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Biomedical subjects

J L Roberts

Publications and source records attributed to J L Roberts.

At least 19 recordsLinked to original sources
PubMed1992

Beta-adrenergic stimulation of cFOS via protein kinase A is mediated by cAMP regulatory element binding protein (CREB)-dependent and tissue-specific CREB-independent mechanisms in corticotrope cells.

Catecholamines stimulate proopiomelanocortin (POMC) gene expression in corticotrope cells, but the molecular mechanisms of these effects are not known. While beta-adrenergic receptors stimulate the protein kinase A (PKA) system, the POMC promoter does not have classical cAMP-response elements (CREs). Therefore, we investigated the induction of the c-fos protooncogen, previously shown to increase POMC transcription in AtT20 cells. In this corticotrope-derived cell line, we show that activation of beta-receptors with isoprenaline (Iso) induces a transient rise in c-fos mRNA levels. Gel mobility shift assays with a labeled AP1 consensus sequence (TGACTCA) showed induction of specific binding activity after Iso treatment. Cotransfection experiments with dominant inhibitory PKA mutants and reporter genes containing c-fos promoter sequences showed that c-fos induction by Iso is entirely dependent on a functional PKA activity. Furthermore, we show that beta-receptor induction of c-fos in corticotrophs is mediated by at least two distinct cAMP-responsive sequences. cAMP regulatory element binding (CREB)-dependent induction is observed on the CRE located at -60 bp on the c-fos promoter. A region located in the vicinity of the dyad symetry element (-290) is also found to mediate tissue-specific cAMP induction. Transcriptional activation by this site, although sensitive to PKA antagonism, is not blocked by CREB mutants.

Animals↗
PubMed1992

Intrahypothalamic injection of a cell line secreting gonadotropin-releasing hormone results in cellular differentiation and reversal of hypogonadism in mutant mice.

GT1 is an immortalized cell line that synthesizes and secretes the neurohormone gonadotropin-releasing hormone (GnRH). We have placed these cells into the brains of adult mutant hypogonadal (hpg) mice, which lack a functional GnRH gene, to determine whether such cells could differentiate in situ and support gonadal development. Immunocytochemical detection of GnRH revealed that these cells migrated widely in the central nervous system and elaborated axonal processes which on rare occasion projected to the normal target, the median eminence. Using a battery of antibodies, we demonstrated that these cells could cleave the GnRH precursor and that the amidated decapeptide as well as other cleavage products were present. The presence of biologically active material and its appropriate secretion were further documented by gonadal growth in both males and females. The morphological differentiation of the GT1 cells correlated with the density of cells injected. Those remaining within the injection site and/or forming a tumor retained a simple, rounded or fibroblastic appearance. Those cells that migrated into the host away from such tumors assumed the simple fusiform shape of normal GnRH neurons with dendrites extending from one or both poles. When cell density was drastically reduced a much more complex dendritic arbor was elaborated. These data suggest that such cell lines can be useful in reversing genetic defects and in studying such processes as GnRH neuronal migration, axonal targeting, and cytological differentiation.

Animals↗
PubMed1992

Expression of Mls determinants in mice exhibiting the severe combined immunodeficiency (scid) mutation or X-linked immunodeficiency (xid) defect.

While Ig+ B cells appear to be the principal cell type expressing immunogenic minor lymphocyte stimulatory (Mls) determinants, both T cells and B cells are capable of mediating deletion of developing Mls-reactive thymocytes. In addition, levels of mouse mammary tumor proviral transcripts are increased after B or T cell stimulation, and expression of functional Mls determinants is augmented by activation of B cells. These findings suggest Mls determinants are present on B and T lymphocytes, and that activation of B and T cells augments Mls expression. In the present study, we wished to determine whether B and T cells were required for expression of Mls determinants by examining mice with severe combined immunodeficiency (SCID) containing no detectable Ig+ B cells or TCR+ T cells, as well as animals that expressed the X-linked immunodeficiency (xid) defect and lacked a subset of mature B cells. We found Mlsa-reactive V beta 6hi T cells were deleted from thymi of male (CBA/NxAKR/J)F1 xid mice, and that spleen cells from these animals stimulated anti-Mlsa mixed lymphocyte responses by unprimed B10.BR spleen T cells. In addition, Mlsc-reactive V beta 3hi AKR/J thymocytes and spleen T cells were deleted in AKR/J----SCID bone marrow chimeras, and spleen cells from SCID mice stimulated proliferation by an Mlsc-specific T cell clone. These results demonstrate that both xid mice and SCID animals express Mls determinants that mediate deletion of developing, Mls-responsive thymocytes and stimulate proliferation of mature, Mls-reactive T cells. Hence, mature B cells and T cells are not essential for Mls expression.

Animals↗
PubMed1992

Positive correlation between proopiomelanocortin and tyrosine hydroxylase mRNA levels in the mediobasohypothalamus of ovariectomized rats: response to estradiol replacement and withdrawal.

We have investigated putative dopaminergic regulation of opiomelanotropinergic activity in the arcuate/periarcuate mediobasohypothalamus (MBH) by assessing the changes in MBH tyrosine hydroxylase (TH; rate-limiting enzyme in catecholamine synthesis) and proopiomelanocortin (POMC; opiomelanotropin precursor) mRNA levels under conditions in which endogenous tuberinfundibular dopaminergic activity exhibits marked changes. Adult Sprague-Dawley rats were sacrificed at 09.00 and 15.00 h, and individual MBH POMC and TH cytoplasmic mRNA levels were simultaneously quantified by multiplex solution hybridization-RNase protection assay with protected fragments separated by polyacrylamide gel electrophoresis. In ovariectomized (OVX) rats treated for 3 days with low-dose estradiol (E2) implants (resulting in 18 +/- 4 pg E2/ml serum), the MBH levels of POMC and TH mRNAs were approximately 17 and 31% lower than those measured in OVX controls, respectively. In OVX rats implanted for 20 days with larger E2 implants (99 +/- 9 pg E2/ml serum), POMC and TH mRNA levels were approximately 29 and 41% lower than in OVX controls, respectively. Additional groups were exposed to the higher E2 dose for 20 days and then killed 10 or 20 days after removal of the E2 implant. In these rats, POMC mRNA levels rebounded to the same level seen in OVX controls, while TH mRNA levels even exceeded control values by 22-27%. TH and POMC mRNA levels did not change significantly between 09.00 and 15.00 h, except 10 days after removal of the E2 implants, when 09.00 h POMC mRNA levels were higher than the 15.00 h levels. MBH POMC and TH mRNA levels were positively correlated with each other within individual animals. This correlation is maintained when both POMC and TH mRNA levels are suppressed in response to both 3-day low-dose and 20-day high-dose E2 treatment. However, although rat MBH opiomelanotropinergic and tuberoinfundibular dopaminergic mRNA biosynthesis thus appear to be positively correlated, the coregulation or functional interactions of these two neuronal systems remain to be determined.

Amino Acid Isomerases↗
PubMed1992

Proopiomelanocortin gene expression in a distinct population of rat spleen and lung leukocytes.

Existence of proopiomelanocortin (POMC) messenger RNA (mRNA) and related peptides in extrapituitary sites has been demonstrated in immune cells, although the particular type of immune cell has been the source of considerable debate. Specifically, double labeling studies have shown that POMC peptide expressing cells in the spleen represent a subpopulation of red pulp macrophages, while splenic lymphocyte areas are POMC negative. In addition, it has also been reported that peripheral blood leukocytes express the POMC gene. Using a sensitive solution hybridization technique with a POMC exon-1 RNA probe, we detected 70 +/- 20 fg and 65 +/- 5 fg POMC mRNA per microgram total RNA in whole spleen and lung, respectively, approximately 20,000-fold lower concentrations than found in the neurointermediate lobe of the pituitary. The presence of nuclease protected full length exon-1 bands, rather than the 5' truncated POMC RNAs seen in many nonpituitary tissues, indicates transcription initiation at the normal pituitary POMC promoter site in lung and spleen. In order to localize POMC gene expression in these tissues we employed an in situ hybridization method. There was an intense signal in a small population of large mononuclear cells scattered throughout the splenic red pulp and lung parenchyma. In the lung, these cells were concentrated in the periarteriolar zone in a manner suggestive of migration from the intravascular lumen. These cells had a histomorphology suggestive of monocyte-macrophages. POMC mRNA was undetectable in the splenic white pulp and bronchus-associated lymphoid tissue, indicating an absence of POMC gene expression in splenic and lung lymphocytes. Immunocytochemical studies suggested that POMC-positive cells made up a subpopulation of cells expressing the rat monocyte-macrophage markers ED1 and ED2. Similarly, the distribution of Jenner-Giemsa stained monocyte-macrophages appeared to overlap with POMC positive cells. Studies with anti-rat beta-endorphin antisera revealed scattered cells in the splenic red pulp and lung parenchyma, suggesting that the POMC mRNA is translated in these cells. In summary, POMC mRNA is expressed in a small population of monocyte-macrophage-like cells in lung and spleen but not in lymphocytes in these tissues.

Animals↗
PubMed1992

Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor.

GnRH plays a pivotal role in the reproductive system, and GnRH analogs have wide therapeutic applications ranging from the treatment of prostatic cancer to infertility. Determination of the predicted structure of the GnRH receptor (GnRHR) would illuminate the mechanisms of receptor activation and regulation and allow directed design of improved GnRH analogs. We report the cloning of a cDNA representing the mouse GnRHR and confirm its identity using Xenopus oocyte expression. Injection of sense RNA transcript leads to the expression of a functional, high affinity GnRHR. Expression of the GnRHR using gonadotrope cell line RNA, however, is blocked by an antisense oligonucleotide. In situ hybridization in the rat anterior pituitary reveals a characteristic GnRHR distribution. The nucleotide sequence encodes a 327-amino acid protein which has the seven putative transmembrane domains characteristic of G protein-coupled receptors, but which lacks a typical intracellular C-terminus. The unusual structure and novel potential regulatory domain of the GnRHR may explain unique aspects of its signal transduction and regulation.

Amino Acid Sequence↗
PubMed1992

6 alpha-hydroxypenicillanic acid-S(S)-oxide and analogues: synthesis and antimicrobial activity.

The synthesis and in vitro antibacterial activity of a series of 6-oxygenated penicillanic acid sulfoxides is described. 6 alpha-Hydroxypenicillanic acid-S(S)-oxide (1a) exhibits weak Gram-negative antibacterial activity and appears to be similar to amdinocillin (5) in its mode of action. 6 alpha-Hydroxypenicillanic acid-S(R)-oxide (4a) has a broader spectrum of activity, but again is rather weak. The corresponding 6 beta-hydroxy series is essentially devoid of activity.

Anti-Bacterial Agents↗
PubMed1991

Alternative transcripts of the rat and human dopamine D3 receptor.

A cDNA for the rat dopamine D3 receptor containing a 113 bp deletion has been isolated. The segment deleted, encompassing the first extracellular loop and third transmembrane domain, alters the reading frame, introducing 19 amino acids not found in the full length receptor followed by a premature stop codon. This novel mRNA encodes a 109 amino acid protein containing two putative transmembrane domains. A similar variant cDNA for the human D3 receptor has also been identified.

Amino Acid Sequence↗
PubMed1991

Distribution of dopamine D2 receptor mRNA splice variants in the rat by solution hybridization/protection assay.

We investigated the distribution of the two dopamine D2 receptor mRNA splice variants in the rat using a sensitive and quantitative solution hybridization/nuclease protection assay. In all brain and endocrine regions studied, both splice variants were detected and the mRNA of the longer form (D2L) was more abundant than that of the shorter form (D2S). The lowest percentages of D2S were found in the pituitary and adrenal glands.

Adrenal Glands↗
PubMed1991

Either isoform of the dopamine D2 receptor can mediate dopaminergic repression of the rat prolactin promoter.

Hypophyseal portal dopamine is a major negative regulator of pituitary prolactin (PRL) production. Dopamine has been reported to repress PRL gene transcription in pituitary cells. To facilitate further study of the effect of dopamine on PRL gene activation, we introduced PRL promoter and D2 receptor (D2R) constructs into GH3 cells. Since two D2R isoforms (termed D2S and D2L) have been cloned previously, we first determined which isoform(s) is present in the lactotroph by measuring the level of each mRNA species in rat prolactinoma. mRNA for each D2R isoform was found to be present, with the D2L mRNA in great (c. 6-fold) excess. Because the lactotroph contains both isoforms, the effect of each on the PRL promoter was investigated. The cDNA for each receptor isoform was synthesized by polymerase chain reaction, and cloned into an RSV-based expression vector. GH3 cells were then transiently co-transfected with either of the resulting RSV-D2R constructs plus a PRL-chloramphenicol acetyltransferase (CAT) construct containing the first 1957 base-pairs of PRL gene 5'-flanking DNA. The cells were then incubated 48 h plus or minus the dopamine agonist ergocryptine (ECR). In the presence of either RSV-D2R isoform, ECR yielded a 4-5-fold decrease in CAT activity, an effect not seen in the absence of the RSV-D2R. The promoter specificity of this effect was demonstrated by the inability of ECR to regulate expression of a control RSV-CAT construct. The PRL promoter repression mediated by each receptor isoform had appropriate pharmacology: the specific D2R agonist, quinpirole, yielded results similar to ECR, and the ECR repression was reversed by the dopamine antagonist spiperone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Expression of luteinising hormone-beta subunit chloramphenicol acetyltransferase (LH-beta-CAT) fusion gene in rat pituitary cells: induction by cyclic 3'-adenosine monophosphate (cAMP).

In this study we determined the activity of the rat luteinising hormone-beta gene promoter in a heterologous rat pituitary cell line (GH3 cells). 1.7 kb of LH-beta 5' flanking sequence and the first 5 bp of the 5' untranslated region were ligated to the chloramphenicol acetyltransferase (CAT) receptor gene (LH-beta-CAT) and transiently transfected by calcium phosphate precipitation into subconfluent cultures of GH3 cells. Basal low-level CAT activity was only detected in GH3 cells, being absent in two non-pituitary cell lines (BeWo and HeLa) RNase analysis revealed that mRNA from transfected GH3 cells protected a fragment of labelled antisense probe of correct size for transcription initiation from the LH-beta CAP site, confirming that promoter activity reflected correctly initiated LH-beta-CAT fusion gene transcripts. CAT activity was consistently induced by an average of 3-5-fold from the full-length 1.7 kb promoter, in a dose- and time-dependent manner, by forskolin, dibutyryl cAMP, and 8-bromo cAMP implying presence of a cAMP-responsive cis-acting domain in the LH-beta promoter region. Transfection of deletion mutants delta-615-CAT, delta-385-CAT and delta-250-CAT each reduced forskolin inducibility to 1.7-fold but did not abolish induction completely suggesting a domain between -1.7 and -0.6 kb contained a cAMP-responsive element(s) (CRE). Further deletion of LH-beta 5' flanking sequences to delta-85-CAT restored forskolin induction to wild-type levels (3-5-fold), suggesting the presence of a weak inhibitory element between -600 and -85 kb, and a cAMP-responsive domain in the proximal promoter region. The LH-beta promoter does not contain perfect tandem repeat palindromic CRE DNA sequences, though there are several octanucleotide sequences differing by only 1 bp from AP-2 binding sites, the consensus CRE, and the vasointestinal peptide gene CRE. Although these data suggest that the LH-beta gene is cAMP responsive this is likely mediated by several and complex protein interactions with multiple DNA sequences in the proximal and distal LH-beta promoter enhancer.

Adenylyl Cyclases↗
PubMed1991

Endopeptidase-24.15 in rat hypothalamic/pituitary/gonadal axis.

Endopeptidase-24.15 (E.C. 3.4.24.15; EP-24.15) cleaves several substrates found in the hypothalamic/pituitary/gonadal axis, including gonadotropin-releasing hormone (GnRH) and the opioid peptides of the dynorphin family. We have examined the activity of EP-24.15 in these tissues as a function of maturation, of the estrous cycle, and in response to ovariectomy and estrogen replacement. A developmental regulation of EP-24.15-specific activity is apparent in anterior pituitary, in hypothalamus, and in the gonads. EP-24.15 is increased in the preoptic area and is decreased in the anterior pituitary in both male and female rats prior to puberty. The specific activity of EP-24.15 was increased following ovariectomy in the anterior pituitary and within medial and lateral preoptic nuclei. Testicular specific activity of EP-24.15 increased with age in a linear fashion, while ovarian EP-24.15 activity increased immediately prior to puberty, but returned to prepubertal levels by 65 days of age. The relevance of EP-24.15 to the metabolism of specific peptides is discussed.

Amino Acid Sequence↗
PubMed1991

Drinking on television: a content analysis of recent alcohol portrayal.

The portrayal of alcohol on British television was examined in a systematic sample of 50 programmes broadcast in 1988. Nearly three-quarters of the programmes contained visual or verbal references to alcohol. On average, there was a reference to alcohol in every six and a half minutes of programming. Alcohol was most prominently featured in fictional programmes, particularly in one soap opera, Eastenders. A third of the programmes showed specific brands of alcohol. It is estimated that the portrayal of alcohol in Britain's most popular programmes is worth pounds 487 million of free promotion to the alcohol industry per annum. Nevertheless, in comparison with an earlier study, carried out in 1986, there appeared to be fewer alcohol images. A number of recommendations are made for further action and monitoring of alcohol portrayal.

Alcohol Drinking↗
PubMed1991

Postnatal development of gonadotropin-releasing hormone and cyclophilin gene expression in the female and male rat brain.

The rat preoptic area-anterior hypothalamic continuum (POA-AH) contains about 400-800 neurons that express the decapeptide GnRH and the 56-amino-acid GnRH-associated peptide. Originating from the olfactory placode, these neurons migrate and establish their final distribution and connections in the POA-AH several days before birth. The aim of the present study was to examine whether the biosynthesis of the mRNA encoding the precursor (proGnRH) common to GnRH and GnRH-associated peptide undergoes postnatal changes corresponding to the development of sexual maturation. The POA-AH content of proGnRH messenger RNA (mRNA) was followed from postnatal day 1 to day 90 in female and male Sprague-Dawley rats killed by decapitation between 1000-1200 h. Cytoplasmic RNA fractionated from individual POA-AH homogenates was purified using proteinase K digestion. Cytoplasmic proGnRH mRNA was quantitated simultaneously with cyclophilin mRNA (an internal standard control) using solution hybridization-RNase protection assay, with the protected fragments separated through polyacrylamide gel electrophoresis. In the POA-AH, the concentrations of proGnRH mRNA (femtograms mRNA per microgram total RNA) increased significantly with age in both sexes (P less than 0.001). In males, proGnRH mRNA levels increased by day 30 some 2-fold over the values of days 5 and 10, and the levels established on day 30 were maintained through adulthood. In females, the first rise in proGnRH mRNA levels occurred on day 30, followed by an additional increase on day 45 to levels seen in adulthood. Levels of proGnRH mRNA established in adulthood were significantly higher in females than in males (P less than 0.03). The concentrations of cyclophilin mRNA (picograms mRNA per microgram total RNA) remained essentially unchanged in both sexes during the same period of time when proGnRH mRNA levels were increasing. These results provide evidence for postnatal sex-related increases in the levels of proGnRH mRNA in the rat POA-AH, which are likely to reflect differential regulation by gonadal steroids.

Amino Acid Isomerases↗
PubMed1991

Adrenocorticotropin-releasing factor down-regulates glucocorticoid receptor expression in mouse corticotrope tumor cells via an adenylate cyclase-dependent mechanism.

Anterior pituitary POMC transcription and peptide release are negatively regulated by glucocorticoids and stimulated by CRF. Although pretreatment of corticotrope cells with CRF markedly inhibits subsequent glucocorticoid effects, the mechanism of this action is unclear. We have thus used a mouse corticotrope tumor (AtT20) cell line, to examine the effects of CRF on glucocorticoid receptor (GR) messenger RNA levels and on GR capacity/nuclear translocation. GR mRNA levels were measured by solution hybridization/S1 nuclease protection, and both total cell binding and nuclear binding were determined with [3H]dexamethasone ([3H]DEX). CRF treatment of AtT20 cells led to a rapid time-dependent decrease in GR mRNA levels which preceded a dose- and time-dependent decrease in GR binding capacity. Scatchard analysis showed a single class of high affinity binding sites (GR) in both control and CRF-treated cultures, and a decrease in the total number of GR after CRF treatment. The relative proportion of nuclear vs. cytoplasmic localized [3H]DEX-bound GR did not differ between control and CRF-treated cultures, indicating that CRF does not interfere with GR nuclear translocation. To investigate whether CRF regulates GR expression through the adenylate cyclase system, as it does POMC, AtT20 cells were treated with either forskolin or 8-bromo-cAMP, and specific nuclear GR binding was determined. Both drugs mimic the CRF-induced decrease in GR binding, and in addition forskolin decreased GR mRNA levels; in contrast, forskolin had no effect on GH3 cell GR levels. These results suggest that CRF can decrease the cellular concentration of GR, and thus potentially the response to glucocorticoids, through the same mechanism by which it stimulates anterior pituitary POMC expression.

8-Bromo Cyclic Adenosine Monophosphate↗