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Biomedical subjects

M E Murphy

Publications and source records attributed to M E Murphy.

At least 37 records · Page 2Linked to original sources
PubMed1999

Influence of redox compounds on nitrovasodilator-induced relaxations of rat coronary arteries.

1 Various classes of nitrovasodilators release nitric oxide (NO) through distinct reaction pathways, many of which involve endogenous reductants and/or oxidants. This study examined relaxations of isolated rat coronary arteries induced by spermine NONOate (SPNO), 3-morpholinosydnonimine (SIN-1), nitroprusside (NP), S-nitroso-N-acetylpenicillamine (SNAP) and nitroglycerin (NTG) in order to assess whether their potency was influenced by any of six redox compounds: 1 mM ascorbate, 1 mM dehydroascorbate, 0.1 mM dithiothreitol, 10 microM diamide, 0.1 mM ferrocyanide, and 0.1 mM ferricyanide. 2 Only SPNO spontaneously generated NO at measurable levels. These levels were decreased by the presence of ascorbate and dithiothreitol, which likewise decreased the potency of SPNO. 3 The potency of SIN-1 was unaffected by any redox compound except ferricyanide, which increased the potency not only of SIN-1, but also of other nitrovasodilators and NO-independent vasodilators. 4 The potency of NP was decreased by two structurally similar multivalent anions, ferrocyanide and ferricyanide, suggesting that NP metabolism requires ionic binding to tissue. 5 SNAP lost its potency in solutions containing ascorbate or dehydroascorbate. SNAP potency was also decreased by the glutathione oxidant, diamide, and by ferrocyanide and ferricyanide, suggesting that glutathione and ionic binding may be required for NO release. 6 NTG appeared to relax arteries via two pathways. One required only low concentrations of NTG and a labile endogenous factor that was preserved by dithiothreitol and eliminated by ferricyanide. A distinct second pathway required higher concentrations of NTG. 7 These distinct attributes of nitrovasodilator metabolism may underlie differences in regional specificity or tolerance development, and therefore might eventually be exploited in the development and use of nitrovasodilators.

Animals↗
PubMed1999

Reducing hospital inpatient lengths of stay.

This article describes the importance of hospital length of stay as an indicator of health care efficiency and provides guidance concerning the development of data for length of stay reduction. It identifies variables involved in length of stay evaluation including the mean stay, median stay, and length of stay standard deviation. It describes how consistent length of stay data can be generated and analyzed for local populations and benchmark communities.

Benchmarking↗
PubMed1999

Using data to reduce hospital readmissions.

This article describes the importance and the development of data concerning hospital readmissions as an outcomes indicator. It emphasizes the need for consistent definition of readmissions according to time intervals and diagnostic categories. It describes the development of readmission information using computer abstract databases to ensure consistency of indicators. It also provides examples of data developed through this approach.

Data Interpretation, Statistical↗
PubMed1999

Vasoactive effects of potassium in kidneys of hypertensive rats fed a high-potassium diet.

DESIGN AND METHODS: Levels of dietary and serum potassium are thought to correlate inversely with vascular resistance and blood pressure. This study examined renal vascular resistance in perfused rat kidneys partially preconstricted with 10 micromol/ phenylephrine, quantifying changes in the resistance when levels of potassium in the perfusate ([K+]o) were varied between 2 and 80 mmol/l. RESULTS: In kidneys from 17-week-old Wistar-Kyoto rats (WKY strain) fed a normal diet (American Institute of Nutrition AIN-76 diet), the resistance decreased when [K+]o was raised from 4 to 6-20 mmol/l, whereas resistance increased when [K+]o was either lowered to 2 mmol/l or raised above 25 mmol/l. The vasodilation that occurred at 13 mmol/l [K+]o was blocked by 100 micromol/l BaCl2 and 10 micromol/l ouabain in an additive manner, suggesting that both the inward rectifier K+ channel and the Na-K-ATPase underlie the dilation. Kidneys from spontaneously hypertensive rats (SHR strain) fed the AIN-76 diet displayed modestly enhanced vasodilations and vasoconstrictions as compared to WKY. A high-potassium diet (AIN-76 supplemented with 3.5% potassium citrate, provided for 8 weeks) led to exaggerated vasoconstrictive effects of [K+]o, and modestly enhanced vasodilations, in WKY rats. In contrast, the diet led to attenuated vasoconstrictions, and dramatically enhanced vasodilations, in the SHR strain. The diet did not affect the blood pressure increase or weight gain of either strain. CONCLUSIONS: Changes in the responsiveness of blood vessels to extracellular potassium might underlie some beneficial effects of high-potassium diets in hypertensive individuals.

Animals↗
PubMed1999

Ascorbate and dehydroascorbate modulate nitric oxide-induced vasodilations of rat coronary arteries.

Soluble guanylyl cyclase (GC) is a heme-containing protein that is a predominant target of nitric oxide (NO). This study examined whether the reductant, ascorbate (ASC), the oxidant, dehydroascorbate (DHAA), or other redox agents modulated the sensitivity of isolated rat coronary arteries to NO-induced vasodilations. Based on NO measurements with a NO-sensitive electrode, NO dilated the arteries with a pEC50 of 8.24 +/- 0.05. The potency of NO was significantly enhanced in the presence of ASC (pEC50 = 8.70 +/- 0.02) but was diminished in the presence of DHAA (pEC50 = 7.91 +/- 0.15). The potency of NO was not affected by other redox agents including dithiothreitol, beta-mercaptoethanol, diamide, 1-chloro-2,4-dinitrobenzene, ferricyanide, or ferrocyanide. Experiments involving the cyclic guanosine monophosphate (cGMP) analog, 8-Br-cGMP, and the phosphodiesterase inhibitor, isobutyl-methylxanthine, indicated that neither ASC nor DHAA has an effect on the degradation or potency of cGMP. ASC and DHAA also failed to affect vasodilations induced by diltiazem or forskolin. However, ASC and DHAA affected the potency of NO in human mesenteric arteries. The results are consistent with other evidence that ASC and DHAA affect the redox state of GC, and through this modulate arterial sensitivity to NO. This suggests that the regulation of the redox state of GC may be an additional site of modulation of the NO/cGMP pathway.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Correlation between renal vascular resistance, pulse pressure, and the resistive index in isolated perfused rabbit kidneys.

PURPOSE: To assess the effect of acute changes in renal vascular resistance (RVR) and pulse pressure on the resistive index (RI) measured by using Doppler ultrasonography (US). MATERIALS AND METHODS: Rabbit kidneys were perfused by using a pulsatile perfusion system in which RVR, systolic and diastolic pulse pressures, and pulse kinetics were controlled and monitored while simultaneously measuring the RI. RESULTS: When RVR was increased fivefold with phenylephrine hydrochloride, the RI increased only slightly (from 0.45 at baseline up to 0.50). There was a virtually linear relationship between the RI and the pulse pressure index ([systolic pressure-diastolic pressure]/systolic pressure) in the range of 0.30-0.80. The RI was not affected by the pulse rate or fraction of time that systolic pressure was applied during the pulse cycle. CONCLUSION: Contrary to conventional teaching, which is based on theoretic considerations, the RI is not readily affected by acute changes in RVR. This indicates a need to reconsider the conventional explanations used to explain increases in RI that are frequently found in patients with renal disease or ureteral obstruction.

Animals↗
PubMed1998

1.8 A crystal structure of the major NAD(P)H:FMN oxidoreductase of a bioluminescent bacterium, Vibrio fischeri: overall structure, cofactor and substrate-analog binding, and comparison with related flavoproteins.

We have solved the crystal structure of FRase I, the major NAD(P)H:FMN oxidoreductase of Vibrio fischeri, by the multiple isomorphous replacement method (MIR) at 1.8 A resolution with the conventional R factor of 0.187. The crystal structure of FRase I complexed with its competitive inhibitor, dicoumarol, has also been solved at 2.2 A resolution with the conventional R factor of 0.161. FRase I is a homodimer, having one FMN cofactor per subunit, which is situated at the interface of two subunits. The overall fold can be divided into two domains; 80% of the residues form a rigid core and the remaining, a small flexible domain. The overall core folding is similar to those of an NADPH-dependent flavin reductase of Vibrio harveyi (FRP) and the NADH oxidase of Thermus thermophilus (NOX) in spite of the very low identity in amino acid sequences (10% with FRP and 21% with NOX). 56% of alpha-carbons of FRase I core residues could be superposed onto NOX counterparts with an r.m.s. distance of 1.2 A. The remaining residues have relatively high B-values and may be essential for defining the substrate specificity. Indeed, one of them, Phe124, was found to participate in the binding of dicoumarol through stacking to one of the rings of dicoumarol. Upon binding of dicoumarol, most of the exposed re-face of the FMN cofactor is buried, which is consistent with the ping pong bi bi catalytic mechanism.

Amino Acid Sequence↗
PubMed1998

Two high-resolution crystal structures of the recombinant N-lobe of human transferrin reveal a structural change implicated in iron release.

The N-lobe of human serum transferrin (hTF/2N) has been expressed in baby hamster kidney cells and crystallized in both orthorhombic (P212121) and tetragonal (P41212) space groups. Both crystal forms diffract to high resolution (1.6 and 1.8 A, respectively) and have been solved by molecular replacement. Subsequent refinement resulted in final models for the structure of hTF/2N that had crystallographic R-factors of 18.1 and 19.7% for the two crystal forms, respectively; these models represent the highest-resolution transferrin structures determined to date. The hTF/2N polypeptide has a folding pattern similar to those of other transferrins, including the presence of a deep cleft that contains the metal-binding site. In contrast to other transferrins, both crystal forms of hTF/2N display disorder at the iron-binding site; model building suggests that this disorder consists of alternative conformations of the synergistically bound carbonate anion, the side chain for Arg-124, and several solvent molecules. Subsequent refinement revealed that conformation A has an occupancy of 0.63-0. 65 and corresponds to the structure of the iron-binding site found in other transferrins. The alternative conformation B has an occupancy of 0.35-0.37; in this structure, the carbonate has rotated 30 degrees relative to the iron and the side chain for Arg-124 has moved to accommodate the new carbonate position. Several water molecules appear to stabilize the carbonate anion in the two conformations. These structures are consistent with the protonation of the carbonate and resulting partial removal of the anion from the metal; these events would occur prior to cleft opening and metal release.

Animals↗
PubMed1998

Relationship between amine-carboxyboranes and TNF alpha for the regulation of cell growth in different tumor cell lines.

The amine-carboxyboranes were shown to be synergistic with tumor necrosis factor alpha (TNF alpha) in cytotoxicity and inhibition of DNA synthesis in select types of cancer cells depending on the presence of a TNF alpha high affinity receptor on the membrane of the cell. Initially both TNF alpha and the amine-carboxyboranes reduce the influx of calcium but later cause a significant increase intracellularly. This influx is not linked with the amine-carboxyborane activating the calcitonin receptor in the tumor cells. Neither the agents nor TNF alpha directly inhibits DNA topoisomerase II activity but both did cause decreased phosphorylation of the enzyme by protein kinase C (PKC). The two agents caused synergistic inhibition. This event correlated with increased DNA protein linked breaks, DNA fragmentation and cell death. These protein linked breaks are additive with etoposide's effects but the latter agent's mechanism is different than phosphorylation of topoisomerase II. There was no evidence that the DNA fragmentation was caused by a calcium induced endonuclease enzyme in these cancer cells. The low-molecular weight amine-carboxyboranes appear to play an identical function as TNF alpha in its role to cause DNA breaks and fragmentation to cause apoptosis.

Animals↗
PubMed1998

Consequences of factor IX mutations in 26 families with haemophilia B.

Haemophilia B is due to a variety of mutations within the factor IX gene. In the Seattle series, 26 additional unrelated families have had a mutation identified within the past 2 years. Of these, 11 were common recurrent point mutations identifiable by rapid restriction digest screening; eight of these probably represent founder mutations. 15 others were identified by sequencing amplified coding region fragments; eight are novel. Two each had frameshift and donor splice mutations and 11 had missense mutations. Five of these mutations associated with normal levels of circulating dysfunctional factor IX were computer modelled into coordinates for factor IXa.

Factor IX↗
PubMed1998

A domain mutations in 65 haemophilia A families and molecular modelling of dysfunctional factor VIII proteins.

A variety of mutations are found in haemophilia A families. Those with circulating, dysfunctional protein can provide insights into structural determinants of factor VIII function. A molecular model based upon the crystal structure of the homologous A domains in caeruloplasmin enables predictions of molecular consequences of mutations. To identify haemophilic mutations in coding regions for three A domains of factor VIII and predict amino acid substitutions important for coagulant cofactor function, amplified DNA fragments from 188 unrelated haemophilia A families were screened for heteroduplex formation. Exons 1-19 were examined. 65 families were positive for 58 distinct mutations (39 novel) on DNA sequencing. 12 were non-missense mutations. 38 missense mutations were found in patients that circulate or potentially circulate dysfunctional factor VIII protein and are in an A domain molecular model. Of these 38, 12 have identical residues among all known species of factors V, VIII and caeruloplasmin. These 38 mutations have been localized onto a factor VIII A domain molecular model. Of these, 19 are in coiled, 15 in beta-pleated sheet, and two each in turns and alpha-helical structures. 15 substituted residues are on the surface, nine are partially on the surface and 14 are buried within the model structure. Mutant side-chain substitutions were inserted to predict changes in surface groups or, for buried residues, potential surface areas whose structure is probably disrupted by the substitution.

Amino Acid Substitution↗
PubMed1998

Influence of contrast media on the response of rat renal arteries to endothelin and nitric oxide: influence of contrast media.

RATIONALE AND OBJECTIVES: Contrast media (CM) such as diatrizoate meglumine (DTZ) or iohexol can cause renal vasoconstriction in vivo, and this may initiate CM-induced nephropathy. Endothelin-1 (ET-1), a vasoconstrictor, and nitric oxide, a vasodilator, are key modulators of renal circulation. We tested the hypothesis that CM enhances arterial responses to ET-1, or diminishes responses to nitric oxide. METHODS: A video dimension analyzer continuously recorded changes in diameter of isolated, pressurized rat interlobar renal arteries (200-400 microm diameter) superfused with combinations of CM, ET-1, nitric oxide, and other vasoactive agents. RESULTS: Superfusion of arteries with 3.3% DTZ, but not with 3.3% iohexol, enhanced their sensitivity to ET-1 by approximately twofold, as assessed by shifts in concentration-response curves. Both DTZ and iohexol decreased the sensitivity of arteries to nitric oxide by approximately threefold. Neither DTZ nor iohexol affected arterial sensitivity to other vasoconstrictors (phenylephrine, potassium) or vasodilators (forskolin, diltiazem). CONCLUSIONS: Diatrizoate meglumine and iohexol may induce or augment renal vasoconstriction in part by causing selective alterations in arterial sensitivity to ET-1 and to nitric oxide.

Analysis of Variance↗
PubMed1998

Impact of a diabetes electronic management system on the care of patients seen in a subspecialty diabetes clinic.

OBJECTIVE: To compare the compliance with diabetes care performance indicators by diabetes specialists using a diabetes electronic management system (DEMS) and by those using the traditional paper medical record. RESEARCH DESIGN AND METHODS: A DEMS has been gradually introduced into our subspecialty practice for diabetes care. To assess the value of this DEMS as a disease management tool, we completed a retrospective review of the medical records of 82 randomly selected patients attending a subspecialty diabetes clinic (DC) during the first quarter of 1996. Eligible patients were defined by the suggested criteria from the American Diabetes Association Provider Recognition Program. During the first quarter of 1996, approximately one half of the providers began using the DEMS for some but not all of their patient encounters. Neither abstractors nor providers were aware of the intent to examine performance in relationship to use of the DEMS. RESULTS: Several measures were positively influenced when providers used the DEMS. The number of foot examinations, the number of blood pressure readings, and a weighted criterion score were greater (P < 0.01) for providers using the DEMS. There was evidence, although not statistically significant, for lower mean diastolic blood pressures (P = 0.043) in patients and for number of glycated hemoglobins documented (P = 0.018) by users of the DEMS. CONCLUSIONS: Performance and documentation of the process of care for patients with diabetes in a subspecialty clinic are greater with the use of a DEMS than with the traditional paper record.

Adult↗
PubMed1997

Structure of nitrite bound to copper-containing nitrite reductase from Alcaligenes faecalis. Mechanistic implications.

The structures of oxidized, reduced, nitrite-soaked oxidized and nitrite-soaked reduced nitrite reductase from Alcaligenes faecalis have been determined at 1.8-2.0 A resolution using data collected at -160 degrees C. The active site at cryogenic temperature, as at room temperature, contains a tetrahedral type II copper site liganded by three histidines and a water molecule. The solvent site is empty when crystals are reduced with ascorbate. A fully occupied oxygen-coordinate nitrite occupies the solvent site in crystals soaked in nitrite. Ascorbate-reduced crystals soaked in a glycerol-methanol solution and nitrite at -40 degrees C remain colorless at -160 degrees C but turn amber-brown when warmed, suggesting that NO is released. Nitrite is found at one-half occupancy. Five new solvent sites in the oxidized nitrite bound form exhibit defined but different occupancies in the other three forms. These results support a previously proposed mechanism by which nitrite is bound primarily by a single oxygen atom that is protonable, and after reduction and cleavage of that N-O bond, NO is released leaving the oxygen atom bound to the Cu site as hydroxide or water.

Alcaligenes↗
PubMed1997

Loss of tolerance to exogenous and endogenous factor VIII in a mild hemophilia A patient with an Arg593 to Cys mutation.

A 42-year-old patient with mild hemophilia A developed spontaneous muscle hematomas 1 month after intense therapy with factor VIII concentrates. Factor VIII clotting activity was less than 1% and his factor VIII inhibitor was 10 Bethesda units (BU)/mL. The titer peaked at 128 BU despite daily infusions of factor VIII; 1 year later, the titer was 13 BU with no spontaneous bleeding for 4 months. The plasma inhibitor was 95% neutralized by factor VIII A2 domain but less than 15% neutralized by light-chain or C2 domain. His inhibitor did not cross-react with porcine factor VIII and was at least 10-fold less reactive to a series of hybrid factor VIII proteins in which human residues 484-508 are replaced by the homologous porcine sequence (Healey et al, J Biol Chem 270:14505, 1995). The inhibitor patient's DNA encoding his A2 domain and flanking sequences showed a C-T transition predicting Arg593 to Cys. Thirteen patients from 5 unrelated families with Cys593 have not developed inhibitors. Factor VIII clotting activity from one of them was inhibited similarly to diluted normal plasma by inhibitor patient plasma. In an homologous structure, ceruloplasmin (Zaitseva et al, J Biol Inorgan Chem 1:15, 1996), the residue equivalent to Arg593, is in a loop distinct from residues 484-508. On solution phase immunoprecipitation with labeled factor VIII fragments, A2, light chain, and C2 domains bound. In contrast to typical immune responses to factor VIII in patients with severe hemophilia A, this patient's inhibitor was almost entirely reactive with common epitopes within the A2 domain whereas by more sensitive immunoprecipitation testing antibodies to light chain epitopes were also present. Accordingly, immune responsiveness to exogenous factor VIII (antigen burden) appears to be more critical than his endogenous, hemophilic factor VIII to his developing high-titer anti-factor VIII antibodies and loss of tolerance to both native and hemophilic factor VIII proteins.

Adult↗
PubMed1997

Structural comparison of cupredoxin domains: domain recycling to construct proteins with novel functions.

The three-dimensional structures of the copper-containing enzymes ascorbate oxidase, ceruloplasmin, and nitrite reductase, comprised of multiple domains with a cupredoxin fold, are consistent with having evolved from a common ancestor. The presence or absence of copper sites has complicated ascertaining the structural and evolutionary relationship among these and related proteins. Simultaneous structural superposition of the enzyme domains and their known cupredoxin relatives shows clearly that there are at least six cupredoxin classes, and that the evolution of the conserved core of these domains is independent of the presence or absence of copper sites. Relationships among the variable loops in these structures show that the two-domain ancestor of the blue oxidases contained a trinuclear-copper interface but could not have functioned in a monomeric state. Comparison of the sequence of the copper-containing, iron-regulating protein. Ferrous transport (Fet3) from yeast to the structurally defined core and loop residues of the cupredoxins suggests specific residues that could be involved in the ferroxidase activity of Fet3.

Amino Acid Sequence↗
PubMed1997

Naphthalene cytotoxicity of differentiating Clara cells in neonatal mice.

Selective Clara cell injury produced by many bioactivated lung toxicants is thought to result from high levels of activating enzymes found in differentiated Clara cells. A recent study found an elevated susceptibility to the Clara cell toxicant 4-ipomeanol in neonatal rabbits when Clara cell P450 activity is low. To determine whether differentiating Clara cells in another species (mouse) are more susceptible to injury by a different bioactivated Clara cell toxicant (naphthalene), adult, 14-day postnatal (DPN) and 7DPN male mice were given a single intraperitoneal dose (25, 50, or 100 mg/kg) of naphthalene and killed 24 hr later. Epithelial damage, as assessed by quantitative histopathology, included cellular swelling, vacuolization, and exfoliation. In 7DPN mice, bronchiolar epithelium was severely injured at the lowest dose of naphthalene tested, 25 mg/kg. Bronchiolar epithelium in 14DPN mice was moderately injured at 25 mg/kg; injury severity was greatest at 50 and 100 mg/kg. Minimal bronchiolar epithelial injury occurred in adult mice at 50 mg/kg and moderate injury at 100 mg/kg. In proximal bronchi, epithelium of 7DPN mice showed signs of injury only at 100 mg/kg. Bronchial epithelium of adult mice was not injured at any dose. Isolated distal airways from 7DPN and 14DPN mice were more sensitive to naphthalene exposure than isolated distal airways from adult mice. Despite the low levels of P450 activity, differentiating Clara cells in neonatal mice are more susceptible to injury by the bioactivated cytotoxicant naphthalene than are differentiated Clara cells in adult mice.

Animals↗