Antimalarial Drugs Exacerbate Rat Liver Microsomal Lipid Peroxidation in the Presence of Oxidants

The study was undertaken to evaluate the effect of prior treatment of rats with the antimalarial drugs amodiaquine (AQ) mefloquine (MQ) and halofantrine (HF) on rat liver microsomal lipid peroxidation in the presence of 1 mM FeSO4, 1 mM ascorbate and 0.2 mM H₂O₂ (oxidants). Ingestion of -tocopheral, a radical chain-breaking antioxidant was also included to assess the role of antioxidants in the drug treatment. In the presence of oxidants AQ, MQ and HF elicited 288%, 175% and 225% increases in malondialdehyde (MDA) formation while the drugs induced 125%, 63% and 31% increases in the absence of oxidants respectively. Similarly, AQ, MQ and HF induced lipid hydroperoxide formation by 380%, 256%, 360% respectively in the presence of oxidants and 172%, 136% and 92% in the absence of exogenously added oxidants respectively. -tocopherol reduced AQ, MQ and HF-induced MDA formation by 40%, 55% and 52% respectively and lipid hydroperoxide formation by 53%, 59% and 54% respectively. Similarly, -tocopherol attenuated the AQ, MQ and HF-induced MDA formation by 49%, 51% and 51% in the presence of oxidants and lipid hydroperoxide formation by 61%, 62% and 47% respectively. The results indicate that rat liver microsomal lipid peroxidation could be enhanced by antimalarial drugs in the presence of reactive oxygen species and this effect could be ameliorated by treatment with antioxidants.     

Stabilization method of an alkaline protease from inactivation by heat,SDS and hydrogen peroxide

An investigation was carried out to evaluate the protective effect of polyhydric alcohols, such as propylene glycol and glycerol on the inactivation of an alkaline protease by sodium dodecyl sulfate (SDS) and H₂O₂. Addition of polyols increased the stability of a Bacillus clausii I-52 alkaline protease towards not only the thermal-induced, but also the SDS and H₂O₂-induced inactivation. Among the polyols examined, the best results were obtained with propylene glycol. The half-life of the enzyme was increased by 43- and >105-fold by the addition of 10% (v/v) propylene glycol to the enzyme preparations containing 5% (w/v) SDS and 5% (v/v) H₂O₂ at 50 °C, respectively. Besides the protection effect of propylene glycol from enzyme inactivation by SDS and H₂O₂, it also improved the hydrolytic efficiency towards substrate like BSA during the protease reaction containing SDS or H₂O₂. This result suggests that propylene glycol has a significant potential as a good stabilizer of an alkaline protease preparation, which finds use as an additive in industrial applications, especially, the detergent industry.     

Ribulose bisphosphate carboxylase/oxygenase from Thiocapsa roseopersicina

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified 80-fold from malate-grown Thiocapsa roseopersicina by salting out the enzyme from the high-speed supernatant between 68–95% saturation with respect to (NH₄)₂SO₄, gelfiltration through Sephadex G-100, and DEAE-cellulose chromatography followed by sedimentation into a 14–34% glycerol gradient. The specific activity of enzyme for the carboxylase reaction was 2.45 mol RuBP-dependent CO₂ fixed/min · mg protein (at pH 8.0 and 30° C) and for the oxygenase reaction was 0.23 mol RuBP-dependent O₂ consumed/min · mg protein (at pH 8.6, and 25° C). The enzyme, which was ultracentrifugally homogeneous in the presence of 4 and 10% v/v glycerol, was stable for at least one year at-80° C in the presence of 10% glycerol. S_{20, w} values obtained in the presence of 4 and 10% glycerol were 19.3 and 16.2, respectively. The enzyme contained both large (53,000-daltons) and mixed small subunits (15,000- and 13,500-daltons).Borate-dependent inactivation of the enzyme by 2,3-butadione, which was greatly reduced in the presence of the product 3-phosphoglycerate, suggested that one or more arginines are at the active site.Abbreviations DTT dithiotreitol - RuBP d-ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate - TCA trichloroacetic acid - TEMBDG buffer (pH 8.0 at 25°C) containing 20 mM Tris, 1 mM disodium EDTA · 2 H₂O, 10 mM MgCl₂·6 H₂O, 50 mM NaHCO₃, 0.1 mM DTT and 10% glycerol (v/v)     

Partitioning of Proteins using a Hydrophobically Modified Ethylene Oxide/SDS Aqueous Two-phase System

Summary A novel aqueous two-phase system containing hydrophobically modified ethylene oxide (HM-EO) and sodium dodecyl sulphate (SDS) was developed to enhance the selectivity of protein partitioning in two phases. Phase diagrams of HM-EO/H₂O and HM-EO/SDS/H₂O were measured, and the mechanism of interaction between HM-EO polymer and the anionic surfactant sodium dodecyl sulphate (SDS) was also discussed. It was found that the improvement of selectivity of protein partitioning was related to the increase of electrostatic potential difference between the two phases because of the charged network formed by mixed micelles of HM-EO and SDS in the bottom phase. With bovine serum albumin (BSA) and lysozyme as model proteins, some factors, such as pH, SDS concentration, conductivity and temperature of the system, were investigated for the influences of protein partition in HM-EO/SDS/H₂O systems. The results showed that the addition of SDS not only changed the phase behaviour, but also played an important role in protein partitioning.     

宁夏陆地生态系统水分利用效率特征及其影响因子

生态系统水分利用效率(Water Use Efficiency, WUE)是表征生态系统碳水耦合程度的重要指标,能反映生态系统碳水循环规律及其相互作用关系。基于MODIS数据以及宁夏生态系统类型数据,分析2000—2017年宁夏不同生态系统WUE的变化特征,探讨了NPP和ET两种因子对WUE年际与年内变化的影响。结果表明:(1)全区陆地生态系统的年均WUE为1.03 g·C/kg·H_2O,值域在0.55—2.98 g·C/kg·H_2O之间,总体上呈现南北高、中部低的特征。(2)不同生态系统的WUE差异较大,由高到低为水体及湿地、森林、农田、草地、聚落、荒漠和其他生态系统,在同类生态系统中,植被生物量和盖度越高的亚类生态系统,其WUE也越高。(3)宁夏陆地生态系统WUE存在着每年0.0141 g·C/kg·H_2O的下降趋势,年内WUE呈典型的单峰形态,变化范围在0.02—2.16 g·C/kg·H_2O之间。(4)年际尺度上,宁夏陆地生态系统WUE与年蒸散(Evapotranspiration,ET)有极显著负相关性(P0.01),而与净初级生产力(Net Primary Production,NPP)没有相关性;年内尺度上,WUE变化与ET呈显著正相关(P0.05),与NPP呈极显著正相关(P0.01),这与植被的年内季节性生长过程有关。(5)根据ET强弱和WUE高低,可将宁夏陆地生态系统水分利用效率特征划分为4类,即低ET低WUE区、低ET高WUE区、高ET低WUE区和高ET高WUE区。宁夏的生态恢复工程在增强植被生产力的同时,也增强了区域水分消耗,致使陆地生态系统整体水分利用效率下降,这为宁夏未来水资源调控和生态重建提供了科学依据。     

Microbial synthesis of 2H-labelled L-phenylalanine with different levels of isotopic enrichment by a facultative methylotrophic bacterium Brevibacterium methylicum with RuMP assimilation of carbon

Using the L-phenylalanine secreting strain of Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via the ribulose-5-monophosphate (RuMP) cycle of carbon assimilation, as an example, we have continued studies on the use of methylotrophic bacteria for the preparative microbial synthesis of amino acids labeled with stable isotopes, including deuterium (²H), suitable for biomedical applications and clinical diagnostics. Here we demonstrate the data on adaptation of the methylotrophic bacterium B. methylicum to the maximal concentration of deuterium in the growth medium with 98% (v/v) ²H₂O and 2% (v/v) [²H]MeOH, and biosynthesis of deuterium labeled L-phenylalanine with different levels of isotopic enrichment. The strain was adapted to ²H₂O by means of plating of initial cells on solid (2% agarose) minimal growth media M9 with an increasing gradient of ²H₂O concentration from 0, 24.5, 49.0, 73.5 up to 98% (v/v) ²H₂O and subsequent selection of individual colonies stable to the action of ²H₂O, which were capable to produce L-phenylalanine. L-phenylalanine was extracted from the growth medium with isopropanol followed by subsequent crystallization in ethanol (output 0.65 g/L). Using the developed method of microbial synthesis it is possible to obtain deuterated L-phenylalanine with different levels of isotopic enrichment, depending on concentration of ²H₂O in growth media, from 17% (the growth medium with 24.5% (v/v) ²H₂O) right up to 75% (the growth medium with 98% (v/v) ²H₂O) of deuterium as evidenced by results of the electron impact (EI) mass-spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfonyl chloride (dansyl) phenylalanine isolated from growth media under different experimental conditions.     

Ultrafast electron transfer in riboflavin binding protein in macromolecular crowding of nano-sized micelle

In this contribution, we have studied the dynamics of electron transfer (ET) of a flavoprotein to the bound cofactor, an important metabolic process, in a model molecular/macromolecular crowding environments. Vitamin B₂ (riboflavin, Rf) and riboflavin binding protein (RBP) are used as model cofactor and flavoprotein, respectively. An anionic surfactant sodium dodecyl sulfate (SDS) is considered to be model crowding agent. A systematic study on the ET dynamics in various SDS concentration, ranging from below critical micellar concentration (CMC), where the surfactants remain as monomeric form to above CMC, where the surfactants self-assemble to form nanoscopic micelle, explores the dynamics of ET in the model molecular and macromolecular crowding environments. With energy selective excitation in picosecond-resolved studies, we have followed temporal quenching of the tryptophan residue of the protein and Rf in the RBP–Rf complex in various degrees of molecular/macromolecular crowding. The structural integrity of the protein (secondary and tertiary structures) and the vitamin binding capacity of RBP have been investigated using various techniques including UV–Vis, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) studies in the crowding environments. Our finding suggests that the effect of molecular/macromolecular crowding could have major implication in the intra-protein ET dynamics in cellular environments.     

Determination of alpha-naphthylisothiocyanate and metabolites alpha-naphthylamine and alpha-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of alpha-naphthylisothiocyanate (1-NITC) and two metabolites alpha-naphthylamine (1-NA) and alpha-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C(18) 5-microm column, a mobile phase of acetonitrile-water (ACN-H(2)O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80 degrees C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 degrees C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.     

Cleavage Of Oligodeoxyribonucleotides From Polymer Supports And Their Rapid Deprotection Under Microwaves

Abstract

Novel conditions for the cleavage of oligodeoxynucleotides from polymer supports and their complete deprotection under microwaves have been developed. The oligonucleotides synthesized using phosphoramidite synthons carrying base labile (Pac, Dmf and t-Bpac) and conventional (Bz for A and C and Pac for G) protecing groups for nucleic bases were deprotected using 0.2M sodium hydroxide (MeOH : H₂O:: 1:1, v/v) = Reagent A and 1M sodium hydroxide (MeOH: H₂O :: 1:1, v/v = Reagent B, respectively under microwaves. The deprotected oligonucleotides were found to be comparable with the corresponding oligonucleotides deprotected under standard conditions (aq. ammonia at 55°C).     

黄河流域植被格局变化对水分利用效率的影响

黄河流域横跨3个气候带,是全球人类活动最为强烈的地区之一,特殊的地理位置和复杂的下垫面导致其碳-水循环过程较为复杂。研究黄河流域碳水循环不仅是区域水资源利用的基础,也是实现气候变化条件下双碳目标的关键。水分利用效率(WUE)作为表征碳水过程的重要指标,可用于反映生态系统碳水耦合规律及其相互作用关系。基于此,利用全球陆表特征参量数据(GLASS)的净初级生产力(NPP)和蒸散(ET)产品以及中国逐年土地利用与覆盖数据集(CLUD-A),分析了黄河流域植被格局变化背景下WUE在1990—2018年的时空变化特征及其驱动力。结果表明：(1)黄河流域全域WUE在29 a的均值处在0.18—1.53 g C/kg H₂O之间,存在明显的空间异质性,上游地区WUE明显高于中下游地区,分别在0.66—0.92 g C/kg H₂O和0.43—0.62 g C/kg H₂O之间波动,二者均存在波动上升态势。(2)黄河流域全域WUE在以2000年为中间点的10 a的增速达到近29 a的峰值,流域植被格局变化所带来的流域内NPP与ET变化速...     

Development and Validation of a Micellar Electrokinetic Chromatography Method for Quantitative Determination of Butenolides in Piper malacophyllum (C. Presl) C. DC.

Introduction – A large number of natural and synthetic compounds having butenolides as a core unit have been described and many of them display a wide range of biological activities. Butenolides from P. malacophyllum have presented potential antifungal activities but no specific, fast, and precise method has been developed for their determination. Objective – To develop a methodology based on micellar electrokinetic chromatography to determine butenolides in Piper species. Methodology – The extracts were analysed in an uncoated fused‐silica capillaries and for the micellar system 20 mmol/L SDS, 20% (v/v) acetonitrile (ACN) and 10 mmol/L STB aqueous buffer at pH 9.2 were used. The method was validated for precision, linearity, limit of detection (LOD) and limit of quantitation (LOQ) and the standard deviations were determined from the standard errors estimated by the regression line. Results – A micellar electrokinetic chromatography (MEKC) method for determination of butenolides in extracts gave full resolution for 1 and 2 . The analytical curve in the range 10.0–50.0 µg/mL (r² = 0.999) provided LOD and LOQ for 1 and 2 of 2.1/6.3 and 1.1/3.5 µg/mL, respectively. The RSD for migration times were 0.12 and 1.0% for peak area ratios with 100.0 ± 1.4% of recovery. Conclusions – A novel high‐performance MEKC method developed for the analysis of butenolides 1 and 2 in leaf extracts of P. malacophyllum allowed their quantitative determined within an analysis time shorter than 5 min and the results indicated CE to be a feasible analytical technique for the quantitative determination of butenolides in Piper extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Inhibition of ethinyloestradiol and tolbutamide metabolism by quinoline derivatives in vitro

The effects of the quinoline derivatives amodiaquine (AQ), chloroquine (CQ), mefloquine (MQ), primaquine (PQ), quinine (Q) and quinidine (QD) on in vitro hepatic metabolism has been studied using as substrates ethinyloestradiol (EE2) and tolbutamide (TOL). The 2-hydroxylation of EE2 and the hydroxylation of TOL were determined in the presence of variable concentrations of each compound. MQ, PQ, AQ and Q significantly inhibited EE2 metabolism at each of the concentrations studied (0.1, 0.2 and 0.5 mM) as shown by an increase in the percentage of unmetabolised EE2. QD significantly inhibited metabolism at 0.2 and 0.5 mM but CQ was without effect. In terms of recovery of 2-OHEE2, PQ was the most potent inhibitor. At an inhibitor concentration of 0.5 mM the order of potency was PQ greater than or equal to MQ greater than or equal to Q greater than or equal to QD greater than or equal to AQ greater than or equal to CQ. TOL hydroxylase activity in control microsomes was 1.52 +/- 0.33 nmol. min-1 X mg protein-1. The order of potency of the inhibitors (0.5 mM) was PQ greater than or equal to MQ greater than or equal to Q greater than or equal to QD greater than or equal to AQ greater than or equal to CQ. These data provide further evidence of the inhibitory potential of some of the quinoline derivatives. PQ, MQ, and to a lesser extent Q produce the most marked inhibitory effects. QD and AQ are of intermediate potency and CQ is essentially non-inhibitory.     

Structural and conformational differences of acylated hyaluronan modified in protic and aprotic solvent system

Acylated hyaluronan (HA) in aqueous (DMSO/H₂O) and nonaqueous (DMSO) solutions was studied by means of nuclear magnetic resonance, differential scanning calorimetry (DSC), mass spectrometry and UV/vis spectroscopy. It has been demonstrated that structural and conformational properties of the acylated hyaluronan derivates are strongly dependent on the nature of reaction solvent. Acylation in DMSO was more selective than that carried out in DMSO/H₂O, though in both cases in average a maximum of one acyl chain was detected per HA dimer. The hydrophobic functionalization of hyaluronan induced its interaction with hydrophobic dye as a consequence of acyl chain aggregation. The higher the degree of acylation the more hydrophobic dye was interacting with HA. For concentrated samples, aggregation was more evident in case of acylated HA in aqueous solution. This phenomenon was explained by its different conformational arrangement in solution which was further supported by DSC data indicating an existence of hydrophobic cavities. The formation of self-aggregated assemblies indicates potential applications of this type of HA derivate as drug delivery system.     

Synthesis,physico-chemical studies of manganese(II), cobalt(II), nickel(II), copper(II) and zinc(II) complexes with some p-substituted acetophenone benzoylhydrazones and their antimicrobial activity

Complexes of the type [M(pabh)(H₂O)Cl], [M(pcbh)(H₂O)Cl] and [M(Hpabh)(H₂O)₂ (SO₄)] where, M = Mn(II), Co(II), Ni(II), Cu(II) and Zn(II); Hpabh = p-amino acetophenone benzoyl hydrazone and Hpcbh = p-chloro acetophenone benzoyl hydrazone have been synthesized and characterized with the help of elemental analyses, electrical conductance, magnetic susceptibility measurements, electronic, ESR and IR spectra, thermal (TGA & DTA) and X-ray diffraction studies. Co(II), Ni(II) and Cu(II) chloride complexes are square planar, whereas their sulfate complexes have spin-free octahedral geometry. ESR spectra of Cu(II) complexes with Hpabh are axial and suggest as the ground state. The ligand is bidentate bonding through >C = N ? and deprotonated enolate group in all the chloro complexes, whereas, >C = N and >C = O groups in all the sulfato complexes. Thermal studies (TGA & DTA) on [Cu(Hpabh)(H₂O)₂(SO₄)] indicate a multistep decomposition pattern, which are both exothermic and endothermic in nature. X-ray powder diffraction parameters for [Co(pabh)(H₂O)Cl] and [Ni(Hpabh)(H₂O)₂(SO₄)] correspond to tetragonal and orthorhombic crystal lattices, respectively. The ligands as well as their complexes show a significant antifungal and antibacterial activity. The metal complexes are more active than the ligand.     

Extracting fumonisins from maize: efficiency of different extraction solvents in multi-mycotoxin analytics

The complete extraction of analytes is of utmost importance when analyzing matrix samples for mycotoxins. Mycotoxins consist of substances with widely different physicochemical properties; therefore, the loss of toxins that occurs in multi-mycotoxin methods due to compromises in the extraction solvent is currently a topic under discussion. With regard to fumonisins, several extractants from recently published multi-mycotoxin methods were investigated when analyzing unprocessed and processed maize matrices. All extractants were tested in a validated on-site method and the extraction yields were compared to those of an HPLC-FLD reference method (EN 14352). Most of the compared multi-mycotoxin methods that have been published were only for analyzing fumonisins in maize or maize-meal; we have applied the extractants of these methods to processed, complex maize matrices for the first time. Our results show that, for extractions with aqueous acetonitrile mixtures with the addition of acid, e.g. MeCN/H₂O/acetic acid (79/20/1, v/v/v), higher extraction yields are obtained than with MeCN/H₂O (80/20, v/v), in both spiked and naturally contaminated maize matrices. But compared to the results of the reference method EN 14352, the two extractants did not show a similar extraction efficiency. Overall, the extractant MeCN/MeOH/H₂O (1/1/2, v/v/v) turned out to be the most appropriate extractant applied in all experiments, obtaining the best and most comparable extraction yields and recoveries. Furthermore, our investigations showed that, with some of the tested extraction solvents, e.g. MeCN/H₂O (75/25) containing 50 mmol/l formic acid, stark differences occur when analyzing spiked and naturally contaminated matrices. With spiked matrices, recoveries of approximately 80–110 % were obtained, but with naturally contaminated matrices no results comparable to the EN method have been achieved. In contrast, a double extraction with MeCN/H₂O/formic acid (80/19,9/0,1, v/v/v), followed by a second polar extraction step with MeCN/H₂O/formic acid (20/79,9/0,1, v/v/v), led, for most naturally contaminated samples, to comparable results with the EN method. However, for spiked samples, the same extractant led to raised recoveries of between 120 and 140 %. For some processed matrices, like taco-chips, all tested extractants showed a poor extraction efficiency for fumonisins. By extending the extraction time from 1 to 15 min, a result comparable to that of the reference method could also be obtained for the extractant using MeCN/MeOH/H₂O (1/1/2, v/v/v). As this extractant has been used in our recently published method (Trebstein et al. Mycotoxin Res 25:201, 2009), this work also presents an update on this method with respect to the extended extraction time.     

Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H₂ to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H₂ evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H₂ evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H₂-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H₂ oxidation or H₂ evolution in whole cells, reversible H₂-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H₂ evolution mode. Our findings suggest that Hyd-2 switches between H₂-consuming and H₂-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H₂ evolution from glycerol requires reverse electron transport.     

Apoptotic cell death in suspension cultures of Taxus cuspidata co-treated with salicylic acid and hydrogen peroxide

Apoptotic cell death in suspension cultures of Taxus cuspidata induced by exogenous salicylic acid and/or H₂O₂ was investigated. H₂O₂ (0.012% v/v) alone changed the permeability of cell membrane while salicylic acid (0.375 mM) not only altered the permeability but also caused nuclei condensation and a small amount of nuclei fragments. The combined use of salicylic acid (0.375 mM) and H₂O₂ (0.012% v/v) changed the cell membrane permeability more significantly and nuclei fragments occurred in ca. 30% of the cells at 48 h. DNA ladders of 180 bp and oligopolymers, characteristics of the apoptotic cleavage of nuclei DNA, were observed by agar electrophoresis. These results show that exogenous salicylic acid and H2O2 could synergistically induce the apoptotic cell death of suspension cultures of Taxus cuspidata.     

Validation of high performance liquid chromatography–electrochemical detection methods with simultaneous extraction procedure for the determination of artesunate,dihydroartemisinin, amodiaquine and desethylamodiaquine in human plasma for application in clinical pharmacological studies of artesunate–amodiaquine drug combination

With the expanded use of the combination of artesunate (AS) and amodiaquine (AQ) for the treatment of falciparum malaria and the abundance of products on the market, comes the need for rapid and reliable bioanalytical methods for the determination of the parent compounds and their metabolites. While the existing methods were developed for the determination of either AS or AQ in biological fluids, the current validated method allows simultaneous extraction and determination of AS and AQ in human plasma. Extraction is carried out on Supelclean LC-18 extraction cartridges where AS, its metabolite dihydroartemisinin (DHA) and the internal standard artemisinin (QHS) are separated from AQ, its metabolite desethylamodiaquine (DeAQ) and the internal standard, an isobutyl analogue of desethylamodiaquine (IB-DeAQ). AS, DHA and QHS are then analysed using Hypersil C4 column with acetonitrile–acetic acid (0.05 M adjusted to pH 5.2 with 1.00 M NaOH) (42:58, v/v) as mobile phase at flow rate 1.50 ml/min. The analytes are detected with an electrochemical detector operating in the reductive mode. Chromatography of AQ, DeAQ and IB-DeAQ is carried out on an Inertsil C4 column with acetonitrile–KH₂PO₄ (pH 4.0, 0.05 M) (11:89, v/v) as mobile phase at flow rate 1.00 ml/min. The analytes are detected by an electrochemical detector operating in the oxidative mode. The recoveries of AS, DHA, AQ and DeAQ vary between 79.1% and 104.0% over the concentration range of 50–1400 ng/ml plasma. The accuracies of the determination of all the analytes are 96.8–103.9%, while the variation for within-day and day-to-day analysis are <15%. The lower limit of quantification for all the analytes is 20 ng/ml and limit of detection is 8 ng/ml. The method is sensitive, selective, accurate, reproducible and suited particularly for pharmacokinetic study of AS–AQ drug combination and can also be used to compare the bioavailability of different formulations, including a fixed-dose AS–AQ co-formulation.     

Cosolvents Induced Unfolding and Aggregation of Keyhole Limpet Hemocyanin

The objective of this study was to examine the effects of 2,2,2 trifluoroethanol (TFE) and acetonitrile (ACN) on the stability, behavior, and structural characteristics of giant multimeric protein Keyhole Limpet hemocyanin (KLH) by combining the circular dichroism (CD) and fluorescence measurements of KLH solution. In concentration range 20–50 % (v/v) TFE, protein at pH 7.4 shows visible aggregation while no aggregation was observed in the entire concentration range of TFE at molten globule (MG) state (pH 2.8) and resulted in stable α-helix. Our result shows that in the presence of 80 % (v/v) and 40 % (v/v) TFE, at native (pH 7.4) and MG state (pH 2.8) occurred in a highly helical state referred to as TFE denatured state I and II, respectively. However, in case of ACN, aggregation starts above 40 % (v/v) for pH 7.4 and at 80 % (v/v) for acid-induced MG (pH 2.8) state, which was dominated by β-sheet structure and referred to as ACN denatured state III and IV. An important object of our investigation is to get more detail study of efficiency of cosolvents in inducing structural changes in KLH. The dependence of scattering intensity and the R _h on alcohol concentrations was investigated at 25 °C.     

A comparative study of lysozyme conformation in various reverse micellar systems

The activity and conformation of lysozyme solubilized in apolar solvents via reverse micelles was investigated. The systems used were sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane/H₂O, cetyltrioctylammoniumbromide (CTAB)/CHCl₃, isooctane/H₂O; tetraethyleneglycoldodecylether (EO₄C₁₂)/isooctane/H₂O, and bulk water. CD spectra of lysozyme in reverse micellar solutions were investigated as a function of w₀ (= [H₂O]/[AOT]) and were compared to the spectra in aqueous solutions. No marked changes were found in the EO₄C₁₂ or in the CTAB systems with respect to water, which indicates that no sizeable conformational changes of the enzyme occurred upon solubilization in the reverse micellar systems. In agreement with previous studies [C. Grandi, R. E. Smith, and P. L. Luisi (1981) J. Biol. Chem. 256 , 837–843] dramatic conformational changes can be inferred in the AOT system on the basis of CD studies. This is taken as an indication that the enzyme denatures in this micellar system. This is particularly striking because the enzyme is fully active in AOT reverse micelles. The apparent paradox is solved by the observation that the native CD spectrum (and by inference, the native conformation) is maintained when lysozyme is bound to NAG or NAG₃, and by inference, when the substrate is bound, e.g., during enzyme turnover. However, in the absence of added NAG, NAG₃, or substrate, the enzyme in the AOT reverse micellar system rapidly denatures. Together with CD studies, fluorescence and nmr data confirm the hypothesis of an irreversible denaturation of lysozyme in the AOT system, the denaturation being slowed down when the substrate is present. The activity of the enzyme has been studied as a function of pH and w₀ using the chromophoric substrate 3,4-dinitrophenyl-tetra-N-acetyl-β-D -chitotetraoside (3,4-DNP-NAG₄). Generally speaking, the kinetic parameters are comparable to those found in bulk water solution. More detailed, in the CTAB system, k_cat tends to be smaller than in aqueous solution (with quite similar K_M), whereas in the EO₄C₁₂ system (at pH 7.0) the turnover number is larger and K_M is smaller than in water. In the AOT system, the kinetic parameters at pH 7.0 are also quite comparable to those found in water.