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D Thomas

Publications and source records attributed to D Thomas.

At least 721 records · Page 40Linked to original sources
PubMed1987

Alteration of the specificity of PvuII restriction endonuclease.

The restriction endonuclease PvuII which cleaves the sequence CAGCTG, at the position indicated by the arrow, was found to decrease its substrate specificity in the presence of organic solvents. Thirty-three sites, that we have named PvuII sites, were identified on the nucleotide sequence of pBR322 DNA. The new recognition sequences cleaved in pBR322 DNA, at the positions indicated by the arrows, were shown to be AAGCTG, GAGCTG, CNGCTG, CANCTG, CAGNTG, CAGCNG, CAGCTC and CAGCTT. (TAGCTG and the complementary sequence CAGCTA are not present in pBR322 DNA). From these recognition sequences, we deduced that PvuII activity recognizes and cleaves degenerate sequences which differ from the standard PvuII sequence CAGCTG at only one of the recognition site. Any substitution can occur at any one of the six positions in the hexanucleotide sequence. The optimum incubation medium for PvuII activity was found to be: 10-50 mM Tris-HCl, pH 8.5, 12-15 mM MgCl2, 50 mM NaCl, 10% ethanol + 10% dimethylsulfoxide (DMSO).

Base Sequence↗
PubMed1987

Interesterification and synthesis by Candida cylindracea lipase in microemulsions.

Unusual reactions of interesterification and synthesis catalyzed by Candida cylindracea lipase have been tested in reverse microemulsions. The microemulsions used are made of fatty acids or triglycerides, the enzyme dissolved in a very low water quantity, Brij 35 used as surfactant and an alcoholic cosurfactant. In such a system, fats and alcohols are both the substrates of the enzyme and the microemulsion components. Incidentally, non specific Candida cylindracea lipase does not catalyze interesterification of short chain triglycerides, revealing a specificity for the chain length. Interesterification reactions tested in the presence of a given water quantity but with varying water activities show that it is the water activity and not the water quantity which is a fundamental parameter of the system. The effect of the surfactant (Brij 35) on the interesterification reaction is studied. Heptyl-oleate synthesis catalyzed by non-specific lipase is obtained in microemulsions at a 98% yield. Synthesis of glycerol esters is also tested in monophasic medium and mono and diglycerides are obtained.

Candida↗
PubMed1987

Reproducibility of rest and exercise left ventricular ejection fraction and volumes in chronic aortic regurgitation.

To assess the variability of rest and exercise radionuclide ventriculography in patients with aortic regurgitation (AR), 22 patients in stable condition with chronic AR underwent radionuclide ventriculography at rest and during exercise for measurement of left ventricular volumes and ejection fraction (EF) on 2 occasions 3 months apart. For the group, there were no significant differences between the findings of the 2 studies except for a slightly lower exercise end-systolic volume on the second study. The inter-study differences and variabilities (expressed as the standard deviation of differences) for rest and exercise EF and change in EF were 0 +/- 0.04, +0.02 +/- 0.05 and +0.01 +/- 0.05, respectively. Thus, although mean differences were minor, considerable individual variability occurred, the magnitude of which was similar to that reported for other patient populations. In 3 patients who had an initial normal response to exercise (increase in EF greater than or equal to 0.05), the response became abnormal and in 2 who initially had an abnormal response, the response normalized. This variability must be considered in clinical decision-making or in research study design for patients with chronic AR.

Adult↗
PubMed1987

Substrate activation and thermal denaturation kinetics of the tetrameric and the trypsin-generated monomeric forms of horse serum butyrylcholinesterase.

Native horse serum butyrylcholinesterase (acylcholine acylhydrolase; EC 3.1.1.8) is a tetrameric enzyme which can dissociate after a limited proteolysis by trypsin into three additional molecular forms, including the monomeric entity. The trypsin-generated monomer of butyrylcholinesterase, isolated by ultracentrifugation on sucrose gradient, is stable and allows the relations between the polymeric structure of butyrylcholinesterase and its kinetic characteristics to be approached, e.g., substrate activation and complex thermal denaturation curves. The trypsin-generated monomer of butyrylcholinesterase behaves with identical kinetic parameter values as the native tetrameric enzyme. On the other hand, the thermal denaturation of the native tetrameric butyrylcholinesterase does not follow first-order kinetics, but may be described by a sum of exponential terms. This behavior is not due to the polymeric nature of butyrylcholinesterase but seems to be related to a structural heterogeneity induced by the heat treatment.

Animals↗
PubMed1987

Kinetics of amino acid esterification catalyzed by hydrophobic derivatives of alpha-chymotrypsin.

Hexyl-alpha-chymotrypsin, a hydrophobic derivative of the enzyme, is explored for the proteinase-catalyzed ester synthesis reaction with N-acetyl-L-tyrosine and ethanol. To guarantee the preservation of the enzyme activity and to allow for the extraction of the product in the organic phase, a biphasic system was used. The Vm increased for the modified enzyme. This phenomenon was linked to the modification of the protein as demonstrated by its chemical denaturation with sodium dodecyl sulfate.

Aldehydes↗
PubMed1987

Hypothesis testing of time-dependent recurrent events.

This paper examines the problem of hypothesis testing for comparison of time-dependent recurrent events between categorical exposure groups. We compare three methods (a summary chi-square test based on a generalization of the simple Poisson distribution, a chi-square test based on a generalization of the compound Poisson distribution, and a test on risk scores based on individual observed to expected ratios), when the dependent variable may be autocorrelated within an individual, and disease risk may be heterogeneous among subjects. We present a simulation study and an application to a cohort of sickle cell anaemia patients. With autocorrelation or heterogeneous risk present, the simple chi-square test is inappropriate, while the other two methods perform well. An attraction of the risk score method is its relative ease of application.

Anemia, Sickle Cell↗
PubMed1987

Digital image capture and analysis for split-drop micropuncture.

A method for automated image capture and analysis of shrinking drop sequences is described. The procedure can be managed by a single operator and allows estimates of proximal tubule volume flux to be calculated during the experiment. Speed of analysis is increased considerably (mean time 1.5 min per sequence) compared with previous methods. The potential exists for measurement of droplets in tubules that follow a tortuous path in the horizontal plane and further increases in capture and analysis rate would allow estimation of volume flux under non-steady state conditions. Film processing is eliminated and the removal of operator errors, bias and fatigue associated with manual measurement, coupled with the greatly reduced time required for analysis of sequences makes the shrinking drop method much more reliable and attractive.

Animals↗
PubMed1987

Immobilization of the restriction endonucleases PvuII and HindIII.

The effects of several chemical reagents on the activity of the restriction endonucleases PvuII and HindIII were investigated. Carbodiimide, which reacts preferentially with carboxyl groups, was found to inactivate these enzymes. This specific effect could be prevented by Mg2+ cation. pBR322 DNA, which contains PvuII and PvuII* sites and HindIII and HindIII* sites, did not protect the enzymes from the carbodiimide. On the other hand, glutaraldehyde, which reacts primarily with lysine residues, inactivates PvuII and HindIII enzymes. This specific effect could not be prevented by pBR322 DNA. Preincubation with high concentrations of N-ethylmaleimide, which reacts with sulfhydryl groups, caused slight inhibition of PvuII activity, but had no effect on the activity of HindIII enzyme. The effects of glutaraldehyde, carbodiimide, and N-ethylmaleimide on other restriction endonucleases were also investigated. Restriction endonucleases PvuII and HindIII were immobilized by covalent coupling to various insoluble carriers. Both immobilized enzymes retained partial enzyme activities, when immobilized through phenolic groups and were stable for at least two months.

Carbodiimides↗
PubMed1987

Cyclophosphamide versus ifosfamide: final report of a randomized phase II trial in adult soft tissue sarcomas.

Ifosfamide (IFOS) 5 g/m2 and its parent analog Cyclophosphamide (CYCLO) 1.5 g/m2 were studied in a randomized phase II study, accruing 171 patients with advanced soft tissue sarcoma. Both drugs were administered as 24 hr infusions, every 3 weeks, with comcomitant Mesna 400 mg/m2 i.v. bolus 4 hourly X 9 doses. Twenty-four patients were ineligible and 12 were not evaluable. The groups were well matched for age, previous chemotherapy (42% of the total) or radiotherapy, the presence of distant metastases and performance status, but there were more females (59% vs. 45%) in the IFOS arm. Among the 68 evaluable patients receiving IFOS, there were 2 CR, 10 PR (overall response 18%), 27 SD and 29 PD. For CYCLO, the corresponding results in 67 patients were 1 CR, 4 PR (overall response 8%), 23 SD and 39 PD. Using the chi-square test the P values for response rate and linear trend were 0.13 and 0.04 respectively. Response rates were higher for females (20% vs. 5%, P = 0.01) and patients who had not received previous chemotherapy (19% vs. 4%, P = 0.01). Fourteen of the 17 responses came from a group of 43 females, who had not received previous chemotherapy, for whom the overall response rate was 37.5%. Remissions were noted in only 4 histological subtypes (centrally reviewed material), i.e., 5 of 17 synovial sarcomas, 7 of 13 mixed mesodermal sarcomas and 2 of 7 fibrosarcomas. One of the 31 leiomyosarcomas responded to Cyclophosphamide. Durations of response did not differ significantly between the 2 arms--median 26, range 10-81+ weeks. Leucopenia was significantly more severe on CYCLO, particularly in patients who had received previous chemotherapy (P = 0.007). Serious infections occurred in approx. 7% of patients with no difference between the two drugs, although there was one toxic death on CYCLO. Nausea and vomiting were significantly worse on IFOS and alopecia, related in extent to dose, was seen in both arms. Other side-effects, such as hematuria or rises in serum creatinine and encephalopathy, were infrequent and mild. A higher response rate with less myelosuppression suggests that IFOS may have advantages over CYCLO in combination therapy.

Adolescent↗
PubMed1987

Adriamycin versus epirubicin in advanced soft tissue sarcomas. A randomized phase II/phase III study of the EORTC Soft Tissue and Bone Sarcoma Group.

The objective of this randomized phase II/phase III study was to investigate the efficacy and toxicity of equimolar doses of adriamycin (ADM) and 4-epiadriamycin (EPI) in patients with locally advanced and/or metastatic soft tissue sarcoma. Doses of ADM and EPI were 75 mg/m2 given as an i.v. bolus injection every 3 weeks. Two hundred and ten patients were entered into the study by 18 institutions. Twenty-eight patients were ineligible and 15 were non-evaluable, leaving 167 evaluable patients. The two treatment groups were well balanced for sex, performance status, age, prior radiotherapy, extent and site of disease. Rates of response were similar, 25% in the ADM group compared to 18% in the EPI group (P = 0.33), and there were no significant differences between the ADM and EPI groups with respect to median duration of response (45 weeks vs. 77 weeks, P = 0.08), time to progression (15 weeks vs. 12 weeks, P = 0.945), and median survival (41 weeks vs. 48 weeks, P = 0.363). Myelotoxicity as shown by leucopenia was significantly more pronounced in the ADM treated patients (P = 0.002). Other toxicities such as alopecia and nausea/vomiting were also more severe in the ADM group (P = 0.02 and 0.06, respectively). In conclusion, the use of equimolar doses of ADM and EPI in advanced soft tissue sarcoma produced response rates which did not differ significantly and were only slightly in favour of ADM. However, this was achieved at the expense of higher toxicity.

Adolescent↗
PubMed1987

Development of a reversible bienzymatic system in immunoenzymatic technique.

The use of specific captors solubilized with a ligand on to proteic membranes together with an automatic-computerized system is proposed for the determination of various haptens or antigens in biological and industrial fluids by an enzyme-linked immunoassay test. Two enzymes are used in this technique: the first enzyme for linking reversibly the immunocomplex to the insoluble matrix, the substrate of this enzyme being immobilized on the matrix; the second (beta-d-glucose oxidase) for labelling the antigen. Its activity is measured by fixing the immunocomplex gelatin membrane on to a pO(2) sensor. After incubation of the antigen labelled with glucose oxidase and the free antigen with specific antibodies linked with the first enzyme in a pre-established concentration, the reaction medium is introduced inside the continuous flow cell. O(2) consumption due to the enzyme reaction is measured in actual time when the electrode is in contact with the glucose standard solution. The signal is correlated by an injection of urea solution. The signal is processed with an automated microcomputer system.

Journal Article↗
PubMed1987

Effect of temperature on the stability of plasmid pTG201 and productivity of xylE gene product in recombinant Escherichia coli: development of a two-stage chemostat with free and immobilized cells.

The effect of temperature on the stability of pTG201, a plasmid carrying the xylE gene (which encodes catechol 2,3-dioxygenase from Pseudomonas putida), and the production of catechol 2,3-dioxygenase in free and immobilized Escherichia coli during continuous culture have been studied at various temperatures. Immobilization of cells increased the stability of pTG201 considerably, even under conditions when expression of the xylE product was enhanced. Since xylE transcription was controlled by the lambda PR promoter and cI857 repressor, increasing derepression temperatures increased catechol 2,3-dioxygenase productivity and decreased pTG201 stability. A two-stage continuous culture system to overcome the impact of the high-level expression of the xylE gene on the stability of pTG201 is described. In the first stage, immobilized cells were grown in the repressed state in order to prevent loss of pTG201, whereas in the second stage, cultures were maintained in the derepressed state.

Catechol 2,3-Dioxygenase↗
PubMed1987

Evoked potentials following diazepam or fentanyl.

The effects of fentanyl or diazepam on somatosensory, visual and brainstem auditory evoked potentials were studied in 13 healthy patients scheduled for elective surgery. Following control recordings of evoked potentials, either diazepam 20 mg or fentanyl 200 micrograms was administered intravenously. Evoked potentials were then recorded twice in the subsequent hour. No significant changes occurred in the latency or amplitude of somatosensory, visual or brainstem auditory evoked potentials. Although dose-related changes in evoked potential latencies and amplitudes have been demonstrated with both the inhalational and intravenous anaesthetics, these changes did not occur with diazepam or fentanyl used alone. An anaesthetic technique based on these two drugs would be suitable when intra-operative evoked potential monitoring is required to assess ischaemia and preservation of evoked responses.

Adult↗
PubMed1987

The pharmacokinetics of nedocromil sodium, a new drug for the treatment of reversible obstructive airways disease, in human volunteers and patients with reversible obstructive airways disease.

1. The plasma concentrations and urinary excretion of nedocromil sodium have been determined following single dose administration in six healthy volunteers dosed orally (1 mg kg-1) and intravenously (0.2 microgram kg-1 for 30 min). Similar parameters were measured in six volunteers and twelve asthmatic patients dosed by inhalation (4 mg). Multiple dose kinetic measurements were also made. 2. The intravenous data demonstrated that nedocromil sodium is a high clearance drug (10.2 +/- 1.3 ml min-1 kg-1). The data were fitted by a two compartment model with very rapid elimination from the central compartment (k10 = 0.088 +/- 0.021 min-1; beta = 0.013 +/- 0.002 min-1). 81% of the dose was excreted in the urine. 3. Oral absorption was low (2-3% of the dose) and contributed negligibly to the plasma profile after inhalation. 4. After inhalation of single doses of 4 mg in volunteers and patients plasma concentration rose rapidly, plateaued and then fell monoexponentially with a half-life of approximately 2 h. The data fitted a 'flip-flop' model with two absorption components. The extent of absorption was up to 6% of the dose with less in patients. 5. After multiple dosing with 4 mg four times daily for 7 days in volunteers negligible accumulation was observed. The pattern was similar in patients treated with 4 mg four times daily after 1, 6 and 12 months.

Administration, Inhalation↗