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Biomedical subjects

G M Cooper

Publications and source records attributed to G M Cooper.

At least 55 records · Page 3Linked to original sources
PubMed1997

A comparison of eltanolone and propofol anaesthesia for minor gynaecological surgery.

We have used eltanolone and propofol to induce anaesthesia and by intermittent injection to supplement nitrous oxide during maintenance in 67 patients undergoing minor gynaecological surgery. This study was a controlled randomized phase III with two parallel groups and a blinded assessment of post-operative recovery. Complications during induction and maintenance were few. Urticaria occurred in two patients given eltanolone and in none of those given propofol. Time to orientation and time to eye opening were significantly shorter in the propofol group (P < 0.001). Systolic and diastolic blood pressure decreased in both groups but the decrease was significantly greater in the propofol group (P < 0.005 systolic blood pressure; P < 0.001 diastolic blood pressure). Heart rate decreased significantly in the propofol group (P < 0.002). We conclude that eltanolone anaesthesia is associated with a greater haemodynamic stability than propofol anaesthesia but that early recovery occurs more slowly.

Adult↗
PubMed1997

Comparison of induction and recovery between sevoflurane and halothane supplementation of anaesthesia in children undergoing outpatient dental extractions.

We have compared sevoflurane and halothane in a double-blind controlled study for supplementation of nitrous oxide and oxygen anaesthesia in 80 children undergoing dental extraction as outpatients. Induction of anaesthesia was more rapid in those who received sevoflurane compared with those who received halothane (89 s compared with 127 s for loss of eyelash reflex). In both groups, mean duration of administration of anaesthesia was less than 4 min. Those who received sevoflurane were slower to awaken (167 s compared with 102 s), although discharge times from hospital were similar. The incidence of complications during induction and maintenance was low in both groups and return to normal appetite and activity occurred in the majority of children on the same day. More children who received halothane suffered nausea after leaving hospital. We conclude that sevoflurane is a suitable alternative to halothane, with more rapid induction of anaesthesia, but in these short procedures, awakening time was slower than after halothane.

Ambulatory Surgical Procedures↗
PubMed1997

Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.

3T3 Cells↗
PubMed1997

Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1.

Activation of growth factor receptors by ligand binding initiates a cascade of events leading to cell growth and division. Progression through the cell cycle is controlled by cyclin-dependent protein kinases (Cdks), but the mechanisms that link growth factor signaling to the cell cycle machinery have not been established. We report here that Ras proteins play a key role in integrating mitogenic signals with cell cycle progression through G1. Ras is required for cell cycle progression and activation of both Cdk2 and Cdk4 until approximately 2 h before the G1/S transition, corresponding to the restriction point. Analysis of Cdk-cyclin complexes indicates that Ras signaling is required both for induction of cyclin D1 and for downregulation of the Cdk inhibitor p27KIP1. Constitutive expression of cyclin D1 circumvents the requirement for Ras signaling in cell proliferation, indicating that regulation of cyclin D1 is a critical target of the Ras signaling cascade.

3T3 Cells↗
PubMed1996

Activation of the CPP32 apoptotic protease by distinct signaling pathways with differential sensitivity to Bcl-xL.

In the absence of growth factors, many types of mammalian cells undergo apoptosis. We and others have shown recently that growth factors promote cell survival by activating phosphatidylinositol 3-kinase (PI 3-kinase) in several cell types. In the present study, we have compared downstream elements of the apoptotic pathways induced by PI 3-kinase inhibitors and other stimuli. In U937 cells, both PI 3-kinase inhibitors (wortmannin and LY294002) and etoposide activated the CPP32 apoptotic protease by cleavage to active p17 subunits. In contrast, treatment with tumor necrosis factor alpha (TNFalpha) resulted in the accumulation of a distinct active CPP32 subunit, p20. Furthermore, overexpression of Bcl-xL blocked DNA fragmentation, CPP32 activation and cleavage of poly(ADP-ribose) polymerase in U937 cells treated with both PI 3-kinase inhibitors and etoposide, but not in cells treated with TNFalpha. Distinct patterns of CPP32 activation and differential sensitivities to Bcl-xL thus distinguish the cell death pathways activated by PI 3-kinase inhibition and DNA damage from that activated by TNFalpha.

Androstadienes↗
PubMed1996

Growth factor-dependent survival of rodent fibroblasts requires phosphatidylinositol 3-kinase but is independent of pp70S6K activity.

A variety of mammalian cells undergo apoptosis when deprived of growth factors, but the intracellular signaling pathways responsible for cell survival remain to be characterized. In the present study, we have investigated the role of PI 3-kinase and pp70S6K in growth factor-dependent survival of rodent fibroblasts. As previously reported for PC12 pheochromocytoma cells, Rat-1 and REF52 cells underwent apoptosis following either serum-deprivation or treatment with the PI 3-kinase inhibitors wortmannin and LY294002. In contrast, NIH3T3 and BALB 3T3 cells were resistant to apoptosis induced by either serum-deprivation or PI 3-kinase inhibition. It thus appears that PI 3-kinase is specifically required to prevent apoptosis of fibroblast cell lines that are dependent upon growth factors for their survival. Consistent with this role of PI 3-kinase, serum and growth factors maintained steady state levels of PI 3-kinase activity that rapidly decreased following serum-deprivation. Serum and growth factors similarly maintained a steady state level of pp70S6K, which is thought to be activated downstream of PI 3-kinase. However, inhibition of pp70S6K activation by rapamycin failed to induce apoptosis in either Rat-1 or PC12 cells. Cell survival thus appears to require a PI 3-kinase signaling pathway that is independent of pp70S6K activation.

3T3 Cells↗
PubMed1996

Two cases of severe postpartum anaemia in Jehovah's witnesses.

Two cases of severe postpartum anaemia are presented in Jehovah's witnesses who refused blood transfusion. Despite haemoglobin concentrations of less than 3 g/dl both women survived. General management was directed to maximizing oxygen delivery and minimizing oxygen consumption. The use of an emulsified perfluorocarbon was organized for one of the cases, but was not administered because a snow storm prevented its delivery to the hospital. In the other case, recombinant human erythropoietin was used to encourage red cell production. The recovery of haemoglobin concentration in the two cases is compared.

Journal Article↗
PubMed1996

Frequency of multiple Epstein-Barr virus infections in T-cell-immunocompromised individuals.

The Epstein-Barr virus (EBV) carrier state is characterized by latent infection of the general B-cell pool and by chronic virus replication at oropharyngeal sites. In Caucasian populations, most healthy carriers seem to harbor one dominant transforming virus strain, usually of type I rather than type 2, which persists over time and is detectable both in the blood and in the throat. This finding implies that once the virus carrier state is established, both viral reservoirs are largely if not completely protected from infection with additional strains. However, it is not known which facets of the immune response offer that protection. Here we address this question by a detailed study of EBV carriage in patients T-cell immunocompromised as a result of chronic human immunodeficiency virus (HIV) infection. Resident EBV strains were rescued from blood and from throat washings by using an in vitro transformation assay which aims to minimize bias toward faster-growing transformants; in this way, a mean of 16 independent isolations were made from each of 35 HIV-positive (predominantly male homosexual) patients. These virus isolates were characterized first at the DNA level by PCR amplification across type-specific polymorphisms in the EBNA2 and EBNA3C genes and across the 30-bp deletion and 33-bp repeat loci in the LMP1 gene and then at the protein level by immunoblotting for the strain-specific "EBNAprint" of EBNA1, -2, and -3C molecular weights. By these criteria, 18 of 35 patients harbored only one detectable EBV strain, usually of type 1, as do healthy carriers. However, the other 17 patients showed clear evidence of multiple infection with different EBV strains. In eight cases these strains were of the same type, again usually type 1, and were more often found coresident in throat washings than in the blood. By contrast, a further nine patients gave evidence of coinfection with type 1 and type 2 strains, and in these cases both virus types were detectable in the blood as well as in the throat. Immunological assays on these HIV-positive patients as a group showed a marked impairment of T-cell responses, reflected in reduced levels of EBV-specific cytotoxic T-cell memory, but an elevation of humoral responses, reflected in raised antibody titers to the EBV envelope glycoprotein gp340 and by the maintenance of virus neutralizing antibodies in serum. We infer that selective impairment of the T-cell system predisposes the host to infection with additional exogenously transmitted EBV strains.

AIDS-Related Opportunistic Infections↗
PubMed1996

Isolation of intertypic recombinants of Epstein-Barr virus from T-cell-immunocompromised individuals.

All wild-type isolates of Epstein-Barr virus (EBV) analyzed to date for allelic polymorphisms of the nuclear antigen EBNA2 gene (in the BamHI YH region of the genome) and of the EBNA3A,-3B, -3C genes (tandemly arranged in the BamHI E region) have proved either uniformly type 1 or uniformly type 2 at all four loci. The absence of detectable intertypic recombination in the wild probably reflects the rarity with which individual carriers, and certainly individual target cells, become coinfected with both virus types. Studying a group of human immunodeficiency virus-positive T-cell-immunocompromised patients known to be at enhanced risk of multiple EBV infections, we have isolated intertypic EBV recombinants from 2 of 40 patients analyzed. These recombinants, whose in vitro transforming capacity appeared at least equal to that of type 1 strains, carried a type 1 EBNA2 allele and type 2 EBNA3A,-3B, and -3C alleles. This was clearly demonstrable at the DNA level by PCR amplification using type-specific primer-probe combinations and was confirmed at the protein level (for EBNA2 and EBNA3C) by immunoblotting with type-specific antibodies. In one patient, the recombinant appeared to be the predominant strain, being the virus most commonly rescued by in vitro transformation both from the blood and from the throat washings on two separate occasions 20 months apart. A regular type 1 virus strain was also present in this individual, but this was not related to the recombinant since the two viruses carried type 1 EBNA2 genes with different patterns of variance from the B95.8 prototype sequence. In the other patient, recombinants were isolated on one occasion from the blood and on a separate occasion, 21 months later, from the throat; these recombinants were almost certainly related, being identical at several genomic polymorphisms and differing only in one facet of the "EBNAprint," the size of the EBNA1 protein. Three different type 1 viruses were also isolated from this patient, two of which carried EBNA2 genes with the same pattern of sequence variation from B95.8 as the recombinant; however, since this is a fairly common pattern of variance, the relationship of these viruses to the recombinant remains an open question. We infer that intertypic recombinants of EBV are not uncommon in HIV-positive T-cell-immunocompromised patients, that they arise in such individuals as a consequence of their increased frequency of mixed-type infections, and that they will prove capable of efficient transmission in the human population.

Amino Acid Sequence↗
PubMed1995

Regulation of the Ras signaling pathway by GTPase-activating protein in PC12 cells.

We have investigated the role of Ras GTPase-activating protein (GAP) in NGF-induced neuronal differentiation by overexpressing both wild-type and membrane-targeted GAP in PC12 cells. Extension of neurites in response to NGF was completely blocked in cells expressing the highest level of membrane-targeted GAP and significantly inhibited in cells expressing either wild-type GAP or lower levels of membrane-targeted GAP. Overexpression of membrane-targeted GAP similarly inhibited induction of differentiation by src, but not by ras or raf oncogenes, indicating that GAP inhibits differentiation of PC12 cells by downregulating Ras function. GAP overexpression also inhibited stimulation of mitogen-activated protein (MAP) kinase and induction of immediate-early genes in response to NGF. In cells expressing wild-type GAP or lower levels of membrane-targeted GAP, the initial activation of MAP kinase and immediate-early gene expression were only partially inhibited. However, GAP expression in these cells resulted in substantial inhibition of sustained MAP kinase activity following NGF treatment, consistent with the inhibition of neurite extension in these cell lines. These results indicate that GAP acts as a negative regulation, rather than an effector, of Ras signaling in PC12 cells.

Animals↗
PubMed1995

Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor.

Nerve growth factor (NGF) induces both differentiation and survival of neurons by binding to the Trk receptor protein tyrosine kinase. Although Ras is required for differentiation, it was not required for NGF-mediated survival of rat pheochromocytoma PC-12 cells in serum-free medium. However, the ability of NGF to prevent apoptosis (programmed cell death) was inhibited by wortmannin or LY294002, two specific inhibitors of phosphatidylinositol (Pl)-3 kinase. Moreover, platelet-derived growth factor (PDGF) prevented apoptosis of PC-12 cells expressing the wild-type PDGF receptor, but not of cells expressing a mutant receptor that failed to activate Pl-3 kinase. Cell survival thus appears to be mediated by a Pl-3 kinase signaling pathway distinct from the pathway that mediates differentiation.

Androstadienes↗
PubMed1995

Comparison of continuous spinal and epidural analgesia for pain relief in labour.

We have compared continuous spinal analgesia with continuous epidural analgesia for pain relief in labour. Twenty-six women were randomly allocated to receive either epidural 0.25% bupivacaine 5-10 ml via a 20 gauge catheter inserted through a 16 gauge Tuohy needle or intrathecal 0.25% bupivacaine 0.5-1.0 ml via a 32 gauge catheter inserted through a 24 gauge Sprotte needle. This was supplemented with fentanyl 5-10 mcg (spinal) or 1 mcg per kg (epidural) if analgesia was unsatisfactory. Outcome was measured by the success and timing of the procedure, time to analgesia, amount of drug given, visual analogue scoring of pain relief by the patient and an observer and degree of motor block. Onset time and dosage were significantly reduced in the continuous spinal group. Two catheters failed to feed in the spinal group. One catheter became displaced in each group. Pain relief was satisfactory in all patients and none had post-dural puncture headache. Continuous spinal analgesia may offer significant advantages over epidural analgesia but technical difficulties remain with the present equipment. The reasons for the withdrawal of the spinal catheters in the United States of America are discussed.

Journal Article↗
PubMed1995

Identification of a candidate c-mos repressor that restricts transcription of germ cell-specific genes.

The c-mos proto-oncogene is specifically expressed in female and male germ cells. Previous studies identified a negative regulatory element (NRE) upstream of the c-mos promoter that suppresses c-mos transcription in transfected NIH 3T3 cells. In this study, we used gel shift assays to detect proteins in nuclear extracts of NIH 3T3 cells that bind to the c-mos NRE in a sequence-specific manner. One protein was found to bind to a region of the NRE which was shown by site-directed mutagenesis to be required for suppression of c-mos transcription. This factor was present in nuclear extracts of several somatic cell lines and tissues but not in male germ cells in which c-mos is transcribed, suggesting that it is a somatic cell repressor of c-mos transcription. The binding site of the candidate repressor within the c-mos NRE consists of sequences related to putative NREs identified in two other male germ cell-specific genes (encoding protamine 2 and phosphoglycerate kinase 2). The c-mos repressor bound and could be UV cross-linked to these protamine 2 and phosphoglycerate kinase 2 gene sequences as a protein with an apparent molecular mass of approximately 30 kDa. The repressor binding site is also conserved in two other germ cell-specific genes (encoding testis-specific cytochrome c and heat shock-like protein 70), suggesting that the c-mos repressor may be generally involved in suppressing transcription of germ cell-specific genes in somatic cells.

3T3 Cells↗
PubMed1995

Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.

Animals↗