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Biomedical subjects

D Thomas

Publications and source records attributed to D Thomas.

At least 577 records · Page 32Linked to original sources
PubMed1991

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli.

The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A-beta-lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5 less than or equal to pH less than or equal to 6.0) and a low temperatures. These conditions were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn2+ ions.

Bacterial Proteins↗
PubMed1991

The synthesis of the two S-adenosyl-methionine synthetases is differently regulated in Saccharomyces cerevisiae.

S-adenosyl-L-methionine (AdoMet) is synthesized by transfer of the adenosyl moiety of ATP to the sulfur atom of methionine. This reaction is catalysed by AdoMet synthetase. In all eukaryotic organisms studied so far, multiple forms of AdoMet synthetases have been reported and from their recent study, it appears that AdoMet synthetase is an exceptionally well conserved enzyme through evolution. In Saccharomyces cerevisiae, we have demonstrated the existence of two AdoMet synthetases encoded by genes SAM1 and SAM2. Yeast, which is able to concentrate exogenously added AdoMet, is thus a particularly useful biological system to understand the role and the physiological significance of the preservation of two almost identical AdoMet synthetases. The analysis of the expression of the two SAM genes in different genetic backgrounds during growth under different conditions shows that the expression of SAM1 and SAM2 is regulated differently. The regulation of SAM1 expression is identical to that of other genes implicated in AdoMet metabolism, whereas SAM2 shows a specific pattern of regulation. A careful analysis of the expression of the two genes and of the variations in the methionine and AdoMet intracellular pools during the growth of different strains lead us to postulate the existence of two different AdoMet pools, each one supplied by a different AdoMet synthetase but in equilibrium with each other. This could be a means of storing AdoMet whenever this metabolite is overproduced, thus avoiding the degradation of a metabolite the synthesis of which is energetically expensive.

Blotting, Northern↗
PubMed1991

Doxorubicin plus ifosfamide with rhGM-CSF in the treatment of advanced adult soft-tissue sarcomas: preliminary results of a phase II study from the EORTC Soft-Tissue and Bone Sarcoma Group.

Doxorubicin and ifosfamide are the two most active agents used in the treatment of advanced inoperable soft-tissue sarcoma, but their use in combination produces dose-limiting myelosuppression. To explore the feasibility of combining optimal doses of both drugs, doxorubicin (75 mg/m2) and ifosfamide (5 g/m2) were given every 3 weeks with recombinant human granulocyte/macrophage-colony-stimulating factor (rhGM-CSF; 250 micrograms m-2 day-1) by subcutaneous injection for up to 14 days after each course. A total of 52 patients with progressive metastatic soft-tissue sarcoma were entered, none having received prior chemotherapy. One patient was ineligible and received no treatment after registration. Preliminary analysis of six cycles of chemotherapy revealed that the full protocol dose intensity had been administered to the majority of patients. Although the median leucocyte and neutrophil counts did not fall with subsequent courses of chemotherapy, the duration of neutropenia increased with each course delivered. Cumulative thrombocytopenia was a major dose-limiting toxicity and was the main reason for any dose modifications that occurred. Although 26 patients experienced infections after one or more courses of treatment, in only 7 was admission required for parenteral antibiotics. One patient died as a result of septicaemia after the first cycle of treatment. To date, there have been 22 responses (43%) with 8% complete remissions. It appears that the administration of rhGM-CSF allows this high-dose regime of chemotherapy to be given safely and the early encouraging response rate adds support to the concept that increasing the dose of doxorubicin improves the outlook for patients with advanced soft-tissue sarcomas.

Adult↗
PubMed1991

Comparison of polarographic and chemical measurements of oxygen uptake in complex media: the example of lipoxygenase reaction.

A polarographic method using a Clark oxygen electrode was used to assess oxygen concentrations in model and complex media before and during the lipoxygenase-catalyzed oxidation of linoleic acid to hydroperoxides. The results were in good agreement with those obtained with the chemical determination of dissolved oxygen. The electrode correctly responded when the dissolved oxygen concentration was decreased by the addition of water activity depressors (sorbitol, sucrose, glucose). The influence of the medium components on the gas solubility was discussed. The linear relationship between partial pressure of oxygen (determined by polarographic method) and the oxygen concentration (determined by chemical method) indicated that the Clark oxygen electrode can be used to study enzyme reactions consuming or evolving oxygen in non-Newtonian media.

Glucose↗
PubMed1991

Acidic fibroblast growth factor is present in regenerating limb blastemas of axolotls and binds specifically to blastema tissues.

The growth of regenerating limbs of amphibians depends upon proliferation of the blastema cells that accumulate beneath the epidermal cap. The epidermal cap is known to be mitogenic for the blastema cells. We have extracted a mitogenic activity from both the mesenchymal and epidermal (epidermal cap) components of cone stage blastemas which is retained on heparin-Sepharose and elutes with 1.15 M NaCl. This fraction stimulates neurite outgrowth of PC12 cells and [3H]thymidine incorporation into CCL 39 cells and is potentiated by heparin. The 2 M fraction was inactive. The heparin-Sepharose-purified growth factor cross-reacts with bovine acidic FGF polyclonal antibodies and shows a Mr of 16,000 on Western blots. Blastema membranes contain specific high affinity binding sites (Kd = 25 pM; capacity = 30 fmole/mg protein) and low affinity binding sites (Kd = 18 nM; capacity = 30 pmole/mg protein) for aFGF as revealed by Scatchard analysis. 125I-aFGF which is bound specifically by both the epidermal cap and mesenchyme of blastema frozen sections is displaced by an excess of unlabeled factor and inhibited by heparin. Heparinase treatment and 2 M NaCl washing which decreased the binding was fourfold more efficient for epidermal cap than for mesenchyme suggesting the presence of high affinity receptors in the latter tissue. The presence of aFGF (or a closely related molecule) in blastemas is consistent with our earlier results that showed stimulation of proliferation of cultured blastema cells by acidic or basic FGF or heparin alone. These results suggest the possibility that aFGF is stored in the epidermal cap during limb regeneration and that it stimulates the proliferation of the underlaying mesenchyme.

Ambystoma↗
PubMed1991

Fecal fat, cyclosporine, and alpha 1-antitrypsin for assessment of small bowel function following transplantation.

Many factors affect the integrity of transplanted small bowel. These include ischemic preservation and immunologic injury as well as the division of intestinal lymphatics during transplantation. This study was undertaken to evaluate the recovery of fat absorption in transplanted small bowel in syngeneic rats. Orthotopic transplantation of the total small bowel with resection of the native intestine was performed. The experimental (n = 11) and a pair-fed, sham-operated control (n = 8) groups were fed a 50% kcal corn oil semipurified diet. Studies of cyclosporine (CSA) absorption, maltose absorption, dietary fat, and fecal alpha 1-antitrypsin (FA1AT) excretion in transplanted animals were performed preoperatively and at 15, 30, and 50 days postoperatively. There was no significant difference in the weight change or fat and maltose absorption in experimental animals compared with control animals at any time point. Peak serum CSA levels were lower at day 15 in transplanted animals than in controls (P = 0.006) and improved but remained lower than those in controls at days 30 and 50 (P = 0.017). FA1AT excretion was increased on postoperative day 15 (accompanied by a decrease in body weight) and returned to control levels at days 30 and 50. Transplanted isogeneic rats had weight recovery and fat and carbohydrate absorption similar to those of controls. Transplanted animals had a protein-losing enteropathy measured by FA1AT at day 15 that resolved by 30 and 50 days, respectively. CSA absorption showed a much more gradual return to control levels and remained abnormal at 50 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

A pilot protein participates in the initiation of P22 procapsid assembly.

The gene 16 protein of the bacteriophage P22 is required as a pilot protein aiding the transfer of DNA from the phage into the Salmonella typhimurium host cell. During assembly 10-20 copies of the 63,000-Da gp 16 protein are incorporated into the procapsid shell prior to DNA packaging. The protein has been purified from isolated procapsids and behaved as a monomer in solution. Upon incubation with purified coat and scaffolding subunits in vitro, it assembled into procapsids with the correct stoichiometry. The addition of physiological quantities of gp 16 resulted in an increased rate of procapsid assembly. Sedimentation of mixtures of coat and gp 16 protein subunits revealed association/dissociation behavior. It is likely that the added gp 16 is acting to stabilize a transient oligomeric coat protein species that functions as the in vitro initiation complex for procapsid assembly.

Capsid↗
PubMed1991

Visualization of a luteovirus in the vector aphid's body by two gold immunolabelling techniques: a comparative study.

Two gold immunolabelling techniques using electron microscopy were compared to examine the in situ localization of a luteovirus, potato leafroll virus (PLRV), inside its main aphid vector, Myzus persicae SULZ. With Gildow's technique, virus particles were labelled prior to fixation, embedding by injecting PLRV-specific IgGs into the living aphids. This facilitated the detection of extracellular particles located between the basal lamina and plasmalemma by trapping them in aggregates. The heavy coating of particles by antibodies and gold indicated good labelling sensitivity. Isometric virus-like particles were also observed inside the cytoplasm, but they were non decorated because the cell membrane prevented labelling reagents from entering the cell. With the second technique, ultrathin sections were immunolabelled after fixation-embedding. Since PLRV lost its antigenicity when aphid tissues were normally treated for electron microscopy, the successful application of this technique required fixation in 4% formaldehyde before embedding in Lowicryl at low temperature; it was also necessary to use PLRV-specific monoclonal antibodies to eliminate non-specific reactions. In these conditions, all intra- and extra-cytoplasmic virions present on the surface of sections were surrounded by gold particles, but the antibody coating was not discernible, and, because the resin limited the access of markers to antigens, the inner virus particles were not labelled. In conclusion, both techniques must be applied on the same material to give complementary information.

Acrylic Resins↗
PubMed1991

Transoesophageal echocardiography in diagnosis of aberrant coronary artery.

Aberrant origin of the main stem of the left coronary artery from the right sinus of Valsalva caused angina of effort in a 40 year-old patient. Diagnosis was revealed by transoesophageal echocardiography and confirmed by selective coronary angiography. Aorto-coronary bypass was performed in order to prevent the recurrence of myocardial ischemia.

Adult↗
PubMed1991

Anomalous coronary arteries coursing between the aorta and pulmonary trunk: clinical indications for coronary artery bypass.

Coronary arteries of anomalous origin with subsequent coursing between the aorta and pulmonary trunk can cause ischaemia, infarction or sudden death. However, reports of surgical correction are sparse due to the rarity of ante-mortem diagnosis. We report two cases in which symptoms were related to anomalous origin of a non-atherosclerotic coronary artery. Surgical repair was performed to prevent sudden death or recurrent ischaemia.

Adult↗
PubMed1991

High-dose DTIC in advanced soft-tissue sarcomas in the adult. A phase II study of the E.O.R.T.C. Soft Tissue and Bone Sarcoma Group.

Forty-four of 50 adult patients with advanced soft-tissue sarcoma who had received previous chemotherapy were evaluable for response after treatment with DTIC. The therapeutic schedule consisted of DTIC 1.2 g/m2 infused over 20 minutes, and repeated every 3 weeks. There were 1 complete and 7 partial remissions (objective activity 18%, 95% C.I. 7%-29%), with a median duration of 8 weeks (range 5-19), with the complete remission lasting 1 yr. Hematologic toxicity was dose-limiting; W.H.O. greater than or equal to 3 values were observed for WBC in 36%, and for platelets in 26% of patients. The non-hematologic toxicity included nausea and vomiting (90%), a flu-like syndrome (49%), diarrhea (35%), pain in the infused vein (28%) and hypotensive episodes (4%). Intermittent high-dose DTIC is active in advanced soft-tissue sarcoma and should be considered for inclusion in combination regimens.

Adult↗
PubMed1991

Methadone levels and neonatal withdrawal.

The purpose of this study was to observe the effects of methadone exposure in utero, with special reference to maternal and neonatal methadone concentrations and neonatal withdrawal. Two groups of mother-infant pairs were studied. In the first group, serum methadone concentrations were determined in infants at 1, 6 and 24 h after delivery. In the second group, blood was obtained at 24, 48, 72 and 96 h after birth. There was no correlation between neonatal serum levels and the intensity of withdrawal symptoms. There was no relationship between maternal methadone dose at delivery or maternal serum levels and neonatal methadone levels. The results of this study may be complicated by the prenatal exposure of the neonates to other drugs of abuse apart from methadone.

Adult↗
PubMed1991

Roxithromycin treatment of mouse chlamydial salpingitis and protective effect on fertility.

We used a mouse model of acute chlamydial salpingitis to evaluate the efficacy of roxithromycin in preventing irreversible inflammatory damage leading to tubal infertility. Female C3H/He mice were genitally inoculated with a human strain of Chlamydia trachomatis and then treated with roxithromycin glutamate subcutaneously. Treatment was initiated either 7 or 10 days postinfection (p.i.) and continued for 7 days at a dosage of 50 or 100 mg/kg of body weight per 24 h. The course of the disease was monitored serologically, bacteriologically, and histologically. At the end of the treatment, the mice were encaged with males and their reproductive capacity was recorded over a 19-week period. The protective effect of roxithromycin was assessed in terms of fertility parameters in comparison with values for noninfected control mice. When treatment was initiated on day 7 p.i. and given in twice-daily 25-mg/kg doses, all the mice remained fertile and the total number of offspring was similar to that of sham-infected mice (17.3 +/- 3.3 versus 17.2 +/- 2.3). When treatment was initiated on day 10 p.i. and given in a single daily dose of 50 or 100 mg/kg, 90 and 70% of the mice, respectively, remained fertile; however, in terms of total offspring, fertility was lower in the group treated with the lower dose (5.6 +/- 1.4 versus 13.0 +/- 3.8). Roxithromycin was found to be effective against C. trachomatis in the mouse genital tract, but fertility was only partially preserved when the time between infection and treatment was prolonged.

Animals↗
PubMed1991

Evaluation of thromboxane production and complement activation during myocardial ischemia in patients with angina pectoris.

BACKGROUND: The complement system and arachidonic acid metabolites are involved in severe myocardial ischemia such as myocardial infarction. Furthermore, there is experimental evidence for C5a participation in thromboxane production. METHODS AND RESULTS: We examined whether C5a and thromboxane are produced during brief and reversible episodes of myocardial ischemia induced in patients with stable angina. Twenty-five patients underwent either atrial pacing or percutaneous transluminal coronary angioplasty associated with arterial and coronary sinus blood sampling. Rapid atrial stimulation of patients with effort angina caused significant ST segment depression (delta ST = -1.7 +/- 0.2 mm), decreased fractional lactate extraction (from +12.8 +/- 2.5% baseline to -13.7 +/- 4.6% at peak ischemia, n = 13, p less than 0.001), and increased coronary sinus plasma thromboxane B2 levels (from 345 +/- 85 pg/ml baseline to 1,684 +/- 64 pg/ml at peak ischemia, p less than 0.01). Changes of fractional lactate extraction correlated significantly with changes of coronary sinus plasma levels of thromboxane B2. There was no change of coronary sinus 6-keto-PGF1 alpha levels. Similar pacing of control subjects (n = 6) did not cause release of lactate or thromboxane. Seventeen other patients underwent exercise testing with noninvasive measurements of thromboxane and prostacyclin metabolites in urinary samples collected before and after the test. No detectable increase of urinary 11-dehydrothromboxane B2 was measured in patients with stable angina after exercise-induced myocardial ischemia. However, basal 11-dehydrothromboxane B2 levels were significantly higher in patients with angina (105 +/- 25 pg/mmol creatinine, n = 9) than in control patients (45 +/- 8 pg/mmol creatinine, n = 8, p less than 0.05 between groups). Coronary sinus plasma levels of the anaphylatoxin C5a always remained below 4 ng/ml in patients undergoing pacing. More severe myocardial ischemia after coronary angioplasty (percent lactate extraction decreased from +24.8 +/- 2.7% baseline to -41.6 +/- 22.4% at peak ischemia, p less than 0.05) was not associated with C3a or C5b-9 generation. In all patients, there was neither platelet sequestration nor platelet alpha-granule release (no changes of beta-thromboglobulin/platelet factor 4 levels) into the coronary sinus plasma. CONCLUSIONS: Patients with stable angina have chronically increased thromboxane synthesis as assessed by excretion of urinary metabolites. Thromboxane is acutely released into the coronary sinus during pacing-induced ischemia without significant intracoronary platelet aggregation. Complement does not appear to be activated in stable angina during brief and reversible episodes of myocardial ischemia and does not contribute to thromboxane production.

Angina Pectoris↗
PubMed1991

Transfer of training of the biofeedback-assisted control of hand temperature.

This study examined the effect of prior biofeedback-assisted training of hand warming or hand cooling on the subsequent ability to train the opposite response. A two-group crossover design employed 43 subjects with one group receiving three days of hand warming followed by three days of hand cooling, while the second group received the opposite sequence. Analysis indicated subjects were able to produce the appropriate changes in hand temperature and were more successful at hand cooling than hand warming. During the transfer of training phase, subjects with prior training in hand cooling were able to produce warming above baseline levels compared with subjects initially trained to hand warm. In contrast, prior training of hand warming had no effect on the subsequent ability to cool hands.

Adult↗