ABSTRACT
Radix Ranuncoli Ternati is clinically effective traditional Chinese medicine for multidrug resistant tuberculosis. Its active components and mechanism of action remain unsolved. Two dimensional gel electrophoresis (2-DE) was employed to address this problem. Globlal proteome of Mycobacterium tuberculosis untreated and treated with Radix Ranuncoli Ternati were compared, and 22 protein spots were found to be expressed differentially. 3 protein spots which remarkably decreased in Mycobacterium tuberculosis treated with Radix Ranuncoli Ternati were subjected to matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. The data obtained from peptide mass finger printing were used for database search. The 3 protein spots in gel were identified as cysA2 (thiosulfate sulfurtransferase), tsf (elongation factor EF-Ts) and hspX (heat shock protein X). These data provide insights into the changed global protein patterns of Mycobacterium tuberculosis treated with Radix Ranuncoli Ternati and may prove useful for further study in the mechanism in how Radix Ranuncoli Ternati influence the life of Mycobacterium tuberculosis. The differentially expressed proteins may be potential novel antituberculosis drug targets.
Subject(s)
Bacterial Proteins/analysis , Medicine, Chinese Traditional , Mycobacterium tuberculosis/drug effects , Plant Extracts/pharmacology , Proteome , Antigens, Bacterial/analysis , Electrophoresis, Gel, Two-Dimensional , Mycobacterium tuberculosis/chemistry , Peptide Elongation Factor Tu/analysis , Peptide Elongation Factors/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The t(11;19)(q23;p13.1) translocation is exclusively associated with myeloid leukemias. Previously, we cloned several species of MLL/MEN chimeric cDNAs in a patient with myeloid leukemia carrying the t(11;19)(q23;p13.1) translocation. The MEN sequence directly followed the 5' region of MLL cDNA in some species and otherwise there presented an inserted sequence of 120 bp between the MLL and MEN sequences in others. Because the insertion sequence contained an in-frame termination codon, they coded only for the NH2-terminal part of MLL (truncated MLL). We also cloned the normal MEN cDNA in full-length with a cDNA library derived from K562 cells. We expressed the normal MEN, MLL/MEN chimeric and truncated MLL proteins in COS7 cells with the corresponding cDNAs and detected them with antibodies raised against the MEN and MLL peptides. Immunostaining and subcellular fractionation showed nuclear localization of all these proteins. These findings suggested that MLL/MEN chimeric cDNAs were actually translated into both MLL/MEN fusion and truncated MLL proteins and that they were localized in the nucleus of leukemic cells. Recently, Conaway et al. reported that MEN is an RNA polymerase II elongation factor. The leukemogenesis by the t(11;19)(q23;p13.1) translocation may have resulted from the alteration of transcription regulation induced by the MLL/MEN fusion protein and/or the truncated MLL protein.
Subject(s)
Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 19/genetics , DNA-Binding Proteins/genetics , Leukemia, Myeloid/genetics , Peptide Elongation Factors/genetics , Proto-Oncogenes , Recombinant Fusion Proteins/genetics , Subcellular Fractions/chemistry , Transcription Factors , Translocation, Genetic , Animals , COS Cells , Cell Transformation, Neoplastic , Chromosomes, Human, Pair 11/ultrastructure , Chromosomes, Human, Pair 19/ultrastructure , Cloning, Molecular , DNA, Complementary/genetics , DNA, Neoplasm/genetics , DNA-Binding Proteins/analysis , DNA-Binding Proteins/biosynthesis , Histone-Lysine N-Methyltransferase , Humans , Leukemia, Myeloid/pathology , Myeloid-Lymphoid Leukemia Protein , Peptide Elongation Factors/analysis , Peptide Elongation Factors/biosynthesis , Recombinant Fusion Proteins/analysis , Zinc Fingers/geneticsABSTRACT
A method for detection of enzymatic activities which use aminoacyl-tRNA is described. It is based on the synthesis of aminoacyl-tRNA in the reaction mixture (in situ) without additional purification. The results are the same as when using purified AA-tRNA.