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Biomedical subjects

G M Cooper

Publications and source records attributed to G M Cooper.

At least 73 records · Page 4Linked to original sources
PubMed1994

Translational control by cytoplasmic polyadenylation of c-mos mRNA is necessary for oocyte maturation in the mouse.

The c-mos proto-oncogene product is a key element in the cascade of events leading to meiotic maturation of vertebrate oocytes. We have investigated the role of cytoplasmic polyadenylation in the translational control of mouse c-mos mRNA and its contribution to meiosis. Using an RNase protection assay we show that optimal cytoplasmic polyadenylation of c-mos mRNA requires three cis elements in the 3' UTR: the polyadenylation hexanucleotide AAUAAA and two U-rich cytoplasmic polyadenylation elements (CPEs) located 4 and 51 nucleotides upstream of the hexanucleotide. When fused to CAT coding sequences, the wild-type 3' UTR of c-mos mRNA, but not a 3' UTR containing mutations in both CPEs, confers translational recruitment during maturation. This recruitment coincides with maximum polyadenylation. To assess whether c-mos mRNA polyadenylation is necessary for maturation of mouse oocytes, we have ablated endogenous c-mos mRNA by injecting an antisense oligonucleotide, which results in a failure to progress to meiosis II after emission of the first polar body. Such antisense oligonucleotide-injected oocytes could be efficiently rescued by co-injection of a c-mos mRNA carrying a wild-type 3' UTR. However, co-injection of a c-mos mRNA lacking functional CPEs substantially lowered the rescue activity. These results demonstrate that translational control of c-mos mRNA by cytoplasmic polyadenylation is necessary for normal development.

Adenosine Monophosphate↗
PubMed1994

Nausea and vomiting after gynaecological laparoscopy: comparison of premedication with oral ondansetron, metoclopramide and placebo.

We have compared the incidence of postoperative nausea and vomiting up to 48 h after day-case gynaecological laparoscopy after oral premedication with ondansetron 4 mg, metoclopramide 10 mg or a placebo allocated randomly and assessed blindly. Emetic symptoms (nausea or vomiting) occurred in 26% of patients who received ondansetron, 42% of those who received metoclopramide and 50% of those given placebo.

Adolescent↗
PubMed1994

Drug-related admissions to an Australian hospital.

This study was conducted to determine the prevalence of drug-related hospital admissions in southern Tasmania, Australia. The causes of consecutive admissions to medical wards of the Royal Hobart Hospital were reviewed. Comprehensive data were collected over a 10-week period on 691 admissions (median age: 67 years and range: 11-97 years; 50.8% males). Sixty-eight (9.8%) of the admissions were classified as being either probably or definitely drug-related. Most of these admissions were attributable to intentional overdose (38.2%) or an adverse drug reaction (30.9%). The overdoses often involved benzodiazepines or antipsychotics. Gastrointestinal bleeding related to the use of nonsteroidal anti-inflammatory drugs was the most common adverse drug reaction (38.1% of all reactions). Other drug-related admission categories were poor compliance (14.7%), dosage decrease or therapy cessation by a doctor producing an exacerbation of symptoms (7.4%), substance abuse (4.4%) and drug interaction (4.4%). Patients with a drug-related admission were, on average, younger than the other medical admissions, with no significant difference in gender. Patients admitted due to an overdose or substance abuse were younger than other drug-related admissions and non-drug related admissions. In conclusion, this study has determined that almost 10% of medical admissions to the hospital are drug-related and it is estimated that 40 to 50 elderly people are admitted each year suffering from gastrointestinal bleeding related to nonsteroidal anti-inflammatory drugs.

Adult↗
PubMed1994

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

Animals↗
PubMed1994

Rapid regeneration of virus from cells infected with a retroviral vector.

Recombinant retroviral vectors usually encode the genes of interest in place of the viral structural genes, which must be provided in trans. These viruses are therefore defective for replication: infected cells cannot produce progeny virus. However, in some cases it may be desirable to generate virus from an infected cell clone displaying a phenotype of interest. We describe a rapid method for producing virus, which involves fusing the infected cells to fresh packaging cells. Stable producer lines are generated after fusion by co-selecting for the Ecogpt+ marker in the packaging cells and the G418 resistance (neor) marker in the infected cells. We have used this method to develop cell lines that produce retroviruses encoding a Leu 61-activated c-Ha-ras oncogene as well as a neor gene. The viruses confer oncogenic transformation on 95%-100% of infected target cells as assayed by altered morphology, focus formation and soft agar growth.

Animals↗
PubMed1994

Expression and potential function of the c-mos proto-oncogene in human eggs.

OBJECTIVE: To investigate the expression and possible function of the c-mos proto-oncogene in human eggs. DESIGN: Eggs obtained as discarded material from assisted reproductive technology procedures were analyzed for c-mos messenger RNA by reverse transcriptase-polymerase chain reaction. As an approach to investigating c-mos function, we measured maturation-promoting factor (MPF) activity (histone H1 kinase) in eggs without and with inhibition of protein synthesis. Detection of RNA transcripts of c-raf was included as control. RESULTS: Transcripts of c-mos were detected in small fractions of individual eggs, indicating that c-mos is abundantly transcribed. Inhibition of protein synthesis resulted in loss of MPF, leading to chromatin decondensation and reformation of a nucleus. C-raf maternal messages were also detectable in individual human eggs. CONCLUSION: The c-mos proto-oncogene is an abundant maternal message in human eggs as in other species. The effects of inhibiting protein synthesis in human eggs are similar to those obtained in mouse and Xenopus eggs, either as a consequence of protein synthesis inhibition or specific ablation of c-mos RNA by injection of anti-sense oligonucleotides. The c-mos gene product is thus likely to play a critical role in human oocyte meiosis by regulating the activity of MPF.

Base Sequence↗
PubMed1993

Expression of proto-oncogenes in mouse eggs and preimplantation embryos.

The expression of several protooncogenes has been investigated in mouse eggs and preimplantation embryos using reverse transcription coupled to amplification of cDNAs by the polymerase chain reaction (RT-PCR). The genes chosen for analysis included both cytoplasmic (c-raf-1, rasH, rasK, and rasN) and nuclear (c-fos and c-myc) proto-oncogenes encoding proteins involved in the transduction of signals from protein-tyrosine kinase growth factor receptors. Transcripts of the cytoplasmic proto-oncogenes were detected both as maternal and embryonic mRNAs at levels (ca. 1,000 copies per egg or embryo) approximately comparable to their levels of transcription in somatic cells. Transcripts of c-fos and c-myc were also detected in both eggs and embryos, although at more variable levels: Maternal transcripts were present at very low levels (ca. 1-10 copies per egg) in growing oocytes and ovulated eggs; embryonic transcription of c-myc increased, reaching mRNA levels of approximately 100-1,000 copies per embryo in four-cell embryos, morula, and blastocysts; in contrast the transcription of c-fos remained at low, barely detectable levels throughout preimplantation development. Although the significance of the low levels of c-fos mRNA is unclear, these results indicate that preimplantation embryos possess the basic intracellular signaling apparatus required to respond to polypeptide growth factors.

Animals↗
PubMed1993

Dural taps revisited. A 20-year survey from Birmingham Maternity Hospital.

The records from 34,819 obstetric epidurals performed at Birmingham Maternity Hospital over the period 1969-1988 were examined. During that time there were 460 dural taps (overall incidence 1.3%). Of the methods used to detect the epidural space, loss of resistance to injection of saline was associated with the lowest incidence of dural tap (0.6%). The incidence of typical postdural puncture headache when managed conservatively was 86%. Provision of an epidural drip after delivery reduced the incidence of headache to 70%. Elective forceps delivery conferred no additional benefit, and tended to delay the onset of headache. Blood patches were performed on 135 patients and provided complete relief of headache in 93 (68%). A further 23 patients (16%) obtained partial relief.

Analgesia, Epidural↗
PubMed1993

Does a bloody tap prevent postdural puncture headache?

The regional anaesthesia records of 274 obstetric patients who had inadvertent dural puncture were reviewed to see whether a bloody dural tap resulted in a lower incidence of postdural puncture headache. No such influence was found.

Anesthesia, Epidural↗
PubMed1993

ras-independent induction of rat brain type II sodium channel expression in nerve growth factor-treated PC12 cells.

Nerve growth factor (NGF) plays an important role in the development of the nervous system, and there is considerable interest in understanding the molecular mechanisms underlying its effects on neuronal differentiation. To determine if the activity of proteins of the ras gene family is necessary for the NGF-mediated induction of sodium channel expression in pheochromocytoma (PC12) cells, sodium channel expression was analyzed in PC12 sublines stably overexpressing the dominant inhibitory mutant c-Ha-ras(Asn-17). Northern blot analysis, RNase protection assays, and whole-cell patch clamp recordings indicate that the NGF-mediated increase in type II sodium channel mRNA and sodium current density can occur independent of ras activity and by doing so provide strong evidence for the importance of ras-independent mechanisms in NGF-mediated neuronal differentiation.

Amino Acid Sequence↗
PubMed1993

Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction.

We have investigated the relationship between hydrolysis of phosphatidylcholine (PC) and activation of the Raf-1 protein kinase in Ras-mediated transduction of mitogenic signals. As previously reported, cotransfection of a PC-specific phospholipase C (PC-PLC) expression plasmid bypassed the block to cell proliferation resulting from expression of the dominant inhibitory mutant Ras N-17. In contrast, PC-PLC failed to bypass the inhibitory effect of dominant negative Raf mutants, suggesting that PC-PLC functions downstream of Ras but upstream of Raf. Consistent with this hypothesis, treatment of quiescent cells with exogenous PC-PLC induced Raf activation, even when normal Ras function was blocked by Ras N-17 expression. Further, activation of Raf in response to mitogenic growth factors was blocked by inhibition of endogenous PC-PLC. Taken together, these results indicate that hydrolysis of PC mediates Raf activation in response to mitogenic growth factors.

3T3 Cells↗
PubMed1992

Oncogenes as markers for early detection of cancer.

Oncogenes are formed in human tumors as a result of mutations or DNA rearrangements leading to the abnormal expression or function of proto-oncogenes. Approximately 20 different oncogenes are reproducibly activated in malignancies of several types, including breast, colon, lung, pancreatic, and thyroid carcinomas, leukemias, and lymphomas. The potential utility of these oncogenes as markers for early detection of cancer is dependent on the stage of tumor development at which they are activated, and on whether the mutated oncogenes are readily distinguished from the corresponding proto-oncogenes by assays that are sufficiently sensitive to detect precancerous lesions.

Biomarkers, Tumor↗
PubMed1992

Hydrolysis of phosphatidylcholine is stimulated by Ras proteins during mitogenic signal transduction.

We have used a dominant inhibitory ras mutant (Ha-ras Asn-17) to investigate the relationship of Ras proteins to hydrolysis of phosphatidylcholine (PC) in the transduction of mitogenic signals. Expression of Ha-Ras Asn-17 inhibited NIH 3T3 cell proliferation induced by polypeptide growth factors or phorbol esters. In contrast, the mitogenic activity of PC-specific phospholipase C (PC-PLC) was not inhibited by Ha-Ras Asn-17 expression. Similarly, cotransfection with a cloned PC-PLC gene bypassed the block to NIH 3T3 cell proliferation resulting from expression of the inhibitory ras mutant. Hydrolysis of PC can therefore induce cell proliferation in the absence of normal Ras activity, suggesting that PC-derived second messengers may act downstream of Ras in mitogenic signal transduction. This was substantiated by the finding that Ha-Ras Asn-17 expression inhibited growth factor-stimulated hydrolysis of PC. Taken together, these results indicate that PC hydrolysis is a target of Ras during the transduction of growth factor-initiated mitogenic signals.

3T3 Cells↗
PubMed1992

Identification of a negative regulatory element that inhibits c-mos transcription in somatic cells.

We have used transient expression assays to identify a cis-acting region in the 5' flanking sequence of murine c-mos which, when deleted, allows expression from the c-mos promoter in NIH 3T3 cells. This negative regulatory sequence, located 400 to 500 nucleotides upstream of the c-mos ATG, also inhibited expression from a heterologous promoter. In addition to NIH 3T3 cells, the c-mos negative regulatory sequence was active in BALB/3T3 cells, PC12 rat pheochromocytoma cells, and A549 human lung carcinoma cells. Site-specific mutagenesis identified three possibly interacting regions that were involved in negative regulatory activity, located around -460, -425, and -405 with respect to the ATG. RNase protection analysis indicated that once the negative regulatory sequences were deleted, transcription in NIH 3T3 cells initiated from the same transcription initiation sites normally utilized in spermatocytes, approximately 280 nucleotides upstream of the ATG. Deletions beyond the spermatocyte promoter, however, allowed transcription initiation from progressively downstream c-mos sequences. Deletion or mutation of sequences surrounding the oocyte promoter at -53 also had little effect on expression of c-mos constructs in NIH 3T3 cells. Therefore, the major determinant of c-mos expression in NIH 3T3 cells was removal of the negative regulatory sequence rather than the utilization of a unique promoter. The c-mos negative regulatory sequences thus appear to play a significant role in tissue-specific c-mos expression by inhibiting transcription in somatic cells.

3T3 Cells↗
PubMed1992

Role of Ras in signal transduction from the nerve growth factor receptor: relationship to protein kinase C, calcium and cyclic AMP.

A dominant inhibitory ras mutant (Ha-ras Asn-17) has been used to investigate the role of Ras in nerve growth factor (NGF)-mediated signal transduction in PC12 cells. Expression of Ha-Ras Asn-17 blocks neuronal differentiation of these cells in response to NGF treatment. The Ha-Ras Asn-17 block was bypassed by treatment with NGF plus dibutyryl cAMP or NGF plus the Ca2+ ionophore ionomycin, but not by NGF plus 12-O-tetradecanoyl phorbol acetate (TPA). Direct stimulation of the cAMP or Ca2+ pathways thus appeared to act synergistically with a Ras-independent NGF signaling pathway. This Ras-independent pathway was also distinct from protein kinase C, since its activity was not affected by protein kinase C down-regulation. It thus appears that NGF stimulation generates a Ras-independent intracellular signal that contributes to neuronal differentiation independently of the cAMP, Ca2+ or protein kinase C second messenger systems. Since TPA did not bypass the Ha-Ras Asn-17 block to differentiation, protein kinase C also did not appear to be sufficient for Ras-dependent pathways mediating NGF-induced differentiation. Down-regulation experiments further indicated that protein kinase C was not required for NGF induction of early response genes via either Ras-dependent or Ras-independent pathways. Moreover, the formation of inositol phosphates and mobilization of intracellular calcium in response to NGF was not inhibited in PC12 cells expressing the Ha-Ras Asn-17 protein. Therefore, although calcium was able to bypass the Ha-Ras Asn-17 block to PC12 differentiation, Ras activity was not required for activation of phospholipase C in response to NGF. It thus appears that both Ras-dependent and Ras-independent signaling pathways contribute to NGF-induced PC12 cell differentiation independently of the cAMP, calcium and protein kinase C second messenger systems.

Animals↗
PubMed1992

Ras controls coupling of growth factor receptors and protein kinase C in the membrane to Raf-1 and B-Raf protein serine kinases in the cytosol.

A dominant negative mutant of Ras, M17 Ras, was used to study the role of Ras in receptor coupling of Raf-1 and B-Raf protein serine/threonine kinases (PSKs). We found that mutant Ras blocks serum- and 12-O-tetradecanoyl phorbol 13-acetate-induced activation of Raf-1 kinase in NIH3T3 cells and Raf-1 as well as B-Raf PSK stimulation by nerve growth factor (NGF) in PC12 pheochromocytoma cells. Mitogen stimulation of Raf kinase was measured by determination of Raf hyperphosphorylation and activity towards exogenous substrates and both of these events were inhibited in cells expressing M17 Ras. In contrast, tyrosine phosphorylation of a direct substrate of activated tyrosine kinase receptors, phospholipase C-gamma 1 (PLC-gamma 1), was unaffected. These data indicate that tyrosine phosphorylation of PLC-gamma 1 is not sufficient for growth induction in NIH3T3 cells and that Ras mediates signal transfer from activated membrane receptors to Raf kinases in the cytosol. As activated Raf induced differentiation in PC12 cells expressing M17 Ras we conclude that Raf kinase activation may be sufficient to account for this aspect of NGF function.

3T3 Cells↗