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Biomedical subjects

E Murphy

Publications and source records attributed to E Murphy.

At least 253 records · Page 14Linked to original sources

Coupled sodium-calcium transport in cultured chick heart cells.

In cultured embryonic chick heart cells, alterations of extracellular Na (Nao) and Ca (Cao), intracellular Na (Nai) and Ca, extracellular pH, and membrane potential resulted in changes in Na and Ca contents that were consistent with sarcolemmal Na-Ca exchange. 24Na efflux measurements revealed a large ouabain-insensitive component, one-third of which was inhibited by removal of Cao. Incubating the cells in Na-free solution resulted in a rapid, 1.5- to 2-fold increase in total cell Ca that remained elevated for at least 15 min. Cells exposed for 15 min to Nao less than or equal to 20 mM became maximally loaded with Ca, whereas Ca loading fell off sharply at values of Nao greater than 20 mM. The movement of Na against its electrochemical gradient was shown to be associated with Ca accumulation. During Na-K pump inhibition (in 10(-4) M ouabain), Na initially rose 2- to 3-fold to a level below its equilibrium value; then, lowering Cao for 30 min from 1.25 to 0.75 mM caused a 26% elevation in Nai, whereas raising Cao from 1.25 to 2.7 mM resulted in a 25% fall in Nai against its electrochemical gradient. These data are consistent with Nai being maintained by a Na-Ca exchange during Na-K pump inhibition. In the presence of ouabain (10(-4) M), Ca uptake into intracellular organelles, e.g., mitochondria, was suggested by an increase in total cell Ca as well as the occurrence of mitochondrial matrix granules, which were shown qualitatively by X-ray analysis to contain Ca. Although matrix granules also occurred in mitochondria during Na-free incubation, they did not contain detectable amounts of Ca when examined under identical conditions of fixation and analysis.

Animals↗

Effects of calcitonin on cytosolic Ca in a suspension of rabbit medullary thick ascending limb tubules.

A basal level of cytosolic free Ca (Caf) of 112 +/- 7 nM (n = 15) was measured in rabbit medullary thick ascending limb tubules that were loaded with the fluorescent Ca indicator quin 2. Calcitonin addition (10(-6) M) caused a greater than twofold increase in Caf. The KD for calcitonin stimulation of Caf was 8 X 10(-11) M. The rise in Caf had a half time of approximately 1.5 min. Neither parathyroid hormone (10(-6) M), dibutyryladenosine 3',5'-cyclic monophosphate (10(-4) M), nor forskolin (2 X 10(-5) M) had any significant effect on Caf levels, consistent with the rise in Caf being independent of an elevation in adenosine 3',5'-cyclic monophosphate. The effects of ouabain and the mitochondrial uncoupler, 1799, were also tested to obtain further information regarding the regulation of Caf. Ouabain (10(-4) M) caused no significant elevation in Caf; in contrast, 15 min of incubation with 1799 (10(-5) M) produced a twofold increase in Caf.

Aminoquinolines↗

Nuclear magnetic resonance measurement of cytosolic free calcium levels in human red blood cells.

Red blood cells were loaded with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (FBAPTA) by incubation with 50 microM of the acetoxymethyl ester (FBAPTA-AM), and cytosolic free Ca2+ was monitored with 19F-nuclear magnetic resonance (NMR). Loading with 50 microM FBAPTA-AM, which results in a final FBAPTA level of approximately 0.5 mM, caused only a 25-30% fall in cell ATP as measured by 31P-NMR when 5 mM pyruvate was present. Leakage of the NMR active Ca2+ indicator, which results from cell lysis, was corrected for with the addition of extracellular Eu3+ ions, extracellular ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), or washing. With this method, we have found basal levels of cytosolic free Ca2+ averaging 61 +/- 6 nM (means +/- SE, n = 19). When the intracellular level of FBAPTA was varied from 0.1 to 1.0 mM, there was no correlation between the level of cytosolic free Ca2+ and the level of loading with FBAPTA. Addition of 10 microM of the Ca2+ ionophore A23187 with extracellular Ca2+ set at different levels by Ca2+-EGTA buffers caused an increase in cytosolic free Ca2+ as expected. Furthermore, ATP depletion caused a two- to three-fold increase in cytosolic free Ca2+, consistent with inhibition of Ca2+ efflux via that Ca2+-ATPase.

Adenosine Triphosphate↗

C-sis and C-abl expression in chronic myelogenous leukemia and other hematologic malignancies.

Cellular oncogenes have been localized at the breakpoints of characteristic chromosomal rearrangements occurring in certain hematologic malignancies. This has been reported to result in aberrant expression of the involved oncogenes. Over 90% of chronic myelogenous leukemia (CML) is characterized by a reciprocal translocation that brings c-abl from chromosome 9 to chromosome 22, and c-sis from chromosome 22 to chromosome 9. To investigate the possible role of these two oncogenes in the leukemic process, we studied their expression in a number of fresh samples obtained from patients with various forms of leukemia, by Northern blot analysis using c-onc probes. Seven of 24 samples obtained from patients with either CML or chronic myelomonocytic leukemia expressed a normal 4.0-kilobase (kb) c-sis transcript. C-sis expression was found only in the accelerated/blast phases but not in the chronic phase of CML. All of the CML Philadelphia chromosome-positive (Ph1+) samples expressed an aberrant 8-kb c-abl transcript. The expression of c-sis in

Humans↗

Sudden death prediction by programmed electrical stimulation following myocardial infarction.

To determine prospectively whether electrophysiologic testing is prognostically useful following a myocardial infarction, 38 patients were studied. Ventricular tachycardia was induced in 32 of 38 (84%) patients (sustained in 12) and was significantly increased with the use of three or four extrastimuli. In 17 months' mean follow-up, 4 patients died suddenly or survived an episode of sustained ventricular tachycardia. Programmed ventricular stimulation was a sensitive but not a specific predictor of these events with an overall 24% predictive accuracy. The use of one or two extrastimuli substantially improved specificity but was insensitive in predicting sudden death or ventricular tachycardia. Programmed ventricular stimulation soon after uncomplicated myocardial infarction was not a useful prognostic indicator for sudden death or subsequent ventricular tachycardia.

Adult↗

Transposon Tn554: complete nucleotide sequence and isolation of transposition-defective and antibiotic-sensitive mutants.

The complete nucleotide sequence of the Staphylococcus aureus transposon Tn554, which encodes resistance to erythromycin and spectinomycin, was determined by the dideoxy chain termination method. The transposon was found to be 6691 bp in length and to contain six open reading frames of greater than 125 amino acids. Small insertion and deletion mutations were obtained in each of these by in vitro mutagenesis at restriction endonuclease cleavage sites and the mutants characterized with respect to transposition functions and antibiotic resistance markers. Three of the reading frames, designated tnpA, tnpB and tnpC, encode functions that are required for transposition of Tn554; genetic analysis indicated that these three genes define distinct complementation groups of transposition-defective mutants. Two of the open reading frames correspond to the resistance determinants spc and ermA, the sixth, designated ORF, has no known function. Tn554-specific peptides corresponding to tnpA, and spc were identified in a coupled transcription-translation system in vitro.

Amino Acid Sequence↗

Acute otitis media: clinical course among children who received a short course of high dose antibiotic.

A prospective study was carried out in 274 children aged 3 to 10 years with acute otitis media. They were randomly allocated to one of two treatment regimens: (a) a seven day course of amoxycillin 125 mg three times a day, and (b) a two day course of amoxycillin 750 mg twice a day. They were followed up by symptom diaries and clinical examination. The findings in the 243 children who completed the trial showed that the short course of treatment was as effective as the seven day course in the speed of resolution of symptoms and signs, irrespective of previous history of otitis media or of episodes in which bulging of the eardrums was observed at presentation. A subgroup of 185 children was followed up for one year after entry to the trial. During this period no appreciable differences emerged between the two antibiotic regimens, either in recurrence rate of otitis media or in the frequency of hearing loss at one month and six months after entry to the study. Side effects of treatment were few, and those that could be attributed to antibiotic use occurred with equal frequency in the two treatment groups.

Acute Disease↗

Nucleotide sequence of a spectinomycin adenyltransferase AAD(9) determinant from Staphylococcus aureus and its relationship to AAD(3") (9).

The nucleotide sequence of the spc determinant of the Staphylococcus aureus transposon Tn554 has been determined. This gene encodes a spectinomycin adenyltransferase, AAD(9), that mediates resistance to spectinomycin but not to streptomycin. The sequence predicts a 260 amino acid protein of molecular weight 28,943. A spectinomycin-sensitive mutant (spc-1) contains a G----A transition resulting in substitution of threonine (ACA) for alanine (GCA) at residue 165. The predicted amino acid sequence is 36% homologous to that of a widely distributed, gram-negative streptomycin/spectinomycin adenyltransferase, AAD(3") (9), specified by the aadA determinant (Holingshead and Vapnek 1985).

Amino Acid Sequence↗

General management of depression in late life.

To summarise, depression in the elderly is often a relapsing, serious illness in which physical treatments by drugs and ECT are the only specific anti-depressant measures we have. Once depressed, patients are likely to suffer further adverse events and are at risk of losing their close emotional supports as a result of the depression itself. The evidence suggests that it is unlikely that a pre-existing close intimate relationship can protect against relapse in the face of further adversity and furthermore it is unlikely that professional social support, as provided by day hospitals and outpatient clinics, is an effective antidote to the adverse effects of continuing problematic social circumstances. We need to learn a great deal more about how to manipulate the social milieu effectively and how to help distressed family carers cope better with the stress of a depressed elderly relative. Admission to an old people's home is rarely the right answer. Finally, let us look more objectively at the ward treatment offered to our seriously depressed patients and not impose a 'therapeutic' regime which is inflexible to individual patient's needs.

Aged↗

Cytosolic free calcium in chick heart cells. Its role in cell injury.

The role of cytosolic free Ca2+ (Caf) in cell injury was investigated using two methods for measuring Caf in freshly disaggregated embryonic chick heart cells. The null-point method, using arsenazo III, is based on determining the extracellular Ca2+ concentration at which no net Ca2+ movement occurs when plasma membrane permeability is increased. With this technique, the null point Caf averaged 0.23 +/- 0.07 microM (n = 6) in the basal state. Using quin2, an intracellular fluorescent dye, to measure Caf a value of 0.05 +/- 0.01 microM (n = 5) was obtained. Elevation of Caf by various agents was associated with an increase in cell injury as measured by the release of the cytosolic enzyme, LDH. However, the relationship between Caf and LDH release was not a direct one under all experimental conditions, indicating that the level of Caf is not the sole determinant of cell injury.

Adenosine Triphosphate↗

MLS-resistance determinants in Staphylococcus aureus and their molecular evolution.

This paper describes the genetic phenomenology of resistance to macrolide-lincosamide-streptogramin B antibiotics (MLSr) in Staphylococcus aureus and attempts to place this phenomenology in a broad evolutionary context. As antibiotic resistance in general and MLS resistance in particular are typical variable traits in bacteria of clinical interest, we shall begin by introducing the concept of variable genetic traits, as outlined in Figure 1. Variable traits are those that are expressed by some strains of a given species but not by others--in comparison to constant traits which are always present as part of the standard genetic make-up of the species and have constant chromosomal locations. Variable traits are often associated with variable and mobile genetic elements and it is suggested that, in general, they are not likely to have evolved as such in the species in which they are found. Rather, they will most probably have evolved as constant (chromosomal) traits in other species and acquired genetic mobility much later as a rare occurrence in that species. These rarely occurring mobile variants would then spread horizontally within a range of new species. The MLSr determinants in Gram-positive bacteria would appear to represent a classic example of this process. Their remarkable variability will be described as the extant end-point of the process and a probable evolutionary pathway will be traced back to the streptomycetes which are a likely primary source.

Anti-Bacterial Agents↗

Nucleotide sequence of ermA, a macrolide-lincosamide-streptogramin B determinant in Staphylococcus aureus.

The complete nucleotide sequence of ermA, the prototype macrolide-lincosamide-streptogramin B resistance gene from Staphylococcus aureus, has been determined. The sequence predicts a 243-amino-acid protein that is homologous to those specified by ermC, ermAM, and ermD, resistance determinants from Staphylococcus aureus, Streptococcus sanguis, and Bacillus licheniformis, respectively. The ermA transcript, identified by Northern analysis and S1 mapping, contains a 5' leader sequence of 211 bases which has the potential to encode two short peptides of 15 and 19 amino acids; the second, longer peptide has 13 amino acids in common with the putative regulatory leader peptide of ermC. The coding sequence for this peptide is deleted in several mutants in which macrolide-lincosamide-streptogramin B resistance is constitutively expressed. Potential secondary structures available to the leader sequence of the wild-type (inducible) transcript and to constitutive deletion, insertion, and point mutations provide additional support for the translational attenuation model for induction of macrolide-lincosamide-streptogramin B resistance.

Amino Acid Sequence↗

Adenosine metabolism in human skin fibroblasts.

When normal fibroblasts were incubated in media containing various initial concentrations of [8-14C]adenosine, ranging from 0.25 to 400 microM, under conditions where product formation was linear, greater than 90% of the intracellular label was found in adenine nucleotides, largely in the form of ATP, less than 1% of the intracellular label appeared in the nucleic acids, the remaining intracellular label was found in adenosine, inosine, and hypoxanthine, and the media contained two labeled products, inosine and hypoxanthine. Production of labeled inosine and hypoxanthine from adenosine was considerably lower in adenosine deaminase (ADA)-deficient cells than in normal cells and virtually eliminated in normal cells by the presence of 1 microM deoxycoformycin (a potent ADA inhibitor), suggesting that labeled inosine and hypoxanthine production requires ADA activity. Initial rates of deamination (inosine and hypoxanthine formation) and phosphorylation (adenine nucleotide formation) were estimated by examining the metabolic fate of [8-14C]adenosine in hypoxanthine phosphoribosyltransferase-deficient cells, which cannot recycle hypoxanthine. The estimate of the initial rate of phosphorylation exceeded that of deamination only at the lowest adenosine concentration examined (0.25 microM). The ratio of deamination to phosphorylation rose from approximately 1 at 0.41 microM to approximately 15 at 400 microM extracellular adenosine.

Adenosine↗

The impact of depression in old age on close social relationships.

Changes in the ratings of intimacy of personal relationships in depressed elderly patients were studied over a 1-year period. Those patients who had recovered by the end of the year were significantly more likely to report an improvement in the quality of their close relationships then were patients who had a poor clinical outcome. Changes in reports of intimacy were due not only to the effect of depression on the quality of relationships but also to independent life events occurring in the follow-up period.

Aged↗

Regulation of cytosolic free calcium in rabbit proximal renal tubules.

The relative role of various Ca2+ transport systems in the regulation of Ca2+ cytosolic free Ca2+ concentration was evaluated using rabbit renal proximal tubules. Intracellular compartmentation was evaluated through Ca2+ releases induced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), A23187, and ruthenium red (RR) alone and in combination. In a Ca2+-free solution after 1 h of incubation, FCCP released 43 +/- 4%, A23187 54 +/- 3%, and RR 29 +/- 5% of total cellular Ca2+; in addition, RR inhibited the rate of FCCP-induced release, confirming its mitochondrial origin. In 1 mM Ca2+, the releases were 57 +/- 9%, 70 +/- 5%, and 34 +/- 10%, respectively. Therefore, the mitochondrial Ca2+ content is 20-60 nmol/mg of mitochondrial protein, sufficiently large to effectively buffer cell Ca2+. To evaluate the role of the plasma membrane Na:Ca exchanger, 10(-4) M ouabain was added and caused a slight decline in total cell Ca2+ content and no change in ionized Ca2+ measured by the null-point method, suggesting that the plasmalemmal Na+:Ca2+ exchanger does not play an important role in Ca2+ extrusion. Cytosolic free Ca2+ increased when 100 mM sodium was replaced with equimolar choline or tetramethylammonium. However, tetramethylammonium replacement released 55% of the mitochondrial Ca2+ content by increasing mitochondrial Ca2+ efflux without affecting the Ca2+ influx pathway. These results suggest that Na+ replacements in this tissue increase ionized Ca2+ by increasing mitochondrial Ca2+ efflux and not by inhibition of Na+:Ca2+ exchange at the plasma membrane.

Adenosine Triphosphate↗

Cloning of the rat endogenous helper leukemia virus DNA sequence and expression of the helper activity encoded by the cloned DNA sequence in normal rat kidney cells by microinjection.

By the use of recombinant DNA technology and microinjection in cultured cells, the molecular genetic elements involved in the evolution of a retrovirus with the multipotential to infect, transform and replicate in host cell, have been critically examined in this investigation. Recently we have identified and purified the integrated and proviral DNA sequences specific for two rat endogenous helper leukemia viruses, WR- RaLV , originated from a chemically induced wild rat fibrosarcoma, and RHHV , isolated from a chemically induced rat hepatoma, HTC-H1 (1). By using a multidisciplinary approach combining restriction endonuclease analysis, reverse phase V-column chromatography, agarose gel electrophoresis, Southern blot transfer and filter nucleic acid hybridization, we were able to demonstrate that the rat helper leukemia viral DNA sequence was approximately 8.4-8.8 kb. The 8.8 kb RHHV DNA was molecularly cloned via the EK-1 certified vector pBR 322 plasmid into E. coli RRI cells. A successful recombinant clone, 8/32, that carried one entire RHHV 8.8 kb DNA sequence was mapped by restriction endonuclease analyses. Restricted DNA fragments of various sizes throughout the complete RHHV genome were isolated and purified for intranuclear microinjection into normal rat kidney cells. Release of type C infectious helper virus in these microinjected cells was investigated by superinfection on K-NRK, Kirsten sarcoma transformed non-producer cells. Recombination of the helper viral DNA sequence, en toto or of subgenomic sizes, carried in microinjected cells, with the sarcomagenic DNA sequence, carried in K-NRK cells, was also studied by genome-rescue and cell-transformation experiments. Our observations led to the conclusion that all critical genetic elements including the 5' LTR helper DNA sequence, gag, pol, and env genes, encoded for the biological activity of the type C helper virus resided within the 6.0 kb proximal to the 5' terminus of the endogenous rat type C helper virus DNA. They proved vitally essential for the recombination with the Src sequence during the evolution of an infectious, transforming and replication-competent retrovirus.

Animals↗