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At least 685 records · Page 38Linked to original sources
PubMed2005

Antisense recognition of the HER-2 mRNA: effects of phosphorothioate substitution and polyamines on DNA.RNA, RNA.RNA, and DNA.DNA duplex stability.

The HER-2 gene is overexpressed in a subset of breast, ovarian, lung, and pancreatic cancers. Antisense oligonucleotides suppress gene expression depending on the stability of the DNA.RNA hybrids formed at the target site. Polyamines, the cellular cations that interact with DNA and RNA, may influence hybrid stability in the cell. Therefore, we studied the ability of natural polyamines (putrescine, spermidine, and spermine) and a series of their structural analogues to stabilize DNA.RNA and RNA.RNA duplexes using melting temperature (T(m)) measurements and circular dichroism (CD) spectroscopy. Phosphodiester (PO) and phosphorothioate (PS) oligonucleotides (ODNs) (15 nucleotides, 5'-CTCCATGGTGCTCAC-3') targeted to the initiation codon region of the HER-2 mRNA, and complementary RNA and DNA ODNs, were used in this study. The relative order of thermal stability was as follows: RNA.RNA > PO-DNA.RNA > PO-DNA.PO-DNA > PS-DNA.RNA > PS-DNA.PO-DNA > PS-DNA.PS-DNA. The ability of polyamines to stabilize the duplexes improved with the cationicity of the polyamine, with hexamines being more effective than pentamines, which in turn were more effective than tetramines and triamines. However, chemical structural effects were clearly evident with isovalent homologues of spermidine and spermine. CD spectra showed B and A conformations, respectively, for the DNA and RNA helices. DNA.RNA hybrids adopted an intermediate structure between the B and A forms. These data help us to understand the role of endogenous polyamines in DNA.RNA hybrid stabilization, and provide information for designing novel polyamines to facilitate the use of antisense ODNs for controlling HER-2 gene expression.

Base Sequence↗
PubMed1997

Hydration effects on the duplex stability of phosphoramidate DNA-RNA oligomers.

Recent studies on uniformly modified oligonucleotides containing 3'-NHP(O)(O-)O-5'internucleoside linkages (3'amidate) and alternatively modified oligonucleotides containing 3'-O(O-)(O)PNH-5'internucleoside linkages (5'amidate) have shown that 3'amidate duplexes, formed with DNA or RNA complementary strands, are more stable in water than those of the corresponding phosphodiesters. In contrast, 5'amidates do not form duplexes at all. There is no steric reason that the 5'amidate duplex should not form. We demonstrate that these differences arise from differential solvation of the sugar-phosphate backbones. By molecular dynamics calculations on models of 10mer single-stranded DNA and double-stranded DNA-RNA molecules, both with and without the phosphoramidate backbone modifications, we show that the single-stranded 3'amidate and 5'amidate backbones are equally well solvated, but the 5'amidate backbone is not adequately solvated in an A-form duplex. These results are supported by quantum chemical free energy of solvation calculations which show that the 3'amidate backbone is favored relative to the 5'amidate backbone.

Amides↗
PubMed1996

Differential expression of the full-length and truncated forms of the epidermal growth factor receptor in the preimplantation mouse uterus and blastocyst.

The present investigation examined the differential expression of the full-length (fl) and the truncated (tr) forms of the epidermal growth factor receptor (EGFr) in the preimplantation mouse uterus and blastocyst. Northern blot hybridization using a complementary RNA probe specific to the full-length form (EGFr-fl) detected a 6.5-kb transcript, whereas that of the truncated form (EGFr-tr) detected a 2.7-kb transcript in the preimplantation mouse uterus on days 1 and 4 of pregnancy (day 1 = vaginal plug). In situ hybridization using these probes detected the EGFr-fl transcripts only in the stroma and myometrium, but not in the epithelium, whereas EGFr-tr transcripts were detected in all major uterine cell-types. To confirm the results of in situ hybridization, RT-PCR was performed on RNA isolated from separated uterine cell-types on day 4 of pregnancy using sequence specific primers for the two forms of the receptor. The results concur that the EGFr-tr transcript is expressed in the epithelium, stroma and myometrium, whereas that of the EGFr-fl transcript is not expressed in the epithelium. In the preimplantation blastocyst, RT-PCR detected the EGFr-fl messenger RNA, but not the EGFr-tr messenger RNA. These results suggest that the blastocyst, not the uterine epithelium, is the target for EGF family of growth factors in embryo-uterine interaction during implantation.

Animals↗
PubMed1999

Ovarian steroid regulation of serotonin-1A autoreceptor messenger RNA expression in the dorsal raphe of rhesus macaques.

It is widely hypothesized that ovarian steroids act on serotonin neurons to modulate mood and alter neuroendocrine function in women. However, information is needed on the molecular consequences of estrogen and progesterone action in serotonin neurons. This study examined the effect of estrogen, with and without progesterone, on the expression of messenger RNA for the serotonin-1A autoreceptor in monkeys using in situ hybridization and a 432-bp serotonin-1A probe generated with polymerase chain reaction. Monkeys were spayed/ovariectomized (control; n=4), estrogen treated (28 days, n=4) and estrogen+progesterone treated (14 days estrogen+14 days estrogen+progesterone, n=4). Perfusion-fixed midbrain sections containing the dorsal raphe (10 microm) were hybridized at 60 degrees C with 35S antisense complementary RNA. After a final wash in 0.1 x standard saline citrate at 70 degrees C, sections were apposed to betamax film for four days and then emulsion fixed. Adjacent sections were immunostained for serotonin to confirm the location of the dorsal raphe. Densitometric analysis of autoradiographs with gray level thresholding was performed at five levels of the dorsal raphe. The number of pixels exceeding background in defined areas was obtained (pixel number), as well as the mean optical density. In the estrogen- and the estrogen+progesterone-treated groups compared to the control group, there was a 38% and 43% decrease in serotonin-1A messenger RNA signal, respectively, represented by pixel number (P<0.05). Mean optical density for serotonin-1A was significantly decreased by estrogen treatment (21%; P<0.05) and then further decreased with the addition of progesterone treatment (45%; P<0.01). Also, the number of positive cells and the grains/cell were counted. There were significantly fewer serotonin-1A messenger RNA-positive cells in the serotonergic neurons of the dorsal raphe in estrogen- and estrogen+progesterone-treated groups (P<0.001) than controls. There were significantly lower single-cell levels of serotonin-1A messenger RNA in serotonergic neurons of the dorsal raphe only in the estrogen+progesterone-treated group (P<0.05). These results suggest that estrogen reduces serotonin-1A gene expression and that the addition of progesterone further reduces serotonin-1A gene expression in non-human primates. If the changes in gene expression are manifested by alterations in protein expression, then, together, these actions of estrogen and progesterone could increase serotonin neurotransmission, thereby elevating mood and/or altering neuroendocrine functions.

Animals↗
PubMed1997

The inhibitory effects of antisense RNA on hepatitis B virus surface antigen synthesis.

Antisense RNA-mediated inhibition of gene expression has the potential for gene therapy of virus infections. We studied the inhibitory effect of antisense RNA directed against the hepatitis B virus (HBV) genome on the expression of the HBV surface antigen (HBsAg). Three prokaryotic antisense RNA expression constructs were produced which expressed antisense RNA complementary to the entire coding region (1.4 kb) and to 1.0 kb and 582 bp of the 5' region of HBsAg mRNA, respectively. In an in vitro translation system, all three antisense RNAs showed concentration-dependent inhibitory effects on translation of HBsAg mRNA. In a coupled in vitro transcription and translation system, concentration-dependent inhibition of HBsAg synthesis was observed for all above mentioned antisense RNAs. Three mammalian antisense RNA expression vectors were then constructed, expressing the same antisense RNAs as used above. Transfection of the vectors into Hep3B cells (an HBsAg secreting cell line) resulted in almost complete blockage of HBsAg production, whereas control vector transfected cells secreted high levels of HBsAg. The inhibitory effect lasted for more than 10 months post-transfection. To examine the possible mechanism of the antisense RNA effect in the cell line, we measured HBV mRNA levels in the transfected cells and found that the mRNA levels in the antisense RNA expressing cells were much lower than those in the control cells. Therefore, in Hep3B cells, the antisense RNAs inhibited HBsAg synthesis, at least partially, through the reduction of HBV mRNA levels.

Gene Expression Regulation, Viral↗
PubMed1994

Gene expression of the type-1 angiotensin II receptor in rat adrenal gland.

To evaluate the biosynthesis of the type-1 angiotensin II (AT1) receptor and the regulation of AT1 receptor subtypes in the rat adrenal gland, we performed non-radioisotope in situ hybridisation histochemistry with an AT1 receptor complementary RNA (cRNA) probe and a messenger RNA (mRNA) probe. The levels of AT1A and AT1B receptor mRNAs were measured by the reverse transcriptase-polymerase chain reaction method after 4-week treatment with a selective AT1 receptor antagonist, TCV-116, and an angiotensin-converting enzyme inhibitor, delapril. Specific hybridisation signals were observed with the cRNA probe in both the cortex and medulla of the rat adrenal gland. An especially strong signal was observed in the zona glomerulosa. TCV-116 did not affect the levels of expression of AT1A and AT1B receptor mRNAs in the adrenal gland. Delapril, on the other hand, significantly reduced the levels of expression of AT1A and AT1B receptor mRNAs. These results indicate that the sites of biosynthesis of the AT1 receptor are mainly distributed in the adrenal zona glomerulosa. The observed differences in levels of expression of AT1 receptor mRNAs following treatment with TCV-116 and delapril suggest the involvement of the AT2 receptor in the regulation of AT1 receptor subtypes.

Adrenal Glands↗
PubMed2001

The endogenous fibroblast growth factor-2 antisense gene product regulates pituitary cell growth and hormone production.

Basic fibroblast growth factor (bFGF; FGF-2) is one of 19 related members of a growth factor family with mitogenic and hormone-regulatory functions. In Xenopus laevis oocytes, a 1.5-kb FGF-2 antisense (GFG) RNA complementary to the third exon and 3'-untranslated region (UTR) of FGF-2 mRNA has been implicated in FGF-2 mRNA editing and stability. The human homolog has been cloned, and we localized this gene by yeast artificial chromosome (YAC), somatic cell, and radiation hybrid panels to the same chromosomal site as FGF-2 (chromosome 4, JO4513 adjacent to D4S430), confirming this as a human endogenous antisense gene. The full-length GFG antisense RNA encodes a 35-kDa protein, which is highly homologous with the MutT family of antimutator nucleosidetriphosphatases (NTPases). We show that human pituitary tumors express FGF-2 and its endogenous antisense partner GFG. While normal pituitary expresses GFG but not FGF-2, pituitary adenomas express FGF-2 and have reduced levels of GFG; aggressive and recurrent adenomas expressed more FGF than GFG mRNA. To examine the effects of this antisense gene in the pituitary, we transfected the pituitary-derived GH4 mammosomatotroph cell line with constructs encoding the full-length human GFG cDNA. Transiently and stably transfected cells expressed the 35-kDa GFG protein that was localized to the cytoplasm. These cells exhibited enhanced PRL expression as documented by transiently transfected PRL-luciferase reporter assay and by endogenous PRL protein. GFG expression in these cells did not alter endogenous FGF-2 expression but increased the proportion of the higher molecular mass 22-kDa form of GH. Moreover, GFG expression inhibited cell proliferation as shown by [(3)H]thymidine incorporation, proliferating cell nuclear antigen (PCNA) nuclear staining, and cell cycle analysis. We conclude that the GFG-encoded protein has divergent hormone-regulatory and antiproliferative actions in the pituitary that are independent of FGF-2 expression. GFG represents a novel mechanism involved in restraining pituitary tumor cell growth while promoting hormonal activity.

Adenoma↗
PubMed2004

Chimeric RNA and 2'-O, 4'-C-ethylene-bridged nucleic acids have stronger activity than phosphorothioate oligodeoxynucleotides in induction of exon 19 skipping in dystrophin mRNA.

Antisense phosphorothioate oligodeoxynucleotides against exon 19 of the dystrophin gene have been shown to induce exon 19 skipping and promote the expression of internally deleted dystrophin by correcting the translational reading frame. Because phosphorothioate oligonucleotides are associated with a variety of toxic nonantisense effects, several modifications of nucleic acid have been introduced to alleviate this toxicity. Recently, a 2'-O, 4'-C-ethylene-bridged nucleic acid (ENA trade mark, Sankyo Lifetech Co., Ltd., Tokyo, Japan) was reported to have high affinity to complementary RNA strands and be resistant to nuclease digestion. Here, we examined the ability of this modified nucleic acid to induce exon skipping. Oligonucleotides having the same sequence as the phosphorothioate oligonucleotides but with some stretches of modified backbone (2'-O-methyl RNA with an ENA5-mer at the 5'-end and 3'-end) (RNA/ENA chimera) were transfected into myocytes, and the expressed dystrophin mRNA was analyzed. The RNA/ENA chimera induced exon 19 skipping in a dose-dependent and time-dependent manner. Remarkably, the exon 19-skipping activity of the RNA/ENA chimera was more than 40 times stronger than that of the corresponding conventional phosphorothioate oligodeoxynucleotide. This is the first report of such strong activity of an RNA/ENA chimera in the induction of exon skipping in the dystrophin gene. This new technology will allow the development of less toxic antisense drugs, making long-term therapy possible.

Base Sequence↗
PubMed1979

Evidence for transcriptional regulation of orotidine-5'-phosphate decarboxylase in yeast by hybridization of mRNA to the yeast structural gene cloned in Escherichia coli.

From a large population of strains of Escherichia coli carrying shear fragments of yeast (Saccharomyces cerevisiae) DNA attached by in vitro recombination to the plasmid vector pMB9, two hybrid plasmids were selected that relieve the pyrimidine requirement of nonreverting pyrF mutants of E. coli. An 1100-base-pair DNA fragment common to the two complementing plasmids was recloned into another plasmid vector, pBR322; these new hybrids retained the ability to specify orotidine-5'-phosphate decarboxylase (orotidine-5'-phosphate carboxy-lyase, EC 4.1.1.23) synthesis in E. coli. Evidence is presented that this common fragment is yeast DNA and thus apparently carried the structural information for yeast orotidine-5'-phosphate decarboxylase, the product of yeast gene ura3. A hybrid plasmid containing the 1100-base-pair fragment was used to measure levels of putative ura3 mRNA from yeast cultures labeled with [3H]adenine, ura3 mRNA was unstable with an apparent half-life of 10.5 min. Under different circumstances previously shown to alter the level of orotidine-5'-phosphate decarboxylase in yeast, a coordinate variation in proportion of labeled RNA complementary to the hybrid plasmid was found. These data support the hypothesis that regulation of the ura3 gene in yeast is at the level of transcription.

Carboxy-Lyases↗
PubMed1999

Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.

Aedes↗
PubMed1982

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold.

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.

Animals↗
PubMed1981

Intestinal pre-prosomatostatin. Identification of mRNA coding for a precursor by cell-free translations and hybridization with a cloned islet cDNA.

Somatostatin, a tetradecapeptide hormone, is produced in numerous organs including the hypothalamus, pancreatic islets, and the gastrointestinal tract. Recently we identified two separate biosynthetic precursors of somatostatin (Mr = 16,000 and 14,000) among the cell-free translation products encoded by mRNAs prepared from the islets of the anglerfish. The nucleotide sequence of a cloned cDNA encoding the larger of the two pre-prosomatostatins revealed the sequence of the tetradecapeptide somatostatin at the COOH terminus of a polypeptide of 119 amino acids. We now have prepared poly(A)RNA from the intestine of the anglerfish and by immunoprecipitation analyses find a single somatostatin-related translation product that co-migrates during electrophoresis on sodium dodecyl sulfate-polyacrylamide gels with the larger islet pre-prosomatostatin (Mr = 16,000). Analyses of the sizes of the intestinal and islet mRNAs by agarose gel electrophoresis and hybridization with 32P-labeled cDNA containing the coding sequence for the large islet pre-prosomatostatin showed that the complementary RNA in the intestine (600 bases) is 30 nucleotides smaller than that in the islet (620-630 bases). These observations indicate that a gene encoding somatostatin is expressed in the intestine and suggest that the intestinal mRNA is distinct from the two mRNAs encoding the islet somatostatins.

Animals↗
PubMed1989

HPV-18 immortalization of human keratinocytes.

The oncogenic potential of human papillomavirus type 18 which is found in a significant number of cervical and penile cancer biopsies was tested in primary human keratinocytes derived from neonatal foreskin. Viral DNA and a gene for resistance to neomycin were introduced into these cells by calcium phosphate transfection. Selection of cells in G418 led to the isolation of resistant colonies which were propagated in culture. Four cell lines termed FE-A, FEH 18L, FEP18-5, and FEP18-11 have been maintained in culture for 1 1/2-2 years and were selected for further analysis. In all cases the viral DNA was integrated into the cellular genome and the early genes were transcribed, including RNA complementary to the E2, E6, and E7 open reading frames. Radioimmunoprecipitation showed that all cell lines synthesized the E6 and E7 proteins. However, none of the cell lines tested were tumorigenic. The differentiation capacity of these cells was analyzed by assessing their ability to proliferate clonally after exposure to 1.2 mM calcium chloride. All four cell lines were resistant to this stimulus and formed colonies upon return to regular growth medium whereas normal cells differentiated terminally. K6a and K14 keratin RNA expression was down-regulated in the HPV immortalized cell lines compared to primary human epithelial cells.

Blotting, Northern↗
PubMed1992

Platelet-derived growth factor and its receptors in human glioma tissue: expression of messenger RNA and protein suggests the presence of autocrine and paracrine loops.

The expression of platelet-derived growth factor (PDGF) and its receptors was analyzed in 14 gliomas of various degrees of malignancy and compared with three gliosis cases by in situ hybridization and immunohistochemistry techniques. Expression of both PDGF A- and B-chains was higher in glioblastomas than in astrocytomas. The PDGF A-chain mRNA was predominantly found in cell-rich areas in glioblastomas. The cognate PDGF-alpha receptor (PDGFR-alpha) mRNA was heterogeneously distributed in gliomas of all grades, and PDGFR-alpha expression was higher in gliomas than in gliosis. Within some glioblastomas probed with PDGFR-alpha complementary RNA, cells heavily loaded with grains were intermingled with others containing low or moderate signals. The heavily labeled cells were often found in the vicinity of proliferating capillaries. Immunostaining with an anti-PDGF antibody and an affinity-purified antiserum against the PDGFR-alpha showed strong staining of most tumor cells with both antibodies in glioblastoma. In addition, the PDGFR-alpha antibodies yielded a strong staining of scattered cells, and the anti-PDGF antibody yielded staining of a few cells within the astrocytoma. Furthermore, high levels of the PDGF-beta receptor (PDGFR-beta) and PDGF B-chain mRNA as well as the beta receptor protein were found in hyperplastic capillaries. These results suggest the presence of autocrine and paracrine loops in glioma, activating the PDGFR-alpha in glioma cells and the PDGFR-beta in endothelial cells.

Adolescent↗
PubMed1976

A plasmid cloning vehicle allowing regulated expression of eukaryotic DNA in bacteria.

We have constructed a plasmid cloning vehicle in which transcription of inserted heterologous DNA fragments can be regulated by a defined bacterial operator and promoter. The lambda plac 5 EcoRIDNA fragment containing the operator, promoter, and beta-galactosidase gene of the lactose operon was linked to the ColE1 derivative plasmid pSF2124, creating a plasmid designated pBGP100, pBGP100 contains one EcoRI site at the lac DNA/pSF2124 DNA junction and another at the lambda DAN/pSF2124 DNA junction. We deleted the latter EcoRI site to generate a plasmid (pBGP120) retaining a single EcoRI site at the lac DNA/nSF2124 DNA junction. To determine whether DNA introduced at the EcoRI site of pBGP120 was expressed under lactose control, we inserted the EcoRI fragment containing 28S ribosomal DNA of Xenopus laevis, creating the hybrid plasmid pBGP123. RNA-DNA hybridization of pulse-labeled RNA from cells containing pBGP123 showed that induction of the lac operon increases the percentage of labeled RNA complementary to Xenopus 28S DNA about 9-fold. This vehicle may be of use for production of eukaryotic gene products in bacteria.

Animals↗
PubMed2001

Identification of G protein-coupled, inward rectifier potassium channel gene products from the rat anterior pituitary gland.

Dopamine (DA) is a physiological regulator of PRL secretion, exerting tonic inhibitory control. DA activates an inward rectifier K(+) (IRK) channel in rat lactotropes, causing membrane hyperpolarization and inhibition of Ca(2+)-dependent action potentials. Both the activation of this effector K(+) channel and the inhibition of PRL release are mediated by D(2)-type receptor activation and pertussis toxin- sensitive G proteins. To study the molecular basis of this physiologically relevant channel, a homology-based PCR approach was employed to identify members of the IRK channel family expressed in the anterior pituitary gland. Nondegenerate primers corresponding to regions specific for IRK channels known to be G protein activated (GIRKs; gene subfamily Kir 3.0) were synthesized and used in the PCR with reverse transcribed female rat anterior pituitary messenger RNA as the template. PCR products of predicted sizes for Kir 3.1, 3.2, and 3.4 were consistently observed by ethidium bromide staining after 16 amplification cycles. The identities of the products were confirmed by subcloning and sequencing. Expression of each of these gene products in anterior pituitary was confirmed by Northern blot analysis. Functional analysis of the GIRK proteins was performed in the heterologous expression system, Xenopus laevis oocytes. Macroscopic K(+) currents were examined in oocytes injected with different combinations of Kir 3.0 complementary RNA (cRNA) and G protein subunit (beta(1)gamma(2)) cRNA. The current-voltage relationships demonstrated strong inward rectification for each individual and pairwise combination of GIRK channel subunits. Oocytes coinjected with any pair of GIRK subunit cRNA exhibited significantly larger inward K(+) currents than oocytes injected with only one GIRK channel subtype. Ligand-dependent activation of only one of the GIRK combinations (GIRK1 and GIRK4) was observed when channel subunits were coexpressed with the D(2) receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent K(d) similar to that for DA binding in anterior pituitary tissue. GIRK1 and GIRK4 proteins were coimmunoprecipitated from anterior pituitary lysates, confirming the presence of native GIRK1/GIRK4 oligomers in this tissue. These data indicate that GIRK1 and GIRK4 are excellent candidate subunits for the D(2)-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.

Animals↗
PubMed1985

Expression of human c-fes onc-gene occurs at detectable levels in myeloid but not in lymphoid cell populations.

Total cellular RNA from a variety of myeloid and lymphoid cell populations, normal and leukaemic, was analysed for the expression of a human cellular onc-gene, c-fes, by northern blot hybridization assays. The probe used was a molecularly cloned human DNA sequence homologous to the 5' terminal sequence of v-fes. All the myeloid cellular populations expressed the c-fes gene. In some cell populations at an advanced stage of differentiation (circulating leucocytes from Chronic myeloid leukaemia (CML), HL60 cells induced to differentiate by retinoic acid) the level of expression was even higher than in early stages of the myeloid lineage (blast cells from AML, uninduced HL60 cells). No transcript of the c-fes gene was detected in the different lymphoid populations studied. The occurrence of an RNA complementary to the c-fes sequence appears sufficiently characteristic of a myeloid population to distinguish it from a lymphoid population, normal and leukaemic.

Cell Differentiation↗