs & derivatives , Leucine/pharmacology , Protein Processing, Post-Translational/drug effects , Repetitive Sequences, Nucleic AcidABSTRACT
The nucleoside analog 2'-nor-2'-deoxyguanosine (2'NDG) effectively inhibits the replication of several laboratory and clinical isolates of human cytomegalovirus. These isolates included viruses obtained from congenitally infected infants and patients suffering from acquired immune deficiency syndrome. The dose of 2'NDG that inhibited cytomegalovirus plaque formation ranged from 0.1 to 1.6 micrograms/ml. At 10 micrograms/ml, 2'NDG completely blocked the production of virus progeny but not the expression of immediate early and early virus gene functions. Cytomegalovirus DNA was not detectable in 2'NDG-treated virus-infected human embryo lung cells when assayed by CsCl density gradient centrifugation. In contrast, the guanosine analog acyclovir at 100 micrograms/ml did not inhibit the production of virus or the synthesis of cytomegalovirus DNA. In virus-infected cells, 2'NDG and acyclovir at 10 and 100 micrograms/ml, respectively, inhibited the incorporation of [3H]thymidine and 32Pi into cellular DNA by ca. 50%. Uninfected human embryo lung cells grown in these concentrations of acyclovir or 2'NDG exhibited a slightly transient lag phase but, overall, cell growth was not retarded, and there was no decrease in cell viability. The extended lag in cell division was not due to inactivation or breakdown of the antiviral compounds but may be due in part to a temporary decrease in cellular DNA synthesis.
Subject(s)
Acyclovir/analogs & derivatives , Antiviral Agents/pharmacology , Cytomegalovirus/drug effects , Virus Replication/drug effects , Acyclovir/pharmacology , Cell Line , Cells, Cultured , DNA/biosynthesis , DNA, Viral/biosynthesis , Embryo, Mammalian , Ganciclovir , Humans , Lung , Thymidine/metabolism , Time FactorsABSTRACT
Experiments were conducted to determine whether any observable short-term biological effects were caused by a static magnetic field created with CoSm magnets similar to those used in the elbow prosthesis described on pages 69-80. Tissue culture studies of several cell lines showed no obvious effects on cell growth rate, morphology, or the ability to grow and remain confluent; and no deleterious effects of the magnetic field were evident as a result of an in-vivo study of wound and bone healing in rats. Long-term effects, if any, have yet to be determined.
