sepharose and thiazolyl-blue

Human interleukin-7 (IL-7) is a member of the interleukin family. Numerous studies have demonstrated IL-7's effect on B- and T-cell development as well as its potential in various clinical applications. Previously, a study reported that IL-7 could be purified from inclusion bodies using a prokaryotic system, however, the required refolding step limits the recovery rate. This study was designed to produce a bioactive recombinant human IL-7 (rhIL-7) in a eukaryotic expression system in order to obtain higher yields of the protein with simpler purification steps. We cloned human IL-7 cDNA and successfully expressed active recombinant protein in yeast using the Pichia pastoris expression system. A simple purification strategy was established to purify the rhIL-7 from the fermentation supernatant, yielding 35 mg/L at 95% purity by the use of a common SP Sepharose FF cation-exchange chromatography. Functional analysis of the purified rhIL-7 by the pre-B cell MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) proliferation assay demonstrated a specific activity comparable to commercial sources. These results suggest that purification of rhIL-7 from yeast provides a sound strategy for large-scale production of the rhIL-7 for clinical applications as well as basic researches.

Topics: Bioreactors; Blotting, Western; Chromatography, Ion Exchange; Cloning, Molecular; Fermentation; Humans; Interleukin-7; Pichia; Protein Renaturation; Recombinant Proteins; Sepharose; Tetrazolium Salts; Thiazoles; Transformation, Genetic

Histone deacetylase inhibitors induce hyperacetylation of the amino-terminal lysine residues of the core nucleosomal histones, which results in chromatin remodeling and altered gene expression. Present studies demonstrate that exposure to a novel hydroxamic acid analogue histone deacetylase inhibitor, LAQ824, induced p21WAF1 and p27KIP1 and caused growth arrest and apoptosis of human breast cancer SKBR-3 and BT-474 cells that possess amplification and overexpression of Her-2/neu. Treatment with LAQ824 depleted the mRNA and protein levels of Her-2/neu-encoded Her-2, which was associated with attenuation of pAKT, c-Raf-1, and phosphorylated mitogen-activated protein kinase levels. LAQ824 also induced the acetylation of heat shock protein (hsp) 90, resulting in inhibition of its binding to ATP, which has been shown to impair the chaperone association of hsp 90 with its client proteins, Her-2, AKT, and c-Raf-1. Consistent with this, treatment with LAQ824 shifted the binding of Her-2 from hsp 90 to hsp 70, promoting proteasomal degradation of Her-2. Thus, LAQ824 depletes Her-2 through two mechanisms: attenuation of its mRNA levels and promotion of its degradation by the proteasome. Following LAQ824 treatment, the cell membrane association, autotyrosine phosphorylation, and colocalization of Her-2 with HER-3 also declined. Cotreatment with LAQ824 significantly increased trastuzumab-induced apoptosis of BT-474 and SKBR-3 cells. This was associated with greater attenuation of Her-2, c-Raf-1, and pAKT levels. LAQ824 also enhanced taxotere-induced, epothilone B-induced, and gemcitabine-induced apoptosis of BT-474 and SKBR-3 cells. These findings suggest that LAQ824 is active against human breast cancer cells and has the potential to improve the efficacy of trastuzumab, taxotere, gemcitabine, and epothilone B against breast cancer with Her-2/neuamplification.

Topics: Annexin A5; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Northern; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Chromatin; Coloring Agents; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Deoxycytidine; Detergents; Docetaxel; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Epothilones; Flow Cytometry; Gemcitabine; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Microscopy, Fluorescence; Multienzyme Complexes; Phosphorylation; Precipitin Tests; Proteasome Endopeptidase Complex; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; Sepharose; Taxoids; Tetrazolium Salts; Thiazoles; Time Factors; Trastuzumab

Topics: Animals; Baculoviridae; Coloring Agents; Moths; Sepharose; Tetrazolium Salts; Thiazoles; Viral Plaque Assay

Rapid and reliable methods for the determination of survival, proliferation, and metabolic activity of immobilized cells in gels are described. The first method is based on an MTT assay that measures qualitatively and quantitatively the metabolic activity of the cells. The second method determines cell number by measuring the amount of DNA available for Feulgen staining. In the third method, two fluorescent dyes are used to differentially stain viable and dead cells. The fourth method involves the use of glutaraldehyde to protect the cells when melting the gel to facilitate hemocytometric count. The presented techniques should help to test the efficiency of the immobilization procedures and to monitor the growth and survival of immobilized cells.

Topics: Animals; Biotechnology; Cell Count; Cell Division; Cell Survival; Coloring Agents; DNA; Fixatives; Fluorescent Dyes; Gels; Glutaral; Hybridomas; Mice; Rosaniline Dyes; Sepharose; Staining and Labeling; Tetrazolium Salts; Thiazoles; Trypan Blue

A rapid and simple colorimetric microassay method for the determination of hematopoietic growth factor activities was established. The assay was used to detect CSF-1, GM-CSF, and IL-3 activities. The assay was based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by metabolically active cells. Results obtained with the colorimetric microassay are comparable with those obtained with the soft agarose assay. Advantages of the colorimetric microassay include the conservation of reagents, the shorter incubation time for the experiment, the shorter assay time, and the ability to evaluate large numbers of samples.

Topics: Animals; Bone Marrow; Cells, Cultured; Colony-Stimulating Factors; Colorimetry; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Hematopoietic Cell Growth Factors; Interleukin-3; Mice; Mice, Inbred C57BL; Recombinant Proteins; Reproducibility of Results; Sepharose; Tetrazolium Salts; Thiazoles