Biomedical subjects

K P Murphy

Publications and source records attributed to K P Murphy.

At least 37 records · Page 2Linked to original sources

PubMed1999

Occurrence of adverse reactions to gadolinium-based contrast material and management of patients at increased risk: a survey of the American Society of Neuroradiology Fellowship Directors.

RATIONALE AND OBJECTIVES: The authors attempted to determine the frequency and severity of adverse reactions to gadolinium-based magnetic resonance (MR) contrast agents and to identify strategies for management of patients at increased risk. MATERIALS AND METHODS: American Society of Neuroradiology program directors were surveyed about adverse reactions at their institutions to gadolinium-based contrast agents, the contrast agents responsible, and the management of patients with allergy-like reactions to iodinated or gadolinium-based agents who required MR contrast agent administration. RESULTS: Fifty-three (50.5%) surveys were received from 105 centers. Of 687,255 gadopentetate dimeglumine injections, 314 (0.046%) nonallergic reactions and 107 (0.016%) mild, 28 (0.004%) moderate, and five (0.001%) severe allergy-like reactions occurred. Of 74,275 gadodiamide injections, 11 (0.015%) nonallergic and 12 (0.016%) mild allergy-like reactions occurred. Of 64,005 gadoteridol administrations, 171 (0.267%) nonallergic reactions and 49 (0.077%) mild, 29 (0.047%) moderate, and 11 (0.017%) severe allergy-like reactions occurred. Twenty-six departments took no precautions for patients with previous allergy-like reactions to iodinated contrast material. Nineteen did not premedicate patients who previously had reactions to gadolinium-based agents before repeat administration of MR contrast agents. CONCLUSION: Although MR contrast agents are safe, adverse reactions occur. Many centers have not adopted policies for the OFF

Contrast Media↗

PubMed1999

In vitro and in vivo bioactivity of single-chain interleukin-12.

Interleukin-12 is a heterodimeric cytokine with potent immunoregulatory properties, making it a potential vaccine adjuvant and an immune response modulator. The study of its function is confounded by its heterodimeric structure. In order to facilitate the study of interleukin-12 in both in vitro and in vivo models, we constructed a single-chain porcine interleukin-12 gene and expressed the recombinant protein in Pichia pastoris. Single-chain porcine interleukin-12 was bioactive in vitro on both human and porcine cells as measured by its ability to induce proliferation of lymphoblasts and interferon-gamma secretion by lymph node cells. In contrast, the p40 subunit of porcine interleukin-12 alone did not induce proliferation or inhibit the activity of the single-chain porcine interkeukin-12. The in vivo bioactivity of single-chain porcine interleukin-12 was demonstrated in an oral immunization model where it increased antigen-specific IgA and IgG in jejunal mucus. These results indicate that binding of interleukin-12 to its receptor and transduction of intracellular signals requires both p40 and p35 subunits. The bioactivity of interleukin-12 expressed as a single polypeptide will facilitate its in vivo delivery and study of its structure and function.

Adjuvants, Immunologic↗

PubMed1999

Medical problems in adults with cerebral palsy: case examples.

The United Cerebral Palsy Association estimates that there are approximately one-half million adults with cerebral palsy in the United States. The number may be growing due to advances in medical care and the increased life expectancy of adults in general. Little has been published regarding their medical issues and rehabilitation needs. What little has been published has indicated almost a total neglect of specialty care and preventive medical services. Five case examples are presented to help illustrate particular medical and surgical problems not identified by either pediatric or adult healthcare providers alone. Each individual shows improvement with comprehensive team-oriented specialty care. Further study is encouraged.

Adult↗

PubMed1999

Treating elder neglect: collaboration between a geriatrics assessment team and adult protective services.

Neglect is the most common type of elder maltreatment in the United States. Currently, the only formal intervention available is provided by each state's adult protective service agency (APS). Elder neglect involves a complicated relationship among an indvidual's medical problems, social situation, and ability to function in the environment. Geriatric assessment teams are facile at dealing with such complex cases while APS caseworkers are expert in their ability to identify and confirm neglect. Forming a geriatric team that includes APS caseworkers is a logical and innovative approach to the growing problem of elder neglect.

Adult↗

PubMed1998

Urea effects on protein stability: hydrogen bonding and the hydrophobic effect.

The effects of urea on protein stability have been studied using a model system in which we have determined the energetics of dissolution of a homologous series of cyclic dipeptides into aqueous urea solutions of varying concentration at 25 degrees C using calorimetry. The data support a model in which urea denatures proteins by decreasing the hydrophobic effect and by directly binding to the amide units via hydrogen bonds. The data indicate also that the enthalpy of amide hydrogen bond formation in water is considerably higher than previously estimated. Previous estimates included the contribution of hydrophobic transfer of the alpha-carbon resulting in an overestimate of the binding between urea and the amide unit of the backbone and an underestimate of the binding enthalpy.

Amides↗

PubMed1998

Expression of human interleukin-17 in Pichia pastoris: purification and characterization.

A Pichia pastoris expression clone has been developed to produce the human cytokine interleukin-17 (hIL-17). Characterization of purified recombinant hIL-17 made with this clone demonstrated that it shared many characteristics with hIL-17 produced in mammalian cells. The hIL-17 produced in Pichia had the correct N-terminus of natural mature hIL-17 and a glycosylation pattern similar to hIL-17 produced in mammalian cells; both Pichia and human cells add approximately 5 kDa of sugars via N-linked glycosylation and both express a mixture of the glycosylated and nonglycosylated forms. Gel filtration provides evidence that the Pichia produced hIL-17 exists as a dimer in solution. A combination of cation-exchange and gel-filtration chromatography yielded 3.5 mg of highly purified and biologically active hIL-17 from a 10-liter fermentation. These results show that P. pastoris is a useful system to produce recombinant hIL-17 in structure/function studies of this molecule.

Amidohydrolases↗

PubMed1998

A phase contrast (PC) rephase/dephase sequence of magnetic resonance angiography (MRA): a new technique for imaging distal run-off in the pre-operative evaluation of peripheral vascular disease.

OBJECTIVE: To prospectively compare a 3-D phase contrast (PC) magnetic resonance angiography (MRA) sequence (rephase/dephase) with digital subtraction angiography (DSA) in pre-operative evaluation for femoropopliteal grafting in peripheral vascular disease. Assessment of distal run-off vessels and suggested siting of distal graft anastomosis to the above or below knee segment of the popliteal artery was made. METHODS: Twenty-two lower extremities booked for infra-inguinal vascular reconstruction were imaged pre-operatively using MRA and DSA. A PC rephase/dephase sequence which includes gradient motion refocusing (Siemens 1.0 T Magnetom Impact TR 50 ms, TE 14/14 ms and 15 degree flip angle) and 3-D MIP algorithm reconstruction was used to obtain the MR images. Standard techniques were used to obtain the DSA images, and the mean and median time between imaging modalities was one month. Blinded review by a consultant vascular radiologist scored nine vessel segments for each limb and assessed which popliteal arterial segment would be most suitable for distal anastomosis. RESULTS: The score from DSA and MRA agreed for 155/198 vessel segments (kappa 0.57) and the suggested siting for distal anastomosis agreed for 19/22 limbs (kappa 0.72). Eighteen limbs had surgery as planned (distal anastomosis to the above knee popliteal eight limbs, below knee popliteal 10 limbs). For three limbs the siting of the distal anastomosis suggested by DSA and MRA disagreed. The more accurate modality was proved for one of three limbs and showed MRA to be superior to DSA. CONCLUSION: Three-dimensional PC rephase/dephase MRA is a promising technique which compared well with DSA in the pre-operative assessment of distal run-off for femoropopliteal grafting.

Aged↗

PubMed1998

Prediction of binding energetics from structure using empirical parameterization.

We have presented an empirical method that can be used to predict the binding energetics for protein-protein or protein-peptide interactions from three-dimensional structures. The approach differs from other empirical methods in yielding a thermodynamic description of the binding process, including delta Cp, delta H degree, and delta S degree, rather than predicting delta G degree alone. These thermodynamic terms can provide a wealth of detail about the nature of the interaction, and, if sufficient experimental data are available for comparison, a greater assessment of the accuracy of the calculations. A recurring theme throughout this article is the need for more complete thermodynamic and structural characterizations of protein-ligand interactions. This includes not only characterization of the binding delta H degree, delta S degree, and delta Cp, but a thorough investigation into equilibria linked to binding, such as protonation, ion binding, and conformational changes. Sufficient data will allow parameterization on binding data rather than protein unfolding data. Further inclusion of information obtained from unfolding studies is not likely to generate significant improvement in the accuracy of the calculations. As additional binding data become available, the parameterization can be further extended to include relationships derived from analyses of these data. Not only will this increase accuracy and thus confidence, but allow extension of the method of additional types of interactions.

Empiricism↗

PubMed1998

Mutation of a putative amphipathic alpha-helix in the third intracellular domain of the platelet-activating factor receptor disrupts receptor/G protein coupling and signaling.

Platelet-activating factor (PAF) is a potent phospholipid mediator that interacts with G protein-coupled PAF receptors to elicit diverse physiological and pathophysiological actions. We recently demonstrated that the third intracellular domain of the rat PAF receptor (rPAFR) is a critical determinant in its coupling to phosphoinositide phospholipase C-activating G proteins. Here, we report identification of a putative amphipathic helix in the third intracellular domain of the rPAFR and the effects of mutational disruption of its amphipathic character on G protein coupling of and signaling by the rPAFR. Modeling of the third intracellular domain and adjacent transmembrane regions of the rPAFR identified a single amphipathic helix located in the amino-terminal region of the third intracellular domain of the receptor. Baby hamster kidney cells were transiently transfected with cDNAs encoding the rPAFR or rPAFR mutants in which nonconserved substitutions were made separately in the hydrophobic or polar face of this amphipathic helix. The number and affinity of binding sites for specific PAF receptor antagonist WEB2086 were identical in membranes prepared from rPAFR and amphipathic helix mutant PAFR transfectants. However, only membranes derived from rPAFR transfectants possessed high affinity PAF binding sites that were sensitive to the G protein-uncoupling effects of guanosine-5'-O-(3-thio)triphosphate. These results show that substitutions into either face of the amphipathic helical domain abolished the ability of the rPAFR to undergo coupling to G proteins to form a high affinity agonist/receptor/G protein ternary complex. To examine the effects of these mutations on rPAFR signaling, PAF-stimulated inositol phosphate accumulation was determined in cells transfected with cDNAs encoding the wild-type or amphipathic helix mutant PAFRs. Although PAF stimulated 10-fold increases in inositol phosphate accumulation in rPAFR transfectants, it had no effects on inositol phosphate accumulation in amphipathic helix mutant PAFR transfectants. These results suggest that an amphipathic helix located in the amino-terminal region of the third intracellular domain of the rPAFR is required for its coupling to and activation of G proteins. This study provides the first insight into the structure of the receptor interface for G protein coupling of a PAFR and suggests a conserved role of amphipathic helices in G protein coupling of receptors ranging from those for biogenic amines to the phospholipid mediator PAF.

Amino Acid Sequence↗

PubMed1997

Activation of NMDA receptors is necessary for the induction of associative long-term potentiation in area CA1 of the rat hippocampal slice.

1. It is commonly assumed that the role of the strongly activated heterosynaptic input during the induction of associative long-term potentiation (LTP) is to relieve the magnesium blockade of NMDA receptors located at the weakly stimulated synapses and thereby allow the weak input to undergo potentiation. We tested this assumption by using a caged form of the NMDA receptor antagonist, D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5) to block the activation of NMDA receptors at the weak input in a conditioning protocol for the induction of associative LTP in area CA1 of the rat hippocampal slice. 2. The effect of releasing D-AP5 by flash photolysis of 100 microM caged D-AP5 (N-[1-(2-nitrophenyl)ethoxycarbonyl]-D-AP5) on pharmacologically isolated NMDA receptor-mediated field EPSPs was examined in area CA1. The slope of the EPSP was reduced by 71% within 50 ms of the initiation of the photolytic reaction when the concentration of released D-AP5 had reached 2.0-2.5 microM and was reduced by 95% within 1 min (10 microM D-AP5 released). 3. Associative LTP was induced by pairing a strong tetanus to one input with a weak tetanus (subthreshold for homosynaptic LTP) to a second input. The strong tetanus preceded the weak by 50 ms. Rapid application of D-AP5, by flash photolysis of caged D-AP5, coincident with the last shock of the strong tetanus, resulted in the blockade of NMDA receptor activation during the period of the weak tetanus. Associative LTP was blocked by photolysis of caged D-AP5 but was normally expressed in experiments using caged L-AP5. 4. We conclude that activation of NMDA receptors at the weakly activated input is an essential requirement for synaptically induced associative LTP.

2-Amino-5-phosphonovalerate↗

PubMed1997

Dissecting the energetics of a protein-protein interaction: the binding of ovomucoid third domain to elastase.

An understanding of the structural basis for protein-protein interactions, and molecular recognition in general, requires complete characterization of binding energetics. Not only does this include quantification of the changes that occur in all of the thermodynamic parameters upon binding, including the enthalpy, entropy and heat capacity, but a description of how these changes are modulated by environmental conditions, most notably pH. Here, we have investigated the binding of turkey ovomucoid third domain (OMTKY3), a potent serine protease inhibitor, to the serine protease porcine pancreatic elastase (PPE) using isothermal titration calorimetry and structure-based thermodynamic calculations. We find that near neutral pH the binding energetics are influenced by a shift in the pKa of an ionizable group, most likely histidine 57 in the protease active site. Consequently, the observed binding energetics are strongly dependent upon solution conditions. Through a global analysis, the intrinsic energetics of binding have been determined, as have those associated with the pKa shift. The protonation energetics suggest that the drop in pKa is largely due to desolvation of the histidine residue. The resulting deprotonation is necessary for the enzymatic function of elastase. Intrinsically, at 25 degrees C the binding of OMTKY3 to PPE is characterized by an almost negligible enthalpy change, a large positive entropy change, and a large negative heat capacity change. These parameters are consistent with a model of the OMTKY3-PPE complex, which shows a large and significantly apolar protein-protein interface. Thermodynamic calculations based upon changes that occur in polar and apolar solvent-accessible surface area are in very good agreement with the measured intrinsic binding energetics.

Animals↗

PubMed1997

Tattooing: another adolescent risk behavior warranting health education.

A cross-sectional, convenient sample of adolescents (N = 2101) from 8 states were queried regarding interest in tattooing. Permanent markings and blood-borne diseases were reasons respondents refrain from tattooing, yet 55% (n = 1159) expressed an interest in tattooing. Tattooed adolescents in the sample (10%, n = 213) responded with their experiences. Tattooing was frequently done around the 9th grade and as early as 8 years of age; over half (56%, n = 120) report academic grades of As and Bs. Potential health risks and definite psychosocial findings of purchase and possession risks were evident, building on data from a similar 1994 study by Armstrong and McConnell. Health providers and educators should initiate applicable health education and become community adolescent advocates regarding this risk-taking behavior. Findings indicate that adolescents who want a tattoo will obtain one, regardless of money, regulations, or risks. Adolescents view the tattoos as objects of self-identity and body art, whereas adults perceive the markings as deviant behavior. Informed decision-making could be promoted in health education by incorporating information about the possibility of blood-borne diseases, permanent markings, and themselves as growing and changing people.

Adolescent↗

PubMed1997

CT-guided brachial plexus biopsy.

We describe a technique for conducting a CT-guided biopsy of the brachial plexus region, report two illustrative cases, discuss potential complications, and conclude that, in selected cases, biopsy of lesions in the region of the brachial plexus can be performed safely with CT guidance.

Adenocarcinoma↗

PubMed1996

Structures of active site histidine mutants of IIIGlc, a major signal-transducing protein in Escherichia coli. Effects on the mechanisms of regulation and phosphoryl transfer.

IIIGlc (also called IIAGlc), a major signal-transducing protein in Escherichia coli, is also a phosphorylcarrier in glucose uptake. The crystal and NMR structures of IIIGlc show that His90, the phosphoryl acceptor, adjoins His75 in the active site. Glutamine was substituted for His-, giving H75QIIIGlc and H90QIIIGlc, respectively (Presper, K. A., Wong, C.-Y., Liu, L., Meadow, N. D., and Roseman, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4052-4055), but the mutants showed unexpected properties. H90QIIIGlc loses regulatory functions of IIIGlc, and the phosphoryltransfer rates between HPr/H75QIIIGlc are 200-fold less than HPr/IIIGlc (Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33440-33445). X-ray crystallography, differential scanning calorimetry, and NMR have now been used to determine the structures of the mutants (phospho-H75QIIIGlc was studied by NMR). The three methods gave completely consistent results. Except for the His to Gln substitutions, the only significant structural changes were in a few hydrogen bonds. H90QIIIGlc contains two structured water molecules (to Gln90), which could explain its inability to regulate glycerol kinase. Phospho-IIIGlc contains a chymotrypsin-like, hydrogen bond network (Thr73-His75-O--phosphoryl), whereas phospho-H75QIIIGlc contains only one bond (Gln75-O--phosphoryl). Hydrogen bonds play an essential role in a proposed mechanism for the phosphoryltransfer reaction.

Binding Sites↗

PubMed1996

Normal spatial learning despite regional inhibition of LTP in mice lacking Thy-1.

The process of learning involves stable changes in synaptic efficacy for which long-term potentiation (LTP) provides a widely adopted mammalian model. Synaptic modification induced by learning or LTP may involve the action of cell adhesion molecules. One such candidate is the ubiquitous neuronal glycoprotein Thy-1. In mice in which the gene encoding Thy-1 has been inactivated, we find a regionally selective impairment of LTP in vivo in the hippocampal formation: LTP is normal in area CA1 but strongly inhibited in the dentate gyrus. Spatial learning by Thy-1-deficient mice, as assessed in the watermaze, is unimpaired. Thus LTP in the cortical input to the dentate gyrus seems not to be required for spatial learning.

Animals↗

PubMed1996

Energetics of hydrogen bonding in proteins: a model compound study.

Differences in the energetics of amide-amide and amide-hydroxyl hydrogen bonds in proteins have been explored from the effect of hydroxyl groups on the structure and dissolution energetics of a series of crystalline cyclic dipeptides. The calorimetrically determined energetics are interpreted in light of the crystal structures of the studied compounds. Our results indicate that the amide-amide and amide-hydroxyl hydrogen bonds both provide considerable enthalpic stability, but that the amide-amide hydrogen bond is about twice that of the amide-hydroxyl. Additionally, the interaction of the hydroxyl group with water is seen most readily in its contributions to entropy and heat capacity changes. Surprisingly, the hydroxyl group shows weakly hydrophobic behavior in terms of these contributions. These results can be used to understand the effects of mutations on the stability of globular proteins.

Amides↗

PubMed1996

Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry.

A theoretical development in the evaluation of proton linkage in protein binding reactions by isothermal titration calorimetry (ITC) is presented. For a system in which binding is linked to protonation of an ionizable group on a protein, we show that by performing experiments as a function of pH in buffers with varying ionization enthalpy, one can determine the pK(a)'s of the group responsible for the proton linkage in the free and the liganded states, the protonation enthalpy for this group in these states, as well as the intrinsic energetics for ligand binding (delta H(o), delta S(o), and delta C(p)). Determination of intrinsic energetics in this fashion allows for comparison with energetics calculated empirically from structural information. It is shown that in addition to variation of the ligand binding constant with pH, the observed binding enthalpy and heat capacity change can undergo extreme deviations from their intrinsic values, depending upon pH and buffer conditions.

Calorimetry↗

PubMed1996

Molecular forces involved in force generation during skeletal muscle contraction.

Recent advances in protein chemistry and the kinetic analysis of tension transients in skeletal muscle fibres have enabled us to elucidate the molecular forces involved in force generation by cross-bridges. On the basis of the temperature effect, we conclude that the elementary step that generates force is an endothermic reaction (the enthalpy change delta H degree = 124 kJ mol-1 at 15 degrees C), which accompanies a large entropy increase (delta S degree = 430JK-1 mol-1) and a reduction in the heat capacity (delta C p = -6.4kJ K-1 mol-1). Thus, it can be concluded that the force-generating step is an entropy-driven reaction. The above results suggest that hydrophobic interactions are the primary cause of force generation, and that polar interactions (hydrogen bonding and charge interactions) are involved to a lesser degree. On the basis of the thermodynamic data, we estimate that during force generation approximately 50 nm2 of surface area is involved for hydrophobic interactions and another 30 nm2 for polar interactions. These data suggest that both the actomyosin interaction and the cleft closure of the myosin head are essential for force generation.

Actins↗