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Biomedical subjects

D R Evans

Publications and source records attributed to D R Evans.

At least 109 records · Page 6Linked to original sources
PubMed1983

Disassembly and characterization of the nuclear pore complex-lamina fraction from bovine liver nuclei.

The nuclear pore complex-lamina (PCL), composed of nuclear pore structures attached to fibrous lamina, was isolated from bovine liver nuclei. We found that the highly aggregated PCL was disrupted and 75% of the constituent polypeptides could be solubilized by extraction for 1 h with 2% deoxycholate (DOC) and 3% 2-mercaptoethanol. While some differential solubilization was observed at lower detergent concentrations, all PCL proteins were solubilized equally at 2% DOC. The reducing agent was necessary to achieve maximum dispersal of the PCL and to prevent aggregation of the solubilized proteins. No tightly bound phospholipid or Triton X-100 could be detected in these preparations. Rapid removal of DOC, by dialysis or gel filtration, resulted in aggregation and precipitation of the PCL proteins, but the detergent could be removed by centrifugation through sucrose gradients. The sedimentation profiles indicated that the three major polypeptides, lamins A, B, and C, each sedimented as a single peak with a shoulder of more rapidly sedimenting material, possibly higher oligomeric forms. The sedimentation coefficient of lamins B and C, in the presence and absence of detergent, was 4.5 S. In the presence of DOC, lamin A had a sedimentation coefficient of 5.6 S, but this value was decreased to 4.1 S, when DOC was omitted from the gradient. These studies suggested that lamins B and C do not interact with or bind DOC, while lamin A may bind appreciable amounts of the detergent. The Stokes radii of lamins A, B, and C were found by gel filtration to be 75, 75, and 70 A, respectively. The molecular weights and frictional ratios estimated from the sedimentation and gel filtration data indicated that the lamins are dimeric, rod-shaped molecules.

Animals↗
PubMed1983

The isolation and characterization of the aspartate transcarbamylase domain of the multifunctional protein, CAD.

The aspartate transcarbamylase activity of mammalian cells is carried by a large multifunctional protein, CAD, that catalyzes the first three steps in the de novo pyrimidine biosynthetic pathway. Controlled proteolysis of CAD cleaved the polypeptide chain into several separate structural domains which carried the individual activities of the complex. The aspartate transcarbamylase activity was associated with a 40,000-dalton proteolytic fragment, which kinetic studies showed was released in one of the first proteolytic cleavages. The species was purified to homogeneity by chromatography on carboxymethyl-Sephadex. The isolated species, designated the aspartate transcarbamylase domain, had a molecular weight under denaturing conditions of 40,000 +/- 1,500, a pI = 9.4, and a sedimentation coefficient (S20,w) of 6.2. The sedimentation coefficient suggested that the isolated domain was an oligomer consisting of two or three identical copies of the 40,000-dalton proteolytic fragment. The aspartate saturation curve obtained at a saturating concentration of carbamyl phosphate gave Km = 2.1 X 10(-2)M and Vmax = 119.5 mumol/min/mg, corresponding to a turnover number of 4,780 min-1. Like the aspartate transcarbamylase activity of CAD, the activity was strongly inhibited by high concentrations of aspartate. The corresponding parameters from the carbamyl phosphate saturation curve were Km = 2.07 X 10(-5)M, Vmax = 52.5 mumol/min/mg, and a turnover number of 2,153 min-1. The similarity of these parameters to those obtained from a steady state kinetic study of CAD indicated that the tertiary structure of this region of the polypeptide chain was largely preserved in the isolated species. In the absence of stabilizing agents, the half-life of aspartate transcarbamylase activity of CAD was 60.2 h, while that of the isolated domain was 10.6 h. This result suggested that there were interactions with other regions of the molecule which stabilized the structure of the aspartate transcarbamylase domain in the intact complex.

Amino Acids↗
PubMed1981

Controlled proteolysis of the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells: evidence for discrete structural domains.

The multifunctional protein that initiates de novo pyrimidine biosynthesis in mammalian cells carries carbamoylphosphate synthetase, aspartate transcarbamylase (aspartate carbamoyltransferase), and dihydro-orotase activities on a single 215,000-dalton polypeptide chain. Kinetic studies of the controlled proteolysis of the molecule by elastase showed that the protein was not attacked at random by the protease but rather was successively cleaved into at least six well-defined proteolytic fragments. The initial cleavage converted the intact molecule into a 190,000-dalton species which appeared to retain all of the catalytic and regulatory functions of the native protein. This species was subsequently cleaved into two fragments, 150,000 and 40,000 daltons. The 40,000-dalton species, which carried the aspartate transcarbamylase activity, was resistant to further proteolysis; the 150,000-dalton polypeptide, which carried carbamoyl-phosphate synthetase and dihydro-orotase activities, underwent further digestion to 140,000 daltons. Continued proteolysis produced two species, 79,000 and 45,000 daltons; like the 40,000-dalton species, these were stable against further elastase digestion. The aspartate transcarbamylase and dihydro-orotase activities and the regulatory functions were preserved throughout the course of digestion; the carbamoylphosphate synthetase activity was more labile. By using sucrose gradient centrifugation and ion exchange chromatography, the 40,000- and 45,000-dalton species have been isolated. The 40,000-dalton fragment was found to have only aspartate transcarbamylase activity; the 45,000-dalton fragment has only dihydro-orotase activity. These experiments showed that this multifunctional protein is organized as discrete structural domains in which regions of the polypeptide chain are autonomously folded into separate functional units.

Amidohydrolases↗
PubMed1980

Programmed learning in the microtraining paradigm with hotline workers.

Twenty-five hotline workers were randomly assigned to three skill-training conditions. Nine microtraining subjects were trained in the use of attending behavior, open inquiry, and reflection of feeling and content using the standard microtraining paradigm. Seven programmed-learning subjects were trained in the same skills using programmed manuals and role-play interviews with feedback. The remaining nine subjects acted as a no-training control group. Pretraining and post-training interviews and telephone calls with pseudoclients were audiotaped for each subject. Subjects were assessed on empathy, the Therapist Error Checklist, attending behavior, and three modes of skill interaction. For interviews the main effect of training on each measure was significant. Both microtraining and programmed learning resulted in superior interview performance over no-training, and differed little from each other. For telephone calls microtraining resulted in higher empathy ratings than progammed learning or no-training.

Adult↗
PubMed1979

Maternal smoking habits and congenital malformations: a population study.

Maternal smoking habits in 67 609 singleton pregnancies were examined. The overall incidence of congenital malformations was 2.8% in both non-smokers and smokers. On analysing congenital defects according to individual systems there was no significant difference in the incidence of malformations according to the number of cigarettes smoked, except for neural tube defects. Further analysis, taking social class into account, showed that neural tube defects were less common in non-smokers in social classes I and II. This study suggests that maternal smoking does not have teratogenic effects in the offspring, except in the case of neural tube defects, where the effect is at most modest.

Congenital Abnormalities↗
PubMed1979

Neural-tube defects: importance of a history of abortion in aetiology.

The overall incidence of anencephaly and spina bifida (ASB) in 69,056 pregnancies was 4.7/1000 births. ASB was more common (8.4/1000 births) among children of mothers who had had two or more abortions, but the increased risk was confined to spina bifida. A history of abortion was more common in older women and women of higher parity, but this was not matched by a similar increase in the incidence of ASB. The incidence of ASB was related to social class, but the prevalence of previous abortions was similar in all classes. The results suggest that expectant mothers with a history of two or more abortions have an increased risk of producing a child with spina bifida. The abortions are considered to be a manifestation of previous abnormal conceptions rather than the primary cause.

Abortion, Spontaneous↗
PubMed1979

Precision in estimating gestational age and its influence on sensitivity of alphafetoprotein screening.

The interpretation of maternal serum alpha-fetoprotein (AFP) concentrations in relation to fetal neural tube defects depends on accurate assessment of the gestational age. In a quadruple-blind study three antenatal methods of assessment--namely, menstrual dates, clinical examination, and ultrasound scanning--were correlated with postnatal assessment using the Dubowitz scoring system. The best agreement to +/- 1 week was obtained using menstrual dates and ultrasound in combination, such agreement being found in 91 (77%) of the 118 women studied. Since serum AFP concentrations vary with gestational age, precise gestational dating is necessary. In many cases, particularly in women who are unsure of their dates or have irregular menstrual cycles, ultrasound examination is needed to supplement clinical findings.

Clinical Trials as Topic↗
PubMed1978

The effect of pH on the cooperative behavior of aspartate transcarbamylase from Escherichia coli.

Saturation curves of activity versus concentration were determined for aspartate transcarbamylase from Escherichia coli (EC 2.1.3.2) for the substrate L-aspartate at saturating carbamyl phosphate (4.8 mM) in buffered solution at pH values from 6.0 to 12.0. Hill coefficients were obtained from the sigmoidal curves. At pH values from 7.8 to 9.1, where substrate inhibition causes difficulties in the Hill approximation, our kinetic scheme includes substrate inhibition and residual activity in the abortive enzyme-substrate complex. The plot of Hill coefficient versus pH has pKalpha values of 7.4 and 9.8 at the half-maximum positions of the curve which has a plateau from pH 8.1 to 9.1. These pKalpha values may be associated with functional groups involved in the allosteric transition which activates the enzyme. A plot of [S]0.5 versus pH shows a pKalpha of 8.5, which may belong to a residue either at or near the aspartate binding site. At 50 mM aspartate concentration the pH-rate profile shows maxima at pH values of 8.8 and 10.0 (cf. Weitzman, P.D.J., and Wilson, I.B.(1966)J. Biol. Chem. 2418 5481-5488, who used 100 mM aspartate). However, when the pH-dependent substrate inhibition is included, the calculated Vmax--H curve is bell-shaped like that of the isolated catalytic subunit.

Aspartate Carbamoyltransferase↗
PubMed1978

Elimination of cooperativity in aspartate transcarbamylase by nitration of a single tyrosine residue.

In a previous report [Landfear, S. M., Lipscomb, W. N. & Evans, D.R. (1978) J. Biol. Chem. 253, 3988--3996] we demonstrated that tetranitromethane can be employed to nitrate a limited number of tyrosine residues in aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2); such modification eliminates cooperativity, feedback inhibition, and enzymatic activity, and reduces binding of the feedback inhibitor cytidine triphosphate. Cooperativity is lost more rapidly than other properties, and this loss correlates with the nitration of a single tyrosine residue. In this paper, we describe the saturation kinetics of hybrid species constructed from nitrated subunits of one type (either catalytic or regulatory) and native subunits of the other type. We conclude that the modification responsible for loss of cooperativity is on the catalytic subunit. The tryptic peptide containing this modification has been isolated and identified.

Allosteric Regulation↗