The origin of the dynamic transition in proteins

Despite extensive efforts in experimental and computational studies, the microscopic understanding of dynamics of biological macromolecules remains a great challenge. It is known that hydrated proteins, DNA and RNA, exhibit a so-called "dynamic transition." It appears as a sharp rise of their mean-squared atomic displacements r2 at temperatures above 200-230 K. Even after a long history of studies, this sudden activation of biomolecular dynamics remains a puzzle and many contradicting models have been proposed. By combining neutron and dielectric spectroscopy data, we were able to follow protein dynamics over an extremely broad frequency range. Our results show that there is no sudden change in the dynamics of the protein at temperatures around approximately 200-230 K. The protein's relaxation time exhibits a smooth temperature variation over the temperature range of 180-300 K. Thus the experimentally observed sharp rise in r2 is just a result of the protein's structural relaxation reaching the limit of the experimental frequency window. The microscopic mechanism of the protein's structural relaxation remains unclear.     

Optimization of solid-phase extraction for the liquid chromatography-mass spectrometry analysis of harpagoside, 8-para-coumaroyl harpagide, and harpagide in equine plasma and urine

Solid-phase extraction cartridges among those usually used for screening in horse doping analyses are tested to optimize the extraction of harpagoside (HS), harpagide (HG), and 8-para-coumaroyl harpagide (8PCHG) from plasma and urine. Extracts are analyzed by liquid chromatography coupled with multi-step tandem mass spectrometry. The extraction process retained for plasma applies BondElut PPL cartridges and provides extraction recoveries between 91% and 93%, with RSD values between 8 and 13% at 0.5 ng/mL. Two different procedures are needed to extract analytes from urine. HS and 8PCHG are extracted using AbsElut Nexus cartridges, with recoveries of 85% and 77%, respectively (RSD between 7% and 19%). The extraction of HG involves the use of two cartridges: BondElut PPL and BondElut C18 HF, with recovery of 75% and RSD between 14% and 19%. The applicability of the extraction methods is determined on authentic equine plasma and urine samples after harpagophytum or harpagoside administration.     

Microwave‐assisted synthesis of new regioisomeric 6,7‐dihydroindeno[1,2‐e]pyrimido[4,5‐b][1,4]diazepin‐5(5aH)‐ones

Novel 11‐amino‐6‐aryl‐6,7‐dihydroindeno[1,2‐e] pyrimido[4,5‐b][1,4]diazepin‐5(5aH)‐ones 4a‐f were prepared regioselectively by the tricomponent reaction of 4,5,6‐triaminopyrimidine 1, 1,3‐indandione 2 and aromatic aldehydes 3a‐f. The bicomponent approach, using 2,4,5,6‐tetraaminopyrimidine 5 and 2‐aryl‐ideneindandiones 6a‐f as reagents, afforded 9,11‐diamino‐6‐aryl‐6,7‐dihydroindeno[1,2‐e]pyrimido[4,5‐b]‐[1,4]diazepin‐5(5aH)‐ones 7a‐f in good yields and the regioisomeric 8,10‐diamino derivatives 8a‐c in lower yields. Both, bi‐ and tricomponent approaches were performed by microwave irradiation and all products were fully characterized by detailed NMR measurements.     

Synthesis of organometallic Ru(II) and Fe(II) complexes containing fused rings hemi-helical ligands as chromophores. Evaluation of non-linear optical properties by HRS

A new family of three-legged piano stool structured organometallic compounds containing the fragment η⁵-cyclopentadienyl-ruthenium(II)/iron(II) has been synthesized to evaluate the existence of electronic metal to ligand charge transfer upon coordination of the novel benzodithiophene ligands (BDT), benzo[1,2-b;4,3-b′]dithiophen-2-carbonitrile (L1) and benzo[1,2-b;4,3-b′]dithiophen-2′nitro-2-carbonitrile (L2). All the compounds were characterized by ¹H, ¹³C, ³¹P NMR, IR and UV-Vis. spectroscopies and their electrochemistry studied by cyclic voltammetry. The X-ray structures of [Ru(η⁵-C₅H₅)(PPh₃)₂(NCC₁₀H₅S₂)][PF₆] (1Ru), [Ru(η⁵-C₅H₅)(PPh₃)₂(NCC₁₀H₅S₂)][CF₃SO₃] (1′Ru), [Ru(η⁵-C₅H₅)(DPPE)(NCC₁₀H₅S₂)][PF₆] 2Ru and [Fe(η⁵-C₅H₅)(DPPE)(NCC₁₀H₅S₂)][PF₆] (2Fe) were determined by X-ray diffraction showing centric crystallization on groups and P2₁/n, respectively.Quadratic hyperpolarizabilities (β) of some of the complexes (2Fe, 2Ru and 3Fe) have been determined by hyper-Rayleigh scattering (HRS) measurements at a fundamental wavelength of 1500 nm, to minimize the probability of fluorescence due to two-photon absorption and to reduce the effect of resonance enhancement, in order to estimate static β values.     

Efficient mining of myxobacterial metabolite profiles enabled by liquid chromatography-electrospray ionisation-time-of-flight mass spectrometry and compound-based principal component analysis

Bacteria producing secondary metabolites are an important source of natural products with highly diverse structures and biological activities. Developing methods to efficiently mine procaryotic secondary metabolomes for the presence of potentially novel natural products is therefore of considerable interest. Modern mass spectrometry-coupled liquid chromatography can effectively capture microbial metabolic diversity with ever improving sensitivity and accuracy. In addition, computational and statistical tools increasingly enable the targeted analysis and exploration of information-rich LC-MS datasets.In this article, we describe the use of such techniques for the characterization of myxobacterial secondary metabolomes. Using accurate mass data from high-resolution ESI-TOF measurements, target screening has facilitated the rapid identification of known myxobacterial metabolites in extracts from nine Myxococcus species. Furthermore, principal component analysis (PCA), implementing an advanced compound-based bucketing approach, readily revealed the presence of further compounds which contribute to variation among the metabolite profiles under investigation. The generation of molecular formulae for putative novel compounds with high confidence due to evaluation of both exact mass position and isotopic pattern, is exemplified as an important key for de-replication and prioritization of candidates for further characterization.     

电感耦合等离子体原子发射光谱法测定钛酸钡纳米粉体中7种杂质元素

钛酸钡纳米粉体样品用盐酸溶解,在所得样品溶液中用电感耦合等离子体原子发射光谱法测定了以氧化物存在于样品中的7种杂质元素(包括镁、锶、钾、钠、铝、硅及铁),对测定的光谱条件进行了试验并优化达到上述各元素的测定上限为0.1%(质量分数).测得方法的检出限(3S/b)为(单位μg·L-1):2.0(铝),1.0(铁),20.0(钾),0.2(镁),4.0(钠),3.0(硅)及0.5(锶).应用此方法分析了两件实样,所得结果的相对标准偏差(n=6)小于等于2.0%,对其中4种杂质元素(镁、钾、钠和锶)同时用HG/T 3587-1999标准中的方法作分析校核,所得结果与此方法结果一致.     

In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis

A capillary electrophoresis (CE)-based method for the in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4/Bradeion β (GST-rDGTPase). This example application demonstrates that the CE technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: v _max = 1.7 μM min⁻¹ ± 0.1, Km = 1.0 mM ± 0.3, and apKcat = 9 × 10⁻³ s⁻¹. In addition the effect of co-factors such as Mg²⁺ and Mn²⁺ on the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze diphosphated and triphosphated forms of other nucleotides.     

Application of design-of-experiments procedures to optimize efficiently pretreatment of lipase for use in a nonaqueous reaction

A variety of different pretreatments can improve the performance of enzymes in nonpolar reaction media. These pretreatments have primarily been studied in isolation; however, interactions between some pairs of pretreatments are known to exist. The presence of these interactions complicates the design of an optimum multifactor pretreatment. Modern design-of-experiments techniques allow the simultaneous optimization of two or more variables. To improve the performance of lipase in a model reaction, we used a technique called the method of steepest ascent to optimize three variables simultaneously: pretreatment pH and sodium phosphate concentration, and the concentration of acetic acid (one of the reactants) in the reaction mixture. In only 26 experimental runs, this optimization process determined a combination of variable settings that yielded a reaction product approx 180 times faster than achieved with untreated enzyme. Evidence is presented to demonstrate that locating this optimum with single-factor experiments would be inefficient. This article demonstrates the efficiency of the method of steepest ascent particularly for evaluation of enzymatic reaction conditions exhibiting significant interactions.     

Modification of polysulfone membranes with polyethylene glycol and lignosulfate: electrical characterization by impedance spectroscopy measurements

Two sets of composite membranes having an asymmetric sulfonated polysulfone membrane as support layer have been obtained and electrically characterized (membranes SPS-PEG and PA-LIGS). The skin layer of the membrane SPS-PEG contains different percentages of polyethylene glycol in the casting solution (5, 25, 40, and 60 wt%), while lignosulfonate was used for manufacturing PA-LIGS membranes (5, 10, 20, and 40 wt%). Membrane electrical characterization was done by means of impedance spectroscopy (IS) measurements, which were carried out with the membranes in contact with NaCl solutions at different concentrations (10(-3) < or = c(M) < or = 5x10(-2)). Electrical resistance and equivalent capacitance of the different membrane samples were determined from IS plots by using equivalent circuits as models. Results show a clear decrease in the membrane electrical resistance as a result of both polysulfone sulfonation and the increase of the concentration of modifying substances, although a kind of limit concentration was obtained for both polyethylene glycol and lignosulfonate (40 and 20%, respectively). Results also show a decrease of around 90% in electrical resistance due to polysulfone sulfonation, while the value of the dielectric constant (hydrated state) clearly increases.