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D R Evans

Publications and source records attributed to D R Evans.

At least 91 records · Page 5Linked to original sources
PubMed1989

Mammalian aspartate transcarbamylase (ATCase): sequence of the ATCase domain and interdomain linker in the CAD multifunctional polypeptide and properties of the isolated domain.

Mammalian aspartate transcarbamylase (ATCase; carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) is part of a 240-kDa multifunctional polypeptide called CAD, which also has carbamoyl-phosphate synthetase and dihydroorotase activities. We have sequenced selected restriction fragments of a Syrian hamster CAD cDNA that are clearly homologous to three prokaryotic ATCases. These studies, combined with previous sequence data, showed that the ATCase domain of CAD is encoded by 924 base pairs and has a mass of 34,323 Da and a pI of 9.8. While the bacterial pyrimidine biosynthetic enzymes are separate proteins, in mammals the ATCase domain is fused to the carboxyl end of the CAD chimera via a 133-amino acid (14-kDa) linker with an unusual amino acid composition, a pI of 10.2, and pronounced hydrophilic character. The fully active domain isolated from proteolytic digests was characterized by partial amino acid sequencing and amino acid analysis. Trypsin cleavage produced the ATCase domain with a 20-residue amino-terminal extension. Hydrodynamic studies showed that the isolated domain is a 110-kDa trimer with a Stokes radius of 41 A. The mammalian ATCase domain and the prokaryotic enzymes have virtually identical active-site residues and are likely to have the same tertiary fold.

Amino Acid Sequence↗
PubMed1989

Phenotypical features of an unique Irish family with severe autosomal recessive osteogenesis imperfecta.

Severe Sillence type II/III Osteogenesis imperfecta (OI) is a lethal or severely crippling disease with either autosomal dominant or recessively inherited type I collagen mutations. Here we describe the detailed clinical features of a thin-ribbed OI variant with deformed limbs. The three consecutively affected children showed no genetic linkage with either of the two type I collagen genes, which implies that a novel mechanism causes this clinical phenotype. It can be prevented using ultrasound to diagnose affected foetuses.

Collagen↗
PubMed1989

Severely heated therapeutic factor VIII concentrate of high specific activity.

A new method for the manufacture of a heated factor VIII concentrate of high specific activity (2-6 IU factor VIII:C/mg protein) has been developed. Addition of heparin to cryoprecipitate extract at acid pH precipitated fibrinogen and fibronectin. Factor VIII was then recovered from the supernatant by precipitation with glycine and sodium chloride. After re-solution and desalting on Sephadex G-25, the concentrate was sterile-filtered and lyophilised. The dried product was stable to heating in the final container at 80 degrees C for 72 h. Data from 25 consecutive batches of concentrate prepared from 1,200-1,500 kg plasma pools are presented. The mean final yield of heated product was 190 IU factor VIII:C/kg plasma. The concentrate has been found to be safe and effective in clinical use.

Chemical Precipitation↗
PubMed1988

Controlled proteolysis of ammonia-dependent carbamoyl-phosphate synthetase I from Syrian hamster liver.

Ammonia-dependent carbamoyl-phosphate synthetase I (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16; formerly EC 2.7.2.5) isolated from hamster liver mitochondria is comprised of identical 160 kDa polypeptide chains. Controlled proteolysis by elastase sequentially cleaved this molecule into a small number of specific fragments. The first cleavage led to a complete loss of enzymatic activity and the formation of a 145 kDa species that was subsequently degraded into 83 kDa and 62 kDa fragments. Very different results were obtained when proteolysis was carried out in the presence of saturating ATP, MgCl2, NH4Cl, and the activator N-acetyl-L-glutamate. These ligands stabilized the molecule 8-fold against elastase digestion. Moreover, only small amounts of the 145 kDa species were generated. Instead, the molecule was initially cleaved into a fully active 120 kDa species and a 40 kDa proteolytic fragment. The same species were found in limit digests conducted in the presence and absence of ligands, indicating that only the sequence of elastase cleavages differed. Comparison of digests conducted in the presence of each ligand alone and in combination, showed that while NH4Cl and N-acetyl-L-glutamate were necessary for maximal stabilization of the molecule, the altered digestion pattern was produced specifically by MgATP. The MgATP-induced change in digestion pattern correlated well with the steady-state ATP saturation curve, suggesting that the production of the 120 kDa species resulted from ATP binding to the active site. The effect of MgATP on the proteolysis of carbamoyl-phosphate synthetase I was not the result of an alteration in oligomeric structure, but the protection of two elastase cleavage sites. The results were interpreted on the basis of the primary structure recently determined elsewhere.

Adenosine Triphosphate↗
PubMed1988

Response specificity revisited: stress and health status.

Numerous authors have obtained significant, but low correlations between life events and specific health status measures. Even with the inclusion of moderating variables, such as coping, the relationship between life stress and health status measures has rarely exceeded .50. It has been argued that failure to enhance the correlation has occurred because response specificity has not been controlled. A representative community sample of 184 adults from London, Ontario, was surveyed with respect to life stress variables, stress-response specificity, and various illness behaviors. First-order factor analysis of the stress-response specificity measure indicated an emotional/psychological response factor, an eating response factor, a drug/alcohol response factor, and several physical response factors. Second-order factor analysis indicated a psychological response and a physical response factor. Each of the illness behaviors was regressed, in a stepwise procedure, upon the life stress measures and related first- and second-order stress-response specificity factors. As predicted, significant relationships were found between the related stress-response specificity and illness behavior measures. These results support the importance of stress-response specificity as a moderator variable in investigations of the relationship between life stress and illness behavior.

Adaptation, Psychological↗
PubMed1988

Intracellular location of the multidomain protein CAD in mammalian cells.

The first three steps of mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein CAD, consisting of glutamine-dependent carbamylphosphate synthetase, aspartate transcarbamylase, and dihydroorotase. The intracellular distribution of CAD in two hamster cell lines, BHK 21 and BHK 165-23 (a strain in which the CAD gene was selectively amplified), was determined by differential centrifugation and by two different cytochemical immunolocalization methods. Ammonia-dependent carbamylphosphate synthetase I was found in both cell types at a concentration of 0.01% of the total cell protein, so its distribution was also determined as a control for possible cross-reactivity of the CAD antibody probes and as a mitochondrial marker. CAD was localized in the cytoplasmic compartment and almost completely excluded from the nucleus. A punctate staining pattern suggested that it was not uniformly dispersed throughout the cytosol (unlike typical soluble proteins) but was associated with subcellular organelles. Although there was a slight tendency for CAD to be localized in the vicinity of the nuclear envelope, the amount of staining was much less than expected from differential centrifugation, which showed that 30% of the protein was found in the nuclear fraction. No interactions with other subcellular components could be detected by centrifugation. It is possible, however, that CAD is associated with subcellular structures that cosediment with the nuclei. Despite a 150-fold increase in CAD concentration in the over-producing cells, the distribution of the protein was unaltered. CAD was not concentrated near the mitochondria where the next enzyme of the de novo pathway, dihydroorotate dehydrogenase, is localized, which indicates that the intermediate dihydroorotate is not channeled, but rather dissociates from CAD and diffuses through the bulk cellular fluid.

Amidohydrolases↗
PubMed1987

The dimensionality of cognitive appraisal and its relationship to physical and psychological well-being.

In current adjustment models, an individual's appraisal of life events has been included as an important determinant in the stress-illness process. In particular, these events have been defined in terms of a variety of unidimensional concepts, such as desirability, controllability, or as having the potential for threat or challenge. The present study investigated the multidimensional nature of cognitive appraisal and its relationship to physical and psychological well-being. Residents of a Canadian community (N = 184) responded to an extensive survey on life-event stress, appraisal, alcohol consumption, health status, and depression. A principal-axis factor analysis of 19 appraisal dimensions revealed five factors accounting for 60% of the variance: Undesirability/Threat, Gain/Challenge, Need for Information, Familiarity, and Need to Accept. Entered with total number of life events and coping behavior as predicted aspects of well-being.

Adaptation, Psychological↗
PubMed1987

Salsolinol in urine following chocolate consumption by social drinkers.

Several studies have shown that there are differential levels of salsolinol excretion between consumers of varying levels of alcohol. The excreted salsolinol may arise during metabolism of alcohol, reflect endogenous biosynthesis of the compound, or result from ingestion of exogenous salsolinol. A previous study identified that salsolinol excretion in urine distinguished light and heavy social drinkers, who had consumed chocolate (containing salsolinol) in combination with a test dose of alcohol. The present study was designed to examine the relationship between social drinking level and the excretion of salsolinol after ingesting dietary salsolinol alone. Participants were 120 volunteers, balanced for gender, social drinking level (abstainers, light, moderate and heavy), and dose of salsolinol. Urine samples were taken on entry, and at 90 minutes and 3 hours after consumption of chocolate. Analysis revealed a dose effect on salsolinol excretion. There were no main or interactive effects of gender or social drinking on salsolinol excretion. The results indicate that the appearance of salsolinol in urine following dietary consumption alone is insufficient to distinguish alcohol consumption levels in social drinkers.

Adult↗
PubMed1986

A pasteurised concentrate of human plasma factor XIII for therapeutic use.

A therapeutic concentrate of factor XIII containing both A and B sub-units has been prepared from 300 kg pools of human plasma. The process starts from a cold-ethanol fraction from cryoprecipitate supernatant and therefore does not interfere with the recovery of other clinically valuable plasma proteins. Factor XIII is purified approximately 600-fold from plasma by precipitation with sodium citrate and by the removal of fibrinogen by brief heating. The product has been pasteurised in sorbitol solution to inactivate blood-borne viruses, ultrafiltered to remove sorbitol, adsorbed with bentonite and freeze-dried in a formulation meeting requirements for intravenous injection.

Drug Contamination↗
PubMed1986

The dihydroorotase domain of the multifunctional protein CAD. Subunit structure, zinc content, and kinetics.

Dihydroorotase (DHOase) catalyzes the third step in eukaryotic de novo pyrimidine biosynthesis. In mammalian cells, this enzyme activity is carried by a large chimeric protein, CAD, that also catalyzes the first two steps in the pathway: glutamine-dependent carbamyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Controlled elastase cleavage of CAD released a 44,000 +/- 2,000-dalton proteolytic fragment which catalyzed only the dihydroorotase reaction. We have devised a rapid and simple method for the isolation of the DHO domain from elastase digests. The domain, which was obtained in 36% yield, was found to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The domain was also characterized by amino acid analysis and analytical high pressure liquid chromatography peptide mapping. The amino terminus of both the DHO domain and intact CAD was blocked suggesting that this domain is located at the extreme amino terminus of the CAD polypeptide, a result consistent with the suspected juxtaposition of domains as DHO-CPS-ATC. The isoelectric point of the DHO domain was 5.1, while that of the ATC domain was 9.4, so that the ends of the CAD polypeptide are oppositely charged at physiological pH. Immunoblotting with DHO domain-specific antibodies showed that a 47-kDa species was generated in the early stages of controlled proteolysis of CAD. Thus there are two elastase cleavage sites within a 3-kDa connecting region that links the DHO and CPS domains. The domain was shown by atomic absorption spectrophotometry and by isolating a 65Zn-containing DHO domain from mammalian cells grown in the presence of the radionuclide to contain 1 g eq of tightly bound zinc in each polypeptide chain. Zinc was not found in any other CAD domain. Chelating agents inhibit dihydroorotase activity of the isolated domain supporting the conclusion, based on studies of intact CAD by others, that zinc participates in catalysis. At moderate protein concentrations the DHO domain was a 88,000 dimer with a Stokes radius of 37.6 A, a S20,w = 5.1 X 10(-13) s, a diffusion coefficient of 3.17 X 10(-7) cm2 s-1, and a frictional ratio of 1.26. On dilution the dimer dissociated and was in rapid concentration-dependent equilibrium with a 43,500 monomer. The hydrodynamic parameters of the monomer have also been estimated (Stokes radius of 29.8 A, D20,w = 4.11 X 10(-7) cm2 s-1, and f/f0 1.21).(ABSTRACT TRUNCATED AT 400 WORDS)

Amidohydrolases↗
PubMed1985

Immunochemical analysis of the domain structure of CAD, the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells.

CAD, is a multidomain polypeptide, with a molecular weight of over 200,000, that has glutamine-dependent carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase activity as well as regulatory sites that bind UTP and 5-phosphoribosyl 1-pyrophosphate. The protein thus catalyzes the first three steps of de novo pyrimidine biosynthesis and controls the activity of the pathway in higher eukaryotes. Controlled proteolysis of CAD isolated from Syrian hamster cells, cleaves the molecule into seven major proteolytic fragments that contain one or more of the functional domains. The two smallest fragments, which had molecular weights of 44,000 and 40,000, corresponded to the fully active dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains, respectively, but the larger fragments have not been previously characterized. In this study, enzymatic assays of partially fractionated digests and immunoblotting with antibodies specifically directed against the purified ATC domain, the purified dihydroorotase domain and an 80-kDa fragment of the putative carbamyl-phosphate synthetase domain established the precursor-product relationships among all of the major proteolytic fragments of CAD. These results indicate that 1) only the intact molecule had all of the functional domains, 2) a species with a molecular weight of 200,000 was produced in the first step of proteolysis which had glutamine-dependent carbamyl-phosphate synthetase and dihydroorotase activity, but neither aspartate transcarbamylase activity nor the antigenic determinants present on the isolated ATC domain, and 3) cleavage of the 200-kDa species produced a species, with a molecular mass of 150,000 which lacked both aspartate transcarbamylase and dihydroorotase domains. This 150-kDa species, containing the postulated carbamyl-phosphate synthetase, glutamine, and regulatory (UTP, 5-phosphoribosyl 1-pyrophosphate) domains, had two elastase-sensitive sites that divided this region of the polypeptide chain into 10-, 65-, and 80-kDa segments. The location of the functional sites on these segments has not yet been established. The immunochemical analysis also revealed the existence of possible precursors of the stable aspartate transcarbamylase and dihydroorotase domains, suggesting that the chain segments connecting the functional domains of CAD are extensive and that the overall size of the intact polypeptide chain has been underestimated. On the basis of these studies we have proposed a model of the domain structure of CAD.

Amidohydrolases↗
PubMed1985

Use of refined perinatal mortality rate trends to evaluate the effect of a confidential inquiry.

Standardised perinatal mortality rates and cause-specific rates were used to evaluate the effect of a confidential inquiry into perinatal deaths in West Glamorgan obstetric units. Cause of death was classified by means of 5 pathological subgroups, and the denominator data were obtained from the computerised standard child-health system. The standardised perinatal mortality rate fell from 103 in 1981 to 85 in 1983, and the perinatal mortality rate fell from 15.9 to 9.4 per 1000 births in that period; 3 of the cause-specific rates declined considerably. The trend indicates an improvement in antenatal care, obstetric management, resuscitation, and care in special-care baby units. An audit in the form of a confidential inquiry contributes to an improvement in health services and a consequent reduction in perinatal mortality.

Birth Weight↗
PubMed1985

The Community Alcohol Use Scale: a scale for use in prevention programs.

The Community Alcohol Use Scale (CAUS) was developed as a continuous, reliable, valid, and acceptable measure for primary prevention studies. Items were written following a comprehensive review of the literature on alcohol dependence and alcoholism. Based upon the responses of 315 respondents to the 100-item initial version of the scale, the 45-item CAUS was developed. The CAUS was then cross-validated on a local sample (n = 274) and a provincial sample (n = 745). Estimates of internal consistency were .91, .96, and .94, respectively, for the initial and cross-validation samples. Correlations of .48 and .69 were obtained between the CAUS and the Michigan Alcohol Screening Test for the initial and local cross-validation samples. In the provincial cross-validation sample a correlation of .62 was obtained between the CAUS and the Usual Weekly Alcohol Index. Preliminary cutting scores were developed to identify those at risk for alcoholism, and alcoholics. The CAUS has potential use as an evaluative and predictive instrument in a variety of primary prevention programs.

Adult↗
PubMed1985

The influences of ethanol and other factors on the excretion of urinary salsolinol in social drinkers.

Salsolinol, a substance that may participate in the development of alcoholism, has been identified in urine and other biological samples from alcoholics. Differentials have been observed between alcoholics and controls. Salsolinol forms when dopamine reacts with acetaldehyde, which may exist in higher concentrations in the blood of alcoholics after alcohol ingestion. Hence, it was postulated that there is a relationship between level of social drinking and the elaboration of salsolinol. Salsolinol is also found in certain food and beverage products. Eighty volunteers, balanced for gender, social drinking level, ethanol dose administered and experimental diet provided urine samples 90 minutes and three hours after ethanol was consumed. Salsolinol levels were analysed in urine using high performance liquid chromatography. A 24 hour carryover effect was observed. Diet, ethanol dose and social drinking level had main and interactive effects on excreted quantities of salsolinol. Gender, situational stress and cigarette smoking had minor if any influence on salsolinol excretion. While there was no evident difference in amounts of salsolinol excreted by light and heavy drinkers in the absence of external sources of salsolinol, heavy social drinkers excreted less salsolinol than did light drinkers after consuming a "salsolinol-enriched" diet, suggesting that they differ in some aspect of absorption, distribution, or metabolism of salsolinol after drinking ethanol. Accordingly, studies that attempt to determine whether salsolinol has any relationship to drinking behaviour in humans should be particularly concerned with salsolinol that occurs in exogenous sources.

Adult↗
PubMed1985

Oligomeric structure of the multifunctional protein CAD that initiates pyrimidine biosynthesis in mammalian cells.

The first three steps in mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein designated CAD. Regions of the single 240-kDa poly-peptide chain are folded into separate structural domains that have discrete functions. Previous studies suggested that CAD forms predominantly trimers. The trimers are found to be in slow equilibrium with hexamers and higher oligomers composed of multiples of three copies of the CAD polypeptide chain. However, quantitative chemical crosslinking studies of CAD with dimethyl suberimidate were used here to show a progressive conversion of monomer to crosslinked hexamer. High levels of the hexamer accumulate in the reaction mixture, suggesting that the major oligomeric form is hexameric, although residual amounts of smaller oligomers remain present. Larger oligomers may form by association of hexamers and are seen after longer crosslinking times. Sucrose gradient centrifugation shows a 20.8S species to be the slowest sedimenting peak, while the larger species sediments at 27.9S. Electron microscopic studies of rotary-shadowed preparations of CAD have confirmed that, while small amounts of other oligomeric forms are present, the CAD monomer is primarily associated into cyclic hexamers with an open planar appearance.

Animals↗
PubMed1985

A pasteurized antithrombin III concentrate for clinical use.

A method for large-scale production of a pasteurized antithrombin III (AT III) concentrate for therapeutic use has been adapted from published methods. It includes the following steps: (1) batchwise adsorption onto heparin-Sepharose from plasma depleted of cryoprecipitate and prothrombin complex; (2) chromatographic elution at high salt concentration; (3) pasteurization for 10 h at 60 degrees C in the presence of added citrate ion; (4) desalting on Sephadex G-25, and (5) sterile filtration and freeze-drying. Seven batches prepared in this manner gave a mean yield of 269 U AT III/kg plasma. The product passed all the usual animal safety and pyrogenicity tests and has been used successfully in several courses of treatment of congenital deficiencies.

Adsorption↗