Solution conformation of a nitrobenzoxadiazole derivative of the polyene antibiotic nystatin: a FRET study

Nystatin is a polyene antibiotic frequently applied in the treatment of topical fungal infections. In this work, a 7-nitrobenz-2-oxa-1,3-diazole (NBD) hexanoyl amide derivative of nystatin was synthesized and its detailed photophysical characterization is presented. The average conformation of the labelled antibiotic in tetrahydrofuran, ethanol and methanol was determined by intramolecular (tetraene to NBD) fluorescence resonance energy transfer measurements. At variance with the literature [Can. J. Chem. 63 (1985) 77-85], it was concluded that there is no need to invoke a solvent-dependent conformational equilibrium between extended and closed conformers of the antibiotic, because the mean tetraene-to-NBD separating distance was found to remain constant (approximately 18 A) in all the solvents studied. In addition, the large solvent dependence of the fluorescence anisotropy observed for the non-derivatized nystatin, was rationalized on the basis of the prolate ellipsoidal geometry of the molecule. It was concluded that the rod shaped and amphipathic antibiotic remains monomeric in different solvents within the concentration range studied (2-20 microM).     

Effect of time on the interfacial and foaming properties of beta-lactoglobulin/acacia gum electrostatic complexes and coacervates at pH 4.2

The electrostatic complexation between beta-lactoglobulin and acacia gum was investigated at pH 4.2 and 25 degrees C. The binding isotherm revealed a spontaneous exothermic reaction, leading to a DeltaHobs = -2108 kJ mol(-1) and a saturation protein to polysaccharide weight mixing ratio of 2:1. Soluble electrostatic complexes formed in these conditions were characterized by a hydrodynamic diameter of 119 +/- 0.6 nm and a polydispersity index of 0.097. The effect of time on the interfacial and foaming properties of these soluble complexes was investigated at a concentration of 0.1 wt % at two different times after mixing (4 min, referred as t approximately 0 h and t = 24 h). At t approximately 0 h, the mixture is mainly made of aggregating soluble electrostatic complexes, whereas after 24 h these complexes have already insolubilize to form liquid coacervates. The surface elasticity, viscosity and phase angle obtained at low frequency (0.01 Hz) using oscillating bubble tensiometry revealed higher fluidity and less rigidity in the film formed at t approximately 0 h. This observation was confirmed by diminishing bubble experiments coupled with microscopy of the thin film. It was thicker, more homogeneous and contained more water at t approximately 0 h as compared to t = 24 h (thinner film, less water). This led to very different gas permeability's of Kt approximately 0 h = 0.021 cm s(-1) and Kt=24 h) = 0.449 cm s(-1), respectively. Aqueous foams produced with the beta-lactoglobulin/acacia gum electrostatic complexes or coacervates exhibited very different stability. The former (t approximately 0 h) had a stable volume, combining low drainage rate and mainly air bubble disproportionation as the destabilization mechanism. By contrast, using coacervates aged for 24 h, the foam was significantly less stable, combining fast liquid drainage and air bubble destabilization though fast gas diffusion followed by film rupture and bubble coalescence. The strong effect of time on the air/water interfacial properties of the beta-lactoglobulin/acacia gum electrostatic complexes can be understood by their reorganization at the interface to form a coacervate phase that is more fluid/viscous at t approximately 0 h vs rigid/elastic at t = 24 h.     

Characterization of N-terminal processing of group VIA phospholipase A<Subscript>2</Subscript> and of potential cleavage sites of amyloid precursor protein constructs by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests

Dysregulation of proteolytic processing of the amyloid precursor protein (APP) contributes to the pathogenesis of Alzheimer's Disease, and the Group VIA phospholipase A(2) (iPLA(2)beta) is the dominant PLA(2) enzyme in the central nervous system and is subject to regulatory proteolytic processing. We have identified novel N-terminal variants of iPLA(2)beta and previously unrecognized proteolysis sites in APP constructs with a C-terminal 6-myc tag by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests. We have developed a Signature-Discovery (SD) program to characterize protein isoforms by identifying signature peptides that arise from proteolytic processing in vivo. This program analyzes MS/MS data from LC analyses of proteolytic digests of protein mixtures that can include incompletely resolved components in biological samples. This reduces requirements for purification and thereby minimizes artifactual modifications during sample processing. A new algorithm to generate the theoretical signature peptide set and to calculate similarity scores between predicted and observed mass spectra has been tested and optimized with model proteins. The program has been applied to the identification of variants of proteins of biological interest, including APP cleavage products and iPLA(2)beta, and such applications demonstrate the utility of this approach.     

Effect of the Secondary Reduction on the Enantioselectivity and Function of Additives in the Chiral Oxazaborolidine‐Catalyzed Asymmetric Borane Reduction of Ketones

The secondary reduction in the direct and oxazaborolidine‐catalyzed asymmetric borane reduction of ketones was investigated by the use of GC/MS tracing titration and control experiments. The results indicate that the secondary reduction affects the enantioselectivity only in noncoordinated solvents at low temperature and not under the usual catalytic reduction conditions because the intermediate alkoxyborane is unstable and quickly converts to borane and dialkoxyborane. The function of an alcohol additive in the asymmetric borane reduction of ketones is to consume excess borane in the reduction system thus inhibiting noncatalytic reduction, which leads to increased enantioselectivity in the catalytic reduction.     

柠檬酸铝/聚丙烯酰胺胶态分散凝胶反应动力学和机理的研究

采用粘度法研究了柠檬酸铝（AlCit)与部分水解取丙烯酰胺（HPAM）胶态分散凝胶体系的反应动力学。结果表明，胶态分散凝胶体系的交联反应是一级反应。并推导出胶态分散凝胶体系的反应动力学方程。     

Phase separation of p53 precedes aggregation and is affected by oncogenic mutations and ligands

Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF). The mechanism of the formation of the aggregates in the nucleus remains uncertain. The present study demonstrated that the DNA-binding domain of p53 (p53C) underwent phase separation (PS) on the pathway to aggregation under various conditions. p53C phase separated in the presence of the crowding agent polyethylene glycol (PEG). Similarly, mutant p53C (M237I and R249S) underwent PS; however, the process evolved to a solid-like phase transition faster than that in the case of wild-type p53C. The data obtained by microscopy of live cells indicated that transfection of mutant full-length p53 into the cells tended to result in PS and phase transition (PT) in the nuclear compartments, which are likely the cause of the GoF effects. Fluorescence recovery after photobleaching (FRAP) experiments revealed liquid characteristics of the condensates in the nucleus. Mutant p53 tended to undergo gel- and solid-like phase transitions in the nucleus and in nuclear bodies demonstrated by slow and incomplete recovery of fluorescence after photobleaching. Polyanions, such as heparin and RNA, were able to modulate PS and PT in vitro. Heparin apparently stabilized the condensates in a gel-like state, and RNA apparently induced a solid-like state of the protein even in the absence of PEG. Conditions that destabilize p53C into a molten globule conformation also produced liquid droplets in the absence of crowding. The disordered transactivation domain (TAD) modulated both phase separation and amyloid aggregation. In summary, our data provide mechanistic insight into the formation of p53 condensates and conditions that may result in the formation of aggregated structures, such as mutant amyloid oligomers, in cancer. The pathway of mutant p53 from liquid droplets to gel-like and solid-like (amyloid) species may be a suitable target for anticancer therapy.

Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF).     

Influence of structure and solubility of chain transfer agents on the RAFT control of dispersion polymerisation in scCO2

Reversible addition–fragmentation chain transfer (RAFT) dispersion polymerisation of methyl methacrylate (MMA) is performed in supercritical carbon dioxide (scCO₂) with 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) present as chain transfer agent (CTA) and surprisingly shows good control over PMMA molecular weight. Kinetic studies of the polymerisation in scCO₂ also confirm these data. By contrast, only poor control of MMA polymerisation is obtained in toluene solution, as would be expected for this CTA which is better suited for acrylates. In this regard, we select a range of CTAs and use them to determine the parameters that must be considered for good control in dispersion polymerisation in scCO₂. A thorough investigation of the nucleation stage during the dispersion polymerisation reveals an unexpected “in situ two-stage” mechanism that strongly determines how the CTA works. Finally, using a novel computational solvation model, we identify a correlation between polymerisation control and degree of solubility of the CTAs. All of this ultimately gives rise to a simple, elegant and counterintuitive guideline to select the best CTA for RAFT dispersion polymerisation in scCO₂.

RAFT dispersion polymerisation of methyl methacrylate is performed in scCO₂ with 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) present as chain transfer agent (CTA) and surprisingly shows good control over PMMA molecular weight.