农牧交错区不同植物群落土壤呼吸的日动态观测与测定方法比较

 采用动态密闭气室法（IRGA）对农牧交错区10种植物群落最大生物量时期的土壤呼吸日动态进行了测定,并将该方法得到的土壤日呼吸速率与碱液吸收法（AA）进行了比较。结果表明：1）10个群落土壤呼吸的昼夜变化比较明显,均为单峰型曲线,主要受土壤温度的驱动,但同时也受到当日降水情况和云量、风速等气象因子的较大影响。因此,这些群落土壤呼吸日动态的一致性较差,规律性并不明显。2）用碱液吸收法和动态密闭气室法测定的10个群落的土壤呼吸速率变化范围分别为394～894 mg C·m-2·d-1和313～2043 mg C·m-2·d-1,其中碱液吸收法测定结果平均为动态气室法的67.5%,明显低于动态密闭气室法。3）两种测定方法具有很好的相关性,R2为0.873 9。本研究中发现,在土壤呼吸速率低的情况下,两种方法的测定结果十分接近甚至碱液吸收法测定结果稍大于动态密闭气室法,而在土壤呼吸速率较高的情况下,动态密闭气室法测定结果则显著高于碱液吸收法。上述结果与国内外同类研究的结果高度一致,从而为校正以往采用碱液吸收法在该区域的测定结果提供了可靠依据。     

Propidium iodide and S1 nuclease: tools for studying DNA reassociation kinetics

A method for the determination of the amount of double-stranded DNA in a reassociation mixture is described. Reassociated DNA resistant to S1 nuclease digestion is measured fluorometrically using propidium iodide. A direct comparison is made between this method and an established method in which radiolabeled Escherichia coli DNA resistant to S1 digestion is measured by scintillation counting after separation of nucleotides by Sephadex G-100 chromatography. Reassociation curves determined for calf thymus and E. coli DNA are presented.     

A method of direct measurement for the enzymatic determination of cholesteryl esters

A direct measurement method for the enzymatic determination of cholesteryl esters (CEs) without measuring total cholesterol (TC) and free cholesterol (FC) is described. In the first step, hydrogen peroxide generated by cholesterol oxidase from FC was decomposed by catalase. In the second step, CE was measured by enzymatic determination using a colorimetric method or a fluorometric method. The measurement sensitivity of the fluorometric method was more than 20 times that of the colorimetric method. Optimal conditions of the assay were determined, and examples of measured CE in human plasma, rat liver, and cultured cells are indicated. The method of directly measuring CE was simple and has exceptional reproducibility compared with the technique of subtracting FC from TC using each measured TC and FC.     

桑沟湾大型底栖植物的光合作用和生产力的初步研究

本文在实验条件下,用O_2法和~(14)C法测定了桑沟湾主要大型底栖植物的光合作用,并根据现场资源调查结果,估算了桑沟湾大型底栖植物的生产力。结果表明:不同底栖植物的光合作用速率的变化范围为:1.53—6.8Amg·g~(-1)·h~(-1)(以碳计,O_2法)或0.29—1.93mg·g~(-1)·h~(-1)(以碳计,~(14)C法);~(14)C法测出的底栖植物的碳同化速率为O_2法测定值的19.0％—40.2％。桑沟湾大型底栖植物的平均年生产力为1060g·m·~2·a·~(-1)(以碳计),每年总碳生产量为9750t。桑沟湾大型底栖植物的生产力与世界其它地区比较,水平较高。     

不同生态环境中铜绿丽金龟幼虫肠道细菌 产消化酶活性比较

目的测定不同生态环境中铜绿丽金龟幼虫肠道细菌产消化酶活性。方法采用平板透明圈法和分光光度计法。结果平板透明圈法测得废弃菜园和花生田中铜绿丽金龟幼虫肠道细菌产蛋白酶和淀粉酶活性差异无统计学意义(F=1.089、0.4963,P0.05),而细菌的产纤维素酶活性有显著差异,其中花生田铜绿丽金龟幼虫肠道中分离到的粘质沙雷菌产酶活性显著高于其他菌株;分光光度计测得的不同生态环境中铜绿丽金龟幼虫肠道细菌的产蛋白酶、淀粉酶和纤维素酶活性差异均有统计学意义(F=461.565、42.349、18.7673,P0.05)。从变异系数来看,分光光度计法测得的蛋白酶、淀粉酶和纤维素酶的变异程度较低,而脂肪酶测定时平板透明圈法的变异系数较低。结论平板透明圈法和分光光度计法均可用于铜绿丽金龟幼虫肠道细菌产消化酶活性测定。通过分析研究比较,分光光度计法适于测定细菌的产蛋白酶、淀粉酶和纤维素酶活性,平板透明圈法适于测定细菌的产脂肪酶活性。     

A method for measuring H2O2 based on the potentiation of peroxidative NADPH oxidation by superoxide dismutase and scopoletin.

NADPH oxidation catalyzed by horseradish peroxidase is considerably increased by scopoletin and superoxide dismutase. These effects were used to develop a method for measuring H2O2 in a horseradish peroxidase, superoxide dismutase, and scopoletin system by measuring the NADPH oxidation rate. The optimal concentration of each reactant was determined. H2O2 could be detected and measured when it was present free in the medium or when it was produced by an H2O2-generating system, such as glucose-glucose oxidase or NADPH oxidase from thyroid plasma membranes. H2O2 was measured either by taking aliquots of the incubation medium or by placing NADPH directly in the medium and following the kinetics of NADPH oxidation. This latter approach required smaller amounts of biological material. In contrast to other methods, the H2O2 which is measured is regenerated. This method is 10 times more sensitive than the standard scopoletin method for H2O2 measurement and will detect a H2O2 production rate as low as 0.2 nmol per hour. The method is particularly suitable for biological systems in which small quantities of biological material are available.     

蒽酮法测定爬山虎果中糖类的含量

用蒽酮法测定爬山虎熟果中糖类的含量,波长620nm,检测线性范围0．01—0．5mg/ml,相关系数为0．9993,RSD＝5．64％回收率为98．56％。此方法操作简便准确、显色灵敏、重现性好、不受还原性物质的干扰。能测定总糖,还能测定可溶性糖,因此适合野生植物中糖类含量测定。     

丙酮和二甲基亚砜法测定植物叶绿素和类胡萝卜素的方法学比较

丙酮和二甲基亚砜作为提取剂测定植物光合色素含量是最为普遍的两种方法,但是对于二者之间的可比性问题探讨很少。本文基于对19中木本植物叶片和树枝内叶绿素含量的测定结果发现如下结果：（1）二甲基亚砜测定叶绿素a、叶绿素b和叶绿素总量的结果均显著高于丙酮测定结果（而对叶绿素a/b比值的影响较小）,与此相反,丙酮对类胡萝卜素提取测定结果要远高于二甲基亚砜的测定结果。（2）两种方法对叶绿素和类胡萝卜素测定结果之间均存在显著线性相关,可以应用相关线性拟合方程进行不同方法测定结果之间的转换,以减少叶绿素含量、类胡萝卜素含量比较过程中产生的测定方法误差。（3）不同单位表示方法,对两种方法测定结果的相关性存在影响,使用鲜重单位表示的相关程度普遍高于以表面积为单位的相关程度。（4）两种方法对树枝和叶片测定结果的影响存在差异：对于叶绿素含量低的树枝,两种方法测定结果的差异较小,而对于叶绿素含量高的叶片,二甲基亚砜测定结果则远高于丙酮的测定结果,可能原因在于丙酮对于叶绿素的提取效果远低于二甲基亚砜。（5）以二甲基亚砜为浸提试剂,通过不同精确度仪器（0.1和1 nm）检测发现尽管不同仪器之间相关关系达到0.99以上,但二者相比精确度为0.1 nm检测叶绿素a、总叶绿素浓度高出15%~33%,而叶绿素b浓度低4%左右。这些研究发现为不同方法之间的比较提供了依据。     

Plant hydraulic conductance measured by the high pressure flow meter in crop plants

A new high pressure flow meter (HPFM) method for measuring plant hydraulic conductances (K) was investigated to examine whether its results are comparable to those from a conventional evaporative flux (EF) method in crops. Hydraulic conductance (K) was measured by the two methods under quasi-steady-state conditions in six crops grown in pots: soybean (Glycine max L. Merr. cv. Tsurunoko daizu), sunflower (Helianthus annuus L. cv. Russian mammoth), kidney bean (Phaseolus vulgaris L. cv. Tsurunashi morocco), tomato (Lycopersicon esculentum Mill. cv. Sekai-ichi), green pepper (Capsicum annuum L. cv. shishitou), and eggplant (Solanum melongena L. cv. Seiguro chunaga nasu). There was a 1:1 agreement between K values measured by the two methods for K values of whole plant, root and stem, and leaf under quasi-steady-state conditions. Leaf water potential (psi leaf) and evaporative flux density (E) in sunflower was curvilinear, indicating whole plant K estimated by the EF method increased with increase of E. Predicted psi leaf (= E divided by whole plant K measured by the HPFM method) agreed with measured psi leaf. Diurnal changes were also found in K measured by the HPFM confirming that K changed in response to temperature and E. The HPFM revealed that variable conductance was located in all organs: roots, stems, petioles, and leaves. These observations indicated that the HPFM is valid for crops as well as for trees (as previously established by Tsuda and Tyree) and has advantages over the EF method because of the speed and ease of the HPFM method.     

An enzymatic method to determine receptor-mediated endocytosis

Many studies have measured receptor-mediated endocytosis using radiolabeled ligands or antibodies. Upon ligation and cross-linking, the labeled ligand or antibody is endocytosed and the internalization of the radioisotope is assayed after stripping the uninternalized ligand from the cell membrane. This study reports on an enzymatic assay to measure receptor-mediated endocytosis and compares it with the radioactive method. The results show that receptor-mediated endocytosis measured using the peroxidase conjugated antibody is two fold higher than that measured with a radiolabeled antibody. Thus, approximately 38% endocytosis of CD3 is measured using an ¹²⁵I-labeled antibody, whereas approximately 79% endocytosis is detected by peroxidase conjugated antibody method. Similar increases are also found with CD2 receptor-mediated endocytosis. Our study has demonstrated that the enzymatic method could be employed in determining receptor-mediated endocytosis. In addition to increased sensitivity, the enzymatic assay eliminates the use of radioactive materials.     

How to measure and predict the molar absorption coefficient of a protein.

The molar absorption coefficient, epsilon, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring epsilon for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-326] and is based on data from Edelhoch [1967, Biochemistry 6:1948-1954]). The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average epsilon values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the epsilon values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average epsilon values for the proteins are less than the epsilon values measured in any of the solvents. For Tyr, the average epsilon values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured epsilon values for 80 proteins, the epsilon at 280 nm of a folded protein in water, epsilon (280), can best be predicted with this equation: epsilon (280) (M-1 cm-1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125) These epsilon (280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict epsilon.     

Fluorometric assays in the study of nucleic acid-protein interactions : II. The use of fluorescamine as a reagent for proteins

Fluorescamine is a useful reagent in monitoring protein-DNA interactions only if a convenient method of separating the complex from free protein is available. Sedimentation of the complex provides such a method at least in the case of histone-like proteins capable of extensive interaction with DNA. This approach is therefore complementary to the filter binding assay. When the interaction of protein and DNA is compared by both methods, a clear-cut distinction between two steps is obtained: (i) a nucleation step that can be measured by the filter binding assay: and (ii) the cooperative growth of the complex that can only be measured by the sedimentation assay. The method is also useful to detect small amounts of protease contamination in DNA preparations.     

Measurement of resistant starch content in cooked rice and analysis of gelatinization and retrogradation characteristics

Digestion-resistant starch (RS) has many physiologic functions. The RS content is measured by enzymatically degrading flour samples according to the method of the Association of Official Analytical Chemists. Experiments have been performed with wheat, corn, and other grains, but there are no data for cooked rice grains in the form ingested by humans. Thus, we investigated a method to measure RS that is suitable for cooked rice grains using rice cultivars that are reported to differentially increase postprandial blood glucose in humans. Using a method for cooking individual rice grains and optimized enzyme reaction conditions, we established an RS measurement method. We also found that the amylopectin crystal condition affects the RS content measured using our method.     

激光时间分辨荧光谱仪＊以及荧光各向异性测量

就时间相关单光子计数荧光谱仪的工作原理、实验装置的建立等问题进行了探讨。利用国产的红离子锁模激光同步泵浦腔倒空输出染料激光器等器件与进口的时幅转换器、定时恒分器建成了激光时间分辨荧光谱仪,时间分辨率为８０ｐｓ。本文还建立了荧光各向异性的测量方法,利用该方法对Ｒ－ＰＥ的旋转弛豫时间进行了测量,并与英国Ｅｄｉｎｂｕｒｇｈ公司生产的２９９Ｔ型荧光谱仪的测量结果进行了比较,结果证明仪器及方法都是可靠的。     

Determination of lysine in pharmaceutical samples containing endogenous ammonium ions by using a lysine oxidase biosensor based on an all-solid-state potentiometric ammonium electrode

A new potentiometric method is proposed to determine lysine in pharmaceutical samples. This method is based on a lysine biosensor consisting of a chemically immobilized lysine oxidase membrane attached to an all-solid-state ammonium electrode. Lysine is degraded in the sensor to release ammonium, which is detected by means of the ammonium electrode. The presence of endogenous ammonium in the samples interferes with these determinations, since the response measured corresponds to the sum of the ammonium generated enzymatically and that present in the sample. This is a general drawback for all biosensors based on the detection of ammonium. Study of samples containing both lysine and ammonium showed that concentration ranges exist in which a near-logarithmic relationship between potentials measured and lysine concentrations is found. Therefore, within these ranges, lysine can be determined by using the standard addition method, with the subsequent data treatment involving an iterative linearization procedure. Results obtained with the proposed potentiometric method are consistent with those given by the standard method for amino acid analysis.     

Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells

Permeabilization was evaluated as a rapid method to prepare mammalian cells for subcellular enzyme activity measurement. It was observed that enzymes can be measured directly in cell suspensions permeabilized by Triton X-100 and digitonin with various concentrations. Total enzyme activities measured in permeabilized cells were identical to those measured in sonicated cells showing that permeabilization can replace the more complicated sonication method. Tuning of digitonin concentration allowed selective permeabilization of plasma and mitochondrial membranes. This was studied by analyzing the release of extramitochondrial and mitochondrial marker enzymes on treatment with different concentrations of the agent. Solely the plasma membrane was permeabilized by using 0.01–0.02% (w/v) digitonin. Access to all cellular enzymes was achieved by using 0.05% (v/v) Triton X-100. This selective permeabilization was further evaluated in a 96-well plate format by testing additional marker enzymes and additional cell lines, Hep G2 and CHO-K1, applying the developed protocol. The presented method is well suited for the high-throughput analysis of subcellular localization and activity of enzymes. The method is simple and enables one to distinguish between mitochondrial and extramitochondrial activities, which is usually achieved only by much more complicated and time-consuming cell preparation.     

A new assay for tRNA aminoacylation kinetics.

An improved quantitative assay for tRNA aminoacylation is presented based on charging of a nicked tRNA followed by separation of an aminoacylated 3'-fragment on an acidic denaturing polyacrylamide gel. Kinetic parameters of tRNA aminoacylation by Escherichia coli AlaRS obtained by the new method are in excellent agreement with those measured by the conventional method. This assay provides several advantages over the traditional methods of measuring tRNA aminoacylation: (1) the fraction of aminoacyl-tRNA is measured directly; (2) data can be obtained at saturating amino acid concentrations; and (3) the assay is significantly more sensitive.     

应用热扩散法测定香蕉树蒸腾速率

香蕉树植株高大,一般采用间接方法确定耗水量,但所得结果受土壤、大气和农艺措施等因素的影响较大.本文于2005年11月15日—12月5日在温室内采用热扩散法（即Granier法）测定香蕉树的茎液流,并与用数字天平（称重法）测定的香蕉树蒸腾速率进行对比试验. 结果表明,Granier法测定的日茎液流量与称重法测定的日蒸腾量相差4％.Granier法测定的茎液流速率一般滞后于称重法确定的蒸腾速率1 h左右.当日蒸腾量小于0.05 L·m^-2（活性叶面积）时,Granier方法不能测定茎液流量.Granier传感器一般在安装2～3 d后即可正常工作,同时在多株植株上安装Granier传感器取其平均流速值计算蒸腾量可以明显减小测量误差.     

A comparison of hole punch and needle punch methods for the measurement of seagrass productivity

Seagrass productivity, as leaf extension, was measured using the hole punch and needle punch techniques. These methods have been widely implemented to determine seagrass leaf extension rates, yet there is no evidence in the literature of a comparison between methods. The hole punch method involves removing part of the basal area of a seagrass leaf and it was proposed that this measurement technique may affect the leaf extension rates that are being measured. Leaf extension rates were measured in Posidonia sinuosa meadows off the coast of Perth, Western Australia. There were no significant differences in seagrass leaf extension between the two methods. The hole punch method is favoured, as measurement of incremental leaf growth is facilitated by the obvious hole left by the punch. The needle punch method leaves lesions on seagrass leaves that are easily confused with other lesions, possibly left by invertebrate grazers. These findings are likely to be applicable to other straplike seagrass species, though a similar comparison is recommended in the initial stages of a study.