Functional assay and high throughput screening for G-protein coupled receptors / 中国药理学通报

G-protein coupled receptor (GPCR) family is the large st group of therapeutic targets. High throughput screening (HTS) plays critical ro les in early stage of drug discovery. Based on signaling pathway stimulated by l igand binding on the GPCRs, many functional assay technologies have been develop ed for HTS. These technologies include Fluorometric microvolume assay technology (FMAT), Fluorescence polarization (FP), Competitive enzyme-linked immunosorben t assay (ELISA), Scintillation proximity assay (SPA), Melanophore assay, Reporte r gene assay and Calcium assay. The principles and applic- ations of these technologies were summarized in this review. We particularly emphasize those techniques of nonradioactive, su bstrate and cofactor free assays, which will be the major approaches for HTS, su ch as reporter gene and calcium assays.

Viability evaluation of engineered tissues

Biohybrid artificial organs encompass all devices capable of substituting for an organ or tissue function and are fabricated from both synthetic materials and living cells. The viability of engineered tissue could be related to the viability of implanted cells. The system of viability assay for mammalian cell culture can be applied to the determination of cell viability for engineered tissue. This review explores various methods of cell viability assay which can be applied to the viability evaluation of engineered tissue. The major criteria employed in viability assays include survival and growth in tissue culture, functional assay, metabolite incorporation, structural altercation, and membrane integrity. Each viability assay method is based on different definitions of cell viability, and has inherent advantages and disadvantages. In order to be able to assess the viability of cells with one assay method, it is desirable to compare the viability measurements from various assays derived from different criteria.

Establishment of Multi-Drug Resistance Functional Assay and Correlation of Multi-Drug Resistance Activity and Chemotherapeutic Outcomes in Patients with Acute Leukemia / 대한혈액학회지

BACKGROUND: Multi-drug resistance (MDR) is one of the most important obstacles in the chemotherapy of acute leukemia, so the modulators of MDR have been developed and tried. METHODS: We measured MDR function and expressoin (surface and cytoplasmic p-glycoprotein and cytoplasmic multidrug-resistance associated protein (MRP)) and inhibitory effects of MDR modulators (cyclosporine and verapamil) by flow cytometry with MDR positive cell line and bone marrow aspirates of patients with acute leukemia (128 specimen). We compared these methods, and tried to clarify the effects of MDR on chemotherapy in patients with acute leukemia. RESULTS: The MDR functional assay and the detection method for inhibitory effects of MDR modulators (cyclosporine and verapamil) by flow cytometry using rhodamine 123 were established. These MDR functional assay was more sensitive, accurate, relatively simple and very economic, compared with the immunofluorescence assays of surface and cytopla-smic p-glycoprotein and cytoplasmic MRP. The positivity of MDR functional assay was observed in about 60% of patients with acute leukemia, and MDR activity (%) was inversely correlated with the complete remission rate and mean survival time. About 60% of the patients showing positive MDR activity revealed MDR inhibitory responses by cyclosporine and/or verapamil, especially all cases of acute myeloid leukemia in persistence after chemotherapy showed MDR inhibitory effect of cyclosporine. CONCLUSION: The chemotherapeutic out- comes of acute leukemia can be expected by MDR functional assay. And it is possible to overcome MDR by the administration of MDR modulator selected according to the results of the functional assay for MDR inhibitory effect in acute leukemia patients with MDR positivity.

p53 Mutation and Functional Analyses by Using Yeast Functional Assay / 대한암학회지

PURPOSE: Mutation of the p53 tumor suppressor gene is the most common genetic defect in all human tumors. Because of the widespread mutations and polymorphism in the p53 gene, the conventional screening methods cannot distinguish between polymorphisms or functionally silent mutations and inactivating mutations. It is well known that plasmids can be generated by homologous recombination in vivo in the yeast by cotransforming the PCR product with a linearized yeast expression vector encoding part of a gene and a selectable marker gene. The aim of this study is to develop more easy and reliable method for functional assay of p53 mutation. MATERIALS AND METHODS: We constructed a gap vector which can reliably and conveniently be used to screen p53 mutations in a simple yeast growth assay. The gap vector was constructed as follows: About 100 bp DNA fragments containing parts of N- and C- terminal portion of p53 were cloned into XbaI/SmaI and HindIII/XhoI sites of yeast expressing vector, respectively. The gap vector was obtained by double cutting with SmaI and HindIII followed by gel elution. Yeast was transformed with the reporter vector containing three tandem copies of the consensus p53 binding site by lithium acetate-mediated method. RT-PCR amplification of p53 transcripts from cell lines or tumor tissues was carried out. To investigate whether p53 gene is mutated or not, yeast containing reporter gene was cotransformed with PCR product and linearized gap vector, plated on SD medium minus histidine, and incubated for 3 days. The colonies on selective media were isolated and characterized. RESULTS: The tumor tissues examined were one hepatocellular carcinoma, three breast cancers, two stomach cancers and two colon cancers. One hepatocellular carcinoma tissue had mutation in both alleles of the p53 gene, and 7 cancer tissues had heterozygous mutations in the p53 gene. The result of functional assay was well correlated with mutational analysis by sequencing. CONCLUSION: p53 functional assay system might be easy and reliable method for functional screening of p53 on tumor tissues and this might be used for screening of other mutated gene. This technique, FASAY, requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.

Immunophenotypes and Proliferative Function of Lymphocytes in Umbilical Cord Blood / 대한임상병리학회지

BACKGROUND: The umbilical cord blood (UCB) is highlighted to be a alternative source of hematopoietic stem cells with a hope of resolving major problems of bone marrow transplantation, which are the lack of suitable HLA-matched donors and the occurrence of graft-versus-host disease (GVHD). In this study we have assessed the immunological potential of lymphocytes in UCB, which could mediate GVDH, by immunophenotypes and functional assays on 20 UCB and 19 adult peripheral blood as controls. METHODS: Immunophenotyping of lymphocytes were performed by one and two color flow cytometry (FACSort, Becton Dickinson, USA) using monoclonal antibodies (CD3, CD4, CD8, CD19, CD16/56, CD45RA, CD45RO, HLA-DR, HLA-DQ, HLA-DP, TCRalphabeta, TCRgammadelta). To assess proliferative responses, UCB lymphocytes were cultured in the presence of phytohemagglutinin (PHA) (5, 20, 100 ug/mL) and concanavalin A (ConA) (1, 5, 10 ug/mL). After 72 hour culture and 3H-thymidine pulsing, specific 3H-thymidine uptake was compared to control cells (without mitogen) by stimulation index (SI). RESULTS: In phenotypic analyses, the percentages of CD4, CD8, CD45RO, TCRgammadelta and HLA- DP positive cells were significantly lower in UCB compared with the adult controls, while the percentage of CD45RA positive cells was increased in UCB. In addition, the percentages of B cell (CD19), NK cell (CD16/56), HLA-DR, HLA-DQ and TCRalphabeta were comparable to those in adult controls. In mitogen stimulated culture, UCB lymphocytes showed a significant lower proliferative responses at 20, 100 ug/mL of PHA, and 5, 10 ug/mL of ConA concentrations. CONCLUSIONS: We have observed that UCB lymphocytes appeared to be phenotypically and functionally immature. Thus, UCB might be a attractive source for hematopoietic stem cells in that these UCB cells may have lower immunological potentials mediating GVHD after transplantation.

The development of a functional assay system of Wnt/?-catenin signal pathway / 重庆医科大学学报

Objective:To develop a Wnt-dependent ?-catenin-mediated functional assay system.Methods:To construct a vector,pCMV-bcGFP,which can express the ?-catenin and green fluorescent protein(GFP) fusion protein;transfect HEK293 with the vector,select the cells with G418 and LiCl and establish the stable cell line,293-bc-GFP by cloning.Results:293-bc-GFP stable cell line was developed;the background expression of ?-catenin-GFP fusion protein was detectable at a very low level in the cell line under fluorescent microscope and via Western blotting analysis;however,the fusion protein was showed to significantly increase in the presence of Wnt1 and lithium.Conclusion:Our results showed that the system is regulated by ?-catenin in a Wnt-responsive fasion.Thus,it can be used as a functional reporter to identify potential upstream factors during tumorigenesis,as well as to screen for the potential anti-cancer agents that specifically inhibit ?-catenin signaling in human tumors.