Ligand-escape pathways from the ligand-binding domain of PPARgamma receptor as probed by molecular dynamics simulations.

Conformational rearrangements of peroxysome proliferator activated receptor (PPARgamma) ligand-binding domain (LBD) that accompany the release and binding of ligands are not well understood. To determine the major events associated with the escape of the partial agonist GW0072, molecular dynamic (MD) simulations were performed using two different methods: reversed targeted molecular dynamics (TMD(-1)) and time-dependent distance restraints (TDR) using as restraints either the root mean square deviation from a reference structure (TMD(-1)) or the distance between the geometrical centers of the binding pocket and of the ligand (TDR). Both methods do not assume any a priori route for ligand extraction. To avoid artifacts, different initial simulation conditions were used and particular attention was paid for giving time to the protein to relax during the extraction process by running 10-12 ns simulations within explicit water. Two distinct exit gates A and B were found, independently of initial conditions and method. During the exit process no interaction between GW0072 and the transactivation AF-2 helix was observed. Our results suggest that the ligand uses the intrinsic flexibility of the protein to move within the receptor. Paths A and B are very similar to those found for other nuclear receptors, suggesting that these routes are a common characteristics of nuclear receptors that are used by different kinds of ligands. Finally, the knowledge of entry/exit pathways of a receptor should be very useful in discriminating between different ligands that could have been favorably docked in the binding pocket by introducing docking along these pathways into computational drug design protocols.

[A familial form of conduction defects associated with a PRKAG2 gene mutation]. / Une forme familiale de troubles conductifs associés à une mutation du gène PRKAG2.

Conduction defects are usually secondary to elective aging of the conduction pathways. However, some familial and genetic forms are now being described. Here we report a particular electrocardiographic pattern in four members of the same family over three generations, naturally leading to the suspicion of a hereditary origin. The ECG appearance is very specific and includes conduction defects (RBBB and occasionally left anterior hemiblock), short PR interval, pseudo appearance of atrial hypertrophy, and occasionally sinus dysfunction or supraventricular extrasystole. Gene analysis identified a R302Q mutation of the gamma2 subunit producing AMP protein kinase, coded by the gene PRKAG2. This is a wrong sense mutation present in the heterozygous state in each of those displaying the ECG anomalies, and is transmitted in an autosomal dominant fashion. The clinical picture here appears to constitute a clinical entity distinct from those previously described as being associated with mutations of the PRKAG2 gene, without any left ventricular hypertrophy or Wolff-Parkinson-White syndrome.

[Management of sudden death in a semi-rural district, Seine-et-Marne: the DEFI 77 study]. / Prise en charge de la mort subite dans un département semi rural, la Seine et Marne: étude DEFI 77.

Sudden death is a major problem in public health, affecting around 50 000 people a year in France. The prognosis for cardiac arrest is abysmal because for every minute lost the chances of survival diminish by 10%. The aim of this work was to prospectively evaluate the characteristics of cardiac arrest victims across an entire 6000 km? area, the Seine-et-Marne district, distinguished by the paradox of lying just outside the capital whilst actually being semi-rural, and to determine the current methods of dealing with this emergency. The DEFI 77 prospective epidemiological survey was carried out with the collaboration of the SAMU emergency medical service, the SDIS fire/ambulance service, the general hospitals and the Paris-Ile-de-France cardiological association. Between January 2001 and December 2005 there were 2001 cardiac arrests (mean age 68 +/- 20 years, 67% male) at home in 80% of cases. The arrest was in front of a witness in 72% of cases, but they performed resuscitation in only 14.3% of cases. The SAMU and SMUR emergency medical services attempted cardio-pulmonary resuscitation in 78% of cases. In 29% of cases, one or more external electric shocks were carried out, using a semi-automatic defibrillator 79% of the time. Only 11.5% of patients arrived at the emergency department alive, the overall hospital survival rate being less than 2%. Only eight patients subsequently underwent automatic defibrillator implantation. The results of this observational study are to a large extent explained by an extremely long delay (12 minutes) before help was called for, the delay between the call and the arrival of medical assistance (9.5 +/- 4 min), the low percentage of active witnesses, and the variability in management (invasive in particular). In conclusion, at the dawn of the third millennium the prognosis of cardiac arrest remains very poor and fully justifies educating the general public about calling for help early and about actions that can save lives, particularly external cardiac massage before the arrival of the emergency services, as well as the benefits of using automated external defibrillators.

Simulation of special bubble detectors for PICASSO.

The PICASSO project is a cold dark matter (CDM) search experiment relying on the superheated droplet technique. The detectors use superheated freon liquid droplets (active material) dispersed and trapped in a polymerised gel. This detection technique is based on the phase transition of superheated droplets at about room temperature and ambient pressure. The phase transition is induced by nuclear recoils when an atomic nucleus in the droplets interacts with incoming subatomic particles. This includes CDM particles candidate as the neutralino (a yet-to-discover particle predicted in extensions of the standard model of particle physics). Simulations performed to understand the detector response to neutrons and alpha particles are presented along with corresponding data obtained at the Montreal Laboratory.

[Cage failure following replacement of the third lumbar vertebral body in Hodgkin's disease]. / Cageversagen nach LWK3-Ersatz bei M. Hodgkin.

AIM: We present a case report and the histological analysis of cage failure following vertebral body replacement in Hodgkin's disease. METHOD: In a 35-year-old patient with a single metastasis of Hodgkin's disease replacement of the third lumbar vertebral body (Harms-titanium-mesh-cage, DePuy Acromed, completely filled with autogenous spongiosa from the iliac crest) was performed. 51 months postoperatively, the patient presented with acute back pain without trauma. The X-ray showed a collapse of the Harms-titanium-mesh-cage. The cage fragments had caused ventral dislocation of the main blood vessels and dura compression without neurological deficits. After the replacement of the failed Harms-cage (Synex, Synthes) histological analysis (light microscope, non-decalcified horizontal cuts, thickness of 150-200 microm, Giemsa und van Gieson staining) was done. RESULT: The histology showed a remodelling of new bone in the area of the endplates without contact to the cage wall. In the region of the cage corpus, bony necrosis and connective tissue rich in cells was found. CONCLUSION: Bridging fusion in vertebral body replacement cages is not proven. As a result cage failure may occur. Therefore, in tumour patients with high live expectancy, Palacos filling of the cages should be discussed.

Direct dark matter search using large-mass superheated droplet detectors in the PICASSO experiment.

The PICASSO experiment investigates the presence and nature of dark matter in the Universe. The experiment is based on the detection of acoustic signals generated in explosive phase transitions induced by dark matter particles. This technique is an alternative more traditional detection technique like scintillation and ionisation, which are largely employed for dark matter search. One of the main advantages of this technique, besides its sensitivity to very low nuclear recoil energies (few keV), is its excellent background suppression features. A pilot experiment consisting of six superheated droplet detectors (40 g of active mass) is presently taking data at the Sudbury Neutrino Observatory (SNO) at a depth of 2000 m. We discuss the operation, calibration and data acquisition of the experiment and also the ongoing work to increase the sensitivity and the active mass of the detectors.

Lateral mobility of proteins in liquid membranes revisited.

The biological function of transmembrane proteins is closely related to their insertion, which has most often been studied through their lateral mobility. For >30 years, it has been thought that hardly any information on the size of the diffusing object can be extracted from such experiments. Indeed, the hydrodynamic model developed by Saffman and Delbrück predicts a weak, logarithmic dependence of the diffusion coefficient D with the radius R of the protein. Despite widespread use, its validity has never been thoroughly investigated. To check this model, we measured the diffusion coefficients of various peptides and transmembrane proteins, incorporated into giant unilamellar vesicles of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) or in model bilayers of tunable thickness. We show in this work that, for several integral proteins spanning a large range of sizes, the diffusion coefficient is strongly linked to the protein dimensions. A heuristic model results in a Stokes-like expression for D, (D proportional, variant 1/R), which fits literature data as well as ours. Diffusion measurement is then a fast and fruitful method; it allows determining the oligomerization degree of proteins or studying lipid-protein and protein-protein interactions within bilayers.

[Differential therapy in radial head fractures]. / Differenziertes Behandlungskonzept der Radiusköpfchenfraktur.

The therapeutic regimen of radial head fractures, especially of displaced and comminuted types is controversial. The radial head resection has been critically reviewed over the past years. From 1984-1993 and 1996-1999, 105 radial head fractures were treated in our hospital. 74 were subject to clinical and radiological follow-up. Fracture-types were classified according to Mason. Undisplaced fractures were treated conservatively, displaced 2-fragment-fractures by an open reduction and screw fixation, and multifragment-fractures by a radial head resection. The results were studied on a functional and radiological basis using the "Functional Rating Index" of Broberg and Morrey and the radiological Score of Albrecht and Ganz. After conservative therapy over 80 % achieved excellent and good as well as 12.5 % satisfactory and 6.3 % unsatisfactory results. After reduction and internal fixation again 80 % had excellent and good results. After radial head resection excellent and good results were achieved in 54.6 % of the cases, satisfactory results in 24.2 % and in 21.2 % unsatisfactory results, however prognosis-influencing concomitant injuries were often present in the latter group. Using the right indication and technique, the radial head resection still is a recommendable therapeutic procedure with an altogether good prognosis. This especially applies to isolated radial head fractures where excellent and good results can be achieved in approximately 70 %.

Dimer interface of transmembrane domains for neu/erbB-2 receptor dimerization and transforming activation: a model revealed by molecular dynamics simulations.

The specific point mutation Val-->Glu664 within the transmembrane domain of the neu/erbB-2 receptor is associated with increased receptor dimerization and increased receptor tyrosine kinase activity resulting in malignant transformation of cells. It is well established that Glu and residues in proximity are necessary for receptor dimerization but many studies suggest that other intramembrane constraints, not yet elucidated, are determinant for transformation. In this work, we investigated dimer models both to understand the structural role of the Glu mutation in the transmembrane domain association and to determine helix-helix contacts required for oncogenic transformation. Different types of helix-helix association based on data resulting from Cys mutational studies of the full wild receptor and spectroscopic data of transmembrane neu peptides have been explored by molecular dynamics simulations. The study leads to propose a model for the dimeric association of the transmembrane domains of the oncogenic neu receptor showing left-handed interactions of the two helices stabilized by symmetrical hydrogen bonding interactions involving the Glu side chain on one helix and the facing carbonyl of Ala661 on the second helix. Contacting residues observed in the symmetric interface explain the transforming activity or the non transforming activity of many neu mutants. Moreover the left-handed coiled coil structure is fully consistent with recent results proving the role of rotational linkage of the transmembrane domain with the kinase domain. Comparison between the predicted dimer model and those presumed from experiments strongly suggests helix flexibility in the extracellular juxtamembrane region.

Self diffusion and spectral modifications of a membrane protein, the Rubrivivax gelatinosus LH2 complex, incorporated into a monoolein cubic phase.

The light-harvesting complex LH2 from a purple bacterium, Rubrivivax gelatinosus, has been incorporated into the Q230 cubic phase of monoolein. We measured the self-diffusion of LH2 in detergent solution and in the cubic phase by fluorescence recovery after photobleaching. We investigated also the absorption and fluorescence properties of this oligomeric membrane protein in the cubic phase, in comparison with its beta-octyl glucoside solution. In these experiments, native LH2 and LH2 labeled by a fluorescent marker were used. The results indicate that the inclusion of LH2 into the cubic phase induced modifications in the carotenoid and B800 binding sites. Despite these significant perturbations, the protein seems to keep an oligomeric structure. The relevance of these observations for the possible crystallization of this protein in the cubic phase is discussed.

Protein turnover modifications induced by the protein feeding pattern still persist after the end of the diets.

This study was undertaken to determine whether the protein feeding pattern could induce chronic adaptation of protein turnover. After a 15-day adaptive period, elderly (68 yr) and young (26 yr) women received, for 14 days, a diet providing 200 KJ x kg fat-free mass (FFM)(-1) x day(-1), where the daily protein intake (1.7 g protein x kg FFM(-1) x day(-1)) was either spread over 4 meals in the spread pattern or mainly (80%) consumed at noon in the pulse pattern. One day after the end of the dietary treatment, whole body leucine kinetics were measured by use of a continuous [(13)C]leucine infusion, both in the postabsorptive state and in the same fed state. The pulse pattern was able to induce, in young as in elderly women, a lower postabsorptive leucine oxidation and endogenous leucine flux than the spread pattern and improved the responsiveness of nonoxidative leucine disposal during 4-h oral feeding. Thus the pulse pattern was able to induce chronic regulation of protein metabolism in young as in elderly women.

Structure prediction of the dimeric neu/ErbB-2 transmembrane domain from multi-nanosecond molecular dynamics simulations.

Dimerization of the neu/ErbB-2 receptor tyrosine kinase is a necessary but not a sufficient step for signaling. Despite the efforts expended to identify the molecular interactions responsible for receptor-receptor contacts and particularly those involving the transmembrane domain, structural details are still unknown. In this work, molecular dynamics simulations of the helical transmembrane domain (TM) of neu and ErbB-2 receptors are used to predict their dimer structure both in the wild and oncogenic forms. A global conformational search method, applied to define the best orientations of parallel helices, showed an energetically favorable configuration with the specific mutation site within the interface, common for both the nontransforming and the transforming neu/ErbB-2 TM dimers. Starting from this configuration, a total of 10 simulations, about 1.4 ns each, performed in vacuum, without any constraints, show that the two helices preferentially wrap in left-handed interactions with a packing angle at about 20 degrees. The resulting structures are nonsymmetric and the hydrogen bond network analysis shows that helices experience pi local distortions that facilitate inter-helix hydrogen bond interactions and may result in a change in the helix packing, leading to a symmetric interface. For the mutated sequences, we show that the Glu side chain interacts directly with its cognate or with carbonyl groups of the facing backbone. We show that the connectivity between interfacial residues conforms to the knobs-into-holes packing mode of transmembrane helices. The dimeric interface described in our models is discussed with respect to mutagenesis studies.

Systemic characteristics of chronic arthritis induced by transfer of human rheumatoid synovial membrane into SCID mice (human/murine SCID arthritis).

Erosive human/murine (hu/mu) SCID arthritis, caused by unilateral engrafting of human rheumatoid arthritis synovial membrane (RA-SM) in the knee joints of SCID mice, was monitored for up to 18 weeks by scintigraphic, radiological, morphological and immunohistochemical analyses.(99m)Tc-DPD scintigraphy and histology revealed secondary, oligoarticular spreading of arthritis to contralateral knees and hips, but not to forelimb joints. Also, there were no extraarticular manifestations. At 18 weeks, surviving human cells were found within the pannus, but not directly at the cartilage erosion front, where fibroblast-like cells and macrophages of murine origin predominated. The latter cells also predominated in secondarily affected joints, where no human cells were detectable. Preventive depletion of murine NK-cells by anti-asialo-GMI antibodies, to check the influence of NK cells independently of strain and MHC system, combined with application of autologous human PBMN cells, had virtually no effects on the disease process. The completeness of the SCID defect was not critical, i.e. T cells were completely absent in the organs examined, and the presence of a few B cells in the spleen did not correspond to particular disease features. The SCID defect itself had a clear impact, since, in the chronic phase, SCID.bg and RAG-2(-/-)knockout mice developed less consistent pathological/scintigraphic signs of disease than SCID mice. Thus, unilaterally-induced hu/mu SCID arthritis is an oligoarticular disorder of the hindlimbs. Murine macrophages and fibroblast-like cells appear responsible for tissue destruction in engrafted and non-engrafted arthritic joints.

Molecular dynamics simulations of the ErbB-2 transmembrane domain within an explicit membrane environment: comparison with vacuum simulations.

Two 500-ps molecular dynamics simulations performed on the single transmembrane domain of the ErbB-2 tyrosine kinase receptor immersed in a fully solvated dilauroylphosphatidyl-ethanolamine bilayer (DLPE) are compared to vacuum simulations. One membrane simulation shows that the initial alpha helix undergoes a local pi helix conversion in the peptide part embedded in the membrane core similar to that found in simulation vacuum. Lipid/water/peptide interaction analysis shows that in the helix core, the intramolecular peptide interactions are largely dominant compared to the interactions with water and lipids whereas the helix extremities are much more sensitive to these interactions at the membrane interfaces. Our results suggest that simulations in a lipid environment are required to understand the dynamics of transmembrane helices, but can be reasonably supplemented by in vacuo simulations to explore rapidly its conformational space and to describe the internal deformation of the hydrophobic core.

[Late potentials in chronic alcoholics]. / Potentiels tardifs chez les alcooliques chroniques.

OBJECTIVE: Cardiac arrest is the most frequent cause of death in chronic alcoholics. Detection of late potentials in this population could be helpful in screening from early signs of myocardial disorders and identifying patients at risk of severe ventricular dysrythmia. PATIENTS AND METHODS: A prospective study of late potentials was conducted in 53 subjects (mean age 49 +/- 10 years) with a history of long-standing alcohol abuse (mean 13.6 +/- 8.5 years, mean daily alcohol intake 86 +/- 30 g). After a period of abstinence, the following explorations were performed: liver tests, liver biopsy, electrocardiogram, echocardiography, Holter recording. RESULTS: Among the 53 patients, 37% were positive for 2 of the 3 criteria for late potentials. There was a strong correlation between the duration of alcohol abuse and presence of late potentials (p = 0.006, r = 0.37). The percentage of hepatic steatosis was higher in alcoholic subjects with late potentials (34% versus 23%; p = 0.05) and was correlated with the number of positive criteria for late potentials (p = 0.05, r = 0.328). Finally, the presence of late potentials was also correlated with the following laboratory results: serum gamma glutamyltranspeptidase (p = 0.031), serum aspartate amino transferase (p = 0.033), serum alkaline phosphatases (p = 0.0025). CONCLUSION: Late potentials can be detected easily although their prognostic value remains to be determined. They could be an early marker of infraclinical myocardial lesions.

[Signal averaging electrocardiography in chronic alcoholism]. / Enregistrement de l'électrocardiogramme haute amplification chez les éthyliques chroniques.

Cardiovascular death is the main cause of mortality in chronic alcoholics, perhaps due to a pro-arrhythmogenic effect of alcohol associated with infraclinical myocardial lesions. The authors investigated prospectively 41 patients (average age: 49.7 years) who were chronic alcoholics but had no acute alcoholic episodes for cardiac disease (ECG, signal averaging for late ventricular potentials, echocardiography and Holter ECG monitoring) and hepatic disease (liver biopsy). The history of alcoholism was 14 +/- 9 years, the quantity of alcohol ingested before they stopped drinking being 89 +/- 31 grammes/day. Thirty per cent of patients displayed 2 or 3 criteria of late ventricular potentials (LP). The authors demonstrated a correlation between the daily quantity of alcohol consumed before stopping drinking and the duration of the filtered QRS complex (p = 0.02). Moreover, the frequency of fatty infiltration found on liver biopsy, greater in alcoholics with LP (35% versus 19%, p = 0.025) correlated with the amplitude of the last 40 ms of the average QRS (p = 0.0485), with the duration of potentials of less than 40 microvolts (p = 0.05) and, above all, with the number of criteria of LP (p = 0.02). Finally, the presence of LP was also related to the following biological abnormalities: GGT (p = 0.027), ASAT (p = 0.046), ALAT (p = 0.039). The ECG abnormalities may reflect early infra-clinical myocardial lesions secondary to cellular metabolic abnormalities perhaps analogous to the fatty hepatic changes. However, the prognostic value of these signal-averaging ECG abnormalities remains unknown.

Time resolved fluorescence properties of phenylalanine in different environments. Comparison with molecular dynamics simulation.

Time resolved fluorescence of the phenylalanine residue (Phe) alone and included in the transmembrane domain (TMD) sequences of the epidermal growth factor receptor (EGFR) and ErbB-2 was studied using the synchrotron radiation source of light, and compared to molecular dynamics (MD) simulations. The fluorescence intensity decay is strongly sensitive to the environment. A mono-exponential decay was obtained for Phe amino acid alone in two different solvents and for Phe included in EGFR transmembrane sequence, with fluorescence lifetime values varying from 1.7 ns (EGFR) to 7.4 ns (Phe dissolved in water). In ErbB-2 transmembrane sequence three lifetimes were detected. The relative amplitude of the shortest one (0.14 ns) is smaller than 10%, whereas the others (0.6 and 2.2 ns) are almost equally represented. They have been attributed to different rotamers exchanging slowly. This interpretation is supported by MD simulations which evidence transitions in time series of the chi 1 dihedral angle of Phe observed in the case of ErbB-2. The anisotropy decays are similar for both peptides and indicate the presence of a correlation time in the nanosecond range (1-4 ns) and the probable existence of a very fast one (< 0.05 ns). Autocorrelation functions computed from MD simulations corroborate these results.

Molecular modeling of c-erbB2 receptor dimerization: coiled-coil structure of wild and oncogenic transmembrane domains--stabilization by interhelical hydrogen bonds in the oncogenic form.

Dimerization models of c-erbB2 transmembrane domains (Leu651-Ile675) are studied by molecular mechanics and molecular dynamics simulations. Both wild and Glu mutated transmembrane helices exhibit the same relative orientation for favorable associations and dimerize preferentially in left-handed coiled-coil structures. The mutation point 659 belongs to the interfacing residues, and in the transforming domain, symmetric hydrogen bonds between Glu carboxylic groups stabilize the dimeric structure. The same helix packing found for the wild dimers, except side-chain-side-chain hydrogen bonds, suggests that the transmembrane domains dimerize according to similar process. Structural and energetical characterization of the models are presented.

Late ventricular potentials and heavy drinking.

OBJECTIVES: To assess the effects of chronic drinking on detection of low amplitude signals, and to determine the relation between late ventricular potentials (LVP) and liver biopsy findings. DESIGN: Prospective study. SETTING: General hospital. PATIENTS: 41 consecutive chronic alcoholics without known pre-existing heart disease. METHODS: About four days after each patient's last alcoholic drink, ECG, echocardiography, signal averaged electrocardiogram, liver biopsy, and blood tests were performed. RESULTS: Twenty eight per cent of patients had evidence of LVP. There was a correlation between the percentage of steatosis of the hepatic biopsy and the amplitude of the last 40 ms of average QRS (P = 0.04), the duration of the terminal low amplitude QRS signal (P = 0.05), and the number of positive criteria of late potentials (P = 0.02). CONCLUSIONS: Chronic drinking sufficient to cause steatosis is associated with positive findings on the signal averaged ECG.

Lipopeptides with improved properties: structure by NMR, purification by HPLC and structure-activity relationships of new isoleucyl-rich surfactins.

The biosynthesis of bacterial isoleucyl-rich surfactins was controlled by supplementation of L-isoleucine to the culture medium. Two new variants, the [Ile4,7]- and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse-phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified by beta-hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two-dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure-activity relationships were discussed in details on the basis of the three-dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu-to-Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.