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P R Evans

Publications and source records attributed to P R Evans.

At least 19 recordsLinked to original sources
PubMed1992

Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase.

Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.

Adenosine Triphosphate↗
PubMed1992

The metabolic output of avian (Sturnus vulgaris, Calidris alpina) adipose tissue liver and skeletal muscle: implications for BMR/body mass relationships.

1. The oxygen uptake rate of avian adipose tissue, liver and skeletal muscle slices were measured. 2. The energy consumption of fat was less than one tenth that of liver and muscle. 3. Thus, interspecific allometric equations for the prediction of basal metabolic rate from body mass will not be accurate throughout the avian annual cycle unless changes in body composition are taken into account.

Adipose Tissue↗
PubMed1991

Steady-state fluorescence of Escherichia coli phosphofructokinase reveals a regulatory role for ATP.

We have investigated the effects of ligands and effectors on the intrinsic fluorescence of Escherichia coli phosphofructokinase (PFK). We have found that the substrate fructose 6-phosphate (Fru6P) or the allosteric activator ADP can quench the fluorescence up to 35%. The response is hyperbolic with Ks[Fru6P] of 20 microM and Ks[ADP] of 13 microM. The allosteric inhibitor phosphoenolpyruvate (PEP) converts the hyperbolic response with respect to Fru6P to a sigmoidal response. AMP-PNP, a nonhydrolyzable analogue of ATP, also inhibits the Fru6P fluorescence response. PFK mutant KA213, which is insensitive to effectors, has a decreased fluorescence response with respect to ADP, and PEP does not convert the Fru6P response to sigmoidicity. However, its fluorescence response with respect to Fru6P is decreased by ATP or AMP-PNP. Taken together, these results suggest that, in the absence of effectors or ligands, E. coli PFK exists in a state with high affinity for Fru6P ("R" state). This state can be altered to a low affinity ("T" state) by PEP binding to the allosteric site or by ATP binding to the enzyme.

Adenosine Diphosphate↗
PubMed1991

Designing an allosterically locked phosphofructokinase.

Six site-directed mutants of Escherichia coli phosphofructokinase (PFK) were made in an attempt to produce an enzyme "locked" in the inactive or "T"-state. The kinetic properties of the mutants were examined as a function of the substrates fructose 6-phosphate (Fru6P) and ATP, the positive effector GDP, and the negative effector phosphoenolpyruvate (PEP). All mutants exhibited lower activity than wild-type PFK. Three mutants (RS63, LV153, and VT246) had apparent dissociation constants for substrates and effectors similar to those of wild type. One mutant, HN160, had a 10-fold reduced affinity for Fru6P and reduced apparent affinity for the effectors. Two mutants, SN159 and T(GS)156, exhibited hyperbolic kinetics consistent with a "locked" T-state protein. Surprisingly, T(GS)156 showed hyperbolic activation in response to the physiological inhibitor PEP. The mutant PFK properties are discussed in terms of the PFK structure. These results suggest that the kinetic properties of PFK are sensitive to interactions in the homotropic interface; residues 156-160 in particular are critical in mediating the interactions between effector and active sites and in the T to R quaternary transition.

Adenosine Triphosphate↗
PubMed1991

Dental laboratory work authorization forms: a national survey of regulatory agency requirements.

This study reports a survey of the dental licensing agencies in the 50 states, Washington, D.C., and Puerto Rico concerning agency legislation and nomenclature of dental laboratory work orders. Differences in both legislation and nomenclature were found. Dental laboratory work authorization was the title most frequently used by the agencies. Nearly all agencies require a work order written by a licensed dentist to accompany all materials transmitted to a dental laboratory. This along with other sources of information may help to increase uniformity in the content of laboratory work authorization forms, thereby improving quality control in dental restorations made in commercial laboratories.

Dental Technicians↗
PubMed1991

No association between susceptibility to multiple sclerosis and HLA-DPB1 alleles in the French Canadian population.

HLA-DPB1 typing was performed using polymerase chain reaction DNA amplification and sequence-specific oligonucleotide probing (PCR-SSOP) which permitted identification of 17 distinct DPB alleles using 15 oligonucleotide probes. The accuracy of this approach was confirmed in an initial study of 26 human B-lymphoblastoid cell lines which demonstrated close agreement between PCR-SSOP and PLT assigned types. A cohort of 47 adult French Canadians was then studied to provide an estimate of DPB1 allelic frequencies in an ethnically homogeneous population. DPB1*0401 was the most frequent phenotype (61.5%) and only DPB1*0101, 0301 and 0402 were also present at frequencies greater than 10%. HLA-DPw4 has been reported to be associated with multiple sclerosis (MS) but our PCR-SSOP analysis of 52 French Canadian MS patients showed no association with either the DPB1*0401 or DPB1*0402 splits of DPw4 or with any other DPB1 allele.

Alleles↗
PubMed1991

Oesophageal intramural pseudodiverticulosis--always benign?

Oesophageal intramural pseudodiverticulosis is a rare condition that presents with dysphagia. The diagnosis is usually not apparent endoscopically and careful radiological evaluation is required. Previous reports suggest a benign course with good response of dysphagia to oesophageal dilatation and complications have not been described. Two cases are reported, one in whom life threatening haemorrhage developed following dilatation, and one complicated by a retro-oesophageal collection. It is possible that oesophageal intramural pseudodiverticulosis results in an increased propensity to trauma and even perforation.

Adult↗
PubMed1991

HLA DPB1 alleles and susceptibility to rheumatoid arthritis.

HLA-DPB1 genotypic and phenotypic frequencies were investigated in a series of 35 adult rheumatoid arthritis (RA) patients and 42 controls. No significant associations between DPB1 alleles and susceptibility to RA were demonstrated, although some non-significant differences in DPB1*0301 and 0401 allele frequencies between patients and controls were observed.

Alleles↗
PubMed1990

Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A.

The crystal structure of the RNA binding domain of the U1 small nuclear ribonucleoprotein A, which forms part of the ribonucleoprotein complex involved in the excision of introns, has been solved. It contains a four-stranded beta sheet and two alpha helices. The highly conserved segments designated RNP1 and RNP2 lie side by side on the middle two beta strands. U1 RNA binding studies of mutant proteins suggest that the RNA binds to the four-stranded beta sheet and to the flexible loops on one end.

Amino Acid Sequence↗
PubMed1990

Active-site mutants altering the cooperativity of E. coli phosphofructokinase.

Crystal structures of the high- and low-activity states of the allosteric enzyme phosphofructokinase implicate three arginines in substrate binding, catalysis and cooperativity. Arginines 162 and 243 reach into the active site from an adjacent subunit and interact with the cooperative substrate fructose 6-phosphate. Mutation of these arginines to serine results in mutant enzymes with reduced substrate binding and lowered cooperativity, but with little change in their catalytic ability (kcat). Arg 72 bridges the two substrates fructose 6-phosphate and ATP, and interacts with the 1-phosphate of the product fructose 1,6-biphosphate. Mutation of this residue to serine reduces the catalytic activity, cooperativity and binding of fructose 6-phosphate and fructose 1,6-bisphosphate. In the reverse reaction, the kinetics of wild-type and the Ser 72 mutant with respect to fructose 1,6-bisphosphate are hyperbolic, whereas those of the Ser 162 and Ser 243 mutants are sigmoidal. These results show that each of the three arginines contributes to cooperativity and to the transmission of allosteric signals between the four subunit of the enzyme.

Adenosine Triphosphate↗
PubMed1990

Structural basis of the allosteric behaviour of phosphofructokinase.

Comparison between the crystal structures of low- and high-affinity forms of phosphofructokinase shows a close coupling between the change of quaternary structure and local changes triggered by binding of the allosteric effectors. These concerted changes link all the substrate and effector sites in the tetramer, and explain the change of affinity for the cooperative substrate.

Allosteric Regulation↗
PubMed1990

Villous adenomas of the duodenum and an unusual variant.

We report two cases of villous adenoma of the duodenum, one arising from the main papilla and the other from the accessory papilla. Both were managed by local resection. In one case endoscopic biopsies and intraoperative frozen sections were negative for carcinoma but histology of the locally resected specimen revealed a focus of invasive adenocarcinoma. Villous adenomas of the duodenum have a high risk of malignant change and foci of carcinoma can be missed on endoscopic biopsy. The literature is reviewed and the clinical, diagnostic, pathological and therapeutic aspects of villous adenomas of the duodenum are discussed.

Adenocarcinoma↗
PubMed1990

Sperm motility, velocity and migration.

Four different methods for evaluating sperm motility were analysed for experimental error: subjective assessment of a wet film preparation, sperm velocity measured by time-lapse photography, sperm velocity measured by computer analysis and sperm migration across a nucleopore membrane. Subjective assessment of motility was found to be inaccurate, within single observer and between 2 observers. Both methods of measuring mean sperm velocity were accurate, particularly that using the computer analysis system; a high technical failure rate was found using time-lapse photography. Sperm migration across a nucleopore membrane was found to be highly inaccurate. Two groups were then analysed for the predictive value of these tests (excluding sperm migration): 104 proven fertile men and 53 infertile men. Although subjective motility was able to predict from which group the sample came at optimum cut-off with 78% accuracy, computer analysed sperm velocity could predict with overall 91% accuracy at optimum cut-off. Computer analysis of sperm velocity offers a rapid, objective and predictive assessment of sperm function.

Humans↗
PubMed1990

Sperm morphological analysis: comparison of two methods in human semen.

Sperm morphological analysis was performed by 2 methods: light microscopy on Papanicolaou stained material and scanning electron microscopy. Both methods were found to have acceptably low levels of observer error and a positive correlation existed between them. Sperm morphology was analysed by both methods in 2 groups: (1) 104 proven "fertile" men, husbands of women who had just given birth and whose paternity had been proven by HLA studies with greater than 95.5% certainty; (2) 53 infertile men from primary infertile marriages in whom no female cause could be found and excluding those with azoospermia. There was no statistically significant difference in medians between the 2 groups when sperm morphology was analysed by light microscopy, but analysis by scanning electron microscopy did show a significant difference.

Humans↗