Structural Properties of Model Phosphatidylcholine Flippases

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Highlights? Model peptides induce sequence-dependent flip of lipids with different headgroups ? Flip activity of peptides depends on the lipid composition of the host membrane ? SNARE proteins have lipid flippase activity     

Kinetics of Complex Formation Between Cobalt(II) and the Polyamine Me2Octaen

Abstract

The kinetics and the equilibria of complex formation between Co(II) and the linear polyamine 1,25-dimethyl-1,4,7,10,13,16,19,22,25-nonaazapentacosane (Me₂Octaen) have been investigated in aqueous solution under anaerobic conditions by the stopped-flow and potentiometric method. Static titrations and kinetic experiments indicate that below pH 5.6 the binding of Co(II) to the ligand gives rise to several 1:1 complexes differing for their degree of protonation, whereas above pH 5.6 binuclear complexes are formed as well. The analysis of the kinetic data reveals that the mostly reactive form of the ligand (denoted in its fully protonated form as H₉L₉₊) is H₆L⁶⁺. The species H₅L⁵⁺ and H₄L⁴⁺ yield only a minor contribution to the overall process of complex formation. Measurements at different ionic strengths have shown positive salt effects, as expected for reactive processes involving particles with charges of the same sign. The analysis of the results shows that Co²⁺ and H₆L⁶⁺ react according to the internal conjugate base mechanism.     

A native electrophoretic technique to study oligomerization and activity of cytosolic 5′-nucleotidase II

The analysis of the oligomeric active state of a native protein usually requires the application of at least two analytical methods such as gel filtration and analytical ultracentrifugation. Both methods require a substantial amount of protein, time and/or expensive equipment. We here describe a native electrophoretic method for the identification of the native molecular weight of the recombinant wild-type cytosolic 5′-nucleotidase (cN-II) and of its mutants in subunit interfaces Y115A, F36R, K311A and G319Q. The protein was stained both with protein dye and with an activity staining method. Our results demonstrated that purified recombinant protein preparations contained substantial amounts of nucleic acids and misfolded, inactive protein. Furthermore, cN-II mutants K311A and G319Q in subunit interface assume a quaternary dimeric active form, while the only active quaternary structure of wild-type cN-II is the tetramer.     

Cellulose Dissolution in Mixtures of Ionic Liquids and Dimethyl Sulfoxide: A Quantitative Assessment of the Relative Importance of Temperature and Composition of the Binary Solvent

We studied the dissolution of microcrystalline cellulose (MCC) in binary mixtures of dimethyl sulfoxide (DMSO) and the ionic liquids: allylbenzyldimethylammonium acetate; 1-(2-methoxyethyl)-3-methylimidazolium acetate; 1,8-diazabicyclo [5.4.0]undec-7-ene-8-ium acetate; tetramethylguanidinium acetate. Using chemometrics, we determined the dependence of the mass fraction (in %) of dissolved cellulose (MCC-m%) on the temperature, T = 40, 60, and 80 °C, and the mole fraction of DMSO, χ_DMSO = 0.4, 0.6, and 0.8. We derived equations that quantified the dependence of MCC-m% on T and χ_DMSO. Cellulose dissolution increased as a function of increasing both variables; the contribution of χ_DMSO was larger than that of T in some cases. Solvent empirical polarity was qualitatively employed to rationalize the cellulose dissolution efficiency of the solvent. Using the solvatochromic probe 2,6-dichloro-4-(2,4,6-triphenylpyridinium-1-yl)phenolate (WB), we calculated the empirical polarity E_T(WB) of cellobiose (a model for MCC) in ionic liquid (IL)–DMSO mixtures. The E_T(WB) correlated perfectly with T (fixed χ_DMSO) and with χ_DMSO (fixed T). These results show that there is ground for using medium empirical polarity to assess cellulose dissolution efficiency. We calculated values of MCC-m% under conditions other than those employed to generate the statistical model and determined the corresponding MCC-m% experimentally. The excellent agreement between both values shows the robustness of the statistical model and the usefulness of our approach to predict cellulose dissolution, thus saving time, labor, and material.     

Synthesis, characterization, DNA interaction and potential applications of gold nanoparticles functionalized with Acridine Orange fluorophores

Two new water-soluble gold nanoparticles (AO-TEG-Au and AO-PEG-Au NPs) are prepared and characterized. They are stabilized by thioalkylated oligoethylene glycols and functionalized with fluorescent Acridine Orange (AO) derivatives. Despite the different core sizes (11.8 and 3.9 nm respectively) and shell composition, they are both well dispersed and are stable in water, even if some self-aggregation is observed in the case of AO-TEG-Au NPs. However, AO-PEG-Au NPs show much lower emission efficiency with respect to AO-TEG-Au NPs. Spectrophotometric and spectrofluorometric experiments indicate that both types of nanoparticle are able to bind to calf thymus DNA, either by external binding or partial intercalation. Preliminary FACS flow cytometry tests seem to indicate that the AO-TEG-Au nanoparticle is able to cross the cell membrane where it is absorbed by Chinese hamster ovary (CHO) cells at the picomolar concentration level.     

Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field-amplified sample injection capillary electrophoresis with UV detection

Alterations in global DNA methylation are implicated in several pathobiological processes. The tissues stored as paraffin blocks represent an important source of DNA for retrospective genetic and epigenetic analysis on a large scale. Therefore, we developed the first capillary electrophoresis method able to measure global methylation in formalin-fixed, paraffin-embedded (FFPE) DNA extracts. A field-amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of cytosine and 5-methylcytosine released following DNA hydrolysis by means of formic acid was employed. Analytes were baseline-separated within 8 min by using 300 mM tris(hydroxymethyl)aminomethane phosphate pH 3.75 as the running buffer. With use of electrokinetic injection the limit of detection (LOD) in real sample was 0.1 nM, thus improving by about 400-fold the LOD of the previously described methods based on capillary electrophoresis. Sample extraction and purification were optimized so that evaluation of the DNA methylation degree was possible starting from 0.5-1 μg of DNA with intra- and interassay relative standard deviations for the 5-methylcytosine to total cytosine ratio of 2.0 and 3.2%, respectively. Because of its high accuracy and throughput, our method will be useful for large-scale applications to determine the implications of genomic DNA methylation levels in tumorigenesis.     

Cellulase Production by <Emphasis Type="Italic">Streptomyces viridobrunneus</Emphasis> SCPE-09 Using Lignocellulosic Biomass as Inducer Substrate

An actinomycete strain, isolated from a soil sample under a sugar cane plantation in Brazil and identified as Streptomyces viridobrunneus SCPE-09, was selected as a promising cellulolytic strain, and tested for its ability to produce cellulases from agro-industrial residues. Sugar cane bagasse or wheat bran was tested as carbon source, and corn steep liquor tested as nitrogen source. Different concentrations of carbon and nitrogen were tested using factorial design to identify optimal cellulose production. The results showed that media containing wheat bran 2.0% (w/v) and corn steep liquid 0.19% (w/v) lead to the highest production, 2.0 U mL⁻¹ of CMCase, obtained on the fifth day of fermentation. The pH and temperature profile showed optimal activity at pH 4.9 and 50°C. As for thermostability, endoglucanases were most tolerant at 50°C, retaining more than 80% of maximal activity even after 2 h of incubation. Zymogram analyses using supernatant from growth under optimized conditions revealed the presence of two CMCase bands with apparent molecular masses of 37 and 119 kDa. The combination of pH tolerance and CMCase production from agro-industrial residues by S. viridobrunneus SCPE-09 offers promise for future bioethanol biotechnologies.     

Direct determination of bioavailable molybdenum in carbon nanotubes

Bioavailable residual metallic impurities within carbon nanotubes (CNTs) are responsible for the toxicity of CNTs. Herein we present a method for fast, sensitive determination of bioavailable molybdenum residual catalyst impurities within CNTs by using electrochemical oxidation in neutral pH buffers at low potentials. This method is unique because no other method can rapidly distinguish between bioavailable/mobilizable impurities from defects in CNTs and between the total amounts of impurities. This method will be indispensable for future toxicological studies of CNTs.