Rapid filtration assays for protein kinase C activity and phorbol ester binding using multiwell plates with fitted filtration discs.

In the conventional approach protein kinase activity and phorbol ester binding associated with protein kinase C (PKC) are measured by initially incubating samples in either test tubes or multiwell plates, followed by filtration of the terminated reaction mixture using either a manifold filtration device or a cell harvester. Here we report a method in which both the incubations and filtrations necessary for the determination of either protein kinase activity or phorbol ester binding are carried out in the same multiwell plate with fitted filtration discs made of polyvinylidene difluoride (Durapore membrane). Due to the very low binding of protein to these filters, there is no interference caused by these filters during the incubation period of the assays. The drawback with these filters compared to commonly used cellulose acetate membrane filters is that they retain less of the phosphate acceptor substrate histone H1 (only 15%) if filtered and washed with standard 5% trichloroacetic acid. However, this can be overcome by increasing the trichloroacetic acid concentration to 25% during filtration. For phorbol ester binding determinations, the samples are incubated with [3H]phorbol 12,13-dibutyrate in the microwells, the ligand bound PKC is adsorbed onto DEAE-Sephadex beads, and the beads then are filtered and washed in the same microwells. Furthermore, this multiwell filtration approach can also be adopted to previously described cytosolic phorbol ester receptor assays, which have the broader conditions for optimal binding to receptors. Durapore membrane filters are found to work well for punching into scintillation vials and there is complete recovery of the radioactivity retained with the filters. In the protein kinase assay the background radioactivity is very low (< 200 cpm) and in the phorbol ester binding assay the nonspecific binding is less than 1%. Thus, these low background values result in at least a fourfold increase in sensitivity for these assays. Since the incubations and filtrations are carried out in the same well without any transfer of the sample, the coefficient of variation in multiple determinations is found to be low. Furthermore, this method is rapid and more convenient for analyzing a larger number of samples than conventional methods which use test tubes, and it is less expensive to set up compared to the automated methods that use a cell harvester.     

Automation of a chloramphenicol acetyltransferase assay

Accurate quantification of chloramphenicol acetyltransferase (CAT) enzyme activity in a large number of samples has been achieved through robotization of a CAT assay on a laboratory workstation (Biomek 1000). The basic principle of this CAT assay relies on the selective diffusion of [3H]acetylchloramphenicol into a water-immiscible liquid scintillation cocktail. This methodology gives unique characteristics to this robotized protocol by allowing complete control over the kinetics of the CAT enzymatic reaction which is a critical parameter in the CAT assay. Thus it has been possible to optimize the CAT assay for every processed sample, through real time monitoring of the enzymatic reaction, and to achieve maximum accuracy in CAT quantification. Moreover the sensitivity of this automated assay is high (detection threshold; 10(-4) CAT unit), and the sample processing is fast (approximately 125 samples per hour). Compared to other CAT assay protocols currently used, our robotized technique offers major advantages in terms of CAT quantification, and sets new standards for CAT assay productivity.     

A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail.

A simple, rapid, sensitive, quantitative, and inexpensive assay for chloramphenicol acetyltransferase (CAT) is described. The assay is based on the direct extraction of the products of the reaction into toluene-based liquid scintillation cocktail. The assay is carried out in 7-ml scintillation vials using 1 mM chloramphenicol and either 100 microM acetyl-CoA and 0.1 microCi of [3H]acetyl-CoA or 1 mM acetyl-CoA and 0.5 microCi of [3H]acetyl-CoA. After incubation, the reaction is terminated with 0.5 ml of 0.1 M sodium borate-5 M NaC, pH 9. The acetylchloramphenicols are extracted with 5 ml of 0.4% 2,5-diphenyloxazole-0.005% 1,4-bis(5-phenyloxazol-2-yl)benzene in toluene by a 30-s shaking. After a short centrifugation to clarify the layers, the vials are counted in a liquid scintillation counter. Extracted products are stable in the organic layer. Under these conditions, nearly 100% extraction of acetylchloramphenicols is shown using nonlabeled compounds and spectrophotometric methods. Using pure enzyme in the assay, linearity of activity with enzyme concentration, time, and temperature of incubation is demonstrated. Assays may even be carried out at 60 degrees C, where the enzyme activity is 3.4-fold higher than that at 23 degrees C. The increase in enzyme activity with increasing temperature is due to the increased formation of predominantly 3-acetyl and 1-acetylchloramphenicols and not to 1,3-diacetylchloramphenicol. The present assay compared very well with the standard assay using [14C]chloramphenicol and TLC. Using this assay, we measured quantitatively the CAT activity in extracts of pSV2-CAT-transfected CV-1 cells in 10 min and NIH 3T3 cell extracts in 60 min at 60 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Herpes simplex virus thymidine kinase enzymatic assay in transient transfection experiments using thymidine kinase-deficient cells.

An enzymatic assay for herpes virus simplex type 1 thymidine kinase (HSV-TK) that was sensitive enough to quantitate intracellular levels of enzyme transiently expressed after transfection of HSV-TK vectors into TK-deficient cells using the DNA-calcium phosphate coprecipitation technique is described. TK activity in extracts of transfected cells was determined by binding of [methyl-3H]thymidylate product to thin layers of polyethyleneimine (PEI)-impregnated cellulose. The assay used high-specific-activity [methyl-3H]thymidine as substrate, which required removal of anionic material on a column of PEI-cellulose to enhance the signal-to-noise ratio. The assay was linear over a wide range with respect to the amount of HSV-TK plasmid transfected or content of HSV-TK enzyme in cell extracts. To validate the assay in transient expression experiments, HSV-TK and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected into NIH/3T3 tk- fibroblasts. Transient TK and CAT levels were concordant in cell extracts prepared from replicate plates of transfected cells. Normalizing the transient TK activity for CAT activity from the cotransfected "internal standard" CAT plasmid improved precision significantly, reducing the sample-to-sample coefficient of variation from 41 to 19%. CAT normalization reduced experimental variability mostly by correcting outlying results in transfection efficiency. The HSV-TK reporter gene system based on TK enzymatic assay was thus subject to experimental variation similar to that of the well-established CAT reporter function, demonstrating its utility in transient gene expression analysis.     

A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferase activity in transfected cells

We describe a simple, rapid yet extremely sensitive assay for chloramphenicol acetyltransferase (CAT) activity in extracts from transfected eukaryotic cells. Using our modified reaction conditions and the mixed-phase assay, less than 0.000010 unit of CAT activity in transfected cells can be reliably detected. The mixed-phase assay is based on the inability of the polar [3H]-acetyl-Coenzyme A (CoA) substrate to partition out of a urea containing aqueous phase into the nonpolar scintillation fluor, while the [3H]chloramphenicol reaction products partition into the toluene scintillation fluor and are quantitated by scintillation counting. The increased sensitivity of this assay is due to the optimization of the acetyl-CoA concentration, to a urea-containing aqueous phase which lowers the assay background, and to the use of extract blanks. The mixed-phase assay is simpler, is quantitative, uses less costly substrates, and is far more sensitive than the most widely used CAT assays, which require solvent extraction followed by thin-layer chromatography to separate the unreacted substrate from product.     

RARβ在胃癌细胞生长调节中的作用

为探讨 RARβ受体介导全反式视黄酸 ( ATRA)抑制胃癌细胞生长的作用机理 ,用 Northern印迹测定 RARβ m RNA表达水平 ,脂质体介导的转染方法将含有 RARβ基因的表达载体转染MKN- 45细胞并稳定表达 ,MTT和软琼脂集落形成等实验测定细胞生长速率和生长状态 ,氯霉素乙酰转移酶活性 ( CAT)测定视黄酸应答元件βRARE的转录活性以及 AP- 1 ( activator protein- 1 )活性 .RARβ在 ATRA敏感细胞株 MGC80 - 3、BGC- 82 3和 SGC- 790 1中表达 ,而在 ATRA抗性细胞株 MKN- 45中不表达 .当 RARβ基因转染 MKN- 45细胞时 ,细胞变为 ATRA敏感 ,由此导致ATRA抑制 MKN- 45细胞生长和软琼脂集落形成 .ATRA可以加强诱导 MGC80 - 3、BGC- 82 3和SGC- 790 1细胞βRARE的转录活性 ,但对 MKN- 45细胞影响不大 ,不能抑制细胞 AP- 1活性 .当RARβ基因转染 MKN- 45细胞后 ,ATRA则能够诱导细胞 βRARE的转录活性 ,并抑制细胞的 AP-1活性 .RARβ表达与 ATRA抑制胃癌细胞生长密切相关 .ATRA诱导 βRARE转录活性和抑制AP- 1活性可能是其调控胃癌细胞生长的机制之一 .     

A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases

A quantitative assay for the analysis of sesquiterpene synthases, wherein each reaction mixture is formulated in glass gas chromatography vials, overlaid with organic solvent such as ethyl acetate, and subsequently vortexed to extract hydrocarbon reaction products into the organic phase after a suitable incubation period, was developed. The product-enriched organic phase is then sampled in an automated fashion and injected directly into a gas chromatograph-mass spectrometer without further workup for analysis and quantification of hydrocarbon products. Application of the vial assay to the analysis of amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, demonstrated the sensitivity of the assay for detection of major and minor reaction products and most notably for the identification of several sesquiterpene products that had escaped previous detection. A steady-state kinetic analysis of tobacco 5-epi-aristolochene synthase (TEAS), another sesquiterpene synthase, validated the quantitative nature of the assay, providing an alternative means to the established method of using radiolabeled substrate, extraction, and scintillation counting. This simplified assay provides a standardized method to facilitate analysis of terpene synthases and diverse mutant enzyme libraries by supplanting the common practice of using larger scale reactions, multiple extractions, and evaporative concentration of the organic phase prior to gas chromatography-mass spectrometry (GC-MS) analysis.     

孤生受体的相互作用对视黄酸应答元件的影响

孤生受体ＣＯＵＰ－ＴＦ和ｎｕｒ７７的功能及其作用机理仍未阐明．以ＤＮＡ瞬时转染和测定氯霉素乙酰转移酶（ＣＡＴ）活性,以及凝胶阻抑测定,分析ＣＯＵＰ－ＴＦ和ｎｕｒ７７的相互作用对视黄酸应答元件（ＲＡＲＥｓ）的影响．实验表明,ＣＯＵＰ－ＴＦ通过降低ＲＡＲＥｓ的基础活性,来增强ＲＡＲＥ对视黄酸（ＲＡ）的敏感性,而ｎｕｒ７７则拮抗ＣＯＵＰ－ＴＦ的作用．ｎｕｒ７７能够加强不同ＲＡＲＥｓ的转录活性,并且与ＲＡ的诱导无关．结果证实,ｎｕｒ７７通过与ＣＯＵＰ－ＴＦ的直接作用而对ＲＡＲＥｓ产生影响,从而抑制ＣＯＵＰ－ＴＦ与ＲＡＲＥｓ结合和ＣＯＵＰ－ＴＦ的转录活性     

Determination of the number of endothelial cells in culture using an acid phosphatase assay

A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After 2 h at 37 degrees C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated.