This study was purposed to investigate the effect of knocking down eukaryotic elongation factor 1A1 (eEF1A1) gene on the proliferation and apoptosis in human acute T lymphocytic leukemia (T-ALL) cell line Jurkat and explore its mechanism. The eEF1A1 mRNA and protein expressions of Jurkat cells and 3 healthy adult peripheral blood mononuclear cells (PBMNC) were detected by real time PCR and Western blot, respectively. eEF1A1-shRNA lentivirus was constructed through molecular biological method, and was used to transfect Jurkat cells. Then, cell eEF1A1 mRNA and protein expressions were detected by real time PCR and Western blot, respectively. Cell proliferation, apoptosis and cycle were detected by MTT method, Annexin V-APC labeling and DNA ploidy analysis, respectively. Cell-related protein expressions of phosphatidylinositol-3-kinase (PI3K)/serine/threonine kinase (Akt) signaling pathway were detected by Western blot. The results showed that eEF1A1 mRNA and protein expression levels of Jurkat cells were significantly higher than that of healthy adult PBMNC, respectively (P < 0.01, P < 0.05). eEF1A1 mRNA and protein expressions of Jurkat cells were significantly knocked down by constructing eEF1A1-shRNA lentivirus. Compared to negative control group (transfected with negative control-shRNA lentivirus), cell proliferation in eEF1A1-shRNA group was significantly inhibited, cell apoptosis was remarkably induced, cell cycle was blocked in G(0)/G(1) phase, and the expression levels of p-Akt (Ser 473), nuclear factor kappa B (NF-κB), p-NF-κB (Ser 468), mammalian target of rapamycin (mTOR) and p-mTOR (Ser 2448) proteins were significantly reduced. It is concluded that eEF1A1 may be a putative oncoprotein in T-ALL cells. Knocking down eEF1A1 gene has noticeable effects on the proliferation inhibition and apoptosis induction of Jurkat cells, which may be mediated by the down-regulation of PI3K/Akt/NF-κB and PI3K/Akt/mTOR signaling pathway.
Apoptosis ; Cell Proliferation ; Gene Silencing ; Humans ; Jurkat Cells ; Peptide Elongation Factor 1 ; genetics ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; genetics

This study was aimed to explore the effects of expressing eukaryotic elongation factor 1A1 (eEF1A1) on proliferation and apoptosis in human acute T lymphocytic leukemia (T-ALL) cell line Jurkat with knocked down eEF1A1 gene and its mechanisms. eEF1A1-expressing lentivirus (LV) was constructed and used to transfect the Jurkat cells with knocked down eEF1A1 gene. Then, the expressions of eEF1A1 mRNA and protein were detected by real time PCR(RT-PCR) and Western blot respectively.Cell proliferation, apoptosis and cycle were detected by MTT method, Annexin V-APC labeling and DNA ploidy analysis respectively. The related protein expressions of phosphatidylinositol-3-kinase (PI3K)/serine/threonine kinase (Akt) signaling pathway were detected by Western blot. The results indicated that eEF1A1 mRNA and protein expressions of Jurkat cells with knocked down eEF1A1 gene were re-established by constructing eEF1A1-expression LV. Compared with negative control group (transfected with negative control LV and eEF1A1-shRNA LV), cell proliferation in eEF1A1 expression group was significantly enhanced, cell apoptosis was remarkably inhibited, percentage of cells in G0/G1 phase was significantly reduced alone with increased percentage of cells in S and G2/M phase, and the expression levels of p-Akt (Ser 473), nuclear factor kappa B (NF-κB), p-NF-κB (Ser 468), mammalian target of rapamycin (mTOR) and p-mTOR (Ser 2448) protein significantly increased. It is concluded that eEF1A1 may have a carcinogenic effect in T-ALL cells. eEF1A1 expression has noticeable effects on the proliferation enhancement and apoptosis inhibition of Jurkat cells, which may be mediated by the up-regulation of PI3K/Akt/NF-κB and PI3K/Akt/ mTOR signaling pathway.
Apoptosis ; Cell Proliferation ; Gene Expression ; Humans ; Jurkat Cells ; Peptide Elongation Factor 1 ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Small Interfering ; genetics ; Signal Transduction

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.
Genes, Essential ; Gelsemium/genetics* ; Peptide Elongation Factor 1/genetics* ; Transcriptome ; Gene Expression Profiling/methods* ; Alkaloids ; Real-Time Polymerase Chain Reaction/methods* ; Reference Standards

Chloride channels belong to a superfamily of ion channels that permit passive passage of anions, mainly chloride, across cell membrane. They play a variety of important physiological roles in regulation of cytosolic pH, cell volume homeostasis, organic solute transport, cell migration, cell proliferation, and differentiation. However, little is known about the functional regulation of these channels. In this study, we generated an integrated transgenic worm strain expressing green fluorescence protein (GFP) fused CLC-type chloride channel 1 (CLH-1::GFP), a voltage-gated chloride channel in Caenorhabditis elegans (C. elegans). CLH-1::GFP was expressed in some unidentified head neurons and posterior intestinal cells of C. elegans. Interacting proteins of CLH-1::GFP were purified by GFP-Trap, a novel system for efficient isolation of GFP fusion proteins and their interacting factors. Mass spectrometry (MS) analysis revealed that a total of 27 high probability interacting proteins were co-trapped with CLHp-1::GFP. Biochemical evidence showed that eukaryotic translation elongation factor 1 (EEF-1), one of these co-trapped proteins identified by MS, physically interacted with CLH-1, in consistent with GFP-Trap experiments. Further immunostaining data revealed that the protein level of CLH-1 was significantly increased upon co-expression with EEF-1. These results suggest that the combination of GFP-Trap purification with MS is an excellent tool to identify novel interacting proteins of voltage-gated chloride channels in C. elegans. Our data also show that EEF-1 is a regulator of voltage-gated chloride channel CLH-1.
Animals ; Animals, Genetically Modified ; Caenorhabditis elegans ; genetics ; metabolism ; Caenorhabditis elegans Proteins ; metabolism ; Chloride Channels ; metabolism ; Green Fluorescent Proteins ; chemistry ; Mass Spectrometry ; Peptide Elongation Factor 1 ; metabolism

Objective To investigate the role and mechanism of eukaryotic translation elongation factor 1(EEF1) family members (EEF1D,EEF1A1,and EEF1A2) in lung adenocarcinoma (LUAD) based on public databases.Methods We examined EEF1 member expression levels in human LUAD samples via The Cancer Genome Atlas in the UCSC Xena browser and the Clinical Proteomic Tumor Analysis Consortium.We analyzed the mRNA and protein levels of EEF1D,EEF1A1,and EEF1A2 and their correlations with pathological variables via the Mann-Whitney U test.The Kaplan-Meier curves were established to assess the prognostic values of EEF1D,EEF1A1,and EEF1A2.The single-sample gene set enrichment analysis algorithm was employed to explore the relationship between the expression levels of EEF1 members and tumor immune cell infiltration.Spearman and Pearson correlation analyses were performed to examine the relationship between the expression levels of EEF1 members and those of the genes in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.The immunohistochemical assay was employed to determine the expression levels of EEF1D,EEF1A1,and EEF1A2 in the LUAD tissue (n=75) and paracancer tissue (n=75) samples.Results The mRNA and protein levels of EEF1D,EEF1A1,and EEF1A2 showed significant differences between tumor and paracancer tissues (all P<0.001).The patients with high protein levels of EEF1A1 showed bad prognosis in terms of overall survival (P=0.039),and those with high protein levels of EEF1A2 showed good prognosis in terms of overall survival (P=0.012).The influence of the mRNA level of EEF1D on prognosis was associated with pathological characteristics.The expression levels of EEF1 members were significantly associated with the infiltration of various immune cells and the expression of key molecules in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.Conclusion EEF1D,EEF1A1,and EEF1A2 are associated with the progression of LUAD,serving as the candidate prognostic markers for LUAD.
Humans ; Peptide Elongation Factor 1/metabolism* ; Proteomics ; Proto-Oncogene Proteins c-akt/metabolism* ; Carcinogenesis ; Adenocarcinoma of Lung ; Lung Neoplasms ; RNA, Messenger/genetics* ; Phosphatidylinositol 3-Kinases ; Prognosis

OBJECTIVETo study the oncogenic potential of mouse translation initiation factor 3 (TIF3) and elongation factor-1delta (TEF-1delta) in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide (NiS).

METHODSAbnormal expressions of human TIF3 and TEF-1delta genes in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were investigated and analyzed by the reverse transcript polymerase chain reaction (RT-PCR) and fluorescent quantitative polymerase chain reaction (FQ-PCR), respectively.

RESULTSRT-PCR analysis primarily showed that both human TIF3 and TEF-1delta mRNA expressions in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were increased as compared with controls. FQ-PCR assay showed that the levels of TIF3 expressions in the transformed cells and tumorigenic cells were 3 and 4 times higher respectively, and the elevated expressions of TEF-1delta cDNA copies were 2.7- to 3.5-fold in transformed cells and 4.1- to 5.2-fold in tumorigenic cells when compared with non-transformed cells, indicating that the over-expressions of human TIF3 and TEF-1delta genes were related to malignant degree of the cells induced by nickel.

CONCLUSIONSThese findings demonstrate that there are markedly abnormal expressions of TIF3 and TEF-1delta genes during malignant transformation of human bronchial epithelial cell lines induced by crystalline NiS. They seem to be the molecular mechanisms potentially responsible for human carcinogensis due to nickel.

Biomarkers ; Bronchi ; cytology ; Cell Line, Transformed ; Cell Line, Tumor ; Cell Transformation, Neoplastic ; metabolism ; DNA, Complementary ; metabolism ; Epithelial Cells ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Nickel ; toxicity ; Peptide Elongation Factor 1 ; genetics ; metabolism ; Prokaryotic Initiation Factor-3 ; genetics ; metabolism

OBJECTIVETo study the alternative expression and sequence of human elongation factor-1 delta (human EF-1 delta p31) during malignant transformation of human bronchial epithelial cells induced by cadmium chloride (CdC12) and its possible mechanism.

METHODSTotal RNA was isolated at different stages of transformed human bronchial epithelial cells (16HBE) induced by CdCl2 at a concentration of 5.0 microM. Special primers and probe for human EF-1 delta p31 were designed and expression of human EF-1 delta mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis.

RESULTSThe expressions of human EF-1 beta p31 at different stages of 16HBE cells transformed by CdCl2 was elevated (P < 0.01 or P < 0.05). Compared with their corresponding non-transformed cells, the overexpression level of EF-1 delta p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed cells and 7.2 folds in Cd-tumorigenic cells. No change was found n the sequence of overexpressed EF-1beta p31 at different stages of 16HBE cells transformed by CdCl2.

CONCLUSIONOverexpression of human EF-1beta p31 is positively correlated with malignant transformation of 16HBE cells induced by CdC12, but is not correlated with DNA mutations.

Cadmium Chloride ; Cell Line ; Cell Transformation, Neoplastic ; chemically induced ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Peptide Elongation Factor 1 ; genetics ; metabolism ; Respiratory Mucosa ; drug effects ; metabolism ; pathology ; Sequence Analysis, DNA

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.
Actins/genetics ; Animals ; Bone Morphogenetic Proteins/genetics ; *Cell Differentiation ; Chickens ; Cytomegalovirus/genetics ; Drug Delivery Systems ; Embryo/*cytology ; *Gene Therapy ; Green Fluorescent Proteins/genetics/*metabolism ; Humans ; Immunoenzyme Techniques ; Microscopy, Fluorescence ; Peptide Elongation Factor 1/genetics ; Pluripotent Stem Cells/*cytology ; Promoter Regions (Genetics)/*genetics ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription, Genetic/genetics

The GABAergic neurons in the parafacial zone (PZ) play an important role in sleep-wake regulation and have been identified as part of a sleep-promoting center in the brainstem, but the long-range connections mediating this function remain poorly characterized. Here, we performed whole-brain mapping of both the inputs and outputs of the GABAergic neurons in the PZ of the mouse brain. We used the modified rabies virus EnvA-ΔG-DsRed combined with a Cre/loxP gene-expression strategy to map the direct monosynaptic inputs to the GABAergic neurons in the PZ, and found that they receive inputs mainly from the hypothalamic area, zona incerta, and parasubthalamic nucleus in the hypothalamus; the substantia nigra, pars reticulata and deep mesencephalic nucleus in the midbrain; and the intermediate reticular nucleus and medial vestibular nucleus (parvocellular part) in the pons and medulla. We also mapped the axonal projections of the PZ GABAergic neurons with adeno-associated virus, and defined the reciprocal connections of the PZ GABAergic neurons with their input and output nuclei. The newly-found inputs and outputs of the PZ were also listed compared with the literature. This cell-type-specific neuronal whole-brain mapping of the PZ GABAergic neurons may reveal the circuits underlying various functions such as sleep-wake regulation.
Animals ; Axons ; physiology ; Brain ; anatomy & histology ; Brain Mapping ; Brain Stem ; cytology ; GABAergic Neurons ; physiology ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Neural Pathways ; physiology ; Peptide Elongation Factor 1 ; genetics ; metabolism ; Rabies virus ; genetics ; metabolism ; Transduction, Genetic ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism