Comparison of different methods for determination of the duodenal and ileal flows of endogenous nitrogen and amino acids in growing goats

The objective of the present study was to compare the isotope dilution, the difference and the amino acid profile (AAP) methods for the quantification of duodenal and ileal flows of endogenous nitrogen (N) and amino acids (AA) in growing goats. Nine growing goats were fed the same diet containing maize stover, ground corn and soybean meal. The duodenal flow of endogenous N determined by the isotope dilution method was significantly (p < 0.01) higher than that determined by the difference and AAP methods, while there was no difference between the difference and the AAP methods. The duodenal flows of individual endogenous AA determined by the isotope dilution method exceed those determined by the difference and the AAP method by 10 to 106%. The endogenous flow at the ileum determined by the isotope dilution method was not significantly different to those determined by the water-soluble method, but tended to be lower for N and all amino acids. It is concluded that the difference method and AAP method underestimate the duodenal flow of endogenous N and AA compared to the isotope dilution method.     

一种新型蚯蚓纤溶酶的部分性质研究

为获得一种高效的溶栓药物。从赤子爱胜蚓(Eiseniafoelida)中分离纯化得到了一种纤溶酶组分。用Lowry法测定蛋白质浓度,SDSPAGE鉴定纯度为98%,表观相对分子质量(Mr)为14850,纤维蛋白平板法测定其总纤溶活性为65.51×103mm2/mg,直接纤溶活性为15.61×103mm2/mg,间接纤溶活性为26.34×103mm2/mg。水解BAEE的米氏常数(Km)为1.82×105mol/L。水解ChromozymPL的米氏常数(Km)3.98×105mol/L,水解ChromozymtPA的米氏常数(Km)5.55×105mol/L活性,N端氨基酸序列测定的结果为VIGGTNAIPGEFPYQ。结果表明该纤溶酶分子量较小,间接活性较高,适宜作为一种新型的溶栓药物。     

Application of capillary zone electrophoresis to study the properties of rhodanese fromAcidithiobacillus ferrooxidans

A new capillary zone electrophoretic method was applied to the assay of enzymic activity of rhodanese from Acidithiobacillus ferroxidans. The enzyme activity determined by capillary zone electrophoresis was compared with that determined by discontinuous spectrophotometry, the values obtained being in good agreement. The method was also used to evaluate Michaelis constants of cyanide and thiocyanate as substrates; a new approach was developed to solve the problem with variable ionic strength of the samples. The pH and temperature optima for the enzyme were also determined.     

Application of the median method to the determination of kinetic constants for competitive enzyme inhibition

A procedure was developed to determine kinetic constants for competitive inhibition (Km, Ki, Vmax) by the median method. Simulated experiments were used to compare the accuracy and precision of kinetic constants determined by the median method with unweighted and weighted least-squares analysis. The median method was superior to unweighted least-squares analysis. The weighted least-squares method was superior to the median method when the error was normally distributed but the median method was superior when two or more outliers were present. The dependence of the accuracy and precision of kinetic constants obtained by the median method on several experimentally important parameters, including the number of experimental points, the number and range of substrate concentrations, and the number and range of inhibitor concentrations, was determined.     

A General Assay Method for Nucleotide Pyrophosphorylases

A general assay method for nucleotide pyrophosphorylases has been investigated. The principle of the method is based on the measurement of consumption rate of 5-phosphoribosylpyrophosphate (PRPP) during the enzyme reaction. In the method, an enzyme preparation for sample was incubated in a reaction mixture containing a purine or pyrimidine base and PRPP for a certain time, and the amounts of PRPP before and after the reaction were determined. The amount of PRPP was determined by an enzymatic method using orotidine-5′-monophosphate (5′-OMP) pyrophosphorylase and 5′-OMP decarboxylase. Nucleotide pyrophosphorylase activity corresponding to each purine or pyrimidine base was determined from the amount of PRPP consumed per unit time.

The present method is generally applicable for determining activities of any kind of nucleotide pyrophosphorylases, and does not need any tedious separation procedure in all cases. Therefore, comparing with the conventional assay methods for nucleotide pyrophosphorylase activities, this method can be said to be much simpler and reliable. As an application of the present method, activities of several nucleotide pyrophosphorylases in Micrococcus glutamicus have been determined.     

反相液相色谱法分析芦荟制品中有机酸

提出了一种反相液相色谱法分析芦荟制品中有机酸的含量。采用C18 RP为色谱分离柱,磷酸盐溶液与乙腈作流动相;芦荟制品经简单处理后直接进行分离定量,在10min内把其中的苹果酸、乳酸和富马酸等完全分离定量,各种酸的回收率均大于98％。经多次实验证明：该方法是测定芦荟制品中有机酸的快速、准确和有效的定量方法。     

Tannins from mimosoid legumes of Texas and Mexico

The percentage of tannins in leaves, bark, wood, and immature fruits of several species of Acacia and related mimosoid legumes from the southwestern U.S. and Mexico, along with a few from Costa Rica and Argentina, was determined by a modified hide powder procedure and by precipitation with casein. The relative percentages of hydrolyzable and condensed tannins were determined by the iodate and the vanillin-HCl methods, respectively. Gallotannins of selected samples were also determined by the rhodanine method. Although the amount of total tannins was similar for the first two methods, values for condensed tannins by the vanillin-HCl method were frequently two to four times greater than the total tannin values.     

A combined method for determining inhibition type, kinetic parameters, and inhibition coefficients for aerobic cometabolism of 1,1,1-trichloroethane by a butane-grown mixed culture.

A combined method for determining inhibition type, kinetic parameters, and inhibition coefficients is developed and presented. The method was validated by applying it to data obtained from batch kinetics of the aerobic cometabolism of 1,1,1-trichloroethane (1,1,1-TCA) by a butane-grown mixed culture. The maximum degradation rates (k(max)) and half-saturation coefficients (K(s)) were independently determined in single compound tests, and compared with those obtained from inhibition tests. The inhibition type was determined using direct linear plots at various substrate and inhibitor concentrations. Kinetic parameters (k(max) and K(s)) and inhibition coefficients (K(ic) and K(iu)) were determined by nonlinear least squares regression (NLSR) fits of the inhibition model determined from the direct linear plots. Initial guesses of the kinetic parameters for NLSR were determined from linearized inhibition equations that were derived from the correlations between apparent maximum degradation rates (k(app)(max)) and/or the apparent half-saturation coefficient (K(app)(s)) and the k(max), K(s), and inhibitor concentration (I(L)) for each inhibition equation. Two different inhibition types were indicated from the direct linear plots: competitive inhibition of 1,1,1-TCA on butane degradation, and mixed inhibition of 1,1,1-TCA transformation by butane. Good agreement was achieved between independently measured k(max) and K(s) values and those obtained from both NLSR and the linearized inhibition equations. The initial guesses of all the kinetic parameters determined from linear plots were in the range of the values estimated from NLSR analysis. Overall the results show that use of the direct linear plot method to identify the inhibition type, coupled with initial guesses from linearized plots for NLSR analysis, results in an accurate method for determining inhibition types and coefficients. Detailed studies with pure cultures and purified enzymes are needed to further demonstrate the utility of this method.     

A study of stability of electrocortical rhythm generators

By means of the autoregressive model the transfer function and the impulse response function were determined and the parameters characterizing electrocortical oscillators were found for the four brain structures of the experimental animals. Different kinds of the representation of these parameters were compared and their sensitivity to the different factors was tested. The advantages of the proposed method over the conventional spectral analysis were demonstrated. Cluster analysis was applied in order to test the stability of the determined basic rhythms. Proposed method seems to be very useful for the investigation of the processes of rhythmical activity generation and control.Military Institute of Hygiene and Epidemiology     

Perfusion method for assaying microbial activities in sediments: applicability to studies of n(2) fixation by c(2)h(2) reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol . g of dry sediment . h by 10 to 20 h. Depletion of interstitial NH(4) was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C(2)H(4) . g of dry sediment . h. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C(2)H(4) production. Initial values obtained by using the perfusion method were 0.66 nmol of C(2)H(4) . g of dry sediment . h for sediments from Zostera communities and 0.70 nmol of C(2)H(4) . g of dry sediment . h for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

Defining the consensus sequences of E.coli promoter elements by random selection.

The consensus sequence of E.coli promoter elements was determined by the method of random selection. A large collection of hybrid molecules was produced in which random-sequence oligonucleotides were cloned in place of a wild-type promoter element, and functional -10 and -35 E.coli promoter elements were obtained by a genetic selection involving the expression of a structural gene. The DNA sequences and relative levels of function for -10 and -35 elements were determined. The consensus sequences determined by this approach are very similar to those determined by comparing DNA sequences of naturally occurring E.coli promoters. However, no strong correlation is observed between similarity to the consensus and relative level of function. The results are considered in terms of E.coli promoter function and of the general applicability of the random selection method.     

Determination of nonheme iron using inductively coupled plasma-atomic emission spectrometry

A technique for the rapid and accurate estimation of nonheme iron using inductively coupled plasma-atomic emission spectrometry is described. Yttrium was used as an internal standard. An external calibration method was used. The standards were prepared in a matrix composed of 2.5N HCl in 10% (w/v) trichloroacetic acid. The supernatant and coagulum fractions of liver nonheme iron were separated by the method of Drysdale and Ramsay with minor modification. The data determined by this procedure was compared and found to be agreement with data determined by the method of Hallgren. To evaluate the iron status of rats, hemoglobin and liver nonheme iron were determined. Hemoglobin and all of the nonheme iron fractions of the rats fed an iron-deficient diet were significantly lower than those of the rats fed an iron-sufficient diet. The blood content in the liver was estimated to be 80 microL/g from the blood iron concentration, and the difference between total and nonheme iron concentration in liver.     

A Rapid Method,Combining Microwave-Assisted Extraction and Gas Chromatography-Mass Spectrometry with a Database,for Determining Organochlorine Pesticides and Polycyclic Aromatic Hydrocarbons in Soils and Sediments

A rapid method for determining organochlorine pesticides and polycyclic aromatic hydrocarbons in soils and sediments was developed to allow pollution surveys to be performed in emergencies. The method involves microwave-assisted extraction and uses an automated identification/quantification system with a gas chromatography mass spectrometry database. A sample (3 g) is extracted with a 3:2 v/v hexane:water mixture (10 mL) for 30 min using a microwave-assisted extraction system at 120°C. The hexane extract is then cleaned using silica gel, then analyzed by gas chromatography mass spectrometry. The total analysis time is approximately 4 h. The precision of the quantitative results and accuracy of the analyte identification were determined. The total analyte concentrations were generally comparable to (61%–110% of) the concentrations determined using a Soxhlet extraction method, but the concentrations of individual high-molecular-weight polycyclic aromatic hydrocarbons were unacceptably low compared with the concentrations determined using the Soxhlet method. However, these compounds (e.g., benzo(ghi)perylene and indeno(1,2,3-cd)pyrene) were subsequently efficiently extracted using a hexane:water:ethanol mixture. The accuracy of identification was evaluated using accurate masses determined by gas chromatography time-of-flight mass spectrometry, and the mass error was 2 ppm for 21 of the 22 compounds identified using the new method.     

海南黎药海巴戟多糖含量测定及其测量不确定度评估

研究和探讨海南黎药海巴戟多糖含量,并对多糖含量测量过程中引入的不确定因素进行评估。采用苯酚-硫酸法测定海巴戟药材中多糖含量,并根据苯酚-硫酸法建立数学模型,找出影响不确定的因素,最终确定测量结果的合成不确定度和扩展不确定度。以D-葡萄糖作为对照品,浓度在0.002 5～0.012 5 g/L范围内线性关系良好(R~2=0.999 4),重现性试验RSD=1.84%,4 h内显色稳定,加样回收率为98.05%(n=9,RSD=2.32%);16批海巴戟药材多糖含量为5.53%～12.63%,其多糖含量测定方法的合成不确定度为0.029 4～0.093 8,扩展不确定度为0.059 0～0.187 6。影响测量不确定度的主要因素有称量、浓度配制和吸光度。本研究建立的海南黎药海巴戟多糖含量测定方法适应性广、灵敏度高,若将测量结果不确定度纳入标准制定有助于对海巴戟药材含量分析方法的建立和质量标准的制定,有利于对海巴戟药材资源的开发。     

Cytochemical model system for microsomal rat liver glucose-6-phosphate.

A method is described for the incorporation of a microsomal rat liver fraction into polyacrylamide films without significant loss of its glucose-6-phosphatase activity. The enzymatic activity was completely lost when the films were prepared with ammonium persulfate as initiator of the polymerization as previously described for alkaline phosphatase, but modification of this method showed that about 90% of the glucose-6-phosphatase activity could be retained. The enzyme in the films prepared with the new method was completely inhibited by alloxan, HgCl2, and preincubation in 0.05 M acetate buffer (pH 5.0) at 37 degrees C, as determined biochemically. Similar results were obtained for the enzyme in films determined histochemically according to the lead method of Wachstein and Meisel. In this respect the behavior of the incorporated enzyme is similar to that in suspension. Films fixed with 1.5% glutaraldehyde showed rapid inactivation of glucose-6-phosphatase. There was good correlation between the biochemical and histochemical activity determined after fixation. A method to embed polyacrylamide films in Epon for electron-microscopical investigation is also described. Dimethyl sulfoxide was used as the dehydrating agent instead of ethanol/acetone.     

Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method

A simple ion-exchange HPLC-UV method was developed for determination of major allergens from mugwort pollen and kiwi fruit extracts in mass-units. The separation of Art v 1 and Act c 1 from other components in the extracts was achieved in one step. The extinction coefficients used in the study were theoretically determined and compared to the extinction coefficients determined by gravimetry. We also reported a close correlation of the major allergen contents with the overall allergenic potency of the extracts determined by inhibition ELISA. This method could be a useful tool for standardization of allergenic extracts for clinical use.     

神经生长因子的发光免疫分析法

介绍了一种灵敏的神经生长因子化学发光免疫测定方法。小鼠神经生长因子（ｍＮＧＦ）的抗体ＩｇＧ用亲和层析纯化并用吖啶酯标记。该法可用于大鼠组织中ＮＧＦ含量的测定。方法的灵敏度为１０ｐｇ／ｍＬＮＧＦ;批内批间变异系数分别为８．７％及１３．２％;回收率平均为１０３％。ｍＮＧＦ抗体对人的ＮＧＦ也有极强的交叉反应,故本方法也可能用于病人血清或脑脊液中ＮＧＦ含量的测定。     

Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis.

A modified direct viable count method to detect living bacteria was used with image analysis for the rapid enumeration of chlorine-injured cells in an Escherichia coli culture. The method was also used for determining chlorine-induced injury in coliform isolates and enteric pathogenic bacteria. Cultures were incubated in phosphate-buffered saline, containing 0.3% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.03% yeast extract, and optimal concentrations of nalidixic acid. Samples were withdrawn before and after incubation and stained with acridine orange, and cell lengths and breadths were measured by computerized image analysis. After incubation, cells which exceeded the mean preincubation length (viable cells) were enumerated and the results were compared with those obtained by the plate count method. Injury in the chlorine-exposed cell population was determined from the difference in viable count obtained with a nonselective Casamino Acids-yeast extract-nalidixic acid medium and a selective Casamino Acids-yeast extract-nalidixic acid medium containing sodium deoxycholate or sodium lauryl sulfate. The levels of injury determined by the direct viable count technique by using image analysis were comparable to those determined by the plate count method. The results showed that image analysis, under optimal conditions, enumerated significantly higher numbers of stressed E. coli than the plate count method did and detected injury in various cultures in 4 to 6 h.     

Nuclear segmentation of bronchial epithelial cells by minimax and thresholding techniques. A comparison

Two nuclear segmentation methods, Baky's minimax algorithm and thresholding, were compared on a sample of 879 atypical bronchial epithelial cells in sputum. Nuclear-cytoplasmic (N/C) ratios for all cells were determined by each segmentation method and compared to a visually determined value. Cells were categorized by atypia class (from metaplastic through malignant), by staining characteristics (orangeophilic and nonorangeophilic) and by method of digitization (either scanning microphotometry or video system). The method of digitization was confounded by subject differences. The results indicated that with most classes of atypia, N/C ratios determined by minimax were closer to the visually derived values than were those of thresholding, particularly with orangeophilic cells. Both methods become progressively less accurate, as compared to the visual procedure, as the degree of atypia increases.     

Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis

A modified direct viable count method to detect living bacteria was used with image analysis for the rapid enumeration of chlorine-injured cells in an Escherichia coli culture. The method was also used for determining chlorine-induced injury in coliform isolates and enteric pathogenic bacteria. Cultures were incubated in phosphate-buffered saline, containing 0.3% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.03% yeast extract, and optimal concentrations of nalidixic acid. Samples were withdrawn before and after incubation and stained with acridine orange, and cell lengths and breadths were measured by computerized image analysis. After incubation, cells which exceeded the mean preincubation length (viable cells) were enumerated and the results were compared with those obtained by the plate count method. Injury in the chlorine-exposed cell population was determined from the difference in viable count obtained with a nonselective Casamino Acids-yeast extract-nalidixic acid medium and a selective Casamino Acids-yeast extract-nalidixic acid medium containing sodium deoxycholate or sodium lauryl sulfate. The levels of injury determined by the direct viable count technique by using image analysis were comparable to those determined by the plate count method. The results showed that image analysis, under optimal conditions, enumerated significantly higher numbers of stressed E. coli than the plate count method did and detected injury in various cultures in 4 to 6 h.