Fluorometric determination of phosphatidylcholine as a measure of phospholipid methylation.

The successive methylation of phosphatidylethanolamine to phosphatidylcholine (phospholipid methylation) has been measured by the incorporation of S-[methyl-3H]adenosylmethionine or colorimetric assay of phosphatidylcholine extracted from adipocyte plasma membranes. A fluorometric assay for phosphatidylcholine was developed to measure phospholipid methylation. This assay is 10 times more sensitive than the colorimetric assay and demonstrates no significant interference with other methylated phospholipids. The fluorometric assay was used to determine a biphasic insulin dose response in adipocyte plasma membranes. This fluorometric assay for phosphatidylcholine represents an alternative method for monitoring phospholipid methylation, especially when increased sensitivity is required.     

A radioactive assay for acetylcholinesterase using anion-exchange disk

A radioactive assay for acetylcholinesterase is described. The assay is based on the separation of [¹⁴C]acetate from [¹⁴C]acetylcholine by differential adsorption of the former on DEAE anion-exchange disks. The procedure is simple and sensitive and eliminates the use of ion-exchange resin columns or organic extractions. Moreover, when unpurified enzyme preparations are assayed, linear steady-state kinetics can be observed with this method as contrasted to the nonlinear colorimetric method using acetylthiocholine and dithiobisnitrobenzoate. This method also permits the detection in biological samples of low levels of acetylcholinesterase activity, which is not detectable by the colorimetric method. Using the present radioactive method, cellular levels of acetylcholinesterase have been surveyed in N4TG1 neuroblastoma cells, NG108-15 neuroblastoma x glioma hybrid cells, H9c2 myoblasts, and 3T3-L1 and 3T3-C2 fibroblasts.     

Reliable Drosophila body fat quantification by a coupled colorimetric assay

Factors and mechanisms controlling lipometabolism homeostasis share a remarkable evolutionary conservation between humans and Drosophila flies. Accordingly, the Drosophila model has been successfully used to understand the pathophysiology of human metabolic diseases such as obesity. Body fat stores in species as different as humans and flies consist of neutral lipids, mainly triacylglycerols. Changes in body fat storage are a diagnostic phenotype of lipometabolism imbalances of genetic or environmental origin. Various methods have been developed to quantify Drosophila body fat storage. The most widely used method adopts a commercial coupled colorimetric assay designed for human serum triacylglycerol quantification, which is based on glycerol content determination after enzymatic conversion of glycerides into glycerol. The coupled colorimetric assay is compatible with large-scale genetic screen approaches and has been successfully applied to characterize central regulators of Drosophila lipometabolism. Recently, the applicability of the coupled colorimetric assay for Drosophila storage fat quantification has been questioned in principle. Here we compare the performance of the coupled colorimetric assay on Drosophila samples with thin layer chromatography, the "gold standard" in storage lipid analysis. Our data show that the presented variant of the coupled colorimetric assay reliably discriminates between lean and fat flies and allows robust, quick and cost-effective quantification of Drosophila body fat stores.     

Enzymic assays for isomers of 2,6-diaminopimelic acid in walls of Bacillus cereus and Bacillus megaterium.

An enzymic assay for individual isomers (meso-, LL- and DD-) of 2,6-diaminopimelate was developed. The enzyme 2,6-diaminopimelate decarboxylase specifically attacked meso-diaminopimelate and was used to measure this isomer manometrically. The meso- and LL-isomers were measured together manometrically in a coupled assay with diaminopimelate decarboxylase and diaminopimelate epimerase (which converts LL-diaminopimelate into meso-diaminopimelate). The DD-isomer was not attacked by either enzyme and was measured, as residual diaminopimelate after the coupled assay, by a colorimetric method, which was also used to measure total diaminopimelate before enzymic treatments. The coupled enzymes were also used to prepare pure DD-isomer from chemically synthesized diaminopimelate. A mixture of diaminopimelate isomers was present in walls of four strains of Bacillus megaterium [in each about 75% (w/w) meso-, 18% LL- and 7% DD-] and in walls of two strains of Bacillus cereus (about 85% meso-, 8% LL- and 7% DD-). One strain of B. cereus contained at least 95% meso-diaminopimelate, with only traces of LL- and DD-isomers. Peptidoglycan from Escherichia coli was assayed as containing at least 95% meso-isomer. The proportion of isomers in the wall of a strain of B. megaterium remained constant after growth in a variety of different media.     

An arsenic hyperaccumulating fern,Pteris vittata L. (Pteridaceae) broadly affects terrestrial invertebrate abundance

1. The Chinese brake fern (Pteris vittata L.; Pteridaceae) can accumulate up to 27 000 mg kg^?1 dry wt. of arsenic (As) from the soil into its above‐ground biomass. They may use this As to deter invertebrate threats. 2. This study explored how As concentrations [As] in the fern, and in soil associated with the fern, influenced the abundance and composition of various invertebrates. 3. Populations of P. vittata were identified in the field. Soils from the base of the fern and from 3 m away of each plant were collected and pitfall traps were installed. Soil and fern arsenic concentrations ([As]) were measured via inductively coupled plasma mass spectrometry and invertebrates were identified to order and classified by feeding guild. 4. Increased [As] did not affect all feeding guilds and orders equally. For example, individual herbivore abundance did not decrease as [As] increased, but predator abundance did. In many cases, the impact of soil [As] on invertebrates depended on the distance from the fern. Fern [As] also influenced components of the community, but only at 3 m away from the fern. Furthermore, the abundances of many invertebrate groups were higher beneath the fern, where [As] was higher. 5. These results suggest that hyperaccumulated As can impact the invertebrate community, but the defensive benefits of hyperaccumulation are more complex than have been previously described. The authors advocate that future studies examining the potential defensive benefits of hyperaccumulation should do so in a natural setting that incorporates this complexity and invertebrate richness.     

A new isotopic assay for purine nucleoside phosphorylase

We have developed a new assay for purine nucleoside phosphorylase which is based on the release of tritium when [2-3H]inosine is used as the substrate and the reaction is coupled with xanthine oxidase. After the reaction is terminated, residual [2-3H]inosine is adsorbed on charcoal and the supernatant solution is assayed for radioactivity by liquid scintillation spectrometry. The new method gave results indistinguishable from those obtained by spectrophotometric determination of uric acid produced by the phosphorylase-xanthine oxidase-coupled reaction or by radioassay of chromatographically isolated [8-14C]hypoxanthine when [8-14C]inosine was used as substrate. The new method is faster than those involving chromatographic isolation of products. In comparison with spectrophotometric methods, it not only requires less manual time, but it also has the advantage that it can be used to study inhibitors whose ultraviolet absorption might interfere with spectrophotometric determination of uric acid.     

沙棘根瘤内生细菌中抑菌促生菌株的筛选和鉴定

【背景】植物内生细菌可产生具有抑菌和促生活性的物质,既能抑制植物病原菌对寄主植物的侵染,也能促进植物的生长。沙棘根瘤内生细菌是根瘤内除共生固氮的弗兰克氏菌外,与沙棘共生的一大类微生物。研究具有抑菌和促生活性的植物内生菌,可为微生物菌肥的研究提供理论基础。【目的】筛选具有优良抑菌和促生活性的沙棘根瘤内生细菌,初步研究其抑菌和促生活性,并对菌株进行鉴定。【方法】采用双层琼脂法、琼脂扩散法、双层平板对峙法、牛津杯法进行沙棘根瘤拮抗性内生细菌的筛选。选取抑菌活性较高的内生细菌,分别采用Salkowski比色法、ChromeAzurolS(CAS)平板检测法和钼锑抗比色法进行产吲哚乙酸、铁载体及溶磷能力的测定。采用发酵液灌根法测定沙棘根瘤内生细菌SR308对黄瓜促生作用的盆栽效果。通过形态和培养特征、生理生化试验及16S rRNA基因序列分析法对菌株TT201和SR308进行鉴定。【结果】从131株沙棘根瘤内生细菌中筛选出9株具有较强抑菌活性的内生细菌,其中菌株TT201抑菌性最佳、抑菌谱广;菌株SR308的促生活性最好,其发酵液对黄瓜的生长具有较强的促进作用。对具有较强抑菌和促生活性的菌株TT201和SR308进行鉴定的结果表明,菌株TT201为侧孢短芽孢杆菌(Brevibacilluslaterosporus),菌株SR308为蕈状芽孢杆菌(Bacillusmycoides)。【结论】获得2株具有优良抑菌和促生活性的沙棘根瘤内生细菌,为进一步开发微生物农药及菌肥提供了资源。     

A spectrophotometric method for the determination of gamma-glutamyl cyclotransferase with alanine dehydrogenase in the presence of anthglutin

gamma-Glutamyl cyclotransferase activity is assayed in tissues by a colorimetric method using gamma-glutamyl alanine as a substrate coupled with alanine dehydrogenase from B. sphericus, to measure the formation of NADH. In order to avoid interference by the reaction catalyzed by gamma-glutamyl transpeptidase, anthglutin, a specific inhibitor of the transpeptidase was included in the reaction mixture. The Km value of rat kidney gamma-glutamyl cyclotransferase with respect to gamma-glutamyl alanine appeared to be the same when determined by either the colorimetric or the radiometric method. This assay presents a reliable alternative to the use of radiolabeled substrate and is used for the assay of gamma-glutamyl cyclotransferase in a variety of physiological and experimental samples.     

Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment

Arsenic is ubiquitous in the biosphere and frequently reported to be an environmental pollutant. Global cycling of arsenic is affected by microorganisms. This paper describes a new bacterial strain which is able to efficiently oxidize arsenite (As[III]) into arsenate (As[V]) in liquid medium. The rate of the transformation depends on the cell density. Arsenic species were separated by high performance liquid chromatography (HPLC) and quantified by inductively coupled plasma-atomic emission spectrometry (ICP-AES). The strain also exhibits high minimum inhibitory concentrations (MICs) for As[III] (6.65 mM (500 mg L^-1)) and other heavy metals, such as cadmium (1.42 mM (160 mg L^-1)) or lead (1.20 mM (250 mg L^-1)). Partial identification of the strain revealed a chemoorganotrophic, Gram-negative and motile rod. The results presented here demonstrate that this strain could represent a good candidate for arsenic remediation in heavily polluted sites.     

An anion-exchange radioassay for glucose 6-phosphate phosphatase: use in topological studies with endoplasmic reticulum vesicles.

A simple procedure is presented for the enzymatic preparation of [2-3H]mannose 6-phosphate (Man 6-P) with purified yeast hexokinase and unlabeled ATP. The enzymatically synthesized [2-3H]Man 6-P is utilized as the radiolabeled substrate in a new rapid assay for glucose 6-phosphate (Glc 6-P) phosphatase. The principle of the assay procedure is that the unreacted substrate, [2-3H]Man 6-P, is retained by the anion-exchange resin, AG 1-X8 (acetate), while the enzymatic product, [2-3H]-mannose, is eluted directly into a scintillation counting vial. When Glc 6-P phosphatase activity associated with mouse liver endoplasmic reticulum (ER) vesicles is assayed by the new chromatographic assay, the same characteristic latency and properties are observed, as determined by the commonly used colorimetric assay of inorganic phosphate produced. The anion-exchange radioassay described should be useful for a variety of topological studies on enzymes associated with membrane vesicles derived from liver and kidney ER.     

The use of beta, gamma-methyleneadenosine 5'-triphosphate to determine ATP competition in a scintillation proximity kinase assay.

A novel method for characterizing the kinetics of protein kinase inhibitors is described. This method uses glycogen synthase kinase beta as the model protein kinase and looks at the shift in IC50 of inhibitors using the nonhydrolyzable ATP analog, beta, gamma-methyleneadenosine 5'-triphosphate, also known as AMP-PCP. Due to its inability to be hydrolyzed, AMP-PCP is being used to characterize known glycogen synthase kinase inhibitors by determining the shift in IC50 at concentrations above its calculated Ki of 490 microM. The assay format for the detection of inhibition is a scintillation proximity assay which is robust and reproducible at very low levels of [gamma-33P]ATP. The use of AMP-PCP coupled with the use of the scintillation proximity assay allows this characterization of inhibition without increasing [gamma-33P]ATP and without significantly diluting the overall assay signal. We have used this method in kinetic analyses to demonstrate that we can detect a significant shift in IC50 with the known ATP competitive inhibitors, staurosporine, Ro 31-8220, and olomoucine. The IC50 for glycogen synthase peptide and lithium chloride, which has been reported to be uncompetitive, remains unchanged.     

On-line reversed-phase liquid chromatography hydride generation emission spectrometry: speciation of arsenic in urine of patients intravenously treated with As2O3

Hydride generation inductively coupled plasma–atomic emission spectrometry (HG ICP–AES) was used as a continuous detection system for the determination of arsenic in the eluate from a high-performance liquid chromatographic (HPLC) system. Four arsenic species [arsenite As(III), arsenate As(V), monomethylarsonate (MMA), and dimethylarsinate (DMA)] present in the urine samples of patients treated intravenously with arsenite, were analyzed separately by HPLC–HG-ICP–AES using a non-polar C₁₈ column. This analytical method allowed the sensitive determination of the arsenic species in the submicrogram per liter range. Urine samples collected on different days after arsenite administration were found to contain arsenite predominantly – monomethylarsonate and dimethylarsinate were also detected.     

Kinetic characterization of d-xylose isomerases by enzymatic assays using d-sorbitol dehydrogenase

The enzymatic and coupled d-xylose isomerase/d-sorbitol dehydrogenase assay is a rapid and specific method, permitting accurate quantification of d-xylose isomerization and of d-xylose. The method is based on the isomerization of d-xylose to d-xylulose, followed by reduction of the latter to xylitol by commercially available d-sorbitol dehydrogenase and NADH. The application of this one-step method cannot be extended to d-glucose isomerization since the conditions for a valid coupled assay are not fulfilled. For quantification of d-glucose isomerization, the two-step procedure with d-sorbitol dehydrogenase is recommended. Kinetic parameters for d-xylose and d-glucose using d-xylose isomerase from Streptomyces violaceoruber are reported. The results are compared with the widely used colorimetric cysteine-carbazole method.     

Adaptation of an [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay to evaluate the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish

AIMS: Adaptation of a colorimetric assay using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) to evaluate the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish. METHODS AND RESULTS: The optimal conditions for the colorimetric assay were determined and this method was compared with the trypan blue exclusion assay. The protein concentration of extracellular products causing the death of 50% of the cell population (CI50) was determined. CONCLUSIONS: This assay enables quantitative and objective comparison of the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish. It was shown to be more accurate than conventional counting with the trypan blue exclusion assay. SIGNIFICANCE AND IMPACT OF THE STUDY: This method may also be useful for characterizing the cytotoxicity of specific components of extracellular products.     

The spectrophotometric determination of indole-3-methanol in small samples with p-dimethylaminocinnamaldehyde on the basis of the formation of an azafulvenium salt

Indole-3-methanol is coupled, at acidic pH, with p-dimethylaminocinnamaldehyde to give a highly colored azafulvenium salt. IR, 1H NMR, and mass spectroscopic evidence indicates that this azafulvenium salt is the 2-[3'-(p-dimethylaminophenyl)-2'-propenyliden]-3-hydroxymethyl-2H- indolenine hydrochloride. This reaction led us to elaborate a rapid colorimetric assay for quantitative determination of indole-3-methanol formed by peroxidases as the product of oxidation of the plant growth regulator indole-3-acetic acid.     

Elemental allelopathy by an arsenic hyperaccumulating fern,Pteris vittata L.

Aims The functional advantages of arsenic (As) hyperaccumulation by plants are poorly understood. One proposed benefit, termed elemental allelopathy, occurs when hyperaccumulated As is cycled from the plant back into the top layer of soil, allowing As hyperaccumulators to gain an advantage over intolerant species by increasing soil As concentrations ([ As]) underneath their canopy. To date, there are no studies that detail the presence of increased soil [ As] associated with As hyperaccumulators. In this study, we documented variation in the soil [ As] associated with the Chinese brake fern, Pteris vittata L. and also compared the effects of environmentally relevant soil and solution [ As] on competitor plant growth.Methods Four populations of P. vittata were identified in central Florida, USA. P. vittata tissue samples and soil samples were collected at the base of and at 3 m away from ferns in each population (n = 36). Five sample locations were randomly selected from each site, and soils from the base and 3 m away from each fern were collected to examine the effects of naturally occurring soil [ As] on the germination and growth of a potential competitor plant (Oxalis stricta). Solutions with increasing [ As] were also used to examine the threshold for negative effects of [ As] on O. stricta growth. [ As] were measured using inductively coupled plasma mass spectrometry (ICP-MS).Important findings Overall, soil [ As] from the base of ferns was nearly twice that of soil 3 m away indicating that ferns hyperaccumulate As. However, ferns and their associated soil, contained different [ As] depending on their collection site, indicating that these populations accumulate and use [ As] differently. O. stricta growth decreased and germination was delayed as solution and soil [ As] increased. However, the relative distance from the fern that the soil was collected from did not affect growth, which would be expected with elemental allelopathy. Our results show that P. vittata is associated with higher soil [ As] and these concentrations are sufficient to inhibit growth of competitors. However, the absence of a strong inhibitory relationship associated with proximity to the fern across all locations suggests that the possible functional advantages of elemental allelopathy may depend on site specific characteristics.     

Colorimetric Measurement of Triglycerides Cannot Provide an Accurate Measure of Stored Fat Content in Drosophila

Drosophila melanogaster has recently emerged as a useful model system in which to study the genetic basis of regulation of fat storage. One of the most frequently used methods for evaluating the levels of stored fat (triglycerides) in flies is a coupled colorimetric assay available as a kit from several manufacturers. This is an aqueous-based enzymatic assay that is normally used for measurement of mammalian serum triglycerides, which are present in soluble lipoprotein complexes. In this short communication, we show that coupled colorimetric assay kits cannot accurately measure stored triglycerides in Drosophila. First, they fail to give accurate readings when tested on insoluble triglyceride mixtures with compositions like that of stored fat, or on fat extracted from flies with organic solvents. This is probably due to an inability of the lipase used in the kits to efficiently cleave off the glycerol head group from fat molecules in insoluble samples. Second, the measured final products of the kits are quinoneimines, which absorb visible light in the same wavelength range as Drosophila eye pigments. Thus, when extracts from crushed flies are assayed, much of the measured signal is actually due to eye pigments. Finally, the lipoprotein lipases used in colorimetric assays also cleave non-fat glycerides. The glycerol backbones liberated from all classes of glycerides are measured through the remaining reactions in the assay. As a consequence, when these assay kits are used to evaluate tissue extracts, the observed signal actually represents the amount of free glycerols together with all types of glycerides. For these reasons, findings obtained through use of coupled colorimetric assays on Drosophila samples must be interpreted with caution. We also show here that using thin-layer chromatography to measure stored triglycerides in flies eliminates all of these problems.     

Radiochemical synthesis of L-[guanidinooxy-14C]canavanine

The initial reaction in this three-step procedure for the radiochemical synthesis of L-[guanidinooxy-14C]canavanine involved the formation of barium [14C]cyanamide by reacting Ba14CO3 with ammonia at 950 degrees C. Barium [14C]cyanamide was converted to radioactive O-methylisourea, a guanidinating agent. L-[guanidinooxy-14C]Canavanine was formed by the reaction between the copper salt of L-canaline and [14C]O-methylisourea under alkaline conditions. The labeled canavanine was racemically pure as determined by enzyme-mediated hydrolysis. Reverse-phase HPLC and a novel colorimetric assay for cyanamide were used to quantify the reaction products. An overall yield for L-[guanidinooxy-14C]canavanine of approximately 25% was obtained.     

Quantitative nuclear magnetic resonance analysis and characterization of the derivatized Haemophilus influenzae type b polysaccharide intermediate for PedvaxHIB

PedvaxHIB is a pediatric vaccine that protects children from severe disease caused by the gram-negative bacterium Haemophilus influenzae type b (Hib). The vaccine is made by chemically conjugating Hib capsular polysaccharide to the outer membrane protein complex of Neisseria meningitidis. The protein-conjugated vaccine has proven to be extremely effective in preventing invasive Hib disease in infants and young children. This paper presents the nuclear magnetic resonance (NMR) methodology for the quantitative characterization of derivatized polysaccharide and its validation closely following ICH guidelines. The assay has been shown to be precise and accurate (relative standard deviation [RSD]相似文献   

One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates

In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43.