XAFS study of the high-affinity copper-binding site of human PrP(91-231) and its low-resolution structure in solution.

Here, we describe the structure of a C-terminal high-affinity copper-binding site within a truncated recombinant human PrP containing residues 91-231, which lacks the octapeptide repeat region. We show that at least two extra co-ordinating groups are involved in binding this copper(II) ion in conjunction with histidine residues 96 and 111 in a region of the molecule known to be critical in conferring strain type. In addition, using X-ray solution scattering, a low-resolution shape of PrP(91-231) is provided. The restored molecular envelope is consistent with the picture where the N-terminal segment, residues 91-120, extends out from the previously known globular domain containing residues 121-231.

Adolescent mothers' beliefs about parenting and injury prevention: results of a focus group.

INTRODUCTION: A nonfatal injury occurs every 2 seconds in the United States, and approximately 30,000 children die each year from injury-related causes. The U.S. Department of Health identified the reduction of unintentional injuries as a major health goal. The current study reports on a focus group discussion conducted with adolescent mothers to examine their beliefs and practices regarding injury prevention. METHOD: Seventeen first-time adolescent mothers participated in the focus group. Participants responded to questions about important aspects of mothering, causes of injury, and strategies to prevent injuries. Responses were categorized. RESULTS: No mother spontaneously identified injury prevention as an important part of mothering. More than half of all participants believed that injuries are unpreventable. Further, mothers identified a limited number of strategies to prevent injury. None of the mothers reported having discussed injury prevention with their child's pediatrician. DISCUSSION: Implications for care are discussed, including the need to address issues of injury prevention during well-baby care, the potential benefits of home visits, and specific challenges in implementing The Injury Prevention Program with young mothers.

Structural and kinetic evidence for an ordered mechanism of copper nitrite reductase.

The crystallographic structures of several copper-containing nitrite reductases are now available. Despite this wealth of structural data, no definitive information is available as to whether the reaction proceeds by an ordered mechanism where nitrite binds to the oxidised type 2 site, followed by an internal electron transfer from the type 1 Cu, or whether binding occurs to the reduced type 2 Cu centre, or a random mechanism operates. We present here the first structural information on both types of Cu centres for the reduced form of NiR from Alcaligenes xylosoxidans (AxNiR) using X-ray absorption spectroscopy. The reduced type 2 Cu site EXAFS shows striking similarity to the EXAFS data for reduced bovine superoxide dismutase (Cu2Zn2 SOD), providing strong evidence for the loss of the water molecule from the catalytic Cu site in NiR on reduction resulting in a tri-coordinate Cu site similar to that in Cu2Zn2 SOD. The reduced type 2 Cu site of AxNiR is shown to be unable to bind inhibitory ligands such as azide, and to react very sluggishly with nitrite leading to only a slow re-oxidation of the the type 1 centre. These observations provide strong evidence that turnover of AxNiR proceeds by an ordered mechanism in which nitrite binds to the oxidised type 2 Cu centres before electron transfer from the reduced type 1 centre occurs. We propose that the two links between the Cu sites of AxNiR, namely His129-Cys130 and His89-Asp92-His94 are utilised for electron transfer and for communicating the status of the type 2 Cu site, respectively. Nitrite binding at type 2 Cu is sensed by the proton abstracting group Asp92 and the type 2 Cu ligand His94, and relayed to the type 1 Cu site via His89 thus triggering an internal electron transfer. The similarity of the type 2 Cu NiR catalytic site to the reduced Cu site of SOD is examined in some detail together with the biochemical evidence for the SOD activity of AxNiR.

Disclosure, stress, and psychological adjustment among mothers affected by HIV.

This study examined patterns of disclosure and psychological adjustment among mothers infected or affected by HIV. All participants were followed through a family AIDS clinic at a midwestern children's hospital. With respect to disclosure, results indicated that high perceived stress (r = 0.50, p = 0.001) and low efficacy related to managing parenting demands (r = -0.42, p = 0.01) were significantly associated with disclosure of seropositive status. Neither time since diagnosis, psychological adjustment, AIDS knowledge, nor health status as indicated by CD4 count were related to disclosure. Results also indicated that 51% of the mothers met DSM-IV diagnostic criteria for a psychological disorder in the preceding year. The most common diagnoses included posttraumatic stress disorder and major depression. Analyses suggested that perceived stress accounted for a significant 43% of the variance in psychological adjustment. Neither disclosure, time since diagnosis, nor CD4 count were related to adjustment. Findings are discussed in terms of mothers' mental health needs and provision of services to families affected by HIV/AIDS. Although the results of this study must be considered preliminary due to a limited number of participants and correlational analyses, they point to several avenues for future research.

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.

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.

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.

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.

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.

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.

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.

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.

Quick Fluorescence-EXAFS: an Improved Method for Collection of Conventional XAFS Data, an Improved Method for Collection of Conventional XAFS Data and for Studying Reaction Intermediates in Dilute Systems.

The quick EXAFS (QuEXAFS) technique provides an alternative way of recording X-ray absorption fine-structure (XAFS) data where the scan time is reduced by moving the monochromator at a constant angular speed and recording the data ;on the fly'. Results are presented to show that the use of fluorescence detection with QuEXAFS is eminently suitable for studying reactions in dilute systems such as metalloproteins at a sub-minute time scale. In addition, we show that the fluorescence-QuEXAFS technique can reduce the overall time for normal data collection by some 50% compared with conventional step-by-step scanning EXAFS using the same optical system, thus reducing the total X-ray exposures of the samples. The use of X-rays for studying in situ redox reactions is demonstrated.

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.

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.

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.

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.