Oligomers of the cytoplasmic fragment from the Escherichia coli aspartate receptor dissociate through an unfolded transition state

The kinetic and equilibrium properties of a clustering process were studied as a function of temperature for two point mutants of a 31 kDa fragment derived from the cytoplasmic region of the Escherichia coli aspartate receptor (C-fragment), which were shown previously to have a greater tendency to form clusters relative to the wild-type C-fragment [Long, D. G., & Weis, R. M. (1992) Biochemistry 31, 9904-9911]. The clustering equilibria were different for the two C-fragments. Monomers of a serine-461 to leucine (S461L) mutant C-fragment were in equilibrium with dimers, while monomers of a S325L C-fragment were in equilibrium with trimers. The positive values for delta H degree, delta S degree, and delta Cp degree of dissociation estimated from a van't Hoff analysis, and the differences in the CD spectra of isolated monomers and oligomers, demonstrated that the monomers were less well-folded than the clustered forms. The oligomer dissociation rate exhibited a marked temperature dependence over the range from 4 to 30 degrees C and was remarkably slow at low temperatures; e.g. t1/2 of dimer dissociation for the S461L C-fragment was 85 h at 4 degrees C. The values for delta H degree +2, delta S degree +2, and delta Cp degree +2 derived from the temperature dependence of the dissociation rate were comparable to the corresponding parameters determined in a DSC study of C-fragment denaturation [Wu, J., Long, D. G., & Weis, R. M. (1995) Biochemistry 34, 3056-3065], which indicated that the transition state resembled thermally denatured C-fragment. Octyl glucoside accelerated the dissociation rate by 3-5-fold presumably by lowering the barrier to dissociation. This acceleration and the positive value of delta Cp degree +2 were interpreted as evidence for an increase in solvent accessible hydrophobic groups in the transition state. The molecular basis for the slow rate of dissociation is proposed to result from the conversion of intermolecular coiled coils in the oligomers to an intramolecular coiled coil in the monomer.     

From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to high-conductance state

The permeability transition pore (PTP) is a channel of the inner mitochondrial membrane that appears to operate at the crossroads of two distinct physiological pathways, i.e., the Ca2+ signaling network during the life of the cell, and the effector phase of the apoptotic cascade during Ca2+-dependent cell death. Correspondingly, two open conformations of the PTP can also be observed in isolated organelles. A low-conductance state, that allows the diffusion of small ions like Ca2+, is pH-operated, promoting spontaneous closure of the channel. A high-conductance state, that allows the unselective diffusion of big molecules, stabilizes the channel in the open conformation, disrupting in turn the mitochondrial structure and causing the release of proapoptotic factors. Our current results indicate that switching from low- to high-conductance state is an irreversible process that is strictly dependent on the saturation of the internal Ca2+-binding sites of the PTP. Thus, the high-conductance state of the PTP, which was shown to play a pivotal role in the course of excitotoxic and thapsigargin-induced cell death, might result from a Ca2+-dependent conformational shift of the low-conductance state, normally participating in the regulation of cellular Ca2+ homeostasis as a pH-operated channel. These observations lead us to propose a simple biophysical model of the transition between Ca2+ signaling and Ca2+-dependent apoptosis.     

Reposition of an implantable cardioverter defibrillator generator from an abdominal pocket to a subpectoral location using the existing electrode

The size of today's implantable cardioverter defibrillator generators makes a subpectoral position feasible even when an existing generator is located abdominally. Elective replacement with reposition of the generator from an abdominal to a subpectoral pocket without implantation of another electrode is described.     

Theoretical studies of an exceptionally stable RNA tetraloop: observation of convergence from an incorrect NMR structure to the correct one using unrestrained molecular dynamics

We report on the results of five independent and unrestrained molecular dynamics simulations of an RNA tetraloop, r(GGACUUCGGUCC), and its related structures with the loop UUCG sugars changed to deoxyribose. Two separate NMR structures have been reported for the loop portion of this molecule, with the second refinement resulting in a slightly different and more accurate conformation for the loop. The root-mean-square deviation (RMSd) between the two NMR structures, for the loop portions only, is 2.5 A. Our simulations, starting from the two NMR structures, demonstrate that this tetraloop is a very stable and rigid structure with both nanosecond length simulations staying very close to the initial structures. Additionally, both simulations preserved most, if not all, of the NMR-derived interactions and violated very few of the nuclear Overhauser effect (NOE)-derived distances used in the structure refinements. However, when the two NMR structures were simulated with deoxyriboses in the loops instead of the native riboses, the flexibility of the systems increased and we observed a conversion from the incorrect to the correct loop conformation in the simulation which started in the incorrect loop conformation. When the riboses were subsequently re-introduced back into the structure which underwent the conversion, the agreement between this simulation and the one starting from the correct NMR structure was a remarkably low 0.5 A, demonstrating an almost complete convergence from the incorrect to the correct structure using unrestrained molecular dynamics.     

Time-resolved EPR studies with DNA photolyase: excited-state FADH0 abstracts an electron from Trp-306 to generate FADH-, the catalytically active form of the cofactor

Photolyase repairs UV-induced cyclobutane-pyrimidine dimers in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains 5,10-methenyltetrahydrofolate, which functions as the light-harvesting chromophore, and fully reduced flavin adenine dinucleotide (FAD), which functions as the redox catalyst. During enzyme preparation, the flavin is oxidized to FADH0, which is catalytically inert. Illumination of the enzyme with 300- to 600-nm light converts the flavin to the fully reduced form in a reaction that involves photooxidation of an amino acid in the apoenzyme. The results of earlier optical studies had indicated that the redox-active amino acid in this photoactivation process was tryptophan. We have now used time-resolved electron paramagnetic resonance (EPR) spectroscopy to investigate the photoactivation reaction. Excitation of the flavin-radical-containing inactive enzyme produces a spin-polarized radical that we identify by 2H and 15N labeling as originating from a tryptophan residue, confirming the inferences from the optical work. These results and Trp-->Phe replacement by site-directed mutagenesis reveal that flavin radical photoreduction is achieved by electron abstraction from Trp-306 by the excited-state FADH0. Analysis of the hyperfine couplings and spin density distribution deduced from the isotopic-labeling results shows that the product of the light-driven redox chemistry is the Trp-306 cation radical. The results strongly suggest that the active form of photolyase contains FADH- and not FADH2.     

Differential contribution of M(r) 120 kDa rasGTPase-activating protein and neurofibromatosis type 1 gene product during the transition from growth phase to arrested state in human fibroblasts accompanied by a unique rasGTPase-activating activity

Production of IgG3 in MRL/Mp-lpr/lpr (MRL/lpr) lupus mice is one of the major factors to develop glomerulonephritis (GN) in these mice. To examine molecular characteristics of IgG3 responsible for GN in these mice, hybridoma clones producing IgG3 antibodies were prepared from one unmanipulated MRL/lpr mouse. Two clones, 2B11.3 and 7B6.8, were nephritogenic; that is, they caused severe glomerular lesions when injected to normal mice, moreover with a different histopathological manifestation. The 2B11.3 clone generated diffuse cell-proliferative lesions, while those induced by the 7B6.8 clone resembled wire loop lesions in human lupus nephritis. The cDNA sequence analysis of 7B6.8 antibody and the other IgG3 antibody, 1G3, non-nephritogenic, revealed that the C regions of the heavy and light kappa chains were completely the same between them. Furthermore, they were identical in deduced amino acid sequences to those from non-autoimmune BALB/c mice, indicating no allelic difference of Igh-8 between these two strains. The V regions of 2B11.3 and 7B6.8 antibodies were composed of different sets of VH, D, JH, Vk and Jk. Although both of the VH belonged to the J558 family, they seemed to use a different VH germline gene. These findings suggest that GN in MRL/lpr mice is generated by the expansion of clonally different B cells producing particular antibodies possibly with a different pathogenetic potency.     

Modalities of transition of diabetic adolescents from pediatrics to the adult care in the Paris-Ile-de-France region: an appeal to cooperative work for improving quality of care. Paris-Ile-de-France Section of DESG (Diabetes Education Study Group) in French language

AIM: The purpose of this study was to evaluate the conditions in which diabetic adolescents are transferred from pediatric to adult health care, and to record the opinions of the physicians about this issue. METHODS: A questionnaire-based study was performed among all the pediatricians in the hospital setting and all the diabetologists from the Paris-Ile-de-France area. Questionnaires from 50 pediatricians and 51 diabetologists were completed (response rate: 68%). RESULTS: 1) Not enough information was transmitted: a quarter of the diabetologists were visiting for the first time without any information on the adolescent, and only half the pediatricians received feedback information from the internists after the first visit. And yet, when considered, it was important to be kept informed after the first visit and the following ones. 2) Medical relationships were poor: more than three out of four pediatricians and diabetologists had none or very few professional meetings, and two thirds of them were not aware of the way the others were working. 3) Eighty percent of pediatricians and diabetologists considered that the transfer of diabetic adolescents had to be organised in order to keep the coherence of medical follow-up, to minimise the psychological effects of the transition, and to avoid a complete break in the patient follow-up. 4) The expectations of the pediatricians were: the validation of their previous follow-up through the feedback information from diabetologists and the continuity of the medical follow-up; those of the diabetologists were: to gain the patient's confidence and to master the patient's previous history, in order to provide a better follow-up. 5) According to the opinion of both pediatricians and diabetologists, the main errors to avoid were, by the paediatricians, to miss the time and the preparation of the transfer and, by the diabetologists, to denigrate the previous pediatric management and to change the insulin regimen immediately. CONCLUSION: This study demonstrates a lack of communication between physicians of pediatric and adult health care centres. But it also underlines their recognition of the importance of the transition's stakes and their common motivations in order to improve it.     

Universal procedure for the recalculation of the interaction parameters of elements from one base of an alloy to another using the theory of quasi-regular solutions: I. Theoretical foundations and the adequacy of the recalculation model

An expression for the indicator-determinant that allows one to estimate the possibility of removal of a certain component of an alloy due to its preferred evaporation when the metal is held in vacuum is obtained for complex multicomponent alloys. The possibility of vacuum evaporative refining of pure nickel and complex nickel superalloys from detrimental impurities is estimated. The influence of the alloying components of these alloys on the evaporation of detrimental impurities is shown.