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Biomedical subjects

D Thomas

Publications and source records attributed to D Thomas.

At least 19 recordsLinked to original sources
PubMed1995

Src homologous and collagen (Shc) protein binds to F-actin and translocates to the cytoskeleton upon nerve growth factor stimulation in PC12 cells.

Immunoprecipitates of metabolically labeled PC12 cells consistently contained a 43-kDa protein that was associated with Shc, a signal-transducing protein with a single SH2 domain. Following affinity chromatography with immobilized recombinant glutathione S-transferase (GST)-Shc fusion protein, the 43-kDa protein was identified as actin by mass spectrometry and immunoblotting. Cosedimentation experiments using purified actin and GST-Shc showed that Shc binds directly to F-actin, confirming Shc-actin interaction in vivo. Various GST-truncated Shc fusion proteins were prepared and used in actin cosedimentation assays. Constructs containing the SH2 and collagen homology domains were not precipitated, and those containing the amino-terminal domain were. Thus, Shc-actin interactions do not occur in the region of tyrosine phosphorylation and leave the SH2 domain free to bind to other tyrosine-phosphorylated molecules. Although the major pool of Shc in unstimulated PC12 cells is soluble, two other pools are associated with the cytoskeleton and the submembranous cytoskeleton. Upon nerve growth factor stimulation, approximately 50% of the soluble Shc translocates to both cytoskeleton environments within 2 min, decreasing thereafter. When cells were pretreated with cytochalasin D, a drug that disrupts actin filaments, Shc translocation to the cytoskeleton was abolished. However, in the submembranous fraction, the Shc level was elevated in resting cells following cytochalasin D treatment. The kinetics of translocation, compared to mitogen-activated protein kinase activation, and the nature of the Shc-actin interaction suggest that the cytoskeletal association of Shc, induced by growth factors, may be related to membrane ruffling and actin fiber reorganization.

Actins↗
PubMed1995

Fibrinogen after coronary angioplasty as a risk factor for restenosis.

BACKGROUND: Fibrinogen is a risk factor for cardiovascular disease and is related to the severity of coronary atherosclerosis. Its role in restenosis after coronary angioplasty remains unknown. Although platelets and thrombosis contribute to the pathogenesis of restenosis, few clinical data are available concerning the relations between restenosis and proteins of the coagulation and fibrinolytic systems. METHODS AND RESULTS: In 107 consecutive patients undergoing coronary angioplasty, we measured plasma levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), von Willebrand factor, and fibrinogen before and immediately after angioplasty and at a 6-month follow-up. The individual changes of intraluminal diameter were measured by quantitative coronary angiography, and patients were classified according to four definitions of restenosis: (1) a final stenosis > 50%, (2) a loss of minimal luminal diameter during the follow-up period greater than the measurement variability in our laboratory (> 0.52 mm), (3) a loss of at least 50% of the gain in luminal diameter achieved by angioplasty, and (4) the combination of definitions 1 and 2. The relations between coagulation variables and each definition of restenosis were assessed univariately; then with the clinical variables included, the relations were analyzed multivariately. Angiographic follow-up was obtained in 92% of patients with a primary success of angioplasty. Global restenosis rates were 38%, 43%, 48%, and 30% for definitions 1 through 4, respectively. Plasma levels of t-PA antigen and PAI-1 antigen were not associated with any of the four definitions of restenosis. Multivariate analysis demonstrated that von Willebrand factor measured immediately after angioplasty predicted restenosis according to definitions 2 and 3. Fibrinogen measured within 6 months of follow-up was significantly increased in all restenosis groups of the four definitions. Patients with a fibrinogen concentration > 3.5 g/L at follow-up had higher restenosis rates than patients with a concentration < 3.5 g/L: 55% versus 22% (P = .001), 68% versus 31% (P = .002), 63% versus 37% (P = .01), and 74% versus 26% (P = .002) for definitions 1 through 4, respectively. The loss index was lower (P = .003) and the net gain higher (P = .03) in patients with a fibrinogen level < 3.5 g/L. There was a significant correlation between fibrinogen level and angiographic loss index (r = .41; P < .0001). Multivariate analysis confirmed that the fibrinogen level predicted restenosis with all definitions. CONCLUSIONS: An independent relation exists between von Willebrand factor measured immediately after angioplasty and restenosis defined by the degree of intraluminal renarrowing. An elevated fibrinogen level during follow-up is a strong biochemical predictor of restenosis. Therefore, fibrinogen should be considered at least as an independent marker of restenosis and perhaps as a common risk factor for both spontaneous coronary atherosclerosis and postangioplasty restenosis, which is an accelerated form of atherosclerosis.

Angioplasty, Balloon, Coronary↗
PubMed1995

Identification of the yeast methionine biosynthetic genes that require the centromere binding factor 1 for their transcriptional activation.

The yeast Centromere binding factor I (Cbf1) belongs to the family of the DNA binding factors that recognize the consensus sequence CACGTG. Phenotypic studies of cells lacking Cbf1 revealed that this factor is actually involved in two cellular processes; the fidelity of the chromosomal segregation and the metabolism of sulfur amino acids. However, the function of Cbf1 in the regulation of the sulfur amino acid metabolism is now a matter of controversy in literature with conflicting reports about its binding to the CACGTG sequences found upstream to the methionine biosynthetic genes. To provide a reliable basis for the functional analysis of Cbf1, we present an analysis of the transcription of the methionine biosynthesic genes in cells lacking Cbf1. Our results prove that Cbf1 is indeed involved in the transcriptional regulation of the sulfur amino acid metabolism.

Base Sequence↗
PubMed1995

Chromatographic resolution of an intracellular calcium influx factor from thapsigargin-activated Jurkat cells. Evidence for multiple activities influencing calcium elevation in Xenopus oocytes.

Acid extracts of thapsigargin-stimulated Jurkat cells revealed both intracellular and extracellular activities stimulating Ca(2+)-dependent Cl- currents on Xenopus laevis oocytes. Chromatographic fractionation of these extracts on gel filtration separated two active fractions of M(r) approximately 600 and 400. Moreover, the M(r) 600 fraction exhibited both intracellular and extracellular activities. However, the intracellular activity was absent from extracts of unstimulated Jurkat cells, suggesting its production was stimulated by thapsigargin. The further purification of this fraction by high performance thin layer chromatography resolved a single fraction which was active only on microinjection and which required calcium entry for activation of current responses. These results suggest that a single authentic calcium influx factor can be resolved by purification from confounding activities detected in crude acid extracts.

Animals↗
PubMed1995

Evaluation of calcium influx factors from stimulated Jurkat T-lymphocytes by microinjection into Xenopus oocytes.

Acid extracts of thapsigargin-activated Jurkat cells have been shown to have intracellular activity in inducing a dose-dependent rapid chloride current upon microinjection in Xenopus laevis oocytes. The extracts act by elevation of calcium through calcium entry. The factor(s) responsible for this activity have been termed calcium influx factor (CIF) and have been found to be small, relatively polar molecules (< 1000 daltons) whose activity is abolished by alkaline phosphatase treatment and potentiated by co-injection of okadaic acid (a protein phosphatase inhibitor). CIF is produced in a time-dependent manner following thapsigargin treatment of Jurkat cells, being first elevated above basal levels by 2 min. Intracellular CIF activity is completely absent from NG115-401L neuronal cells, which lack capacitative entry. On this basis, it appears that Jurkat cells, activated by stimuli that deplete internal calcium stores, produce one or more CIF activities acting intracellularly, and Xenopus oocytes may be a powerful tool to purify and characterize CIFs.

Animals↗
PubMed1995

[Evaluation of the coronary risk in sclerotic arterial diseases of the lower limbs].

The prevalence of coronary artery disease is very high among patients with peripheral arterial disease. The fate of these patients mainly depends on their coronary risk. Most often, the evaluation of coronary risk is performed when a surgical intervention on the aorta or lower limb arteries is decided. However, the late risk, at medium and long term, of coronary disease is much higher than its immediate perioperative risk. Diagnostic work-up is complex. Symptoms of coronary insufficiency may be masked by the limited capacity to physical exercise, but remain essential indices. None of the available complementary investigations has a satisfactory predictive value. Diagnostic explorations and therapeutic decisions for coronary heart disease have to be discussed in each case, according mainly to age and symptoms, and to whether peripheral arterial surgery is required.

Arterial Occlusive Diseases↗
PubMed1995

Bone progenitor cell deficits and the age-associated decline in bone repair capacity.

Aging bone shows a progressive decline in mass and strength. Previous studies have suggested that bone marrow stem cells are reduced with aging and that this could be responsible, in part, for age-associated bone deficits. We measured the number of osteoprogenitor cells present in the bone marrow from adult and aged rats as well as their ability to differentiate in vitro and to form bone in vivo. We found that the number of adherent colony-forming cells was significantly lower (65%) in marrow cells isolated from aged compared with adult rats. Furthermore, 88% of the colonies obtained from aged rats were alkaline phosphatase (AP) positive, whereas virtually all the colonies from adult rats were positive. The addition of dexamethasone to the culture medium decreased the proliferation of the adherent cells and reduced the number of colonies obtained from both adult and aged bone marrow, all of which were AP positive. No significant differences were found in the expression of certain major bone cell marker genes as a function of donor age. However, dexamethasone treatment increased expression of osteopontin (OP) by fivefold. Adult stromal cells not treated with dexamethasone and implanted subcutaneously in recipient rats exhibited about 10-fold greater formation of bone compared with cells from aged rats. In contrast, dexamethasone-treated cells exhibited high levels of bone formation, irregardless of donor age or the age of the recipient into which the cells were grafted. These studies are consistent with a deficit of osteoprogenitor cells in the bone marrow site as a contributing, perhaps correctable factor in the decline in bone repair and bone mass with age.

Aging↗
PubMed1995

Stability of reamed and unreamed intramedullary tibial nails: a biomechanical study.

We examined the correlation of bone mineral density and bone strength in the cadaver tibia, and looked in vitro at the relative stability of tibial fractures fixed with either reamed or unreamed tibial nails. Bone-mineral density correlated well with bone strength (r = 0.946), but paired tibias did not correlate closely. The unreamed nail-bone construct was less stable than the reamed construct in each pair tested (P < 0.05), and a comparison of all bones showed it to be less stable at all levels of our testing regimen, including failure (P < 0.01). Bending and breakage was seen in four of the smaller unreamed interlocking screws (4 mm).

Biomechanical Phenomena↗
PubMed1995

Buprenorphine increases intake of freely available and operant-contingent food in satiated rats.

Opiate administration increases short-term free feeding in satiated rats. The feeding effects of the mixed opioid receptor agonist/antagonist buprenorphine were examined in both free-feeding and operant chamber paradigms. Buprenorphine (0.1 and 0.3 mg/kg) produced significant increases in short-term free feeding (i.e., 4 h), an effect enhanced by repeated administration. Buprenorphine's effects on operant responding were examined in satiated rats using a fixed ratio (FR) 80 (initial pellet) FR 3 (subsequent pellets) reinforcement schedule. Buprenorphine (0.03-0.3 mg/kg) decreased latency to begin responding for food, and increased total number of pellets consumed in a 1-h session. Increases in food intake relative to control were caused by continued responding for food as sessions progressed. Naloxone suppressed both the free-feeding and operant-contingent intake induced by buprenorphine. Thus, buprenorphine increases both freely available and lever-press contingent food intake.

Animals↗
PubMed1995

Screening for colorectal cancer: what are the costs?

We examined a screening program for colorectal cancer in South Australia in terms of its overall direct costs to society and costs to participants. The best estimate of the cost per cancer detected was $18,924 (Australian dollars). Potential improvements in health outcome through screening are discussed in light of these costs.

Colorectal Neoplasms↗
PubMed1995

Expression of retinoblastoma gene product and p53 protein in bladder carcinoma: correlation with Ki67 index.

OBJECTIVES: To determine whether loss of the tumour suppressor gene retinoblastoma (Rb) and increased expression of the p53 protein were associated with increased tumour cell growth fraction. PATIENTS AND METHODS: Tumours from 105 patients (72 men, 33 women; median age 69 years, range 35-89) with newly diagnosed primary transitional cell bladder carcinoma were studied. Tumour samples were taken by means of cystoscopic resection. Expression of the retinoblastoma (Rb) and p53 gene products was assessed immunohistochemically in 98 of the carcinomas. The proportion of cells expressing the Ki67 antigen (Ki67 index which is a measure of growth fraction) was determined in 64 cases. RESULTS: p53 protein was detectable in 50% and Rb protein in 82% of the tumours. Staining for p53 and lack of staining for Rb protein were associated with muscle-invasive growth and high tumour grade (G3). The Ki67 index varied over a wide range (1-47%), but there were significant differences between mean indices for poorly differentiated (G3) and well or moderately differentiated (G1/G2) tumours, and between indices for muscle-invasive and the remaining tumours. The mean Ki67 indices for Rb-negative tumours and p53-positive tumours were approximately twice those for Rb-positive and p53-negative tumours. Only 10% of the tumours expressed high levels of p53 protein and failed to express Rb. CONCLUSION: These observations are consistent with the hypothesis that loss of Rb and mutation and overexpression of p53 are associated with an increased tumour cell growth fraction and that such changes may play a role in the de-regulation of cell proliferation in transitional cell carcinoma of the bladder.

Adult↗
PubMed1995

Activity of synchronized cells of a steady-state biofilm recirculated reactor during xenobiotic biodegradation.

The maintenance of a steady-state biofilm in a continuous-flow fixed-bed reactor, as a consequence of the reproduction-detachment of cells (an interfacial cell physiology phenomenon of steady-state biofilm) during the biodegradation of 2,4,6-trichlorophenol by Pseudomonas cells, was determined. After cell adhesion on an open-pore glass support, the biofilm was formed in a packed-bed recirculated reactor. After the steady-state biofilm was reached, the mechanisms of the interfacial cell detachment (at the biofilm-liquid interface) were determined. It was established that (i) the hydrophobicity of immobilized sessile cells (parent cells) increased (from 50 to 80%) as the dilution rate increased, while the hydrophobicity of detached suspended cells (daughter cells) remained constant (about 45%); and (ii) the immediately detached suspended cells showed a synchronized growth in about three generations. These results indicate that (i) the immobilized sessile and suspended detached cells grew synchronically at the end and at the beginning of the cell cycle, respectively; and (ii) the hydrophobicity difference of immobilized sessile and suspended detached cells permitted the cells detachment. Therefore, it is probable that independent of shear stress (due to recirculated flow), the synchronized growth and hydrophobicity of cells (which vary during the cell cycle) are the main factors permitting the maintenance of a steady-state xenobiotic-degrading biofilm reactor (in which the overall accumulation of biofilm is determined by the average growth rate of the biofilm cells minus the rate of detachment of cells from the biofilm).

Bacterial Adhesion↗
PubMed1995

Functional analysis of Met4, a yeast transcriptional activator responsive to S-adenosylmethionine.

Transcription of the genes necessary for sulfur amino acid biosynthesis in Saccharomyces cerevisiae is dependent on Met4, a transcriptional activator that belongs to the basic region-leucine zipper protein family. In this report, we show that one mechanism permitting the repression of the sulfur network by S-adenosylmethionine (AdoMet) involves inhibition of the transcriptional activation function of Met4. Using a wide array of deleted LexA-Met4 fusion proteins as well as various Gal4-Met4 hybrids, we identify the functional domains of Met4 and characterize their relationship. Met4 appears to contain only one activation domain, located in its N-terminal part. We demonstrate that this activation domain functions in a constitutive manner and that AdoMet responsiveness requires a distinct region of Met4. Furthermore, we show that when fused to a heterologous activation domain, this inhibitory region confers inhibition by AdoMet. Met4 contains another distinct functional domain that appears to function as an antagonist of the inhibitory region when intracellular AdoMet is low. On the basis of the presented results, a model for intramolecular regulation of Met4 is proposed.

Basic-Leucine Zipper Transcription Factors↗
PubMed1995

Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA-binding and transcriptional activities of NF-kappa B.

The transcription factor NF-kappa B is exploited by many viruses, including the human immunodeficiency virus, for expression of viral genes, but its primary role appears to be in the rapid induction of cellular genes during immune and inflammatory responses. The inhibitor protein I kappa B alpha maintains NF-kappa B in an inactive form in the cytoplasms of unstimulated cells, but upon cell activation, I kappa B alpha is rapidly degraded, leading to nuclear translocation of free NF-kappa B. However, NF-kappa B-dependent transcription of the I kappa B alpha gene leads to rapid resynthesis of the I kappa B alpha protein and inhibition of NF-kappa B-dependent transcription. Here we demonstrate a new regulatory function of I kappa B alpha exerted on NF-kappa B in the nuclear compartment. Although normally found in the cytoplasm, I kappa B alpha, newly synthesized in response to tumor necrosis factor or interleukin I, is transported to the nucleus. In the nucleus I kappa B alpha associates with the p50 and p65 subunits of NF-kappa B, inhibiting DNA binding of the transcription factor. Furthermore, nuclear expression of I kappa B alpha correlates with transcription termination of transfected NF-kappa B-dependent luciferase genes. Following the appearance of I kappa B alpha in the nuclei of activated cells, a dramatic reduction in the amount of nuclear p50 occurs, suggesting that NF-kappa B-I kappa B alpha complexes are cleared from the nucleus.

Cell Nucleus↗