A growing body of evidence, including studies using genetically engineered mouse models, has shown that Ca2+ cycling and Ca2+ -dependent signaling pathways play a pivotal role in cardiac hypertrophy and heart failure. In addition, recent studies identified that mutations of the genes encoding sarcoplasmic reticulum (SR) proteins cause human cardiomyopathies and lethal ventricular arrhythmias. The regulation of Ca2+ homeostasis via the SR proteins may have potential therapeutic value for heart diseases such as cardiomyopathy, heart failure and arrhythmias.
Animals ; Animals, Genetically Modified ; Arrhythmia/genetics ; Calcium/*metabolism ; Calcium Channels/genetics/*physiology ; Calcium-Binding Proteins/genetics/*physiology ; Cardiac Output, Low/genetics ; Cardiomyopathies/genetics ; Heart Diseases/*etiology/genetics/metabolism ; Humans ; Mutation/genetics ; Research Support, Non-U.S. Gov't ; Sarcoplasmic Reticulum/metabolism

OBJECTIVETo explore the mechanism of Notch signaling transduction system and its effects on hematopoietic system.

METHODSNotch ligands transfected CHO cells were added into Notch1 and Notch2 transfected CHO cells, which were transiently transfected with reporter gene TP1. PGL-100 was used as substrate to test the interaction between Notch and Notch ligands. CHO, Jagged2-CHO and Delta 4-CHO cells were seeded in the petri dish containing G-CSF, and then Notch 1-32D cells were added in it to observe the differentiation of Notch1-32D cell after incubation and staining.

RESULTSAll of the five Notch ligands binding to Notch1 could induce TP1 activity, it increased significantly the Jagged2-CHO, Delta 4-CHO1-4 and Delta 4-CHO1-5 cells. For Notch2, the TP1 activity induced by the five ligands in these cells was much higher than that of CHO. At the presence of G-CSF, Notch1-32D could differentiate to mature granulocyte. Jagged2 could inhibit G-CSF induced Notch1-32D cell differentiation, but Delta 4 could not.

CONCLUSIONJagged2 and Delta 4 are the ligands of Notch1. Jagged2 can inhibit G-CSF induced Notch1-32D cell differentiation, but Delta 4 can not.

Animals ; CHO Cells ; Calcium-Binding Proteins ; genetics ; metabolism ; physiology ; Cell Differentiation ; physiology ; Cricetinae ; Cricetulus ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; physiology ; Membrane Proteins ; genetics ; metabolism ; physiology ; Receptor, Notch1 ; genetics ; metabolism ; physiology ; Receptor, Notch2 ; genetics ; metabolism ; physiology ; Receptors, Notch ; genetics ; metabolism ; physiology ; Serrate-Jagged Proteins ; Signal Transduction ; physiology ; Transfection

Human K(v) channel interacting protein 1 (KCHIP1) is a new member of the neural calcium binding protein superfamily. Theoretically KCHIP1 has several calcium binding domains and two myristoylation sites. In this study, we demonstrated that the calcium binding domains and myristoylation sites were functional. The first, through running SDS-PAGE gel, we testified its ability of binding Ca(2+) with obvious discrepancy of the electrophoresis migrating rate after binding Ca(2+). Then, through the techniques of fused green fluorescence protein and site-directed mutagenesis, we demonstrated that wild type KCHIP1 protein accumulated in the secretory vesicles of Golgi body. In contrast, its two mutated forms without myristoylation sites accumulated throughout the whole cytoplasm. These observations indicate that KCHIP1 protein has a myristoylation motif mediating the interaction between KCHIP1 protein and membrane.
Animals ; COS Cells ; Calcium ; metabolism ; Calcium-Binding Proteins ; genetics ; metabolism ; Cercopithecus aethiops ; Humans ; Kv Channel-Interacting Proteins ; chemistry ; physiology ; Potassium Channels ; metabolism ; Potassium Channels, Voltage-Gated ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; metabolism ; Transfection

The BCCIP (BRCA2- and CDKN1A-interacting protein) is an important cofactor for BRCA2 in tumor suppression. Although the low expression of BCCIP is observed in multiple clinically diagnosed primary tumor tissues such as ovarian cancer, renal cell carcinoma and colorectal carcinoma, the mechanism of how BCCIP is regulated in cells is still unclear. The human INO80/YY1 chromatin remodeling complex composed of 15 subunits catalyzes ATP-dependent sliding of nucleosomes along DNA. Here, we first report that BCCIP is a novel target gene of the INO80/YY1 complex by presenting a series of experimental evidence. Gene expression studies combined with siRNA knockdown data locked candidate genes including BCCIP of the INO80/YY1 complex. Silencing or over-expressing the subunits of the INO80/YY1 complex regulates the expression level of BCCIP both in mRNA and proteins in cells. Also, the functions of INO80/YY1 complex in regulating the transactivation of BCCIP were confirmed by luciferase reporter assays. Chromatin immunoprecipitation (ChIP) experiments clarify the enrichment of INO80 and YY1 at +0.17 kb downstream of the BCCIP transcriptional start site. However, this enrichment is significantly inhibited by either knocking down INO80 or YY1, suggesting the existence of both INO80 and YY1 is required for recruiting the INO80/YY1 complex to BCCIP promoter region. Our findings strongly indicate that BCCIP is a potential target gene of the INO80/YY1 complex.
Calcium-Binding Proteins ; genetics ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Chromatin Assembly and Disassembly ; physiology ; DNA Helicases ; genetics ; metabolism ; HeLa Cells ; Humans ; Multiprotein Complexes ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Promoter Regions, Genetic ; physiology ; Transcription, Genetic ; physiology ; YY1 Transcription Factor ; genetics ; metabolism

OBJECTIVETo study the effect of human calcyclin binding protein (CacyBP) encoding gene on the development of multiple drug resistance in gastric cancer.

METHODShCacyBP sense nucleic acid eukaryotic expression vector (pcDNA3.1/hCacyBP +) was constructed and then transfected steadily into the gastric cancer drug sensitive cell (SGC7901) mediated by lipofectamine ( trade mark ) 2000. RT-PCR was used to measure the CacyBP mRNA expression level. MTT was used to measure the adriamycin (ADR) drug sensitivity of SGC7901 and SGC7901 after transfection. FCM was used to measure the average ADR accumulation concentration and cell cycle of SGC7901 and SGC7901 after transfection.

RESULTSThe hCacyBP mRNA expression level of SGC7901 transfected with pcDNA3.1/hCacyBP + was higher than SGC7901 transfected with pcDNA3.1 or SGC7901, with the higher survival rate in the former. The average ADR accumulation concentration in SGC7901 and SGC7901 transfected with pcDNA3.1 or pcDNA3.1/hCacyBP + was 5.64, 5.49 and 5.17, respectively. The G(1) phase cell proportion of SGC7901 transfected with pcDNA3.1/hCacyBP + or pcDNA3.1 was reduced slightly but G(2) and S phases increased slightly as compared with SGC7901.

CONCLUSIONCalcyclin binding protein may play a certain role in gastric cancer drug resistance.

Base Sequence ; Calcium-Binding Proteins ; genetics ; physiology ; DNA, Complementary ; analysis ; Drug Resistance, Multiple ; physiology ; Drug Resistance, Neoplasm ; physiology ; Drug Screening Assays, Antitumor ; Gene Transfer Techniques ; Humans ; Molecular Sequence Data ; Plant Lectins ; Stomach Neoplasms ; pathology ; Tumor Cells, Cultured

To investigate the effects of HIV-1 infection on the expression of host factors TSG101 (Tumor Susceptibility Gene 101) and Alix (ALG-2-interacting protein X). HIV-1 infectious clone pNL4-3 was used to infect TZM-bl, PM1, Jurkat cell lines and human peripheral blood mononuclear cells (PBMC). Twenty-four hours post-infection, the infected or uninfected cells were harvested respectively for extraction of total RNAs and total cellular proteins, which were subsequently used in RT-PCR and Western-blotting respectively to quantify TSG101 and Alix, respectively. Our data showed that HIV-1 infection resulted in various influences on the expression of TSG101 and Alix in the cell lines and the primary PBMC. A down-regulation was mainly observed in the cell lines, whereas an up-regulation of TSG101 was identified in primary PBMC. Three patterns were observed for down-regulation, including dual down-regulation of TSG101 and Alix for Jurkat cells, single down-regulation of Alix for TZM-bl cells and marginal or no influence on PM1 cells. The dual down-regulation of Alix and TSG101 in Jurkat cells coincided with less expression of HIV-1 p24 protein. This is the first-line evidence that HIV-1 infection affects the expression of host factors TSG101 and Alix, the down-regulation of these molecules may influence the HIV-1 replication. The underlying mechanism remains to be addressed.
Calcium-Binding Proteins ; genetics ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Endosomal Sorting Complexes Required for Transport ; genetics ; metabolism ; Gene Expression Regulation ; HEK293 Cells ; HIV-1 ; physiology ; Humans ; Jurkat Cells ; Leukocytes, Mononuclear ; metabolism ; virology ; RNA, Messenger ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism

BACKGROUNDNotch activation leads to transcriptional suppression of lineage-specific genes, inhibiting differentiation in response to inductive signals. The Notch signal system contains three parts: Notch molecules, Notch ligands and effectors. Delta4 is a newly-discovered Notch ligand which has received the attention of few detailed studies. This study sought to explore the biological function of Delta4 and observe its effects on 32D cell differentiation.

METHODSDelta4-expressing vector pTracer.CMV.Delta4.FLAG was constructed using molecular biological techniques. CHO cells stably transfected with pTracer.CMV.Delta4.FLAG were confirmed to have a Delta4 protein band via Western blotting. High-expression Delta4-CHO clones were selected for the following functional studies. Notch1-CHO and Notch2-CHO were used as host cells. After transiently transfecting with transition protein 1 (TP1), Delta4 activity was compared in both cell lines by means of luciferase analysis. CHO cells were incubated with Notch1-32D cells that had been transfected with Notch1 and were observed for granulocyte colony-stimulating factor (G-CSF)-induced differentiation. Jagged2-CHO and Delta4-CHO cells transfected with the Notch ligands Jagged2 and Delta4, respectively, were incubated with Notch1-32D cells to observed inhibition of Notch on G-CSF-induced differentiation.

RESULTSThe vector pTracer.CMV.Delta4.FLAG was constructed successfully. CHO cells were stably transfected with the vector pTracer.CMV.Delta4.FLAG. Two CHO cell lines expressing Delta4 at high levels were selected for use in the study. Delta4 was found to induce signal activity via both Notch1 and Notch2 and the induction of signaling activity was stronger in Notch2 cells than in Notch1 cells. Compared with other Notch ligands, Delta4 was slightly weaker than Jagged2, but stronger than Delta1 and Jagged1 in terms of Notch1 ligands. In terms of Notch2, Delta4 had a strong signaling activity, but was weaker than Delta1, Jagged1, and Jagged2. Jagged2 could inhibit Notch1-32D cell differentiation induced by G-CSF, but Delta4 could not.

CONCLUSIONSDelta4 induces both Notch1 and Notch2 activity and is a ligand for both of them. The effect of Delta4 is stronger on Notch2 than that on Notch1. Jagged2 can inhibit Notch1-32D cell differentiation induced by G-CSF, but Delta4 cannot.

Animals ; CHO Cells ; Calcium-Binding Proteins ; Cell Differentiation ; Cricetinae ; Intercellular Signaling Peptides and Proteins ; Intracellular Signaling Peptides and Proteins ; Jagged-1 Protein ; Jagged-2 Protein ; Membrane Proteins ; genetics ; physiology ; Mice ; Receptor, Notch1 ; Receptor, Notch2 ; Receptors, Cell Surface ; physiology ; Serrate-Jagged Proteins ; Signal Transduction ; Transcription Factors ; physiology

Animals ; Calcium ; metabolism ; DNA-Binding Proteins ; metabolism ; Enzyme Activation ; Focal Adhesion Kinase 2 ; metabolism ; Gene Expression Regulation, Viral ; Hepatitis B virus ; genetics ; physiology ; Humans ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Virus Replication

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.
Caco-2 Cells ; Calcium-Binding Proteins ; Calpain/genetics/metabolism ; Caspase 3/genetics/metabolism ; Caspases ; *Cell Death ; Colon/cytology ; Entamoeba histolytica/*physiology ; Epithelial Cells/cytology/parasitology ; Humans ; I-kappa B Proteins/metabolism ; Intestinal Mucosa/cytology ; NF-kappa B/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor/genetics/*metabolism ; STAT5 Transcription Factor/genetics/*metabolism ; Signal Transduction

OBJECTIVEPancreatic cancer is one of the most deadly cancers, which is characterized by its high metastatic potential. S100A4 is a major prometastatic protein involved in tumor invasion and metastasis which precise role in pancreatic cancer has not been fully investigated. We knocked down the S100A4 gene in the Bxpc-3 pancreatic cancer cell line via RNA interference to study the changes in cell behavior.

METHODSReal-time polymerase chain reaction and western blotting were used to detect mRNA and protein expression levels of S100A4, matrix metalloproteinase (MMP)-2, E-cadherin and thrombospondin (TSP)-1. Transwell chambers were used to detect the migration and invasion abilities; a cell adhesion assay was used to detect adhesion ability; colony forming efficiency was used to detect cell proliferation; flow cytometry was used to detect apoptosis.

RESULTSS100A4 mRNA expression was reduced to 17% after transfection with S100A4-siRNA, and protein expression had a similar trend. mRNA and protein expression of MMP-2 was reduced and that of E-cadherin and TSP-1 was elevated, indicating that S100A4 affects their expression. S100A4-silenced cells exhibited a marked decrease in migration and invasiveness and increased adhesion, whereas overall proliferation and apoptosis were not overtly altered.

CONCLUSIONS100A4 and its downstream factors play important roles in pancreatic cancer invasion, and silencing A100A4 can significantly contain the invasiveness of pancreatic cancer.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cadherins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Pancreatic Neoplasms ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; S100 Calcium-Binding Protein A4 ; S100 Proteins ; genetics ; metabolism ; Thrombospondin 1 ; genetics ; metabolism