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Biomedical subjects

L M Murphy

Publications and source records attributed to L M Murphy.

17 recordsLinked to original sources
PubMed1998

Modulating the redox potential and acid stability of rusticyanin by site-directed mutagenesis of Ser86.

The expression of rusticyanin in Escherichia coli and a number of mutants for Ser86 is reported. Mutations of Ser86 to Asn, Asp, Gln, and Leu were undertaken as this is an Asn residue in other structurally characterized cupredoxins, and it has been suggested that this may be partly responsible for the high redox potential (680 mV) and extreme acid stability of rusticyanin. N-Terminal sequence analysis, together with other biochemical and spectrochemical characterization, shows that the recombinant wild-type protein is indistinguishable from native rusticyanin. All four mutants retain the rhombic nature of the EPR spectra and a significant absorption maximum at approximately 450 nm, thus confirming that the overall geometry of the Cu ligands is essentially maintained. The oxidized form of all four mutants is less acid stable than the wild-type protein, although the detailed mechanism of lability varies. Ser86Leu readily loses copper as the pH is reduced from 4.0, but the protein does not denature. A significant proportion (approximately 30%) of Ser86Gln is denatured at lower pH values, whereas Ser86Asn and Ser86Asp are stable as the reduced (CuI) protein. The redox potential also varies by approximately 110 mV (590-702 mV) upon these single point mutations, thus providing direct experimental support to the idea that this residue is at least in part responsible for the acid stability and the highest redox potential of rusticyanin in the cupredoxin family.

Amino Acid Sequence↗
PubMed1998

The nature of ligand-induced conformational change in transferrin in solution. An investigation using X-ray scattering, XAFS and site-directed mutants.

Ligand-induced conformational change in transferrins has been studied by site-directed mutagenesis of human serum half molecule (N-lobe), X-ray absorption fine structure (XAFS) spectroscopy and X-ray solution scattering. Use of recent advances in data analysis has been made for extracting model-independent molecular shapes from X-ray solution scattering data for the intact, the half molecule and its mutants. Clear evidence is provided that the transferrin molecule (intact as well as N-lobe), in its apo and holo forms, exists for the majority of the time in well-defined specific conformations representing the "fully opened" and "closed" states of the molecule, respectively. Evidence is also provided for the existence of an additional conformation, referred to here as the "intermediate" conformation for simplicity, which is trapped in the case of some of the mutants in the iron-bound form. We suggest that domain closure in the transferrin molecule is a two-step process, with the intermediate conformation representing the first stage of domain closure (approximately 20 degrees hinge-twist of domain II). Our data are not inconsistent with the ligand-free molecule sampling the closed states occasionally (< or = 10%) but are not in support of a continuous conformational search between the fully opened and closed states in the absence of iron.

Animals↗
PubMed1998

Illness specific patterns of psychological adjustment and cognitive adaptational processes in children with cystic fibrosis and sickle cell disease.

Illness-specific patterns of adjustment and cognitive adaptational process were identified in children (7-12 years of age) with cystic fibrosis (CF, n = 40) or sickle cell disease (SCD, n = 40). Anxiety diagnoses were most frequent for both illness subgroups but children with CF had a higher rate of oppositional disorder (27.5%) than did children with SCD (2.5%). Significant portions of the variance in adjustment were accounted for by stress appraisal (19%), expectations of efficacy (9%) and health locus of control (9%) for children with CF and by stress appraisal (21%) and self-worth (12%) for children with SCD. The interaction of general and specific illness tasks and adaptational process with developmental tasks in delineating intervention opportunities is discussed.

Adaptation, Psychological↗
PubMed1997

A critical assessment of the evidence from XAFS and crystallography for the breakage of the imidazolate bridge during catalysis in CuZn superoxide dismutase.

BACKGROUND: Copper-zinc superoxide dismutase (CuZn SOD) protects cells from the toxic effects of superoxide radicals. Key steps in the catalytic mechanism of CuZn SOD are thought to be the breakage of the imidazolate bridge between copper and zinc upon reduction of the copper site and the subsequent proton donation from the bridging histidine. This view has been recently challenged by a crystallographic study at 1.9 A resolution where evidence for a five-coordinate copper site in the reduced enzyme was provided. In contrast, a crystallographic study of yeast CuZn SOD at 1.7 A has confirmed the breaking of the bridging histidine in reduced crystals. We have examined the nature of the changes in metal coordination which result upon reduction of the enzyme using the X-ray absorption fine structure (XAFS) technique. RESULTS: The copper and zinc K-edge XAFS data of bovine SOD, recorded in the buffer systems used in the two crystallographic studies, were analyzed by constrained refinement using fast curved wave theory, taking full account of multiple-scattering effects. The study confirms that in the oxidized form of the enzyme the copper atom is five coordinate, with four histidine ligands at 1.99 +/- 0.02 A and a water molecule at 2.18 +/- 0.03 A. In the reduced form of the enzyme, one of the histidine ligands and the water molecule are lost from the inner coordination sphere of the copper, with the three remaining histidines ligated at 1.97 +/- 0.02 A. The X-ray absorption near edge structure (XANES) of the reduced enzyme is consistent with an approximate trigonal planar geometry at the copper site. The XAFS at the zinc K-edge is essentially identical in both the oxidized and reduced enzyme and is accounted for by three histidines coordinated at 2.01 +/- 0.02 A and an aspartate ligand at 1.96 +/- 0.03 A. CONCLUSIONS: The existence of a three-coordinate cuprous ion in bovine CuZn SOD is demonstrated and is a key feature of catalytic degradation of superoxide substrate by SOD involving alternate Cu(I) and Cu(II) states of the enzyme. Only subtle changes in the zinc K-edge XAFS take place upon reduction. Thus the reaction mechanism which involves breakage of the bridging histidine is unambiguously supported by the XAFS data.

Animals↗
PubMed1997

Adherence behavior among adolescents with type I insulin-dependent diabetes mellitus: the role of cognitive appraisal processes.

Guided by a transactional stress and coping model, this study examined the contribution of cognitive appraisal processes to diabetes adherence behavior among adolescents 12 to 18 years old (n = 40). Multiple hierarchical regression analyses indicated that esteem related to physical appearance accounted for a significant 16% of the variance in checking one's blood sugar. Perceived control when ill and attributional style for negative events each accounted for significant increments of variance as well (10 and 6%, respectively), yielding a total of 32% of the variance explained by appraisal processes. Results suggest that adolescents who (a) have a negative perception of their bodies, (b) perceive little internal control over health when ill, and (c) have an external attributional style for negative events were at greatest risk for poor compliance as indicated by less frequent checking of blood sugar.

Adaptation, Psychological↗
PubMed1996

Effect of pH and ligand binding on the structure of the Cu site of the Met121Glu mutant of azurin from Pseudomonas aeruginosa.

A pH-dependent X-ray absorption fine structure (XAFS) study has been undertaken to provide a structural interpretation of the spectroscopic properties of the Met121 Glu mutant of azurin from Pseudomonas aeruginosa (Azp). Ligand binding studies have been carried out to investigate the effect of the cavity formed at the Cu site as a result of the mutation. The optical spectrum at pH 4 exhibits an intense band at approximately 600 nm and a weaker band at approximately 450 nm, typical for the blue copper proteins. As the pH is increased, these bands decrease in intensity and shift to 570 and 413 nm, respectively, with the latter becoming the more intense of the two [Karlsson, B.G., et al. (1991) Protein Eng. 4 (3), 343-349]. These changes are accompanied by a change in the EPR spectrum from a rhombic type 1 Cu spectrum at pH 4 to a spectrum with the rhombic splitting decreasing to zero and the hyperfine coupling increasing from 25 to 83 G. X-ray absorption a the Cu K-edge shows that this change results from the lengthening of the Cu-His (by 0.07 A) and Cu-Cys (by 0.06 A) bonds and the coordination of one of the oxygen atoms of the glutamate ligand at pH 8, at a distance as close as 1.90 A. The copper site thus changes from a normal type 1 copper center with three strong bonds at pH 4 to a copper site with four strong bonds at pH 8, with Cu-His distances significantly longer than known distances for type 1 copper centres measured using the XAFS technique. The XAFS of the azide derivative measured at pH 8 shows a similar Cu coordination, with azide replacing glutamate as the fourth ligand. Azide binding at pH 8 is accompanied by a further increase in the EPR hyperfine coupling to 110 G. This structural information when taken together with recent structural sudies on copper proteins points toward the need for a reexamination of the basis on which copper proteins are classified.

Absorptiometry, Photon↗
PubMed1996

Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein.

PTH-related protein (PTHrP) is the principle mediator of the syndrome of humoral hypercalcemia of malignancy and has potential paracrine actions on smooth muscle, epithelial cell growth, and placental calcium transport. The human PTHrP gene is complex: a combination of three promoters, one 5' alternative splicing event and alternative 3' splicing, which produces three PTHrP isoforms (139, 141, or 173 amino acids), results in multiple PTHrP messenger RNA (mRNA) species. We employed the RT-PCR technique to identify promoter usage and splicing patterns in a range of human cell lines. Cell line-specific utilization of the promoters and the 3' alternative splicing pathways was detected among bone, breast, kidney, and lung cell lines, although each cell line could potentially produce the three PTHrP isoforms. We also determined whether some of the known regulators of PTHrP differentially modulate promoter usage or splicing patterns. Dexamethasone decreased the abundance of each of the alternative mRNA species. In contrast, epidermal growth factor and transforming growth factor-beta treatment increased the abundance of each PTHrP mRNA species, with particularly marked effects on promoter 1- and promoter 2-initiated transcripts, especially those containing exon VII or VIII. Epidermal growth factor treatment was found to alter PTHrP splicing patterns in a manner consistent with increased transcription from promoters 1 and 2 and stabilization of exon VII- and IX-containing transcripts.

Base Sequence↗
PubMed1995

Structural and spectroscopic studies of the copper site of stellacyanin.

The structure of the copper site in oxidized and reduced Rhus vernicifera stellacyanin has been studied by X-ray absorption (XAFS) spectroscopy at different pH values. Data for the oxidized protein are consistent with the fourth ligand being an O- or N-donating ligand rather than a cysteine from the disulfide bridge. The fourth ligand is not present in the inner coordination sphere, but makes a more distant interaction 2.7 A from the copper atom. Only minor changes in the details of the Cu(II) coordination occur when the pH is varied. Direct structural information on reduced stellacyanin is provided. Upon reduction, one of the histidine ligands moves away from the copper atom by at least 0.2 A. A low-Z (O or N) scatterer is present approximately 2.4 A from the Cu(I) atom in the protein at low pH, and this ligand is lost at high pH. There is no evidence for an S-donating fourth ligand in the reduced protein. The XAFS results are presented in relation to the spectroscopic and structural information available for some methionine-121 mutants of azurin. The data reveal that there are spectroscopic similarities between stellacyanin and some of the mutant proteins, but distinct structural differences exist that preclude these proteins as suitable models for the copper site of stellacyanin.

Azurin↗
PubMed1993

Structural characterization of azurin from Pseudomonas aeruginosa and some of its methionine-121 mutants.

Azurin from Pseudomonas aeruginosa and two mutants where the methionine ligand has been mutated have been studied in order to directly investigate the functional and structural significance of this ligand in the blue copper proteins. Reduction potentials, X-ray absorption fine structure (XAFS), electron paramagnetic resonance (EPR), and optical spectra are obtained in an attempt to provide a direct correlation between the spectrochemical properties and the immediate structure of this redox center.

Absorptiometry, Photon↗
PubMed1993

Nutritional support of the cardiopulmonary patient.

In this article, the consequences of malnutrition on the cardiopulmonary system are reviewed. Methods of identification of malnourished patients, means of calculating nutritional requirements, and procedures for providing adequate and safe nutritional repletion are discussed. Complications of nutrition support and methods to prevent such complications are reviewed.

Critical Illness↗
PubMed1987

Transvascular protein movement in the intact ischemic hindlimb.

Postischemic limb swelling following reperfusion may be related to microvascular changes associated with ischemia. We used lymph-to-plasma total protein concentration ratios (L/P) and lymph flow (QL) as an index of transvascular exchange in the intact dog hindlimb during steady state (C) (1 hr), ischemia (I) (6 hr), and reperfusion (R) (3 hr). Central pressures, femoral arterial and venous pressures (PA, PV) and QL were recorded every 15 min. Lymph was collected from a femoral lymphatic in the passively flexed leg (50 cycles/min). Three groups of animals were studied: GI, sham-operated (N = 5); GII, moderate ischemia (N = 7, PA = 30-45% C); and GIII, severe ischemia (N = 7, PA = 5-20% C). In GI, QL gradually increased over 10 hr without change in L/P. Moderate ischemia produced a decrease in QL, 3.55 +/- 2.02 mg/hr to 0.92 +/- 0.53 mg/hr (P less than 0.0001), and QL remained below baseline during R with no change in L/P over the 10 hr. Severe ischemia produced a similar decrease in QL, 1.91 +/- 2.05 mg/hr to 0.15 +/- 0.1 mg/hr (P less than 0.01); however, an increase to 2.56 +/- 2.14 mg/hr occurred during R. Severe ischemia increased L/P 0.42 +/- 0.08 to 0.64 +/- 0.23 (P less than 0.001) and remained elevated during R at 0.63 +/- 0.18 (P less than 0.001). An increase in the wet-to-dry weight ratio of ischemic to nonischemic muscle after reperfusion was noted only in GIII, 3.82 +/- 1.17 vs 2.60 +/- 0.45 (P less than 0.04). Severe ischemia produces changes in vascular integrity which augment protein flow. Prevention of these vascular changes may help to minimize the muscle swelling of reperfusion.

Animals↗
PubMedDate not supplied

Central venous catheter care in parenteral nutrition: a review.

Central venous catheter care in parenteral nutrition has been described in numerous publications. These descriptions include care of both short- and long-term catheters. Important aspects in the prevention of infection in central venous catheters used for parenteral nutrition have included: the method of central venous access, subcutaneous tunneling, dressing change techniques, therapeutic uses of the catheter, and catheter-intravenous tubing connection care. Methods of predicting infection and evaluating catheter sepsis have been described. The efficacy of intervention by specialized nutrition support teams in infection control related to these catheters has been demonstrated. Newer partially implantable and completely implantable venous access devices have been used for parenteral nutrition. Methods of infection control and prevention of mechanical damage of these types of catheters have been examined with various results. Care and composition of central venous catheters have been shown to have some role in thrombus formation in the central veins. The use of heparin to prevent thrombosis and catheter occlusion, and the varying degrees of success obtained, have been described. Medical treatment of the occluded catheter with urokinase may be a necessary alternative to discontinuance of that catheter. Overall consideration of research methods used to reach conclusions for catheter care should be considered in the evaluation of appropriate care in each situation.

Catheters, Indwelling↗