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Biomedical subjects

E Murphy

Publications and source records attributed to E Murphy.

At least 127 records · Page 7Linked to original sources
PubMed1996

Measurement of free Ca2+ in sarcoplasmic reticulum in perfused rabbit heart loaded with 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid by 19F NMR.

Measurements of free calcium ion concentration in the sarcoplasmic reticulum ([Ca2+]SR) and an evaluation of its relationship to changes in cytosolic free calcium and energy state of the cell, as well as heterogeneity of the SR calcium pool, were performed using 19F NMR in Langendorff perfused rabbit hearts loaded with acetoxymethyl ester of 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid. We report a base-line time-average [Ca2+]SR value of 1.5 mM (n = 13) in the beating heart, similar to the value measured at diastole. We further report that [Ca2+]SR decreases by approximately 30% at the start of systole and that there is no evidence of spacial heterogeneity in [Ca2+]SR during the contraction cycle. However, there appears to be a heterogeneous response to SR calcium channel release activator (caffeine) and SR calcium-ATPase inhibitor (cyclopiazonic acid), consistent with studies suggesting that there are subpopulations of SR. Raising cytosolic free calcium by depolarizing the cell with 30 mM extracellular KCl, resulted in an increase in [Ca2+]SR; however, the calcium gradient was unchanged. Lowering cell phosphorylation potential, which would reduce the free energy available for the SR Ca2+-ATPase, leads to a decrease in the calcium gradient across the SR, but this reduced gradient was primarily due to an increase in cytosolic free calcium and not a net release of SR calcium.

Animals↗
PubMed1996

Reversibility of T helper 1 and 2 populations is lost after long-term stimulation.

Commitment of T helper 1 (Th1) or Th2 populations developing during an immune response to a pathogen, or an inappropriate immune response to an allergen or autoantigen, may determine the difference between health and chronic disease. We show that strongly polarized Th1 and Th2 populations assessed by immunoassay are heterogeneous using flow cytometry to detect single cells producing interferon gamma (IFN-gamma) and interleukin 4 (IL-4). Th1 populations arising after 1 wk of stimulation in IL-12 plus anti-IL-4 antibodies could convert to Th2 cells when restimulated in IL-4. Th2 populations resulting from stimulation for 1 wk in IL-4 could give rise to Th1 cells upon restimulation in IL-12 plus anti-IL-4. In contrast, the cytokine profiles of long-term Th1 and Th2 populations arising originally from repeated stimulation in IL-12 or IL-4 appeared more homogeneous and were not reversible, although IL-4 dramatically reduced the number of IFN-gamma-producing Th1 cells. This may explain previous reports that Th1 cells can be converted to Th2 cells.

Allergens↗
PubMed1996

Effect of ischemic preconditioning and PKC activation on acidification during ischemia in rat heart.

Ischemic preconditioning (PC) has been shown to attenuate intracellular acidification during a subsequent period of ischemia, to minimize stunning, and to decrease infarct size, PKC activation has been suggested to be involved in this phenomenon. The present study is designed to test whether PKC activation could mimic and PKC inhibition could block the PC effects on intracellular acidification during ischemia and on stunning during reflow in Langendorff perfused rat hearts. Prior to 20 min of sustained global normothermic ischemia, groups of hearts were treated with the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoyl-srt-glycerol (DOG), a group of hearts was treated with the PKC inhibitor chelerythrine (CH), a group was treated with DOG plus CH, a group was preconditioned with four cycles of 5 min of ischemia and 5 min of reflow, and a group was treated with CH during PC. Recovery of left ventricular developed pressure (% of initial, pretreatment, preischemic LVDP), measured after 20 min of reflow, was improved in hearts treated with DOG, but not PMA (80 +/- 3% (DOG), 55 +/- 3% (PMA) v 51 +/- 3% (control), P < 0.05 between DOG and control), although both caused a similar degree of PKC translocation (measured by fractionation followed by an assay of PKC activity using incorporation of 32P into histone). The improved recovery of LVDP in the PC group and in the DOG group was blocked by chelerythrine. Measurement of pH (by 31P NMR) showed that DOG reduced acidification at 15-20 min of ischemia, although the effect was not as great as PC, while PMA did not reduce acidification. The effect of DOG on pHi was attenuated by CH; however, the PC-induced attenuation of the fall in pHi, was not affected by CH. High energy phosphates (measured by 31P NMR) were not significantly different between any of the groups during ischemia or reflow. This study confirms that the protective effect of ischemic preconditioning on stunning in rat heart can be eliminated by inhibition of PKC, but suggests that the effect of PC on the fall in pHi during sustained ischemia is not mediated by PKC.

Animals↗
PubMed1995

Characterization of the variable regions of a chimpanzee monoclonal antibody with potent neutralizing activity against HIV-1.

The variable (V) regions of C108G, a potent neutralizing chimpanzee mAb against a glycan-dependent epitope in the V2 region of HIV-1 gp120, have been characterized for reactivity with human VH and VK family-specific antisera, and their nucleotide sequences have been determined and analysed. To our knowledge, this is the first study characterizing expressed chimpanzee VH and VK genes. Results show that C108G expresses members of the VH3 and VK1 families, the largest VH and VK families in humans, respectively. Nucleotide and amino acid sequence analyses reveal that C108G VH is most homologous to the human VH3 germline gene, hsigdp33 or V3-43, and the human JH4 minigene. The human germline VK1 gene that is most homologous to C108G VK, hsigk1012, was previously observed in unmutated form in a human autoantibody with anti-i red blood cell antigen specificity and in seven human Fabs and a mAb directed against epitopes overlapping the CD4-binding site of HIV-1 gp120. This germline gene was unmutated in three of the human Fabs and was somatically mutated in the other four Fabs and the mAb. In addition, the JK minigene was used in C108G VK, JK2, is apparently over-represented in anti-HIV-1 mAbs/Fabs; this minigene was used in 61% of the anti-gp120 human Fabs recently described and in three other anti-CD4-binding site human mAbs derived by EBV transformation. While the significance of these findings is unclear, they may suggest a bias in VK/JK gene usage and/or network regulation involving an hsigk1012/JK2 idiotope(s) in the antibody response to HIV-1. Both the C108G VH and VK genes showed evidence of somatic mutation and antigen selection that apparently occurred in vivo during chronic exposure to HIV-1 and its antigens. Surprisingly, this somatic mutation was most profound in the CDR3 region of C108G VK; this region shared only 48% nucleotide homology with hsigk1012 contrasted with a homology of 94% over the remainder of these two V gene sequences. Perhaps the most significant finding of this study is that the expressed VH and VK genes of chimpanzee mAb C108G are no more divergent from their most homologous human germline genes than are the expressed V genes of several recently characterized human anti-HIV-1 mAbs/Fabs from their apparent human germline genes. This suggests that chimpanzee mAbs are no more likely to elicit deleterious anti-immunoglobulin responses in humans than are human mAbs and emphasizes the potential for development of chimpanzee mAbs as immunotherapeutic agents.

Amino Acid Sequence↗
PubMed1995

No symptoms, no problem? Patients' understandings of non-insulin dependent diabetes.

Depth interviews were carried out with 46 people with non-insulin dependent diabetes. In the course of narrative accounts, respondents displayed their thinking about the nature of diabetes and their understandings of how one ought to respond to it. Two variations in patients' interpretations of diabetes are discussed here: the extent to which patients primarily orientated themselves toward symptom control or toward prevention of complications, and their perceptions of the seriousness of the condition. Most patients believed diabetes to be a serious condition, which could cause complications. However, many were able to reconcile such beliefs with a less than whole-hearted adherence to medical advice about lifestyle. The analysis suggests a range of specific areas of diabetes care where patients could be more fully informed. Most importantly it describes a range of ways in which patients present their non-adherence to medical advice about lifestyle as entirely rational, given their perceptions of diabetes and its personal implications for each of them. Better understanding of such perceptions by health professionals may improve therapeutic alliances.

Adaptation, Psychological↗
PubMed1995

Oral iloprost as a treatment for Raynaud's syndrome: a double blind multicentre placebo controlled study.

OBJECTIVE: To compare the efficacy, tolerance and safety of 50-150 micrograms orally administered iloprost given twice a day versus placebo in patients with Raynaud's syndrome. METHODS: The study was multicentre (n = 3), double blind and placebo controlled. Sixty three patients who had eight or more vasospastic attacks per week were enrolled. After a one week run-in period, all patients received either iloprost or placebo treatment to a maximum tolerated dose of 150 micrograms twice a day for 10 days. Diary cards assessed the duration and severity of the vasospastic attacks. Side effects were monitored by direct questioning. A global assessment of treatment efficacy was made by the patient at the end of treatment and two weeks later. RESULTS: Patient opinion tended to favour iloprost at the end of the 10 day treatment phase (p = 0.09) and this was significant at day 24 (the follow up visit) (p = 0.011). Although the duration and severity of attacks tended to decrease in the iloprost treated group, these results tended not to reach statistical significance (for severity p = 0.06 at end of treatment, p = 0.09 on day 24). CONCLUSION: Iloprost administered intravenously has been shown to be of benefit in the treatment of the Raynaud's syndrome associated with systemic sclerosis, but this route of administration is inconvenient. This study evaluated the use of iloprost administered orally to patients with Raynaud's syndrome. Patient documented improvement was significantly improved by iloprost. Diary card analysis showed a trend in favour of iloprost, but these results did not reach statistical significance.

Administration, Oral↗
PubMed1995

Role of lipoxygenase metabolites in ischemic preconditioning.

Preconditioning with brief intermittent periods of ischemia before a sustained period of ischemia has been shown to reduce infarct size and improve recovery of function in rat hearts. The mediators of this protective response are unknown in rats. We tested the hypothesis that a lipoxygenase metabolite might be involved in preconditioning, since lipoxygenase metabolites such as 12-hydroperoxyeicosatetraenoic acid have been shown to increase K+ channel activity and to decrease Ca2+ channel activity, which could have a protective effect on ischemic injury. In support of this hypothesis, we report that the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA, 5 mumol/L) and eicosatetraynoic acid (7 mumol/L) added just before and during preconditioning blocked the protective effects of preconditioning on recovery of function during reflow after 30 minutes of global ischemia. In addition, these lipoxygenase inhibitors partially blocked the ability of preconditioning to attenuate the rise in cytosolic free calcium during sustained ischemia. We also investigated the effects of preconditioning on eicosanoid metabolism by using high-performance liquid chromatography and found that 12-hydroxyeicosatetraenoic acid (12-HETE), the stable product of the lipoxygenase pathway, was made during the preconditioning protocol and that 12-HETE accumulation was blocked by NDGA. Thus, there is a correlation between functional recovery after ischemia and stimulation of the lipoxygenase pathway of arachidonic acid metabolism before the sustained period of ischemia; inhibition of the lipoxygenase pathway eliminates the protective effect of preconditioning on recovery of function after ischemia.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

A redox-based mechanism for cardioprotection induced by ischemic preconditioning in perfused rat heart.

Recent studies have suggested that mild redox alterations can regulate cell function. Therefore, we tested the hypothesis that alteration in the thiol redox state might be responsible for the cardioprotective effects conferred by ischemic preconditioning in the perfused rat heart. We find that preconditioning with four 5-minute periods of ischemia, each separated by 5 minutes of reflow, is associated with a significant loss of glutathione (3.98 +/- 0.32 mumol/g dry wt, n = 8) compared with no preconditioning (6.38 +/- 0.24 mumol/g dry wt, n = 14). We further find that the addition of N-acetylcysteine (NAC, a glutathione precursor and antioxidant) during the preconditioning protocol not only blocks the loss of glutathione (5.60 +/- 0.31 mumol/g dry wt, n = 9) but also blocks the protective effects of preconditioning. It is observed that after 20 minutes of ischemia followed by 20 minutes of reflow, untreated hearts recover 38 +/- 7% (n = 5) of their initial preischemic contractile function, whereas preconditioned hearts recover 91 +/- 11% (n = 7). Hearts preconditioned in the presence of NAC recover 24 +/- 3% (n = 7) of their preischemic function. Similarly, the addition of NAC reverses the protective effect of preconditioning on creatine kinase release. On reflow after 60 minutes of ischemia, creatine kinase release from control hearts was 271 +/- 20 IU.20 min-1.g dry wt-1 (n = 5), whereas preconditioned hearts release only 170 +/- 26 IU.20 min-1.g dry wt-1 (n = 6), and hearts preconditioned in the presence of NAC release 361 +/- 30 IU.20 min-1.g dry wt-1 (n = 5). We also find that hearts preconditioned in the presence of NAC have less attenuation of the decline in pHi than hearts preconditioned in the absence of drug. Thus, a redox-sensitive mechanism may be involved in the protection afforded by ischemic preconditioning.

Acetylcysteine↗
PubMed1995

Cerebral edema after temporary and permanent middle cerebral artery occlusion in the rat.

BACKGROUND AND PURPOSE: The potential of thrombolytic agents to improve outcome after ischemic stroke could be negated if recanalization of an occluded artery exacerbates cerebral edema. We examined whether infarctions associated with reperfusion have more edema than those without reperfusion and whether the time course for the development of cerebral edema varied with and without reperfusion. METHODS: Infarct volumes were measured 24 hours after permanent and 1, 2, and 3 hours of temporary right middle cerebral artery (MCA) occlusion in spontaneously hypertensive rats. Hemispheric volume, water, sodium, and potassium were measured 3, 6, 12, 24, 36, and 48 hours after permanent and 3 hours of temporary MCA occlusion and also determined 24 hours after permanent and 2 and 3 hours of temporary MCA occlusion. RESULTS: Minimal tissue damage occurred after 1 hour of temporary ischemia. Infarct sizes were similar after permanent and 3 hours of temporary MCA occlusion and significantly greater than after 2 hours of temporary ischemia. Hemispheric volume, water, and sodium from the infarcted right hemisphere were significantly greater than those from the left hemisphere beginning 6 hours after MCA occlusion and continuing for 48 hours, with a peak at 24 hours. Right hemispheric water measured 24 hours after 2 hours of temporary ischemia was significantly less than after permanent or 3 hours of temporary ischemia. CONCLUSIONS: This study demonstrates that cerebral edema after focal stroke is related to infarct size and is independent of reperfusion status. The results suggest that exacerbation of cerebral edema will not occur after thrombolytic treatment or spontaneous recanalization of occluded cerebral vessels.

Analysis of Variance↗
PubMed1995

The effects of different stretch velocities on average force of the shortening phase in the stretch-shorten cycle.

The function of muscle is thought to be enhanced through the use of a stretch-shorten cycle. Although this pattern of muscle function is commonly used, optimal parameters of the cycle are not well understood. The purpose of this paper is to review the stretch-shorten cycle pattern of muscle function, as it applies to human movement, and to present a research study that investigated the effects of different stretching speeds on the average concentric forces during stretch-shorten cycles. Twenty-two women performed repeated plantar flexor stretch-shorten cycles on a Kin-Com dynamometer at approximately 60% of their isometric plantar flexor maximum. Sequential trials were recorded for 15 seconds at three different speed combinations (eccentric speed/concentric speed): 50/180 degrees/sec, 100/180 degrees/sec, and 150/180 degrees/sec. Eighteen cycles from each speed combination were chosen for analysis based on contributing to an equal plantar flexor activity among the speed combinations. Analysis of variance indicated that the speed combination that most closely approximated normal walking (50/180 degrees/sec) resulted in the highest average concentric forces. Analysis of covariance indicated that the highest forces were due, at least in part, to a higher averaged interphase force.

Adult↗
PubMed1995

Social support. Among elderly in two community programs.

Elderly individuals lacking adequate social support have shown greater deficits and difficulties in illness recovery, length of hospitalization, and development of cognitive and emotional changes than those who do receive adequate social support. This study evidenced a very meaningful finding for nurses--older adults who are often dependent upon others for support in some or most facets of daily living specified love and respect as the type of support most needed, rather than tangible aid. In most communities, a wide array of educational and support group programs are offered to older adults. Social networking and the enhancement of social support among participants should be primary goals of such programs.

Aged↗
PubMed1995

Primary care research ethics.

Research activity in primary care is increasing rapidly, and raises a range of specific ethical issues. Many of these relate to the involvement of individuals in the community who are not seeking medical care and to the impact of research participation on relationships between general practitioners and their patients. The ethical issues pertinent to a range of quantitative and qualitative research methodologies in primary care are identified and considered.

Ethics, Medical↗
PubMed1994

Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B.

Excessive cytokine expression induced by superantigen may be one aspect of the pathophysiology associated with Gram positive bacteremia. We have undertaken a study of the kinetics of cytokine production in lymph nodes obtained from in vivo Staphylococcus enterotoxin B (SEB) treated animals. This study was designed to evaluate the short term cytokine profile observed using immunohistochemistry (IHC) in BALB/c mice injected intraperitoneally (i.p.). The observed immunohistochemical kinetic profiles were corroborated using reverse transcription-polymerase chain reaction (RT-PCR) RNA analysis. We report here that TNF, IL-2, and IFN-gamma are the principal cytokines which were detected within hours of SEB administration, and that other cytokines such as IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF and M-CSF were undetectable. TNF and IL-2 appeared very early following SEB priming, and were observed by 1 h. IFN-gamma which appeared later (maximally at 14 h) was produced predominantly by CD8+ cells. In contrast, the TNF and IL-2 were produced primarily by CD4+ cells. Identical results were obtained by IHC and RT-PCR; the kinetics of mRNA expression slightly preceded the appearance of protein. The TNF and IFN-gamma staining patterns observed in lymph node sections were indicative of Golgi-localized cytokine. The IL-2 staining pattern observed in lymph node sections was distinctive, covering a significant local area of cells. This local regional concentration of IL-2, which may result from cytokine attached to extracellular binding components, may be an important aspect of the activation phase of a developing immune response. Rapid induction and excessive cytokine production elicited by superantigen in vivo, may ultimately help to explain the shock and death associated with SEB.

Animals↗
PubMed1994

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages.

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q beta, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.

Antiviral Agents↗
PubMed1994

NMR-sensitive fluorinated and fluorescent intracellular calcium ion indicators with high dissociation constants.

A new series of high-dissociation constant (KD) Ca2+ indicators has been developed to reduce perturbations due to buffering of transients, to carry out measurements in cells and organelles with high basal Ca2+ concentrations, and to measure cytosolic Ca2+ levels in the presence of perturbations that may significantly increase these levels. A tetrafluorinated derivative of the chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA), has a KD of 65 microM and exhibits two fluorine nuclear magnetic resonances, one of which is insensitive to Ca2+ chelation and the second of which shifts by approximately 10 ppm upon Ca2+ binding. TF-BAPTA has pK values of approximately 5.0 and Mg2+ dissociation constants > 50 mM. At a field of 8.5 T, the Ca(2+)-sensitive resonance is in fast-intermediate exchange. Correction factors for the effects of intermediate exchange and for the effect of protonation (pK approximately 5.0) and Mg2+ complexation are discussed. An analogous approach has been used to synthesize 2-[2-(5-carboxyoxazole)]-5-[2-(2-bis(carboxymethyl) amino-5,6-difluorophenoxy)]ethoxy-6-bis(carboxymethyl)aminobenz ofuran (fura F), a structural analogue of fura 2, which exhibits fluorescence characteristics similar to those of fura 2, but has a KD of 20 microM.

Calcium↗