Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue

1. ATP sulphurylase was purified up to 1000-fold from spinach leaf tissue. Activity was measured by sulphate-dependent [(32)P]PP(i)-ATP exchange. The enzyme was separated from Mg(2+)-requiring alkaline pyrophosphatase (which interferes with the PP(i)-ATP-exchange assay) and from other PP(i)-ATP-exchange activities. No ADP sulphurylase activity was detected. 2. Sulphate was the only form of inorganic sulphur that catalysed PP(i)-ATP exchange; K(m) (sulphate) was 3.1mm, K(m) (ATP) was 0.35mm and the pH optimum was 7.5-9.0. The enzyme was insensitive to thiol-group reagents and required either Mg(2+) or Co(2+) for activity. 3. The enzyme catalysed [(32)P]PP(i)-dATP exchange; K(m) (dATP) was 0.84mm and V (dATP) was 30% of V (ATP). Competition between ATP and dATP was demonstrated. 4. Selenate catalysed [(32)P]PP(i)-ATP exchange and competed with sulphate; K(m) (selenate) was 1.0mm and V (selenate) was 30% of V (sulphate). No AMP was formed with selenate as substrate. Molybdate did not catalyse PP(i)-ATP exchange, but AMP was formed. 5. Synthesis of adenosine 5'-[(35)S]sulphatophosphate was demonstrated by coupling purified ATP sulphurylase and Mg(2+)-dependent alkaline pyrophosphatase (also prepared from spinach) with [(35)S]sulphate and ATP as substrates; adenosine 5'-sulphatophosphate was not synthesized in the absence of pyrophosphatase. Some parameters of the coupled system are reported.     

云南丽江山慈菇遗传多样性的DALP分析

采用DALP (Direct amplification of length polymorphism) 分子标记技术, 对产自云南的药用植物丽江山慈菇Iphigenia indica (L.) Kunth的9个居群进行DNA指纹检测。筛选出5个引物组合, 扩增共产生131条DNA片段, 其中104 条谱带具有遗传多态性, 约占79 39%, 平均每组引物扩增所得多态条带为20 8, 9个居群平均多态百分率为42 21%。9个居群平均观察等位基因数Na为1 4224, 总Na为1 7939; 平均有效等位基因数Ne 为1 3141, 总Ne 为1 4810; 平均遗传多样性指数H为0 1745, 总H为0 2831; 平均Shannon 多样性指数I 为0 2527, 总I为0 4231; 总基因多样性Ht为0 2831, 居群内多样性Hs 为0 1745, 居群间基因分化系数Gst为0 3834, 即丽江山慈菇有61 66%的遗传变异来自居群内, 38 34%来自居群间, 居群间存在较高水平的遗传分化。滇西北居群的遗传多样性明显高于滇中居群的遗传多样性, 这与滇中地区丽江山慈菇野生资源被大规模挖掘有着直接的关系。     

Absence of uterokinetic effects of prostaglandin F 2 alpha on oxytocin-reactive uterus in the mare

In most cyclic females, prostaglandin F(2alpha) (PGF(2alpha)) triggers a uterine motility response resembling that of oxytocin (OT). To determine if PGF(2alpha) is a uterokinetic substance in the cycling mare, uterine motility was measured by intrauterine balloon technique in 12 conscious, normally cyclic mares. After 60 min of saline infusion, continuous intravenous (i.v.) infusion with OT (1 i.u./min) was followed by PGF(2alpha) (200 mug/min) for 60 min each. The experiment was repeated 3 wk later except with PGF(2alpha) preceeding OT. A second group of mares was administered OT (60 i.u.) either i.v., intramuscularly (i.m.), or intrauterinely (i.u.). Plasma samples were studied for progesterone concentration. Control uterine motility for the first group of mares was (mean +/- SEM) 545.83 +/- 45.10 mm(2). Significant (P<0.05) elevation in uterine motility was recorded for OT (1118.60 +/- 70.56 mm(2)) regardless if PGF(2alpha) preceded OT infusion or vice-versa. No significant difference (P>0.05) was seen in motility after PGF(2alpha) (423.33 +/- 31.12 mm(2)) infusion. The uterokinetic effect of OT was greatest when OT was administered i.v. (1696.50 +/- 195.46 mm(2)) followed by i.m. (819.82 +/- 39.96 mm(2)), and it was least effective when administered i.u. (607.83 +/- 21.56 mm(2)) as compared to control uterine motility (279.78 +/- 22.33 mm(2)). Skin electrical resistance values rose from 0 to 2000 ohms with PGF(2alpha) infusion (but not with OT), indicating that PGF(2alpha) was bioactive. It was concluded that PGF(2alpha) was not a uterokinetic substance in the cyclic mare.     

Maillard reaction rate in various glassy matrices

The Maillard Reaction (MR) rate below the glass transition temperature (T(g)) for various model glassy food systems was studied at temperatures between 40 degrees C and 70 degrees C. As a sample, freeze-dried glucose and lysine systems embedded in various glassy matrices (e.g., polyvinylpyrrolodone and trehalose) were used, and the MR rate below the T(g) was compared among the various glassy matrices. The extent of MR was estimated spectrophotometrically from the optical density at 280 nm (OD(280)), and the MR rate (k(280)) was determined as a pseudo zero order reaction rate from the time course of OD(280). Although k(280) was described by the Arrhenius plot, the temperature dependence of k(280) was almost the same and the intercept was different among the matrices. From the comparison of k(280), it was suggested that the MR rate in glassy matrix was affected not only by the T(g), but also by the hydrogen bonding between MR reactants and glassy matrix.     

中国林蛙变态蝌蚪对pH、盐度和碱度的适应性

在水温16～18℃的野外条件下,采用单因子急性毒性实验法,研究了水环境中pH、盐度和碱度对中国林蛙(Rana chensinensis)变态蝌蚪的毒性效应．结果表明,中国林蛙变态蝌蚪对pH的适应范围为4．3～9．7,最低耐受限3．6,最高耐受限10．7;对盐度的最高耐受限为9．98g·L^-1,适应盐度上限7．14g·L^-1,安全盐度上限1．70g·L^-1;对碱度的最高耐受限为19．96mmol·L^-1,适应碱度上限8．76mmol·L^-1。安全碱度上限2．41mmol·L^-1．野外变态蝌蚪饲养池水体pH应控制在6．5～8．5,盐度控制在2．0g·L^-1以下,碱度不超过4．0mmol·L^-1．中国林蛙变态蝌蚪是一种狭酸碱、低耐盐、低耐碱生物。     

Carbon Dioxide Fixation by Cells of Streptococcus faecalis var. liquefaciens

Fixation of NaH(14)CO(3) by a heavy cell suspension of Streptococcus faecalis var. liquefaciens was studied. Several nutrients, pyridoxal, riboflavine, adenine, uracil, and O(2) stimulated (14)CO(2) incorporation into cells only under conditions that were adequate for synthesis of cell macromolecules. Biotin increased CO(2) incorporation in the absence of extensive synthesis of macromolecules, whereas O(2) inhibited incorporation under these conditions. When (14)CO(2) fixation was occurring during synthesis of macromolecules, 71% of the (14)C was incorporated into cells and 29% occurred extracellularly. Ninety-three per cent of the cellular (14)C was in protein and 5.5% was in nucleic acid. Aspartic acid was the only amino acid in the protein fraction that was radioactive. Eighty-three per cent of the extracellular (14)C was resistant to precipitation by trichloroacetic acid. When (14)CO(2) fixation was occurring in cells that were not carrying on extensive synthesis of macromolecules, 38% of the (14)C was incorporated into cells and 59% occurred in the supernatant fluid. Sixty-nine per cent of the cellular (14)C was in protein, 21% was in low-molecular-weight compounds, and 9% was in nucleic acid. Addition of unlabeled aspartate to the medium inhibited incorporation of (14)CO(2). Based on studies of the rate of (14)CO(2) fixation, the cells fix CO(2) into a pool of intermediates which are either used for synthesis, primarily protein, or are excreted into the medium.     

The formation of choline O-sulphate by Pseudomonas C12B and other Pseudomonas species

Pseudomonas C(12)B and other Pseudomonas species released larger amounts of a (35)S-labelled metabolite into the medium when cultured on growth-limiting concentrations of Na(2)SO(4) as opposed to growth in SO(4) (2-)-sufficient media. The metabolite was found at all stages of the culture cycle of Pseudomonas C(12)B and maximum quantities occurred in stationary-phase culture supernatants. The metabolite was not detected when the bacterium was cultured on growth-limiting concentrations of potassium phosphate. The amount of the metabolite present in the medium greatly exceeded that which could be extracted from intact cells and, except for choline chloride, it was independent of the carbon source used for growth. If choline chloride was present in high concentration, then larger amounts of the metabolite were found in the culture medium. The metabolite was not detected extracellularly or intracellularly when the bacterium was grown in SO(4) (2-)-deficient media containing 5mm-l-cysteine. The same metabolite was also synthesized in vitro only when Pseudomonas C(12)B extracts were incubated with choline chloride, ATP, MgCl(2) and Na(2) (35)SO(4). The metabolite-forming system was not subject to repression by Na(2)SO(4) and was completely inhibited by 0.5mm-l-cysteine and activated by Na(2)SO(4) (up to 1.0mm). The metabolite was identified as choline O-sulphate by electrophoresis, chromatography and isotope-dilution analysis. Another (35)S-labelled metabolite was also detected in culture supernatants, but was not identified.     

Effect of H(2)-CO(2) on Methanogenesis from Acetate or Methanol in Methanosarcina spp

Growth of Methanosarcina sp. strain 227 and Methanosarcina mazei on H(2)-CO(2) and mixtures of H(2)-CO(2) and acetate or methanol was examined. The growth yield of strain 227 on H(2)-CO(2) in complex medium was 8.4 mg/mmol of methane produced. Growth in defined medium was characteristically slower, and cell yields were proportionately lower. Labeling studies confirmed that CO(2) was rapidly reduced to CH(4) in the presence of H(2), and little acetate was used for methanogenesis until H(2) was exhausted. This resulted in a biphasic pattern of growth similar to that reported for strain 227 grown on methanol-acetate mixtures. Biphasic growth was not observed in cultures on mixtures of H(2)-CO(2) and methanol, and less methanol oxidation occurred in the presence of H(2). In M. mazei the aceticlastic reaction was also inhibited by the added H(2), but since the cultures did not immediately metabolize H(2), the duration of the inhibition was much longer.     

Effects of alpha-Hydroxy-2-Pyridinemethanesulfonic Acid on Photosynthetic Carbon Dioxide Uptake and Stomatal Movements in Excised Tomato Leaves

The effects of 10(-2)m alpha-hydroxy-2-pyridinemethanesulfonic acid (alphaHPMS) on the CO(2) compensation point, photosynthetic CO(2) uptake, CO(2) evolution into CO(2)-free air in light, and stomatal movement, in excised tomato leaves (Lycopersicon esculentum Mill. Eurocross BB-F(1) Hybrid) were studied. It was found that alpha-HPMS had a transient lowering effect on the CO(2) compensation point of treated leaves within the first 5 minutes of application. The net photosynthetic CO(2) uptake was inhibited by alpha-HPMS treatment. The inhibition increased with time and was enhanced in an O(2)-free atmosphere. The CO(2) evolution into CO(2)-free air in light was inhibited by alpha-HPMS. The inhibition was O(2)-dependent because the effect was observed only in 21% O(2) but not in O(2)-free N(2). Stomatal apertures were affected by alpha-HPMS, but the effect was transient and was observed 15 to 30 minutes after the application. The time course of this closure did not account for the observed inhibition of net CO(2) uptake.     

Yeast orotidine-5'-phosphate decarboxylase: steady-state and pre-steady-state analysis of the kinetic mechanism of substrate decarboxylation

The catalytically active form of monofunctional yeast orotidine-5'-phosphate decarboxylase was a dimer (E(2)). The dimer equilibrium dissociation constant was 0.25 microM in 0.01 M MOPS Na(+) at pH 7.2. The bimolecular rate constant for dimer formation was 1.56 microM(-1) s(-1). The dimeric form of the enzyme was stabilized by NaCl such that the enzyme was E(2) in 100 mM NaCl at all concentrations of enzyme tested. The kinetics of binding of OMP to E(2) was governed by two ionizations (pK(1) = 6.1 and pK(2) = 7.7). From studies with substrate analogues, the higher pK was assigned to a group on the enzyme that interacted with the pyrimidinyl moiety. The value of the lower pK was dependent on the substrate analogue, which suggested that it was not exclusively the result of ionization of the phosphoryl moiety. During the decarboxylation of OMP, the fluorescence of E(2) was quenched over 20%. The enzymatic species with reduced fluorescence was a catalytically competent intermediate that had kinetic properties consistent with it being the initial enzyme-substrate complex. The stoichiometry for binding of OMP to E(2) was one OMP per enzyme monomer. The value of the first-order rate constant for conversion of the enzyme-substrate complex to free enzyme (36 s(-1)) calculated from a single turnover experiment ([E] > [S]) was slightly greater than the value of k(cat), 20 s(-1) (corrected for stoichiometry), calculated from steady-state data. In the single turnover experiments, the enzyme was E(2)*S, whereas in the steady-state turnover the experiment enzyme was E(2)*S(2). The similarity of these values suggested that the subunits were catalytically independent such that E(2)*S(2) could be treated as E*S and that conversion of the enzyme-substrate complex to E was k(cat). Kinetic data for the approach to the steady-state with OMP and E(2) yield a bimolecular association rate complex of 62 microM(-1) s(-1)and a dissociation rate constant for E*S of 60 s(-1). The commitment to catalysis was 0.25. By monitoring the effect of carbonic anhydrase on [H(+)] changes during a single turnover experiment, the initial product of the decarboxylation reaction was shown to be CO(2) not HCO(3-). UMP was released from the enzyme concomitantly with CO(2) during the conversion of E*S to E. Furthermore, the enzyme removed an enzyme equivalent of H(+) from solvent during this step of the reaction. The bimolecular rate constants for association of 6-AzaUMP and 8-AzaXMP, substrate analogues with markedly different nucleobases, had association rate constants of 112 and 130 microM(-1) s(-1), respectively. These results suggested that the nucleobase did not contribute significantly to the success of formation of the initial enzyme-substrate complex.     

Determination of oseltamivir carboxylic acid in human serum by solid phase extraction and high performance liquid chromatography with UV detection

A new straightforward method based on cloud-point extraction (CPE) has been developed, optimized and validated for the determination of venlafaxine in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection. The non-ionic surfactant Triton X-114 (polyethylene glycol tert-octylphenyl ether) was chosen as the extract solvent. Separation was obtained using a reversed-phase Diamonsil column (C(18), 250mmx4.6mm I.D., 5mum) and a mobile phase composed of acetonitrile-phosphate buffer solution (pH 3.0)-triethylamine (33.5:66.5:0.4). Fluorescence detection was used (lambda(ex) 276nm, lambda(em) 598nm). Maprotiline was used as the internal standard. Under the optimum conditions, the linear range of venlafaxine in human plasma was 10-800ngmL(-1) (r(2)=0.9995). The limit of detection (LOD) was less than 2ngmL(-1) (S/N=3) and the limit of quantification (LOQ) was less than 10ngmL(-1) (S/N=10). The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine capsules in nine healthy volunteers.     

Determination of serum glucose by horseradish peroxidase-catalysed imidazole chemiluminescence coupled to a micro-flow-injection system.

The reactivity of flow-injection (FI)-horseradish peroxidase (HRP)-catalysed imidazole chemiluminescence (CL) was studied for continuous determination of hydrogen peroxide (H(2)O(2)) and serum glucose with immobilized glucose oxidase. Light emission by the HRP-catalysed imidazole CL was obtained when immobilized HRP, alkaline imidazole (in Tricine solution, pH 9.3) and H(2)O(2) were reacted at room temperature. The optimal pH for the CL reaction was 9.3 and the optimal concentration of imidazole was 100 micromol/L. When no imidazole was added, the light intensity of the same H(2)O(2) specimen decreased to a level that could not be quantitatively determined. The spectrum of the light emitted by imidazole CL was in the range 400-600 nm with a peak at 500 nm. The calibration equation for determination of H(2)O(2) was y = 9860x(2) + 3830x + 11,700, where y = light intensity (RLU) and x = concentration of H(2)O(2) (micromol/L). The detection limit of H(2)O(2) was 5 pmol, and the reproducibility of the H(2)O(2) assay was 2.3% of the coefficient of variation (H(2)O(2) 48 micromol/L, n = 13). The CL method was successfully applied to assay glucose after on-line generation of H(2)O(2) with the immobilized glucose oxidase column, resulting in good reproducibility (CV = 3.3% and 1.0% for the standard glucose and the control serum, respectively).     

不同含水量下尖叶拟船叶藓光合速率对光温的响应及其模型

对不同大气温度、藓体含水量及光照条件下尖叶拟船叶藓光合速率测定研究结果表明,光合速率(Pn)与光照强度(PAR)、大气温度(Ta)及藓体含水量(PWC)之间密切相关,光合速率的光响应曲线为直角双曲线,温度、藓体含水量影响图形的曲度参数,在低含水量、高气温组合和高含水量、低气温组合的藓体高光强下都使光合速率降低．弱光下(PAR<200μmol·s^-1·m^-2),光合速率最大值Pmax出现在PWC：为50％～80％,但随着Ta的升高而增大,当Ta>25℃,Pmax随Ta升高而降低;随着光照强度的增大,Pmax出现的PWC水平随之提高,当PAR<200μmol·s^-1·m^-2时,光合速率最大值Pmax出现在Ta比较高的范围(20～25℃),并随PWC的升高而增大,当PWC>80％时,Pmax随PWC升高而降低;随着光照强度的增大,Pmax出现的Ta水平降低、在230     

Ecological Life Table of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

An ecological life table was constructed, aiming to determine the critical stages and key mortality factors of Tuta absoluta (Lepidoptera: Gelechiidae). The total population mortality of this tomato leafminer was 92.3%. During the egg stage the mortality was 58.7%, mainly due to egg inviability. A total of 8.6% egg parasitism by Trichogramma pretiosum (Riley) (Hymenoptera: Trichogrammatidae) and 5.0% egg predation by Xylocoris sp. (Heteroptera: Anthocoridae), Cycloneda sanguinea (L.) (Coleoptera: Coccinellidae) and members of the family Phlaeothripidae (Thysanoptera) was observed. The mortality of the larval stage was 33.0%. This was considered to be the critical stage as it showed the highest apparent mortality (79.8%). Larval parasitism was low (0.1%), and was only found with Goniozus nigrifemur Ashmead (Hymenoptera: Bethylidae). Predators were responsible for 79.4% of larval mortality. Therefore, their attraction to and maintenance in the target area are important management tactics to be considered for T. absoluta control. The first and second instars were considered to be the most critical, and predation by the above mentioned species was the key mortality factor. The mortality at the pupal stage was low (0.6%) and was due to malformation.     

Electrodeposition of gold-platinum alloy nanoparticles on ionic liquid-chitosan composite film and its application in fabricating an amperometric cholesterol biosensor

An electrodeposition method was applied to form gold-platinum (AuPt) alloy nanoparticles on the glassy carbon electrode (GCE) modified with a mixture of an ionic liquid (IL) and chitosan (Ch) (AuPt-Ch-IL/GCE). AuPt nanoparticles were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and electrochemical methods. AuPt-Ch-IL/GCE electrocatalyzed the reduction of H(2)O(2) and thus was suitable for the preparation of biosensors. Cholesterol oxidase (ChOx) was then, immobilized on the surface of the electrode by cross-linking ChOx and chitosan through addition of glutaraldehyde (ChOx/AuPt-Ch-IL/GCE). The fabricated biosensor exhibited two wide linear ranges of responses to cholesterol in the concentration ranges of 0.05-6.2 mM and 6.2-11.2 mM. The sensitivity of the biosensor was 90.7 μA mM(-1) cm(-2) and the limit of detection was 10 μM of cholesterol. The response time was less than 7 s. The Michaelis-Menten constant (K(m)) was found as 0.24 mM. The effect of the addition of 1 mM ascorbic acid and glucose was tested on the amperometric response of 0.5 mM cholesterol and no change in response current of cholesterol was observed.     

养殖密度对史氏鲟消化率、摄食率和生长的影响

以体重（43.90±1.75）g史氏鲟为研究对象,研究了0.525、1.171和2.138 kg·m^-2 3种养殖密度对史氏鲟幼鱼生长、摄食率和消化率的影响,实验时间为60 d.结果表明,养殖密度对史氏鲟的生长、摄食率和消化率具有显著影响.高养殖密度不利于史氏鲟的生长,低密度组中史氏鲟的特定生长率和日增重显著高于高密度组,食物转化率显著低于高密度组;特定生长率和日增重随养殖密度的降低而显著增高.低密度组、中密度组中史氏鲟的消化率无显著差异,但均显著高于高密度组.中密度组摄食率显著低于高密度组和低密度组,低密度组摄食率介于两者之间;食物转化率和消化率呈显著负相关,特定生长率与消化率呈显著正相关.     

Electrochemiluminescence determination of pipemidic acid using sulfite as energy transfer mediator

An electrochemiluminescence (ECL) based on energy transfer from electro-generated triplet sulfur dioxide to pipemidic acid (PPA) was studied. A weak ECL from triplet sulfur dioxide (3)SO2 * was observed when sulfite was electrochemically oxidized in sulfuric acid solution on a Pt electrode. When PPA was present, the weak ECL was enhanced. The enhanced ECL was attributed to energy transfer from (3)SO2 * to PPA. Based on the enhanced ECL, a flow-injection (FIA) ECL method for the determination of PPA was proposed. The proposed method allowed the measurement of PPA over the range of 1.0x10(-7) to 2.0x10(-5)moll(-1). The detection limit was 3.9x10(-8)moll(-1), and the relative standard deviation for 1.0x10(-6)moll(-1) PPA (n=9) was 1.3%. This method was evaluated by the analysis of PPA in pharmaceutical preparations and urine samples.     

橄榄星室木虱实验种群生命表

从 2 0 0 2年 7月 1 8日到 9月 3 0日 ,在莆田市城厢区组建了橄榄星室木虱实验种群生命表 ,并对生命表进行了分析。实验结果表明 ,橄榄星室木虱的种群趋势指数I为 3 7 .2 2 ,世代死亡率为 68 0 7% ,内禀增长力rm 为 0 . 1 1 1 ,周限增长速率λ为 1 . 1 1 7,净繁殖率R0 为 41. 5 64,世代平均周期T为 3 3. 7d ,双倍时间为 7d。     

Biosorptive removal of cadmium from contaminated groundwater and industrial effluents

The cadmium removing capacity of a biosorbent Calotropis procera, a perennial wild plant, is reported here. The biomass was found to possess high uptake capacity of Cd(II). Adsorption was pH dependent and the maximum removal was obtained at two different pH i.e. pH 5.0 and 8.0. Maximum biosorption capacity in batch and column mode was found to be 40 and 50.5 mg/g. The adsorption equilibrium (> or =90% removal) was attained within 5 min irrespective of the cadmium ion concentration. Interfering ions viz. Zn(II), As(III), Fe(II), Ni(II) interfered only when their concentration was higher than the equimolar ratio. The Freundlich isotherm best explained the adsorption, yet the monolayer adsorption was also noted at lower concentrations of Cd(II). The FTIR analysis indicates the involvement of hydroxyl (-OH), alkanes (-CH), nitrite (-NO(2)), and carboxyl group (-COO) chelates in metal binding. The complete desorption of the cadmium was achieved by 0.1M H(2)SO(4) and 0.1M HCl. The C. procera based Cd(II) removal technology appears feasible.