Biomedical subjects
Publications and source records attributed to D Thomas.
Transcription factors are often targeted for rapid degradation by the ubiquitin-proteasome system. Recent evidence points to a correlation between the potency and instability of transcriptional activators, suggesting a possible direct role for ubiquitin-dependent proteolysis in transcriptional activation.
Progression through the cell cycle requires the coordination of basal metabolism with the cell cycle and growth machinery. Repression of the sulfur gene network is mediated by the ubiquitin ligase SCF(Met30), which targets the transcription factor Met4p for degradation. Met30p is an essential protein in yeast. We have found that a met4Deltamet30Delta double mutant is viable, suggesting that the essential function of Met30p is to control Met4p. In support of this hypothesis, a Met4p mutant unable to activate transcription does not cause inviability in a met30Delta strain. Also, overexpression of an unregulated Met4p mutant is lethal in wild-type cells. Under non-permissive conditions, conditional met30Delta strains arrest as large, unbudded cells with 1N DNA content, at or shortly after the pheromone arrest point. met30Delta conditional mutants fail to accumulate CLN1 and CLN2, but not CLN3 mRNAs, even when CLN1 and CLN2 are expressed from strong heterologous promoters. One or more genes under the regulation of Met4p may delay the progression from G(1) into S phase through specific regulation of critical G(1) phase mRNAs.
BACKGROUND: Patients with mechanical heart valves require life-long anticoagulation. We report here the first large and comparative series of consecutive patients anticoagulated with low molecular weight heparin (LMWH) after mechanical heart valve replacement. METHODS AND RESULTS: In this comparative, nonrandomized study, 208 consecutive patients who underwent a single or double heart valve replacement with mechanical prostheses were anticoagulated subcutaneously with unfractionated heparin (UH) in the first period (n=106) and LMWH in the second phase (n=102) of the study. Baseline characteristics were similar in the 2 groups. The mean durations of UH and LMWH treatments were 13.6+/-0.5 and 14.1+/-0.6 days, respectively (not significant). On the second day of treatment, 87% of patients treated with LMWH had an anti-Xa activity within the range of efficacy (0.5 to 1 IU/mL), but only 9% of UH-treated patients had an activated partial-thromboplastin time value within the therapeutic range (1.5 to 2.5 times control, P<0.0001 between the 2 groups). On the last day of prescription, all LMWH-treated patients had anti-Xa activity above 0.5 IU/mL, but 19% were above 1 IU/mL. In the UH group, 27% of patients had an activated partial-thromboplastin time above 1.5 times control, but 62% were overanticoagulated. Two major bleedings occurred in each group, and one stroke occurred in the UH group. CONCLUSIONS: In this first comparative study, anticoagulation with LMWHs after mechanical heart valve replacement appears feasible, provides adequate biological anticoagulation, and compares favorably with UH anticoagulation. Randomized studies are now needed to further evaluate this new therapeutic approach.
Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and developmental processes, including glycogen metabolism, muscle contraction, and the cell cycle [1] [2] [3] [4] [5]. Drosophila and humans both have multiple genes encoding PP1 isoforms [3] [6] [7]; each has one beta and several alpha isoform genes (alpha(1), alpha(2), alpha(3) in flies, alpha and gamma in humans; mammalian PP1beta is also known as PP1delta). The alpha/beta subtype differences are highly conserved between flies and mammals [6]. Though all these proteins are >85% identical to each other and have indistinguishable activities in vitro, we show here that the Drosophila beta isoform has a distinct biological role. We show that PP1beta9C corresponds to flapwing (flw), previously identified mutants of which are viable but flightless because of defects in indirect flight muscles (IFMs) [8]. We have isolated a new, semi-lethal flw allele that shows a range of defects, especially in muscles, which break away from their attachment sites and degenerate.
Clinical studies demonstrated a reduction of acute complications by high-pressure stenting. This study was performed to correlate the histomorphologic changes of the vessel wall after coronary stenting with stent expansion pressure. We studied the effects of intravital and postmortem stenting on coronary morphology in human hearts. Artifact-free analysis and morphometry of the artery segments' cross section was performed after plastic resin embedding and cutting and grinding sectioning. By comparing intra- and postmortem findings we demonstrated that postmortem stent implantation can serve as an adequate model to study the mechanical effects of coronary stenting. A consistent histologic feature was eccentric stent expansion. Larger calcified areas of the vessel wall were not deformed by implanted stents. The highest degree of vessel injury and deformation was apparent in anatomically "nondiseased" or only slightly fibrotic parts of the arterial wall. Dissections were predominantly located directly adjacent to calcified plaques and appeared as "half-moon"-like tears reaching into the arterial media. A statistically significant stent lumen gain was found when the implantation pressure was increased up to 15 atm. Stent symmetry was not influenced by the applied implantation pressure but depended mostly on local coronary morphology. Thus, increasing implantation pressures during coronary stenting seemed to improve the stenting result up to 15 atm. When applying histomorphologic criteria, the higher pressures (>15 atm) did not cause further optimization of stent expansion. Morphometric analysis of stents implanted postmortemly and intravitally revealed comparable results. Postmortem stenting seems to be an appropriate model for studying stent expansion and stenting results in human coronary arteries.
OBJECTIVES: To assess the contribution of expiratory flow limitation (FL) in orthopnea during acute left heart failure (LHF). BACKGROUND: Orthopnea is typical of acute LHF, but its mechanisms are not completely understood. In other settings, such as chronic obstructive pulmonary disease, dyspnea correlates best with expiratory FL and can, therefore, be interpreted as, in part, the result of a hyperinflation-related increased load to the inspiratory muscles. As airway obstruction is common in acute LHF, postural FL could contribute to orthopnea. METHODS: Flow limitation was assessed during quiet breathing by applying a negative pressure at the mouth throughout tidal expiration (negative expiratory pressure [NEP]). Flow limitation was assumed when expiratory flow did not increase during NEP. Twelve patients with acute LHF aged 40-98 years were studied seated and supine and compared with 10 age-matched healthy subjects. RESULTS: Compared with controls, patients had rapid shallow breathing with slightly increased minute ventilation and mean inspiratory flow. Breathing pattern was not influenced by posture. Flow limitation was observed in four patients when seated and in nine patients when supine. In seven cases, FL was induced or aggravated by the supine position. This coincided with orthopnea in six cases. Only one out of the five patients without orthopnea had posture dependent FL. Control subjects did not exhibit FL in either position. CONCLUSIONS: Expiratory FL appears to be common in patients with acute LHF, particularly so when orthopnea is present. Its postural aggravation could contribute to LHF-related orthopnea.
Turbulence is significantly reduced in a tokamak plasma as a result of neon seeding of an L-mode discharge. Correspondingly, confinement is improved and cross-field ion thermal transport reduced. Fully saturated turbulence in the range 0.1</=k( perpendicular)rho(s)</=0. 6 is measured at rho = 0.7 and exhibits a factor of 5 reduction in total power after neon injection, with almost complete suppression for k( perpendicular)rho(s)>0.35. These observations are consistent with a reduction in the calculated linear growth rate for k( perpendicular)rho(s)>0.5 and an increase in the measured ExB flow shearing rate.
Explore the source record for details and available documents.
The measles virus (MV) P gene encodes three proteins: the P protein and two nonstructural proteins, C and V. Because the functions of both the C and V protein are unknown, we used MV C (C-) and V (V-) deletion recombinants generated by the MV reverse genetics system (F. Radecke, P. Spielhofer, H. Schnieder, K. Kaelin, M. Huber, C. Dotsch, G. Christiansen, and M. A. Billeter 1995. EMBO J. 14, 5773-5784). Compared to parental vaccine strain, Edmonston (Ed) MV, both had normal growth and cytopathic effects in Vero cells and showed similar growth kinetics in human neuroblastoma SK-N-MC cells and in primary mouse neurons expressing the MV receptor, CD46. However, in vivo, using YAC-CD46 transgenic mice as a model for MV induced CNS disease (M. B. A. Oldstone, H. Lewicki, D. Thomas, A. Tishon, S. Dales, J. Patterson, M. Manchester, D. Homann, D. Naniche, and A. Holz 1999. Cell 98, 629-640), C- and V- viruses differed markedly from wt Ed(V(+)C(+)) virus. Newborn mice inoculated with as little as 10(3) PFU of Ed strain became ill and died after 10-15 days. In contrast, those inoculated with 10(3) or 10(4) PFU of MV C- or MV V- showed significantly fewer and milder clinical symptoms and had a lower mortality. A total of 10(5) PFU V- virus were required to kill most YAC-CD46 mice, and less than half (44%) were killed with a corresponding dose of MV C-. Immunohistochemical staining for MV antigens showed similar extents of spread for MV C- and MV Ed but restricted spread for MV V- throughout the brain. Viral load and transcription were markedly reduced for V- but not for C-. Multiple cytokines and chemokines were equivalently upregulated for all three viruses. Therefore, MV C and V proteins encode virulence functions in vivo and likely operate via separate mechanisms.
Saccharomyces cerevisiae SCF(Met30) ubiquitin-protein ligase controls cell cycle function and sulfur amino acid metabolism. We report here that the SCF(Met30 )complex mediates the transcriptional repression of the MET gene network by triggering degradation of the transcriptional activator Met4p when intracellular S-adenosylmethionine (AdoMet) increases. This AdoMet-induced Met4p degradation is dependent upon the 26S proteasome function. Unlike Met4p, the other components of the specific transcriptional activation complexes that are assembled upstream of the MET genes do not appear to be regulated at the protein level. We provide evidence that the interaction between Met4p and the F-box protein Met30p occurs irrespective of the level of intracellular AdoMet, suggesting that the timing of Met4p degradation is not controlled by its interaction with the SCF(Met30) complex. We also demonstrate that Met30p is a short-lived protein, which localizes within the nucleus. Furthermore, transcription of the MET30 gene is regulated by intracellular AdoMet levels and is dependent upon the Met4p transcription activation function. Thus Met4p appears to control its own degradation by regulating the amount of assembled SCF(Met30) ubiquitin ligase.
BACKGROUND: Elementary Ca2+ signals, such as 'Ca2+ puffs', that arise from the activation of clusters of inositol 1 ,4,5,-trisphosphate (InsP3) receptors are the building blocks for local and global Ca2+ signalling. We previously found that one, or a few, Ca2+ puff sites within agonist-stimulated cells act as 'pacemakers' to initiate global Ca2+ waves. The factors that distinguish these pacemaker Ca2+ puff sites from the other Ca2+ release sites that simply participate in Ca2+ wave propagation are unknown. RESULTS: The spatiotemporal properties of Ca2+ puffs were investigated using confocal microscopy of fluo3-loaded HeLa cells. The same pacemaker Ca2+ puff sites were activated during stimulation of cells with different agonists. The majority of agonist-stimulated pacemaker Ca2+ puffs originated in a perinuclear location. The positions of such Ca2+ puff sites were stable for up to 2 hours, and were not affected by disruption of the actin cytoskeleton. A similar perinuclear distribution of Ca2+ puff sites was also observed when InsP3 receptors were directly stimulated with thimerosal or membrane-permeant InsP3 esters. Immunostaining indicated that the perinuclear position of pacemaker Ca2+ puffs was not due to the localised expression of InsP3 receptors. CONCLUSIONS: The pacemaker Ca2+ puff sites that initiate Ca2+ responses are temporally and spatially stable within cells. These Ca2+ release sites are distinguished from their neighbours by an intrinsically higher InsP3 sensitivity.
The topic of nuclear Ca2+ signalling is beset by discrepant observations of substantial nuclear/cytoplasmic gradients. The reasons why some labs have recorded such gradients, whilst other workers see equilibration of Ca2+(cyt) and Ca2+(nuc) using the same cells and techniques, is unexplained. Furthermore, how such gradients could arise across the NE that possesses many highly-conductive NPCs is a mystery. Although nuclei may have the capacity to be autonomous signalling entities, with functional Ca2+ release channels and an inositide cycle, the balance of evidence suggests that Ca2+ release on the inner NE does not occur during physiological stimulation. Our work suggests that elementary Ca2+ release events originating in the cytoplasm can give rise to Ca2+ signals without causing elevation of the bulk cytoplasm. Clearly, the many Ca2+ signalling mechanisms that may impinge on Ca2+(nuc) will remain a topic of controversy and debate for some time.
OBJECTIVE: To evaluate the efficacy of single doses of intravenous diclofenac sodium (25, 50 and 75 mg) in patients with post-operative pain after third-molar surgery in a randomised, placebo-controlled study. METHODS: Two hundred and sixty-nine patients (168 females) who required the removal of their impacted third molars participated in the study, which had received prior ethical approval. Surgery was completed under general anaesthesia and, during the early post-operative period, patients received either a single intravenous dose of diclofenac (25, 50 or 75 mg) or matched placebo in random, double-blind order. Pain intensity was assessed on 10-cm visual analogue scales at fixed time points throughout a 4-h investigation period. Other efficacy variables obtained included time until rescue medication and overall assessment at 4, 6, 12 and 24 h after dosing. RESULTS: Throughout the 4-h investigation period, patients treated with diclofenac reported significantly less pain than those treated with placebo (P < 0.001). No differences were observed among the three doses of diclofenac (P = 0.22). Similar results were observed at 6, 12 and 24 h after dosing. Significant differences were also noted between the placebo group and all the diclofenac treatment groups with respect to time until rescue medication (P < 0.001) and the proportion of patients taking such medication. CONCLUSION: Single doses of i.v. diclofenac (25, 50 and 75 mg) provide significant pain relief after third-molar surgery. The efficacy of this preparation does not appear to be dose related.
The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.
OBJECTIVES: We tested the hypothesis that different anticoagulant treatments may produce different platelet effects and von Willebrand factor (vWf) release in unstable angina. BACKGROUND: The early increase of vWf has been reported to be a risk factor for adverse outcome in unstable angina. Anticoagulant drugs play a key role in stabilization of unstable angina, but they may not have the same efficacy and the same effects on acute vWf release. METHODS: We studied 154 patients enrolled in several clinical trials testing four different anticoagulant treatments in unstable angina or non-Q-wave myocardial infarction. Patients were treated during at least 48 h by either intravenous unfractionated heparin, one of two different low molecular weight heparins (enoxaparin or dalteparin) or the direct thrombin inhibitor PEG-hirudin. All patients received aspirin but no Ib/IIIa inhibitors. RESULTS: The release of vWf over the first 48 h (delta vWf) did not relate to the baseline clinical characteristics. At 30 days of follow-up, delta vWf was sevenfold higher in patients with an end point (death, myocardial infarction, revascularization) than in patients free of events (+53 +/-7% vs. +7 +/-14%, p = 0.004). The same trend was present for each component of the composite end point with the highest levels for one-month mortality (+87 +/- 32% vs. +26 +/- 8%, p = 0.09). The vWf values did not increase over 48 h in patients receiving either enoxaparin or PEG-hirudin (+10 +/- 9% and -5 +/- 20%, respectively). A serious rise ofvWf was measured in unfractionated heparin-treated patients (+87 +/- 11%), which differed significantly from the enoxaparin group (p = 0.0006) and PEG-hirudin group (p < 0.0001). In dalteparin-treated patients, delta vWf was elevated (+48 +/- 8%) and did not differ from the unfractionated heparin group (NS). CONCLUSIONS: We confirm that, in unstable angina patients, a rise of vWf over the first 48 h is associated with an impaired outcome at 30 days. Moreover, the four different anticoagulant treatments tested here do not provide the same protection with regards to vWf release, which may have important prognostic implications and explain different results observed in recent clinical trials.
OBJECTIVES: Review the acute and late results of percutaneous transluminal coronary angioplasty (PTCA) in heart transplant recipients and examine the factors predictive of restenosis. BACKGROUND: Coronary graft disease (CGD) is the main factor responsible for late graft loss. Medical treatment, surgical revascularization, or retransplantation gives only suboptimal results in this regard. Therefore, PTCA has been attempted in this situation. METHODS: More than 332 heart transplantations in our institution have been performed since 1992, the date of the first PTCA in our patients. We are currently in charge of 450 patients. All the characteristics, procedure-related information, and clinical outcome of patients needing PTCA were assessed by review of each patient's clinical records. All coronary angiograms were reviewed by an independent cardiologist. RESULTS: Since 1992, 53 coronary sites have been dilated in the course of 39 procedures in 29 patients. Indication for PTCA was asymptomatic angiographic coronary graft disease in 35 sites (64.8%), angina in 9 (16.6%), silent ischemia in 2 (3.7%), acute myocardial infarction in 1 (1.8%), and CHF in 7 (12.9%). Primary success (< 50% residual stenosis) was obtained in 50 (94.3%) of 53 lesions. No periprocedural death occurred. Procedural complications were 1 transient acute renal failure and 1 persistent bleeding at the puncture site. Six months restenosis rate (defined as percent stenosis > 50%) was 32.5% (14/43). Mean follow-up was 1.27 year +/- 1.2 (SD). Five deaths (17. 2%) occurred in follow-up and were all in relation to coronary graft disease. Mean time separating PTCA from death was 0.9 year +/- 1.3 (SD). We also sought to look at factors predictive of restenosis. By multivariate analysis, a positive recipient's serology for cytomegalovirus (CMV) before the graft was the only factor found protective against restenosis (odds ratio 22.4; confidence interval 1.1 to 443.4). CONCLUSION: PTCA in heart transplant recipients allows a high level of primary success with a low periprocedural-complication rate. Restenosis rate seems equivalent to restenosis rate in native coronary arteries. Mortality during follow-up is increased in this population and is the consequence of a high level of coronary events. Recipient positivity for CMV before the graft is associated with a protective effect from restenosis.
Single-nucleotide polymorphisms (SNPs) have been the focus of much attention in human genetics because they are extremely abundant and well-suited for automated large-scale genotyping. Human SNPs, however, are less informative than other types of genetic markers (such as simple-sequence length polymorphisms or microsatellites) and thus more loci are required for mapping traits. SNPs offer similar advantages for experimental genetic organisms such as the mouse, but they entail no loss of informativeness because bi-allelic markers are fully informative in analysing crosses between inbred strains. Here we report a large-scale analysis of SNPs in the mouse genome. We characterized the rate of nucleotide polymorphism in eight mouse strains and identified a collection of 2,848 SNPs located in 1,755 sequence-tagged sites (STSs) using high-density oligonucleotide arrays. Three-quarters of these SNPs have been mapped on the mouse genome, providing a first-generation SNP map of the mouse. We have also developed a multiplex genotyping procedure by which a genome scan can be performed with only six genotyping reactions per animal.
Physical methods of molecule observation and manipulation will prove useful, not only as research tools for investigating biomolecular structure and behavior, but also for the creation of nanostructures. Supramolecular and self-assembling structures are able to generate nanostructures, with many such systems being of biological origin. They form the interface between nanotechnology and biotechnology. Whereas biotechnological processes usually involve populations of cells or molecules, nanotechnological methods operate at the level of individual molecule manipulation. This article considers what advances have been made through cross-fertilisation between nanotechnology and biotechnology to develop for the next millennium new analytical tools at the microscale, using nanostructures as the sensitive part and with the ability to detect individual molecules.