Application of immunoproteomics to rapid cytokine detection

Cytokines are pivotal to a balanced innate or cell-mediated immune response, and can be indicative of disease progression and/or resolution. Methods to measure key cytokines rapidly with accuracy, precision, and sensitivity are consequently important. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative, semi-quantitative approach described here, uses an immunological capture step and a mass spectral readout. The goal of the assay is speed rather than sensitivity or precision.     

药物不良反应造成急性肾损害的临床分析

目的：研究药物不良反应（ADR）所致急性肾损伤的相关因素并探讨其诊疗思路。,方法：对56例ADR造成急性肾损害的病例进行归类统计和分析评价。结果：56例病例中,男性略多于女性,男女之比为1．24：1,平均年龄53．1岁;抗生素发生率居首位,其次为中成药;途径以静脉注射为主。结论：合理用药,早期干预治疗,预防药源性急性肾损害的发生。     

一批逆转录酶活性检测假阳性的鉴别

目前逆转录病毒的检测方法被普遍应用的是逆转录酶活性检测,自从PCR方法被应用到逆转录酶活性检测中后,使检测的灵敏度及特异性得到极大地提高。同时,假阳性结果也带给各实验室很大困扰,包括试验过程中的实验用试剂及检测样品本身带来的假阳性。实验中观察了一批逆转录酶活性检测阳性样品和两个依赖DNA的DNA聚合酶的假阳性,通过对检测的严密监控以及改变反应体系pH值,抑制了由于细胞死亡后所释放的细胞内DNA聚合酶以及实验用DNA聚合酶的类逆转录酶样作用,从而达到鉴别结果的真实性的目的。     

Influence of chain length on mono- versus di-alkylation in the reactivity of [Pt2(μ-S)2(PPh3)4] towards α,ω-dihalo-n-alkanes; a synthetic route to platinum(II) ω-haloalkylthiolate complexes

The reactions of [Pt₂(μ-S)₂(PPh₃)₄] with α,ω-dibromoalkanes Br(CH₂)_nBr (n = 4, 5, 6, 8, 12) gave mono-alkylated [Pt₂(μ-S){μ-S(CH₂)_nBr}(PPh₃)₄]⁺ and/or di-alkylated [Pt₂(μ-S(CH₂)_nS}(PPh₃)₄]²⁺ products, depending on the alkyl chain length and the reaction conditions. With longer chains (n = 8, 12), intramolecular di-alkylation does not proceed in refluxing methanol, with the mono-alkylated products [Pt₂(μ-S){μ-S(CH₂)_nBr}(PPh₃)₄]⁺ being the dominant products when excess alkylating agent is used. The bridged complex [{Pt₂(μ-S)₂(PPh₃)₄}₂{μ-(CH₂)₁₂}]²⁺ was accessible from the reaction of [Pt₂(μ-S)₂(PPh₃)₄] with 0.5 mol equivalents of Br(CH₂)₁₂Br. [Pt₂(μ-S){μ-S(CH₂)₄Br}(PPh₃)₄]⁺ can be cleanly isolated as its BPh₄⁻ salt, but undergoes facile intramolecular di-alkylation at −18 °C, giving the known species [Pt₂(μ-S(CH₂)₄S}(PPh₃)₄]²⁺. The reaction of I(CH₂)₆I with [Pt₂(μ-S)₂(PPh₃)₄] similarly gives [Pt₂(μ-S){μ-S(CH₂)₆I}(PPh₃)₄]⁺, which is fairly stable towards intramolecular di-alkylation once isolated. These reactions provide a facile route to ω-haloalkylthiolate complexes which are poorly defined in the literature. X-ray crystal structures of [Pt₂(μ-S){μ-S(CH₂)₅Br}(PPh₃)₄]BPh₄ and [Pt₂(μ-S(CH₂)₅S}(PPh₃)₄](BPh₄)₂ are reported, together with a study of these complexes by electrospray ionisation mass spectrometry. All complexes fragment by dissociation of PPh₃ ligands, and the bromoalkylthiolate complexes show additional fragment ions [Pt₂(μ-S){μ-S(CH₂)_n−2CHCH₂}(PPh₃)_m]⁺ (m = 2 or 3; m ≠ 4), most significant for n = 4, formed by elimination of HBr.     

Kinetics of Sulfate Reduction in a Coastal Aquifer Contaminated with Petroleum Hydrocarbons

An integrated field and laboratory study was conducted to quantify the effect of environmental determinants on the activity of sulfate reducers in a freshwater aquifer contaminated with petroleum hydrocarbons (PHC). Within the contaminated zone, PHC-supported in␣situ sulfate reduction rates varied from 11.58±3.12 to 636±53 nmol cm⁻³ d⁻¹ and a linear increase (R ²=0.98) in reduction rate was observed with increasing in situ sulfate concentrations suggesting sulfate limitation. Half-saturation concentration (K _s) for sulfate reduction coupled to PHC mineralization was determined for the first time. At two different sites within the␣aquifer, maximum sulfate reduction rate under␣non-limiting conditions (R _max) was 5,000 nmol cm⁻³ d⁻¹, whereas the retrieved K _s values were 3.5 and 7.5 mM, respectively. The K _s values are the highest ever reported from a natural environment. Furthermore, the K _s values were significantly higher than in situ sulfate concentrations confirming sulfate limited growth. On addition of lactate and formate, sulfate reduction rate increased indicating that reactivity and bioavailability of organic substrate may also have played a role in rate inhibition in certain parts of the aquifer. Experiments with sulfide amendments show statistically minor decrease in sulfate reduction rates on addition of sulfide and analogous increase in sulfide toxicity with increasing sulfide concentrations (0.5–10 mM) was not apparent.     

Relations between biochemical thermodynamics and biochemical kinetics

The parameters in steady-state or rapid-equilibrium rate equations for enzyme-catalyzed reactions depend on the temperature, pH, and ionic strength, and may depend on the concentrations of specific species in the buffer. When the complete rate equation (i.e. the equation with parameters for the reverse reaction as well as the forward reaction) is determined, there are one or more Haldane relations between some of the kinetic parameters and the apparent equilibrium constant for the reaction that is catalyzed. When the apparent equilibrium constant can be calculated from the kinetic parameters, the equilibrium composition can be calculated. This is remarkable because the kinetic parameters all depend on the properties of the enzymatic site, but the apparent equilibrium constant and the equilibrium composition do not. The effects of ionic strength and pH on the unoccupied enzymatic site and the occupied enzymatic site have to cancel in the Haldane relation or in the calculation of the apparent equilibrium constant using the rate constants for the steps in the mechanism. Several simple enzymatic mechanisms and their complete rate equations are discussed.     

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369–7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473–21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

Convection Effects in the BIAcore Dextran Layer: Surface Reaction Model

The BIAcore is a surface plasmon resonance (SPR) device used to measure rate constants, primarily for biochemical reactions. It consists of a flow channel containing one reactant adjoining a dextran gel containing the other. In order to explain anomalous measurements from the device, it has been proposed that some flow penetrates into the dextran layer, thus enhancing transport. A model is presented that accounts for such behavior, and typical velocity fields in the dextran are constructed. The system is analyzed in the limit of the surface reaction model, which corresponds to the limit of thin dextran layers. Asymptotic and singular perturbation techniques are used to analyze association and dissociation kinetics. Linear and nonlinear integral equations result from the analysis; explicit and asymptotic solutions are constructed for physically realizable cases. The results indicate that the effects of such penetration are bound to be small, regardless of the flow model used.     

Kinetic studies on the first dihydrogen aquacomplex, [Ru(H2)(H2O)5]: Formation under H2 pressure and catalytic H/D isotope exchange in water

The ruthenium(II) hexaaqua complex [Ru(H₂O)₆]²⁺ reacts with dihydrogen under pressure to give the η²-dihydrogen ruthenium(II) pentaaqua complex [Ru(H₂)(H₂O)₅]²⁺.The complex was characterized by ¹H, ²H and ¹⁷O NMR: δ_H = −7.65 ppm, J_HD = 31.2 Hz, δ_O = −80.4 ppm (trans to H₂) and δ_O = −177.4 ppm (cis to H₂).The H-H distance in coordinated dihydrogen was estimated to 0.889 Å from J_HD, which is close to the value obtained from DFT calculations (0.940 Å).Kinetic studies were performed by ¹H and ²H NMR as well as by UV-Vis spectroscopy, yielding the complex formation rate and equilibrium constants: k_f = (1.7 ± 0.2) × 10⁻³ kg mol⁻¹ s⁻¹ and K_eq = 4.0 ± 0.5 mol kg⁻¹.The complex formation rate with dihydrogen is close to values reported for other ligands and thus it is assumed that the reaction with dihydrogen follows the same mechanisn (I_d).In deuterated water, one can observe that [Ru(H₂)(H₂O)₅]²⁺ catalyses the hydrogen exchange between the solvent and the dissolved dihydrogen.A hydride is proposed as the intermediate for this exchange.Using isotope labeling, the rate constant for the hydrogen exchange on the η²-dihydrogen ligand was determined as k₁ = (0.24 ± 0.04) × 10⁻³ s⁻¹.The upper and lower limits of the pK_a of the coordinated dihydrogen ligand have been estimated:3 < pK_a < 14.     

Synthesis and theoretical analysis of the structures and bonding in five new neutral square planar bis[2,4-di(aryl)-1,3,5-triazapentadienato]nickel(II) complexes

Solvothermal reactions in methanol of nickel acetate tetrahydrate, Ni(OAc)₂ · 4H₂O, with benzonitrile derivatives NC(C₆H₄)X, where X is one of the electron withdrawing substituents -CN, -NO₂, or -CF₃, located at the m- or p-positions relative to -CN, yield complexes of the general formula Ni{HNC(R)-NC(R)-NH}₂. More specifically, 3-nitrobenzonitrile, 4-nitrobenzonitrile, 1,3-dicyanobenzene, 1,4-dicyanobenzene, and ααα-trifluoro-p-toluonitrile are found to react with Ni(OAc)₂ · 4H₂O to yield Ni{HNC(R)-NC(R)-NH}₂, where R = 3-(NO₂)C₆H₄, 4-(NO₂)C₆H₄, 3-(CN)C₆H₄, 4-(CN)C₆H₄, or 4-(CF₃)C₆H₄, respectively. Analogous reactions of nitriles lacking electron withdrawing groups do not occur under similar conditions. Solid-state structures have been determined for the complexes with p-NO₂, p-CN, and p-CF₃ substituents on the phenyl rings. In addition, we describe density functional theory (DFT) and natural bonding orbital theory (NBO) studies on a simplified analog of these compounds, aimed at understanding their molecular bonding. It is shown that the new compounds for which solid-state structures have been determined are model examples of coordination compounds containing robust ω-bonds.