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Biomedical subjects

W Parris

Publications and source records attributed to W Parris.

16 recordsLinked to original sources
PubMed2001

Painless electrodiagnostic current perception threshold and pain tolerance threshold values in CRPS subjects and healthy controls: a multicenter study.

The purpose of this study is to evaluate both painless and painful sensory transmission in patients with Complex Regional Pain Syndrome (CRPS) using the automated electrodiagnostic sensory Nerve Conduction Threshold (sNCT) test. This test generates reliable, painless Current Perception Threshold (CPT) and atraumatic Pain Tolerance Threshold (PTT) measures. Standardized CPT and PTT measures using constant alternating current sinusoid waveform stimulus at 3 different frequencies 5 Hz, 250 Hz, and 2 kHz (Neurometer CPT/C Neurotron, Inc. Baltimore, MD) were obtained from CRPS subjects at a distal phalange of the affected extremity and at an ipsilateral asymptomatic control site. Matched sites were tested on healthy subjects. Detection sensitivities for an abnormal PTT and CPT test were calculated based on specificity of 90% as determined from data obtained from healthy controls. A Spearman rank correlation was used to test for a significant association between presence of allodynia and an abnormal PTT or CPT at any frequency tested. Thirty-six CRPS subjects and 57 healthy controls were tested. The highest detection sensitivity of the PTT test from symptomatic test sites was 63% for the finger and 71% for the toe. PTT abnormalities were also detected, to a lesser degree, at the asymptomatic control site (41% finger control site, 16% toe control site). The highest CPT detection sensitivity at the symptomatic site was 37% for the finger site and 53% for the toe site. CPT abnormalities were also detected at the asymptomatic control site (29% finger control site, 37% toe control site). Eighty-six percent of the CRPS subjects had either a PTT or CPT abnormality at any frequency at the symptomatic site. There was a significant correlation between presence of allodynia and presence of an abnormal CPT and PTT, respectively (P < .01). The correlation coefficient was lower for CPT than for PTT, ie, 0.34 versus 0.6 for the finger and 0.48 versus 0.67 for the toe, respectively. In studied CRPS patients an abnormal PTT was detected with higher sensitivity than an abnormal CPT. Assessing PTT may become a useful electrodiagnostic quantitative sensory test for diagnosing and following the course of neuropathic pain conditions.

Journal Article↗
PubMed1997

Do triage nurse-initiated X-rays for limb injuries reduce patient transit time?

Patients with isolated limb injuries are often required to wait a long time for treatment and investigation in emergency departments. It was hypothesized that allowing triage nurses to initiate X-rays would reduce transit times for these patients. A prospective, randomized comparison trial of 175 patients was conducted, comparing transit times between a group of patients who had X-rays initiated at triage and a group which did not. No statistically significant reduction in transit time was demonstrated by this change in practice, either for a group who had sustained fractures or for one which had not. Despite this finding, staff and patient satisfaction with this change in procedure was high. This justifies continuation of the practice and further research.

Adolescent↗
PubMed1995

The in vitro translocase activity of lambda terminase and its subunits. Kinetic and biochemical analysis.

The terminase holoenzyme of bacteriophage lambda is a multifunctional protein composed of two subunits, gpNu1 and gpA. In vitro, under certain conditions, terminase can render DNAs from various sources, of varying lengths and termini, resistant to degradation by high concentrations of DNase I. This reaction is completely dependent on the presence of terminase, proheads, a hydrolyzable triphosphate, and a divalent metal ion, and we propose that it is the result of translocation of DNA into proheads by terminase. This reaction is stoichiometric with respect to terminase, DNA, and proheads and can be supported by all deoxyribo- and ribonucleoside triphosphates, but not by the corresponding diphosphates or nonhydrolyzable ATP analogs. Mg2+ and Ca2+ promote the reaction, but Mn2+ and Zn2+ do not. In the absence of spermidine, translocase activity is low, but addition of the Escherichia coli protein integration host factor (IHF) promotes specific translocation of only those DNA fragments containing the terminase-binding site, cosB. When spermidine is present, nonspecific translocation of DNA from any source is stimulated. Under these conditions IHF no longer promotes specificity, but translocation of only cosB-containing DNA fragments can be restored by addition of small amounts of a dialyzed and RNase-treated E. coli extract, suggesting that additional host factor(s) may be involved in determination of packaging specificity. To a limited extent, gpA alone can promote translocation, but gpNu1, which has no translocase activity on its own, must be added to approach the holoenzyme-like activity levels. Formation of viable phage cannot be accomplished by gpA in the absence of gpNu1.

Animals↗
PubMed1994

A new procedure for the purification of the bacteriophage lambda terminase enzyme and its subunits. Properties of gene product A, the large subunit.

New methods for the purification of highly active bacteriophage lambda terminase holoenzyme, and its individual subunits, gene products (gp) A and gpNu1, have been developed. These methods are rapid, simple, reproducible, and give high yields of unaggregated protein from small volumes of culture. The procedures involve fractionation of extracts of Escherichia coli strains harboring plasmids engineered to overproduce the respective proteins. All purified proteins exist as monomers or dimers at moderate concentrations. At concentrations where holoenzyme efficiently promotes in vitro cosN-cleavage and lambda DNA packaging, gpA displays neither of these activities unless supplemented with gpNu1 and the E. coli protein integration host factor. At high protein concentrations, however, gpA can promote cos-cleavage by itself. Although gpNu1 itself cannot promote either cosN-cleavage or DNA packaging, it does modulate these activities of gpA. GpA is a DNA-stimulated ATPase whose catalytic parameters closely resemble those of the holoenzyme. Like the holoenzyme, gpA displays a DNA helicase activity which is able to melt the annealed cosN overhangs. Certain preparations of gpA appear to undergo a time-dependent amino-terminal clipping at discrete sites even in the presence of as many as four protease inhibitors and at low temperature.

Adenosine Triphosphatases↗
PubMed1994

The in vitro endonuclease activity of gene product A, the large subunit of the bacteriophage lambda terminase, and its relationship to the endonuclease activity of the holoenzyme.

The reaction requirements and kinetic properties of the in vitro endonuclease activity of the bacteriophage lambda terminase and its large subunit, gene product (gp) A, have been analyzed. Optimal cleavage reaction activity for both proteins requires Mg2+, a pH between 8.5 and 9.0, and is enhanced by ATP or ATP analogs. Under these conditions both terminase and gpA generate aberrant nicks in and around cosN. Optimal nicking specificity of terminase is observed under conditions of 50-100 mM salt, 5 mM spermidine, 1.5 mM ATP, and a pH between 7.0 and 7.5. Specific activity of terminase is greatly reduced under these conditions, and gpA is completely inactive at all protein concentrations tested. Under optimal reaction conditions, gpA endonuclease activity differs from that of the holoenzyme in that it can only be detected at high concentrations, is strongly protein concentration-dependent, and can not be stimulated by the Escherichia coli protein integration host factor. Addition of purified gpNu1 partially, but not completely, minimized these differences, suggesting that the role of gpNu1 in the holoenzyme is to modulate the basal endonuclease of gpA.

Bacteriophage lambda↗
PubMed1994

The in vitro ATPases of bacteriophage lambda terminase and its large subunit, gene product A. The relationship with their DNA helicase and packaging activities.

The bacteriophage lambda terminase is composed of two subunits, gpNu1 and gpA. In vitro, the holoenzyme is a site-specific endonuclease, helicase, ATPase, and can package lambda DNA into proheads. gpA possesses ATPase and helicase activities which are similar to those of the holoenzyme. Both terminase and gpA can hydrolyze a wide range of deoxyribo- and ribonucleoside triphosphates to inorganic phosphate and the corresponding diphosphate. Nucleoside diphosphates are not substrates for either protein. ATPase of both proteins is stimulated by double-stranded DNA. The ATPase of gpA is protein concentration-dependent, while that of terminase is not. Helicase activity of both proteins is not concentration-dependent, and requires a hydrolyzable triphosphate. ATP, dATP, and GTP supported helicase activity, while adenosine 5'-(beta, gamma-methylene)triphosphate, adenosine 5'-3-O-(thio)triphosphate, ADP, CTP, and UTP did not. The kinetic parameters of ATPase and helicase activities were similar for both proteins, but packaging with terminase was optimal only at a significantly higher level of ATP. Packaging was detectable at significant levels with CTP and UTP, but not with GTP. Packaging also differed from ATPase and helicase in the utilization of divalent metal cations and susceptibility to various inhibitors.

Adenosine Triphosphatases↗
PubMed1993

Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding.

Threonine 246 is an active site residue that is conserved in all known L-lactate dehydrogenase (LDH; EC 1.1.1.27) sequences. In order to investigate the role of Thr246 in Bacillus stearothermophilus LDH, this residue was altered by site-directed mutagenesis to valine, alanine, leucine, and serine, respectively. The effects of these mutations, as observed in both steady-state and single-turnover kinetic measurements with different substrates, demonstrated the importance for catalysis of a hydroxyl group in the 246 amino acid residue. In contrast, no significant contribution of the OH group of Thr246 to productive pyruvate binding was observed. Instead, it is proposed that the role of Thr246 may be to facilitate hydride transfer from the nicotinamide ring of the NADH cofactor to the pyruvate carbonyl group.

Base Sequence↗
PubMed1992

THR246 mutations decrease substrate inhibition in lactate dehydrogenase.

Threonine 246 in Bacillus stearothermophilus L-lactate dehydrogenase has been changed to valine, serine, and alanine by site-directed mutagenesis. Kinetic analyses show a decrease in substrate inhibition for pyruvate reduction with the T246S mutant and virtual elimination of substrate inhibition for the T246A and T246V mutants. The results indicate that the absence of substrate inhibition in the 246A/V-catalyzed reactions is due to the elimination of a key hydrogen bond between the hydroxyl group of threonine and pyruvate in the wild-type complex that is an important contributor in the formation of the abortive enzyme-NAD(+)-pyruvate complex responsible for substrate inhibition.

Escherichia coli↗
PubMed1992

Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.

Bacterial Proteins↗
PubMed1992

Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase.

Lactate dehydrogenases are of considerable interest as stereospecific catalysts in the chemical preparation of enantiomerically pure alpha-hydroxyacid synthons. For such applications in synthetic organic chemistry it would be desirable to have enzymes which tolerate elevated temperatures for prolonged reaction times, to increase productivity and to extend their applicability to poor substrates. Here, two examples are reported of significant thermostabilizations, induced by site-directed mutagenesis, of an already thermostable protein, the L-lactate dehydrogenase (EC 1.1.1.27, 35 kDa per monomer subunit) from Bacillus stearothermophilus. Thermal inactivation of this enzyme is accompanied by irreversible unfolding of the native protein structure. The replacement of Arg171 by Tyr stabilizes the enzyme against thermal inactivation and unfolding. This stabilizing effect appears to be based on improved interactions between the subunits in the core of the active dimeric or tetrameric forms of the enzyme. The thermal stability of L-lactate dehydrogenase variants with an active site Arg residue, either in the 171 (wild-type) or in the 102 position, is further increased by sulfate ions. The two stabilizing effects are additive, as found for the Arg171Tyr/Gln102Arg double mutant, for which the stability of the protein in 100 mM sulfate solution reaches that of L-lactate dehydrogenases from extreme thermophiles. All mutant proteins retain significant catalytic activity, both in the presence and absence of stabilizing salts, and are viable catalysts in preparative scale reactions.

Calorimetry↗
PubMed1990

Substance P, acetylcholinesterase, and beta-endorphin levels in the plasma and pericardial fluid of patients with and without angina pectoris.

We measured substance P-like immunoreactivity (SPLI), beta-endorphin-like immunoreactivity (BELI), acetylcholinesterase activity, and total protein content in pericardial fluid and plasma of patients with angina pectoris and patients with no angina pectoris. SPLI and BELI levels, acetylcholinesterase activity, and total protein content were determined by radioimmunoassay, a colorimetric method, and by the method of Lowry et al. (J Biol Chem 1951; 193:265-75), respectively. In the pericardial fluid, patients with angina had SPLI, BELI, acetylcholinesterase, and total protein values of 1.69 +/- 0.23 fmol/mg protein, 0.16 +/- 0.13 fmol/mg protein, 0.06 +/- 0.02 units, and 25.7 +/- 3.2 mg/ml, respectively. Patients with no angina had SPLI, BELI, acetylcholinesterase, and total protein values of 0.93 +/- 0.17 fmol/mg protein, 0.19 +/- 0.10 fmol/mg protein, 0.16 +/- 0.02 units, and 44.6 +/- 5.3 mg/ml, respectively. SPLI levels were significantly higher (p less than 0.03), and acetylcholinesterase (less than 0.002) and total protein content (less than 0.004) were significantly lower in the pericardial fluid of patients with angina when compared with those of patients with no angina. BELI levels were not significantly different between the two groups. In the plasma, no significant differences were found in SPLI, BELI, acetylcholinesterase, and total protein values between the two groups of patients. Patients with angina had SPLI, BELI, acetylcholinesterase, and total protein values of 0.47 +/- 0.26 fmol/mg protein, 0.06 +/- 0.06 fmol/mg protein, 0.29 +/- 0.15 units, and 68.2 +/- 8.7 mg/ml, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1988

The Nu1 subunit of bacteriophage lambda terminase.

The maturation and packaging of bacteriophage lambda DNA are catalyzed by the phage terminase enzyme. Terminase is composed of two protein subunits, gpNu1 and gpA. The holoenzyme is multifunctional in vitro; it binds to and cleaves lambda DNA at the cos site (where cos represents cohesive-end site), packages DNA into lambda proheads, and is also a DNA-dependent ATPase. The genes of the two subunits have been cloned separately into powerful expression vectors which allow for very high levels of protein overproduction. The gpNu1 protein has been purified to homogeneity and has a monomeric molecular weight of 21,200, in close agreement with the Mr of 20,444 expected from its amino acid sequence. Both gel filtration and sedimentation velocity centrifugation indicate that the native gpNu1 protein exists as a Mr greater than 500,000 aggregate. The sequence of the first 20 amino acids and the overall composition both match those predicted by the nucleotide sequence of the Nu1 gene. Purified gpNu1 is able to complement gpA-containing extracts in both lambda DNA packaging and cos cleavage assays. The Nu1 gene amino acid sequence predicts DNA binding by the protein, and gpNu1 does show specific binding to lambda DNA by filter binding assays. Also, as predicted from its sequence, gpNu1 exhibits ATPase activity; but in contrast to the holoenzyme, this activity is DNA-independent.

Amino Acids↗
PubMed1988

Terminase host factor: a histone-like E. coli protein which can bind to the cos region of bacteriophage lambda DNA.

Terminase Host Factor (THF), an E. coli protein capable of fulfilling the host factor requirement for in vitro bacteriophage lambda terminase activity, displays properties characteristic of the prokaryotic type II DNA-binding or "histone-like" proteins. It is a 22 K basic, heat- and acid-stable protein which binds non-specifically to various DNAs. Conditions can be established, however, where THF binds preferentially to the cohesive end site (cos) of lambda DNA forming several distinct complexes as visualized by band retardation in polyacrylamide gels. DNase I footprinting reveals that THF can protect several regions of the top strand on the right side (+) of cos but does not bind as well to the left side (-). The binding regions are separated either by unprotected or by DNase I- hypersensitive bases. Under the conditions used in these experiments, DNA which does not contain cos lambda sequences does not show this pattern of protection. Several repeated motifs in the cos lambda nucleotide sequence may represent a consensus sequence for THF interaction. THF may be similar to other "histone-like" proteins which display both non-specific and selective DNA-binding capacities.

Bacteriophage lambda↗
PubMed1988

Late stages in bacteriophage lambda head morphogenesis: in vitro studies on the action of the bacteriophage lambda D-gene and W-gene products.

The in vitro maturation of bacteriophage lambda can be divided into discrete steps. Concatemers of lambda DNA bind terminase to form complex I. This DNA-terminase complex then binds a prohead to form a ternary complex (II). Complex II in turn can be converted to infectious phage by the addition of extracts containing the products of the phage genes D, W, FII, as well as phage tails. By using in vitro complementation assays gpD and gpW have been partially purified and their interactions with complex II studied. gpD can bind to complex II in vitro to form a new complex (III) which can be isolated by sedimentation on neutral sucrose gradients. This complex requires only the addition of gpW, gpFII, and phage tails to form mature phage particles. The sedimentation of complex III is virtually identical to that of complex II; however, the resistance of the former to inactivation by DNase is higher, likely due to the partial packaging of the DNA. In similar experiments it was shown that gpW cannot bind to complex II but can effectively interact with complex III. This latter reaction converts complex III to a DNase-resistant form which sediments in a manner identical to that of full phage heads (complex IV). After isolation of the complex IV only gpFII and tails are required for mature phage formation in vitro. gpW is a heat-stable protein of molecular weight approximately 10,000.

Bacteriophage lambda↗
PubMed1986

A bacterial protein requirement for the bacteriophage lambda terminase reaction.

The bacteriophage lambda terminase enzyme cleaves the cohesive-end sites of lambda DNA to yield the protruding 5'-termini of the mature molecule. In vitro, this endonucleolytic event requires a protein factor which has been isolated and purified from extracts of uninfected E. coli. The terminase host factor (THF) is a heat stable basic protein of M.W. approximately 22,000. The integration host factor (IHF) protein of E. coli can efficiently substitute for THF in the terminase reaction; however, THF can be demonstrated to be physically present in, and isolated with full biological activity from extracts of cells defective or deficient in IHF.

Bacterial Proteins↗