Prediction of site-specific amino acid distributions and limits of divergent evolutionary changes in protein sequences

We derive an analytic expression for site-specific stationary distributions of amino acids from the structurally constrained neutral (SCN) model of protein evolution with conservation of folding stability. The stationary distributions that we obtain have a Boltzmann-like shape, and their effective temperature parameter, measuring the limit of divergent evolutionary changes at a given site, can be predicted from a site-specific topological property, the principal eigenvector of the contact matrix of the native conformation of the protein. These analytic results, obtained without free parameters, are compared with simulations of the SCN model and with the site-specific amino acid distributions obtained from the Protein Data Bank. These results also provide new insights into how the topology of a protein fold influences its designability, i.e., the number of sequences compatible with that fold. The dependence of the effective temperature on the principal eigenvector decreases for longer proteins, as a possible consequence of the fact that selection for thermodynamic stability becomes weaker in this case.     

Role of IL-6 and CD23 in the resistance to growth arrest and apoptosis in LCL41 B lymphoma cells

Interleukin-6 (IL-6) is a growth and survival factor in Epstein-Barr virus (EBV)-infected B lymphoma cells and IL-6 antagonists have been used in clinical practice for this pathology. We thus wanted to investigate the effect of the IL-6 receptor antagonist Sant7 on proliferative and anti-apoptotic signals in the IL-6-secreting LCL41 B lymphoid cells, taken from a patient with EBV-induced lymphoproliferative disorder. Results show efficient inhibition of constitutive Stat3 activation by Sant7. However, this inhibition is associated with marginal induction of apoptosis and with minor decrease of cell proliferation, contrary to the effect of the Jak kinase inhibitor AG490, which down-regulates both proliferation and Stat3 activation. Anti-apoptotic markers such as Bcl-xL or Mcl-1 are constitutively expressed in these cells, and their expression is not affected by Sant7 treatment. Inhibition of Stat3 activation is therefore not sufficient to prevent proliferation and to induce apoptosis in these cells. In addition, low cell density is a condition favouring inhibition of cell clustering and anti-proliferative Sant7 activity. A marked inhibition of cell cluster formation and proliferation is achieved by antibody treatment against the CD23 mature B cell surface marker expressed in LCL41 cells. These findings may thus contribute to the identification of possible resistance mechanisms to growth arrest in B cell lymphoproliferative conditions.     

Analytical precision,biological variation,and mathematical normalization in high data density metabolomics

Metabolic serotypes sensitive to caloric intake may enable sera metabolomic profiles to validate epidemiological parameters and predict disease risk in humans. This long-range goal is complicated by the lack of known state markers and the requirement for simultaneous monitoring of multiple small changes. Therefore, analytical precision for appropriate high data density studies using HPLC separations coupled with coulometric array detectors was evaluated over a two month period in pooled rat sera samples (previously collected and stored at –80 °C), and in authentic biochemical standards. In sera, mean coefficients of variation (CV) of retention time and ratio accuracy within the established metabolic serotype varied within ±1% and ±3%, respectively. In sets of purified standards, the same parameters fluctuated, correspondently, in ranges of ±0.1% and ±1%. Median CV of the metabolite concentrations were ~13% in standards and ~11–19% in sera, and varied non-monotonically with the analytical system status and experimental design. These parameters were shown to be sufficiently controlled so as not to dominate intra-group biological variability in serum metabolomics studies. Continuation of experimental runs across an analytical breakpoint (column replacement) was associated with disproportionate changes in metabolite concentrations, independent of maintained analytical precision. These changes were sufficient to shift overall profile localization in megavariate projection analyses. We developed a mathematical approach to normalize this break and use partial least squares projection to latent structure discriminant analysis to confirm validity of this normalization approach. This generally applicable mathematical correction helps enable longer term high data density studies by removing a critical source of systemic variation.     

Towards the synthesis of new dideoxy δ-dicarbonyl heptoses

The preparation of a δ-dicarbonyl sugar thorough ring-opening, by a methoxymercuration-demercuration procedure, of a 5-spirocyclopropanated d-galactose derivative, is reported. This method constitutes a new route for the transformation of a hexose into new and interesting δ-dicarbonyl sugars, synthetic precursors of cyclitols, carba- and azasugars. Moreover this is, to our best knowledge, the first reported example of an elongation to a higher sugar starting from a spirocyclopropanated saccharide.     

Sequence determinants of protein folding rates: positive correlation between contact energy and contact range indicates selection for fast folding

In comparison with intense investigation of the structural determinants of protein folding rates, the sequence features favoring fast folding have received little attention. Here, we investigate this subject using simple models of protein folding and a statistical analysis of the Protein Data Bank (PDB). The mean-field model by Plotkin and coworkers predicts that the folding rate is accelerated by stronger-than-average interactions at short distance along the sequence. We confirmed this prediction using the Finkelstein model of protein folding, which accounts for realistic features of polymer entropy. We then tested this prediction on the PDB. We found that native interactions are strongest at contact range l = 8. However, since short range contacts tend to be exposed and they are frequently formed in misfolded structures, selection for folding stability tends to make them less attractive, that is, stability and kinetics may have contrasting requirements. Using a recently proposed model, we predicted the relationship between contact range and contact energy based on buriedness and contact frequency. Deviations from this prediction induce a positive correlation between contact range and contact energy, that is, short range contacts are stronger than expected, for 2/3 of the proteins. This correlation increases with the absolute contact order (ACO), as expected if proteins that tend to fold slowly due to large ACO are subject to stronger selection for sequence features favoring fast folding. Our results suggest that the selective pressure for fast folding is detectable only for one third of the proteins in the PDB, in particular those with large contact order.     

A small molecule SMAC mimic LBW242 potentiates TRAIL- and anticancer drug-mediated cell death of ovarian cancer cells

Background

Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays.

Principal Findings

LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis.

Conclusion

LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.     

Human Haemato-Endothelial Precursors: Cord Blood CD34+ Cells Produce Haemogenic Endothelium

Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144−), triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45−) capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level.     

Homogeneous asymmetric catalysis in fragrance chemistry

Opposite enantiomers of a chiral fragrance may exhibit different olfactory activities making a synthesis in high enantiomeric purity commercially and scientifically interesting. Accordingly, the asymmetric synthesis of four chiral odorants, Fixolide, Phenoxanol, Citralis, and Citralis Nitrile, has been investigated with the aim to develop practically feasible processes. In the devised synthetic schemes, the key step that leads to the formation of the stereogenic center is the homogeneous asymmetric hydrogenation of a prochiral olefin. By an appropriate choice of the catalyst and the reaction conditions, Phenoxanol, Citralis, and Citralis Nitrile were obtained in high enantiomeric purity, and odor profiles of the single enantiomers were determined.     

Anaerobic and aerobic relative contribution to total energy release during supramaximal effort in patients with left ventricular dysfunction.

Energetic metabolism during effort is impaired in patients with left ventricular dysfunction (Dysf), but data have been lacking up to now on the relative anaerobic vs. aerobic contribution to total energy release during supramaximal effort. Recently, the maximal accumulated oxygen deficit (MAOD) has been shown to be measurable in Dysf patients, making it possible to evaluate the anaerobic/aerobic interaction under conditions of maximal stress of both anaerobic and aerobic metabolic pathways in this population. Nineteen Dysf patients and 17 normal patients (N) underwent one ramp cardiopulmonary, three moderate-intensity constant-power, and three supramaximal constant-power (1- to 2-min, 2- to 3-min, and 3- to 4-min duration) exercise tests. MAOD was the difference between accumulated O(2) demand (accO(2)dem; estimated from the moderate-intensity O(2) uptake/watt relationship) and uptake during supramaximal tests. Percent anaerobic (%Anaer) and aerobic (%Aer) energetic release were [(MAOD/accO(2)dem).100] and 100 - %Anaer, respectively. MAOD did not vary between 1-2, 2-3, and 3-4 min supramaximal tests, whereas accO(2)dem increased significantly with and was linearly related to test duration in both Dysf and N. Consequently, %Anaer and %Aer decreased and increased, respectively, with increasing test duration but did not differ between Dysf and N in 1-2 min, 2-3 min, and 3-4 min tests. Our study demonstrates a similar relative anaerobic vs. aerobic contribution to total energy release during supramaximal effort in Dysf and N. This finding indicates that energetic metabolism during supramaximal exercise is exercise tolerance independent and that relative anaerobic vs. aerobic contribution in this effort domain remains the same within the physiology- or pathology-induced limits to individual peak exercise performance.