Biomedical subjects

Stephen C Robson

Publications and source records attributed to Stephen C Robson.

23 records · Page 2Linked to original sources

PubMed2002

Alternative splicing of the adenylyl cyclase stimulatory G-protein G alpha(s) is regulated by SF2/ASF and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) and involves the use of an unusual TG 3'-splice Site.

The factors involved in regulating alternative splicing of the human adenylyl cyclase stimulatory G-protein G alpha(s) in different cell types remain undefined. We have designed a G alpha(s) minigene that retains the signals required for G alpha(s) alternative splicing in vivo. Employing transient transfection of human myometrial smooth muscle cells and HeLa cells, as well as in vitro splicing assays, we have provided evidence that the antagonistic splicing factors SF2/ASF and hnRNPA1 act as potent regulators of G alpha(s) isoform expression in these cells. Both SF2/ASF and hnRNPA1 control the selection of competing 5'-splice sites and respectively promote inclusion or skipping of the small cassette-type exon 3 of G alpha(s) transcripts, resulting in the generation of G alpha(s)-long and G alpha(s)-short mRNA isoforms. We have also provided evidence that SF2/ASF and hnRNPA1 play a role in 3'-splice site selection involving the use of a non-canonical TG 3'-splice site preceding exon 4. Using a score-matrix analysis to identify putative exonic enhancer sequences (ESEs), we found multiple high score ESE motifs for SF2/ASF, SC35, and SRp40 in exon 3 of G alpha(s). These results suggest that tissue-specific expression of SF2/ASF and hnRNPA1 governs the expression of alternative isoforms of G alpha(s) in these different cells types.

Adenylyl Cyclases↗

PubMed2002

Flow-mediated nitric oxide activity in the forearm vasculature of premenopausal women.

AIMS: Nitric oxide (NO) is involved in acute flow-mediated vasodilatation in various vascular beds. We determined whether acutely increasing flow in the human forearm of premenopausal women increases vascular NO activity. METHODS: Forearm blood flow (FBF) was measured by venous occlusion plethysmography. Responses to brachial artery infusion of noradrenaline (a control vasoconstrictor, 20, 50, and 100 ng min(-1), each for 5 min) and NG-monomethyl l-arginine (L-NMMA), an NO synthase inhibitor (200, 400, and 800 microg min(-1), each for 5 min), were determined in eight premenopausal women before and following elevation of basal FBF with glyceryl trinitrate (GTN) on two separate occasions. RESULTS: Flow elevation with GTN increased responses to L-NMMA (summary measure 103 +/- 12 vs 65 +/- 12 arbitrary units, P<0.05), but not to noradrenaline (95 +/- 35 vs 74 +/- 12, P=0.50). CONCLUSIONS: Acute elevation of FBF in nonpregnant women is associated with enhanced responses to NO synthase inhibition, consistent with flow-mediated increased NO activity.

Adult↗

PubMed2002

Punch biopsy of the human placental bed.

OBJECTIVE: The purpose of this study was to report our experience with placental bed biopsy with the use of forceps. STUDY DESIGN: Placental bed biopsies were undertaken transcervically under ultrasound guidance in 313 women who underwent termination of pregnancy between 7 and 20 weeks of gestation, in 104 women with a missed abortion who underwent evacuation of retained products of conception between 7 and 21 weeks of gestation, and in 13 women after vaginal delivery. Placental bed biopsies were also undertaken in 139 women who underwent caesarean delivery. Frozen sections were immunostained with monoclonal antibodies to cytokeratin, factor VIII-related antigen, and desmin. RESULTS: Of the 417 cases attempted at <22 weeks, the placental bed was successfully sampled 281 women (67%); in 229 women (55%), at least one of the biopsy specimens contained a uterine spiral artery. Success was correlated with gestational age. Figures for the late cases that were sampled during caesarean delivery were 108 of 139 cases (78%) and 66 of 139 cases (47%) and after vaginal delivery were 11 of 13 cases (84%) and 6 of 13 cases (46%), respectively. The sampling procedure did not result in any significant morbidity. CONCLUSION: With the use of forceps, uterine spiral arteries can be sampled successfully throughout pregnancy in approximately 50 % of cases.

Arteries↗

PubMed2002

Differential expression of the adenylyl cyclase-stimulatory guanosine triphosphate-binding protein G(s)alpha in the human myometrium during pregnancy and labor involves transcriptional regulation by cyclic adenosine 3',5'-monophosphate and binding of phosphorylated nuclear proteins to multiple GC boxes within the promoter.

G(s)alpha is the G protein subunit that stimulates adenylyl cyclase activity in the myometrium during pregnancy, raising intracellular levels of the smooth muscle relaxant cAMP. The promoter region of the gene encoding G(s)alpha is GC rich and contains multiple putative binding sites for the specificity protein (Sp) transcription factor family. In electrophoretic mobility shift assays, four of these Sp sites were bound by recombinant Sp1 protein. Binding was dependent on phosphorylation of Sp1 by protein kinase A. Phosphorylated Sp1-4 proteins were observed in extracts of cultured human myometrial cells, but in electrophoretic mobility shift assays G(s)alpha promoter sequence binding by Sp1 was not apparent. Instead, these assays showed phosphorylation-dependent G(s)alpha promoter binding by lower molecular weight myometrial proteins that could not be supershifted by antibodies specific to Sp1-4 proteins. To investigate the regulation of G(s)alpha expression, the GC-rich promoter region was used to direct transcription of a firefly luciferase reporter gene in transient transfection assays of primary human myometrial cell cultures, COS-7 and HEK 293 cells. Reporter gene expression was found to follow a biphasic response to forskolin and 8-bromo-cAMP, with an initial, concentration-dependent increase in luciferase activity, followed by a prolonged decrease. In myometrial cells, this pattern was also seen in response to treatment with human chorionic gonadotropin.

Adenylyl Cyclases↗

PubMed2002

Characterization and functional analysis of cAMP response element modulator protein and activating transcription factor 2 (ATF2) isoforms in the human myometrium during pregnancy and labor: identification of a novel ATF2 species with potent transactivation properties.

There is now extensive evidence to indicate that components of the cAMP signaling pathway are up-regulated in the human myometrium during pregnancy so as to potentiate the maintenance of uterine quiescence until term. In many tissue and cell types, increased signaling of the cAMP pathway results in profound changes in gene expression that are catalyzed via stimulation of PKA and activation of cAMP-dependent transcription factors that bind cAMP response elements (CREs) within the promoter regions of affected genes. In the myometrium, these CRE containing genes include beta2-adrenoceptor, cyclo-oxygenase 2, oxytocin receptor, and connexin-43. In preliminary investigations, we reported the differential expression of members of the cAMP bZIP protein family in the myometrium during pregnancy and labor. In this present study, we have now identified and functionally characterized these proteins with respect to myometrial gene expression. We report the identification of a 39,000 mol wt CRE response element modulator protein (CREM)tau2alpha protein having both transactivation and transrepressor properties whose expression is sequentially decreased in the myometrium during gestation and parturition. In contrast, expression of a myometrial 28,000 mol wt CREMalpha protein having only transrepressor actions progressively increased in the myometrium during pregnancy and labor. Similarly, we have isolated two ATF2 proteins of 60,000 and 28,000 mol wts, which represent full-length ATF2 and a novel small isoform of ATF2 that we have termed ATF2-small (ATF2-sm). These proteins are potent transactivators of gene expression and appear to be spatially expressed within the myometrium of the upper and lower uterine regions. The identification and functional characterization of these basic region/leucine zipper proteins in the myometrium may provide further insight into the molecular mechanisms regulating uterine activity during fetal maturation and parturition.

Activating Transcription Factor 2↗