ABSTRACT
The human basic Krüppel-like factor (hBKLF) is a newly cloned human transcription factor from the cDNA library of fetal liver. It belongs to the Krüppel-like transcription factor family. Previous expression study showed that it is a hematopoietic related factor. This study was aimed to investigate the effect of hBKLF on cell proliferation, differentiation and hemoglobin synthesis by using K562 cell line as model. The sense and antisense expression plasmids of hBKLF were constructed, and transfected into K562 cells by lipofectamine. After G418 selection for 4 weeks, the cell line with stable expression of the gene was obtained. Then the hBKLF expression level, proliferation ability, colony formation and hemoglobin production were detected by RT-PCR and Western blot, MTT method, methyl cellulose semisolid culture method and benzidine test respectively. The morphologic change of cell was observed with inverted microscope. The results showed that the sense plasmid could increase hBKLF level and antisense plasmid could decrease hBKLF expression. When hBKLF level was down-regulated, K562 cells could proliferate more quickly and synthesize more hemoglobin. But there were no differences in colony formation ability and no apparent morphologic change. It is concluded that hBKLF can inhibit hematopoietic cell proliferation and hemoglobin synthesis. It is suggested that hBKLF plays an important role in the proliferation and differentiation of hematopoietic cells.
Subject(s)
Animals , Humans , COS Cells , Cell Differentiation , Physiology , Cell Proliferation , Cell Transformation, Neoplastic , Chlorocebus aethiops , Hemoglobins , K562 Cells , Kruppel-Like Transcription Factors , Genetics , Pharmacology , Transcription Factors , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To identify the single nucleotide polymorphisms(SNPs) in the regulatory and coding regions of human Toll-like receptor 4(TLR4) gene and to search for its new genetic makers.</p><p><b>METHODS</b>The 5' flank region, exons, parts of the introns, as well as 3' flank region of TLR4 gene were sequenced to identify and characterize the SNPs in Chinese population. SNP genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism for 2 highly distributed SNPs.</p><p><b>RESULTS</b>Five novel SNPs were identified through a 4.98 kb sequencing of TLR4 gene. Among them, three were in 5'flank region, two in 3'UTR. In the sample of Han population from Chongqing, the minor allele frequencies of two highly distributed SNPs were 0.266 and 0.404 respectively.</p><p><b>CONCLUSION</b>Sampling analysis in Han population of Chongqing showed that the two highly distributed SNPs of TLR4 were common in Chinese population and could be used for genetic marker of TLR4 gene.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Asian People , Genetics , Base Sequence , China , Gene Frequency , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Polymorphism, Single Nucleotide , Sequence Analysis, DNA , Toll-Like Receptor 4 , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To identify the single nucleotide polymorphisms (SNPs) in the regulatory and coding regions of human interleukin-1 receptor type I (IL-1R1) gene and to assess their potential effect on the function of IL-1R1.</p><p><b>METHODS</b>The 5' flank region, exons, parts of the introns, as well as 3' flank region of IL-1R1 gene were sequenced to identify and characterize the SNPs in Chinese population. Effects of the SNP on the structure and function of IL-1R1 were analyzed by computational methods.</p><p><b>RESULTS</b>Sixteen SNPs were identified through a 9643 bp sequencing of IL-1R1 gene. Among them, four were in 5' flank region, four in intron region, one in coding region, and seven in 3' untranslated region. A novel SNP in Chinese population was involved in a structural change in IL-1R1, which may influence the signal transduction of IL-1R1.</p><p><b>CONCLUSION</b>The SNP in the IL-1R1 gene might influence its function as an important receptor of IL-1 family.</p>
Subject(s)
Humans , Amino Acid Sequence , Asian People , Cell Membrane , Metabolism , Computational Biology , Exons , Genetics , Hydrophobic and Hydrophilic Interactions , Introns , Genetics , Molecular Sequence Data , Polymorphism, Single Nucleotide , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Interleukin-1 Type I , Chemistry , Genetics , Metabolism , Sequence Homology, Amino AcidABSTRACT
Scansite is a short linear motif-based scanning approach established in the latest two years. It's accessible over the World Wide Web and can be used to identify sequence motifs likely to be phosphorylated by specific protein kinases or likely to bind to specific protein domains such as 14-3-3, SH2 and SH3 domains. The usage and function of the potent approach were reviewed and compared with previously established tools for phosphorylation prediction. The facing problems and application outlook of Scansite in prediction of cell signaling networks within proteomes were also presented.
Subject(s)
Amino Acid Motifs , Amino Acid Sequence , Databases, Factual , Internet , Molecular Sequence Data , Phosphorylation , Protein Structure, Tertiary , Proteins , Chemistry , Metabolism , Signal TransductionABSTRACT
<p><b>OBJECTIVE</b>To study the function of IL-18 in promoting metastasis of lung cancer.</p><p><b>METHODS</b>The differential expression of IL-18 protein or mRNA level between highly and poorly metastatic sublines of human lung giant cell carcinoma metastatic model was detected by Western blot, semi-quantitative RT-PCR and northern blot analysis. The poorly metastatic PLA801C subline or highly metastatic PLA801D subline was transfected with constructed IL-18 sense or IL-18 antisense expressed plasmid by lipofectamine stable transfection technique. The metastasis-related effect mediated by IL-18, the metastatic phenotype differences, cell motility and cell invasion potential in vitro determined by MICS system and the expression level of metastasis-associated biomarkers detected by Western blot analysis, were compared between IL-18 stably transfectants and mock control, i.e. between PLA801C/IL-18(S) and PLA801C/pcDNA3.1, or between PLA801D/IL-18(As) and PLA801D/pcDNA3.</p><p><b>RESULTS</b>IL-18 was only present in highly metastatic PLA801D subline at either protein or mRNA level, which implied that IL-18 might play a role in promoting metastasis of lung cancer. After IL-18 sense expressed plasmid was transfected into poorly metastatic PLA801C subline, IL-18 fused protein with myc tag detected by Western blot analysis using either IL-18 or myc tag monoclonal antibody. In addition, cell motility ability in vitro was significantly increased about 3 times and E-cadherin protein was significantly down-regulated at about 50% in PLA801C/IL-18(S) transfectants compared with mock control. While IL-18 expressed plasmid was transfected into highly metastatic PLA801D subline, IL-18 protein and mRNA were simultaneously decreased by 30%. In addition, cell invasion ability in vitro was significantly decreased at about 75% and E-cadherin protein was significantly up-regulated in PLA801D/IL-18(As) transfectants compared with mock control.</p><p><b>CONCLUSION</b>IL-18 might play a role in enhancing tumor metastasis of lung cancer by down-regulating E-cadherin protein expression.</p>
Subject(s)
Humans , Cadherins , Metabolism , Carcinoma, Giant Cell , Metabolism , Cell Line, Tumor , Cell Movement , DNA, Antisense , Genetics , Gene Expression Regulation, Neoplastic , Interleukin-18 , Genetics , Lung Neoplasms , Metabolism , Pathology , Neoplasm Invasiveness , Neoplasm Metastasis , Genetics , Plasmids , RNA, Messenger , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To study the metastasis-associated molecules differentially expressed in highly and poorly metastatic sublines and the mechanism of metastasis in lung giant cell carcinoma.</p><p><b>METHODS</b>Highly and poorly metastatic sublines (PLA801D and PLA801C)were used as metastasis model. Cell motility and invasion assay in vitro were first compared between the two sublines. Then, gelatin zymography analysis was used to determine the MMP-2 and MMP-9 activity. The protein expression level of secreted MMP-2, MMP-9, TIMP-1, TIMP-2 and intracellular expression level of p53, p16, PCNA, CD44(V6) isomeride, E-cadherin, CK18, nm23-H1 as well as the mRNA expression level of MMP-2, MMP-9, TIMP-1, TIMP-2, VEGF were compared through Western blot. Semi-quantitative RT-PCR analysis was used to determine the intracellular mRNA expression of MMP-2, MMP-9, TIMP-1, TIMP-2 and VEGF.</p><p><b>RESULTS</b>The in vitro cell invasion potential of highly metastatic subline PLA801D was significantly higher than that of poorly metastatic subline PLA801C by about 4 folds, while the cell motility potential was similar. The secreted MMP-2 activity was notably higher in PLA801D, which was initiated by the higher expression of MMP-2 at protein and mRNA level. In addition, the expression level of p53, PCNA, CK18 protein and VEGF mRNA were significantly higher, while the expression level of p16, E-cadherin and nm23-H1 protein were significantly lower in PLA801D. Some molecules such as MMP-9, TIMP-1, TIMP-2, CD44(V6) isomeride, which had been reported to be associated with tumor metastasis, were not observed to change significantly between the two sublines.</p><p><b>CONCLUSION</b>There are significant differences in metastatic potential and phenotypes between highly and poorly metastatic sublines of lung giant cell carcinoma. Some differentially expressed molecules might be playing roles in promoting or inhibiting metastasis of lung giant cell carcinoma, which may be useful to elucidate the mechanism of metastasis.</p>
Subject(s)
Humans , Carcinoma, Giant Cell , Metabolism , Pathology , Cell Line, Tumor , Interleukin-8 , Genetics , Lung Neoplasms , Metabolism , Pathology , Matrix Metalloproteinase 2 , Genetics , Metabolism , Matrix Metalloproteinase 9 , Genetics , Metabolism , Neoplasm Invasiveness , Neoplasm Metastasis , RNA, Messenger , Tissue Inhibitor of Metalloproteinase-1 , Vascular Endothelial Growth Factor A , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of recombinant human hepatopoietin (rhHPO) and partial hepatectomy on rapidly induced expression of immediate early gene.</p><p><b>METHODS</b>We investigated the different gene expression within 1 hour after 2/3 partial hepatectomy by representational difference analysis and in primary cultured hepatocytes system.</p><p><b>RESULTS</b>In the expressed sequence tag (EST) library, we identified that most of these genes were immediate early gene, and found one new gene PC3 that might be associated to liver regeneration in the EST library. Moreover, PC3 gene was rapidly induced after 2/3 partial hepatectomy and the expressing peak was within 1~2 hours after operation. HPO can rapidly induce the expression of these genes (c-fos, LRF-1, and PC3, etc.) in primarily cultured rat hepatocyte, which might be one of HPO molecular mechanism on stimulating hepatocyte proliferation.</p><p><b>CONCLUSIONS</b>rhHPO and partial hepatectomy can rapidly induce the expression of immediate early gene. PC3 gene is immediate early gene related to liver regeneration.</p>
Subject(s)
Animals , Rats , Aspartic Acid Endopeptidases , Genetics , Blotting, Northern , Gene Expression Regulation , Genes, Immediate-Early , Hepatectomy , Hepatocyte Growth Factor , Pharmacology , Liver Regeneration , Genetics , Proprotein Convertases , RNA, Messenger , Rats, Wistar , Recombinant Proteins , PharmacologyABSTRACT
In the post-genomic era, with the accomplishment of the sequence mapping of human genome, one of the most important tasks for life science is the explanation and identification of human genome, that is, about 1/3 genes of human genome and their functions need further revealment and verification on the level of protein. In the field of functional proteomics, the human disease proteomics shows great potential in the discovery of new molecular targets and biomarkers for medicine and biopharmacy. In this article, we have made a concise discussion on the current status, existing problems and future development in the research of human disease proteomics both in and out of China.