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Biomedical subjects

S Bradley

Publications and source records attributed to S Bradley.

At least 19 recordsLinked to original sources
PubMed1991

Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins.

The C-terminal catalytic domain (residues 704-1047) of the human ras GTPase-activating protein (GAP) has been engineered so as to incorporate the tripeptide, Glu-Glu-Phe, at its C terminus. This motif is recognized by the commercially available YL1/2 monoclonal antibody to alpha-tubulin and has previously been used for the immunoaffinity purification of HIV enzymes engineered to contain this epitope (Stammers, D. K., Tisdale, M., Court, S., Parmar, V., Bradley, C., and Ross, C. K. (1991) FEBS Lett. 283, 298-302). The engineered GAP catalytic domain (GAP-344) was obtained in high yield and purity from Escherichia coli extracts by means of a single affinity column of immobilized YL1/2, eluted under mild conditions with the dipeptide, Asp-Phe. The protein had similar activity to that previously described for full-length GAP, suggesting that the addition of the epitope did not grossly affect the activity. R903K and L902I mutants of GAP-344 were constructed, and the immunoaffinity purification procedure allowed their rapid characterization. The R903K mutant had less than 3% the activity of the normal protein, whereas the L902I substitution had less than 0.5% of normal activity, suggesting an important role for Leu-902 and Arg-903, residues absolutely conserved among GAP-related proteins. This work exemplifies the general utility of the C-terminal Glu-Glu-Phe motif for the rapid purification of proteins whose function is not altered by C-terminal modification.

Amino Acid Sequence↗
PubMed1991

Characterization of recombinant human Kirsten-ras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems.

Kirsten-ras is the oncogene most frequently activated in human tumors. Studies of its biological function have been limited by the nonavailability of significant amounts of the major protein product, Kirsten-ras (4B) p21. When expressed in Escherichia coli K12, the recombinant protein was rapidly cleaved upon cell lysis in the lysine-rich C terminus region, probably by the ompT protease. However, soluble full-length protein was obtained when the Kirsten-ras gene was expressed in an E. coli strain lacking the ompT gene, and also in a baculovirus/insect cell expression system. Additionally, the baculovirus/insect cell system produced about half of the Kirsten-ras protein in a membrane-associated form, which was post-translationally modified by polyisoprenylation and carboxyl-methylation. A C-terminally truncated form (residues 1-166) was also expressed at high levels in E. coli for x-ray crystallographic studies. The kinetics of GDP release and of GTP hydrolysis of the purified proteins are similar to those of the corresponding Harvey-ras proteins, though there are small differences in the relative affinities for GDP and GTP. Biological activity of full-length Kirsten Val-12 p21 was demonstrated by microinjection into Swiss 3T3 cells, resulting in morphological transformation, with a lower potency than that of Harvey Val-12 protein.

Animals↗
PubMed1990

Reduced-size lung transplantation in neonatal swine: technique and short-term physiological response.

Lung transplantation is now a clinical reality in adults but is limited by the scarcity of appropriate donors. The donor shortage is even more acute for neonatal and pediatric patients. Reduced-size lung grafts would expand the pool of appropriate cadaveric donors and allow HLA-matched living related lobar or segmental lung transplants. To evaluate this experimentally, we developed a model of pulmonary lobar transplantation in neonatal pigs and studied the acute hemodynamic response after transplanting the left lower lobe from a more mature donor to a neonatal recipient. Technical considerations included using the recipient atrial appendage for the pulmonary venous anastomosis. Nine pairs of pigs underwent left lateral thoracotomy. The recipient left atrial and pulmonary arterial pressures, cardiac output, and pulmonary vascular resistance were measured before pneumonectomy and after left lower lobe transplantation. Although the left atrial and pulmonary arterial pressures remained unchanged after transplantation, there was a 15% increase in pulmonary vascular resistance and a 23% reduction in cardiac output. Neither change was statistically significant. The distribution of blood flow through the left and right pulmonary arteries was unchanged after transplantation. We conclude that lobar transplantation is technically feasible in immature animals and that the pulmonary venous anastomosis to the left atrial appendage facilitates the procedure. This model may prove useful in studying lung transplantation in immature recipients and expedite implementation of reduced-size lung transplantation in neonatal and pediatric patients.

Anastomosis, Surgical↗
PubMed1990

Complete heart block in fetal lambs. I. Technique and acute physiological response.

Prenatally diagnosed complete heart block (CHB), without associated cardiac anomalies, is often refractory to attempts to increase heart rate and cardiac output by medical therapy, and results in fetal death. We developed a model of CHB in fetal lambs in order to evaluate the acute physiological consequences of CHB and to assess the feasibility of fetal cardiac ventricular (V) and atrioventricular (AV) pacing. After hysterotomy and fetal sternotomy AV-sequential pacing leads were placed on the right atrium and ventricle (RV). Electromagnetic flow transducers were placed around the pulmonary artery and the ascending aorta to measure combined ventricular output (CVO) in normal sinus rhythm (NSR), CHB, AV, and V pacing. CHB block was successfully induced in 19 of 21 (90%) fetal lambs at 100 to 110 days' gestation by injection of formalin into the interventricular septum. Two fetuses were refractory to CHB despite multiple injections. The mean ventricular escape rate was 52 +/- 3 (+/- SEM) beats/min (range, 45 to 60 beats/min). CHB lasted for the period of study (1 to 8 hours), and spontaneously reverted to NSR in one fetus after 3 hours. Combined ventricular output in NSR was 470 +/- 31 mL/min (+/- SEM), which fell to 213 +/- 13 mL/min in CHB, 45% of normal CVO (P less than .0001). CVO during AV-sequential and V pacing was 77% (P = .02) and 62% (P less than .001), respectively, of combined ventricular output in NSR. Both RV (27%) and left ventricles (LV) (37%) showed compensatory increase in stroke volume during CHB.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Relation of global ratings of functioning with behaviour and development in delayed and disturbed preschoolers.

Ratings on the Global Assessment Scale for Children (GASC) were assessed in relation to a battery of objective developmental and behavioural measures. Children aged three to six years (N = 55) attending a day treatment program for developmental delays, language disorders, and behavioural and emotional problems comprised the sample. A battery of tests was administered to the children. Parents and teachers completed standardized questionnaires to measure developmental, behavioural and emotional problems. The results indicated that the GASC score correlated significantly with child developmental level, impulse control, and with teacher ratings of social withdrawal. The GASC was unrelated to ratings of behavioural problems. Ratings on the GASC also were highly consistent over time. These results are discussed with respect to the strengths and limitations of using global ratings with preschool children.

Affective Symptoms↗
PubMed1988

The purification and properties of two soluble reduced nicotinamide: acceptor oxidoreductases from Trichomonas vaginalis.

The occurrence of soluble reduced nicotinamide nucleotide:acceptor oxidoreductases has been reported in a number of strains of the oxygen-tolerant anaerobe Trichomonas vaginalis and other trichomonad species. The quantitatively more important enzyme in most strains of T. vaginalis is an NADH oxidase which produces water from the reduction of oxygen. This enzyme has been purified by a combination of gel filtration, chromatofocusing, Cibacron Blue chromatography and high pressure gel permeation chromatography. It is a monomeric protein with an estimated molecular mass from sodium dodecyl sulphate gel electrophoresis of 98 kDa; an isoelectric point of approximately pH 5.5 and a Km for NADH of 5.4 microM. The purified NADH oxidase is significantly inactivated during turnover under air (t1/2 3.65 min) and rapidly inactivated by microM levels of hydrogen peroxide. The NADPH-dependent minor activity requires a flavin. It has been partially purified by gel filtration and chromatofocusing. The apparent molecular mass of this enzyme is 36 kDa by gel filtration; it has an isoelectric point of approximately pH 5.2 and Km values for NADPH and FMN of 16.6 microM and 6.1 microM respectively. The product of oxygen reduction by this enzyme, using FMN as acceptor is hydrogen peroxide. The possible role of these two enzymes in the cell and their affinity with related enzymes from other organisms is discussed.

Animals↗
PubMed1988

Light-induced conductivity changes of purple membrane suspensions in strong electrolytes.

Measurements have been made of the modulated light-induced changes in conductivity and the associated relaxation times of bacteriorhodopsin in a variety of strong electrolytes, both unbuffered and buffered. The effects of pH and temperature variation have been studied as well as the effect of adding valinomycin. Two relaxation times can be distinguished: a fast lifetime associated with protonation-deprotonation, and a slow lifetime associated with ion binding. The ion-binding effects appear to be cation specific.

Bacteriorhodopsins↗
PubMed1988

A randomised controlled trial of adjuvant hormono-chemotherapy in Stage II breast cancer.

From 1979 to 1984 women with histologically proven Stage II breast cancer were entered into a randomised controlled trial of adjuvant hormono-chemotherapy following mastectomy. Adjuvant therapy was started within 2 weeks of surgery and was given for 6 months as continuous 'Tamoxifen' and 6-monthly cycles of vincristine, adriamycin and cyclophosphamide (VAC). Of the 157 women randomised 82 were assigned to the control group and 75 to the adjuvant group. Comparing the two groups by life table analysis the rate of recurrence was found to be significantly greater in the control group (n = 46) than in the adjuvant group (n = 25) (Peto log rank test chi 2 = 7.46 P less than 0.01). Although there were more deaths amongst the control patients (n = 31) than in those randomised to receive adjuvant therapy (n = 18), this difference did not achieve statistical significance (chi 2 = 2.80 P less than 0.1). This analysis has shown that although adjuvant hormono-chemotherapy may significantly delay recurrence the trial has failed to demonstrate any significant improvement in the survival of women with Stage II breast cancer following mastectomy.

Adult↗
PubMed1987

Surgical wound infections documented after hospital discharge.

Shorter lengths of hospitalization may result in more surgical wound infections being documented after hospital discharge. The current investigation analyzed 1644 surgical procedures performed over a 3-month period, and documented surgical wound infections both before and for 1 month after hospital discharge. Physician and patient questionnaires were used. One hundred eight infections were noted, of which 50 (46%) were seen after hospital discharge by either the patient or the surgeon. Rates of infection were 5.2%, 7.5%, and 7.5% for clean, clean-contaminated, and contaminated-dirty categories, respectively. Had postdischarge surveillance not been used, rates would have appeared to be 2.5%, 6.5%, and 6.8% for the same surgical classes. Infections following clean and clean-contaminated procedures were more likely to be noticed after hospital discharge. Excluding those that were patient-documented, wound infection rates would have been 4.2% (clean), 6.3% (clean-contaminated) and 6.8% (contaminated-dirty). Postdischarge surveillance is imperative to meaningfully document true rates of surgical wound infection, inasmuch as increasing numbers are likely to occur only after patients leave the hospital.

Follow-Up Studies↗
PubMed1985

A comparison of infections in different ICUs within the same hospital.

Infections identified between 1981 and 1983 in a hospital's medical/surgical, pediatric, neonatal, coronary care, and cardiac surgery ICUs were compared. Among 14,360 admissions, 1840 infections occurred in 1360 patients. Total infection rates ranged from 1.0% (cardiac surgery ICU) to 23.5% (medical/surgical ICU). Rates of ICU-acquired infection ranged from 0.8% (cardiac surgery ICU) to 11.2% (medical/surgical ICU), indicating that only about half of infections in the latter unit were acquired from within. Primary bacteremias comprised 14.5% of neonatal ICU infections, a rate 500% higher than in other ICUs. Meningitis and genitourinary infections were more common in pediatric and coronary care ICUs. Candida and Pseudomonas species and Klebsiella-Enterobacter-Serratia were most common in the medical/surgical ICU. Survival rate of infected patients was over 87% in pediatric and neonatal ICUs, compared with only 55.4% in the medical/surgical ICU. These differences in types and rates of infection have an important bearing on infection-control activities in the ICU, and also provide a yardstick against which similar institutions can gauge their ICU infection status.

Bacteria↗