Requirements for reliable determination of binding affinity constants by saturation analysis approach

Accurate calculation of the equilibrium association constant (K) and binding site concentration (N) related to a receptor (R)/ligand (L) interaction, via R saturation analysis, requires exact determination of the specifically bound L concentration (B(S)) and the unbound L concentration (U) at equilibrium. However, most binding determinations involve a procedure for separation of bound and unbound L. In such situations, it was previously shown that correct calculation of B(S) and U from binding data requires prior determination of alpha, i.e. the procedure parameter representing the proportion of equilibrium B(S) recovered after running the separation process, and of kn, i.e. the equilibrium nonspecific binding coefficient. For the simplest model of R/L interaction, the consequences of alpha neglect and/or kn neglect on determination of K and N, via R saturation analysis, are investigated. When alpha but not kn has been determined, B(S) can be accurately calculated, whereas U is overestimated by factor (kn + 1). Consequently the type (linear or hyperbolic) of theoretic curves obtained by usual representations (such as the Scatchard, the Lineweaver-Burk or the Michaelis-Menten plot) of the R/L binding is unchanged; these curves afford correct N and underestimation of K by factor (kn + 1). When alpha (alpha < 1) has not been determined B(S) and U are underestimated and overestimated, respectively. Then erroneous representations of the R/L binding result (e.g. instead of regular straight line segments, Scatchard plot and Lineweaver-Burk plot involve convex-upward and convex-downward hyperbola portions, respectively, suggestive of positive cooperativity of L binding), which leads to incorrect N and K. Errors in N and K would depend on (i) the binding (K, N and kn) and method (alpha) parameters and (ii) the expressions used to calculate approximate B(S) and U values. Simulations involving variable alpha, KN and kn values indicate that: (1) the magnitude of error in N determination (mainly involving moderate underestimation) directly depends on the alpha value; (2) the magnitude of K underestimation mainly depends on the KN value; it is moderate (usually < two-fold) with KN values < 1, but could become very high (e.g. > 100-fold), when KN > 10(2). In this case, the K underestimation is modulated by the alpha and kn values. Practical situations which afford high KN and thus might result in very marked underestimation of K are discussed. A single R dilution method is proposed to assess the validity of K determinations using the R saturation analysis approach.     

Soil potassium mobility and uptake by corn under differential soil moisture regimes

This study examined the effects of soil moisture on soil K mobility, dynamics of soil K, soil K fixation, plant growth and K uptake. A pot experiment, with and without corn (Zea maysL.), was conducted over a 16-d duration using a Yolo silt loam treated with two soil moisture regimes, i.e. constant moisture vs. wetting–drying (W–D) cycles. Soil K dynamics were determined using both ion exchange resin and direct extraction of soil solution. Soil K mobility increased significantly with soil moisture content (θ_v) and there was a positive curvilinear relationship between θ_v and effective diffusion coefficient (D_e), suggesting that more K⁺ can diffuse to the plant roots at sufficient soil moistures. Increase in D_e could be attributed to the decrease of impedance factor. During W–D cycles, soil solution K concentration increased as soil solution volume decreased, but soil solution K and NH₄ ⁺-extractable K pools decreased. In the constant moisture regime, available K pools decreased over the 16-d duration, but to a lesser extent than in W–D regime. The W–D cycles significantly enhanced K fixation and reduced available K pools in the soil in contrast to the constant moisture regime. Potassium fixation by the soil showed a biphasic pattern under the W–D regime, with a rapid fixation within the first 2 d after re-wetting, followed by a slower fixation. In the soil with constant moisture, K fixation was rapid during the first 8 h after wetting the soil, and then proceeded so slowly that no significant K fixation was observed after 4 d. The W–D cycles decreased root and shoot growth and K uptake by corn compared to constant moisture condition. Our results support the hypothesis that W–D cycles enhance soil K fixation, reduce soil K mobility and plant growth, and therefore reduce plant K⁺ uptake. This revised version was published online in June 2006 with corrections to the Cover Date.     

Erythrocyte aggregation: approach by light scattering determination

Erythro-aggregometer is a Couette viscometer which was developed to measure aggregation parameters of red blood cells. The system is based on the analysis of the light intensity backscattered by a blood suspension. It allows to approach aggregation phenomenon in terms of kinetics, structural and rheological parameters. The measurement system designed for use with a microcomputer is suitable for both research and clinical investigations.     

Measurement technique for the determination of blood oxygen saturation

A new optical measurement technique based on spectral scanning is described for the determination of oxygen saturation of whole blood. The principles of this technique are outlined, together with a calibration procedure used to test its feasibility in vitro. The preliminary results show that the accuracy of the new technique is of the order ± 2% over the full range of oxygen saturation. This degree of accuracy is comparable with commercial CO-oximeters.     

人工小叶锦鸡儿(Caragana microphylla)灌丛土壤水分动态研究

采用由植被空间序列断时间系列的方法。分析了1984,1987,1995和1999年建立的人工小叶锦鸡儿固沙植被土壤水分的时空变化特征,结果表明,受沙地土壤机械组成的影响,沙地土壤组成以物理性沙粒为主,＞0.01mm沙粒占97％以上,凋萎湿度为1．55％,田间持水量5．5%,大有效水为3．95％,随着固沙植被优势度的增加,小叶锦鸡儿灌丛下土壤含水量持续下降,1984年建立的植被区土壤含水量明显低于1999年建立的植被区;在年内土壤水分季节变化中,4个阶段植被区土壤水分状况在4－6月不断降低,到6月达到最低值,7－10月逐渐回升;对于建立较早的植被区（1984和1987年）,土壤水分垂直变化表现出随着土层深度的增加,土壤含水量逐递减的趋势,特别是70cm下土层中,土壤含水下降明显,含水量低于凋萎湿度,由于对根际区域土壤水分的利用,加剧了固沙植被区深层土壤（70cm下）水分的亏缺,进而影响植被物种的组成,随着小叶锦鸡儿灌丛年龄的增加,浅根性植物所占比重增加。     

Soil carbon saturation: concept,evidence and evaluation

Current estimates of soil C storage potential are based on models or factors that assume linearity between C input levels and C stocks at steady-state, implying that SOC stocks could increase without limit as C input levels increase. However, some soils show little or no increase in steady-state SOC stock with increasing C input levels suggesting that SOC can become saturated with respect to C input. We used long-term field experiment data to assess alternative hypotheses of soil carbon storage by three simple models: a linear model (no saturation), a one-pool whole-soil C saturation model, and a two-pool mixed model with C saturation of a single C pool, but not the whole soil. The one-pool C saturation model best fit the combined data from 14 sites, four individual sites were best-fit with the linear model, and no sites were best fit by the mixed model. These results indicate that existing agricultural field experiments generally have too small a range in C input levels to show saturation behavior, and verify the accepted linear relationship between soil C and C input used to model SOM dynamics. However, all sites combined and the site with the widest range in C input levels were best fit with the C-saturation model. Nevertheless, the same site produced distinct effective stabilization capacity curves rather than an absolute C saturation level. We conclude that the saturation of soil C does occur and therefore the greatest efficiency in soil C sequestration will be in soils further from C saturation.

Accurate automatic determination of acid phosphatase substrate saturation curves

An automatic method for the determination of acid phosphatase substrate saturation curves is described; the curves are recorded continuously in a Technicon AuteAnalyzer in which the enzyme, at a constant pH, reacts with a gradient of substrate made using the equalized continuous flow rate technique in a closed vessel. A slightly modified method for the determination of the substrate saturation curves on the same enzyme in cell suspension, by continuous dialysis, is also reported. The method is highly accurate and makes it possible to evaluate very small kinetic variations.     

Soil moisture effects on uptake of metals by Thlaspi, Alyssum, and Berkheya

Most commonly used hyperaccumulator plants for phytoextraction of metals evolved on soils where moisture is limited throughout much of the year. As these plant species are commercialized for use, they are frequently moved from the point of evolution to locations where environmental conditions may be significantly different. Greatest among these potential differences is soil moisture. The objective of this study was therefore to determine whether these plants could grow in soils with much higher soil moisture and whether they would continue to hyperaccumulate metals as soils approach saturation. We examined extractable soil metal concentrations, plant growth, and metal accumulation for the Ni hyperaccumulators, Alyssum murale and Berkheya coddii and the Zn hyperaccumulators Thlaspi caerulescens cultivars AB300 and AB336. Non-hyperaccumulating control species for each were also examined. In general, extractable soil concentrations of Ni decreased with increasing soil moisture content. Few significant effects related to Zn extractability were observed for any of the soil moisture treatments. The biomass of all tested species was generally greater at higher soil moisture and inhibited at low soil moisture. Further, plants accumulated large amounts of metals from soil at higher soil moisture. Highest foliar concentrations of Zn or Ni were found at the two highest WHCs of 80 and 100%. These results show that hyperaccumulators grow well under conditions of high soil moisture content and that they continue to hyperaccumulate metals. Thus, growing Thlaspi, Alyssum, and Berkheya for commercial phytoextraction under nonnative conditions is appropriate and suggests that this technology may be applied to a wide and diverse range of soil types, climatic conditions, and irrigation regimes.     

An improved dual-tube megaprimer approach for multi-site saturation mutagenesis

Saturation mutagenesis is a powerful tool in protein engineering. Even though QuikChange site-directed mutagenesis method is dominantly used in laboratories, it could not be successfully applied to the generation of a focused mutant library of human glutathione transferase A2-2. In the present study, we further developed an improved versatile dual-tube approach of randomizing difficult-to-amplify targets, exhibiting significant improvement towards equal distribution of nucleotides at randomized sites compared to other published methods.     

Quantitative determination method of nucleic acids by enzymatic amplification (PCR method) to saturation]

We describe here conditions under which the enzymatic amplification of DNA using the polymerase chain reaction (PCR) is quantitative, even when the amplification reaction is run to saturation. DNA in the sample to analyze is co-amplified with known quantities of an internal standard, namely a DNA molecule whose sequence or length differs from that of the sample DNA by only a few base pairs. The two amplification products are detected as run-off products elongated from one or several additional labelled, primers. The ratio between the two signals provides a precise estimate of the amount of specific DNA in the sample to analyze.     

Soil moisture influence on micronutrient cation availability under aerobic conditions

Summary The three major soil series comprising the Gezira scheme (Sudan) are Hosh, Suleimi, and Laota. Surface soil samples from each soil series were employed to study the effect of soil moisture on the DTPA-extractable micronutrient cation under aerobic conditions. The study continued for 8 weeks using an incubation technique at two levels of soil moisture (continuously moist and moist/dry cycles). The DTPA-extractable Cu, Fe, Mn and Zn from air-dry soil samples were much higher compared to values from their incubated counterparts. For the three soils the CO₂ production (microbial activity) reached the maximum in 5 weeks and then levelled off while the lowest values of micronutrient cation from the incubated soils were obtained between 2 to 8 weeks.Generally, the study suggests that the hot dry months preceding crop growth should increase clay surface acidity and hence availability of mironutrient cations.Contribution from the Texas Agricultural Experiment Station, Texas A&M University, College Station. TX 77843, U.S.A.