Dehydration-responsive element binding proteins (DREBs) are an important class of transcription factors related to plant stress tolerance. Ammopiptanthus mongolicus is an evergreen broadleaf shrub endemic to desert areas of northwest China, and it has a very high tolerance to harsh environments. In order to reveal the functions and mechanisms of the AmDREB1F gene from this species in enduring abiotic stresses, we performed subcellular localization test, expression pattern analysis, and stress tolerance evaluation of transgenic Arabidopsis harboring this gene. The protein encoded by AmDREB1F was localized in the nucleus. In laboratory-cultured A. mongolicus seedlings, the expression of AmDREB1F was induced significantly by cold and drought but very slightly by salt and heat stresses, and undetectable upon ABA treatment. In leaves of naturally growing shrubs in the wild, the expression levels of the AmDREB1F gene were much higher during the late autumn, winter and early spring than in other seasons. Moreover, the expression was abundant in roots and immature pods rather than other organs of the shrubs. Constitutive expression of AmDREB1F in Arabidopsis induced the expression of several DREB-regulated stress-responsive genes and improved the tolerance of transgenic lines to drought, high salinity and low temperature as well as oxidative stress. The constitutive expression also caused growth retardation of the transgenics, which could be eliminated by the application of gibberellin 3. Stress-inducible expression of AmDREB1F also enhanced the tolerance of transgenic Arabidopsis to all of the four stresses mentioned above, without affecting its growth and development. These results suggest that AmDREB1F gene may play positive regulatory roles in response to abiotic stresses through the ABA-independent signaling pathways.
Arabidopsis/metabolism* ; Droughts ; Ectopic Gene Expression ; Fabaceae/genetics* ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Plants, Genetically Modified/genetics* ; Stress, Physiological/genetics*

Drought stress affects the growth and development of rice, resulting in severe loss in yield and quality. Ectopic expression of the bacterial RNA chaperone, cold shock protein (Csp), can improve rice drought tolerance. Archaeal TRAM (TRM2 and MiaB) proteins have similar structure and biochemical functions as bacterial Csp. Moreover, DNA replication, transcription and translation of archaea are more similar to those in eukaryotes. To test if archaeal RNA chaperones could confer plant drought tolerance, we selected two TRAM proteins, Mpsy_3066 and Mpsy_0643, from a cold-adaptive methanogenic archaea Methanolobus psychrophilus R15 to study. We overexpressed the TRAM proteins in rice and performed drought treatment at seedling and adult stage. The results showed that overexpression both TRAM proteins could significantly improve the tolerance of rice to drought stress. We further demonstrated in rice protoplasts that the TRAMs could abolish misfolded RNA secondary structure and improve translation efficiency, which might explain how TRAMs improve drought tolerance transgenic rice. Our work supports that ectopic expression of archaeal TRAMs effectively improve drought tolerance in rice.
Droughts ; Ectopic Gene Expression ; Gene Expression Regulation, Plant ; Oryza ; Plant Proteins ; Plants, Genetically Modified ; Stress, Physiological

PURPOSE: Adipogenic differentiation of adipose tissue-derived mesenchymal stem cells (AMSCs) is critical to many disease-related disorders, such as obesity and diabetes. Studies have demonstrated that miRNA-138 (miR-138) is closely involved in adipogenesis. However, the mechanisms affected by miR-138 remain unclear. This work aimed to investigate interactions between miR-138 and lipoprotein lipase (LPL), a key lipogenic enzyme, in AMSCs. MATERIALS AND METHODS: Human AMSCs (hAMSCs) isolated from human abdomen tissue were subjected to adipogenic differentiation medium. Quantitative real-time polymerase chain reaction and Western blot assay were applied to measure the expressions of miR-138, LPL, and the two adipogenic transcription factors cytidine-cytidine-adenosine-adenosine-thymidine enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). The relationship between miR-138 and LPL was predicted utilizing the miRTarBase database and validated by dual luciferase reporter assay. RESULTS: Showing increases in C/EBPα and PPARγ expression levels, hAMSCs were induced into adipogenic differentiation. During adipogenesis of hAMSCs, miR-138 expression was significantly downregulated. Overexpression of miR-138 by transfection inhibited hAMSCs adipogenic differentiation in vitro. Mechanically, LPL was a target of miR-138. LPL expression was upregulated during adipogenesis of hAMSCs, and this upregulation was reversed by miR-138 overexpression. Functionally, silencing of LPL by transfection exerted similar inhibition of the expressions of C/EBPα and PPARγ. Meanwhile, LPL ectopic expression was able to partly abolish the suppressive effect of miR-138 overexpression on adipogenic differentiation of hAMSCs. CONCLUSION: Upregulation of miR-138 inhibits adipogenic differentiation of hAMSCs by directly downregulating LPL.
Abdomen ; Adipogenesis ; Blotting, Western ; Carrier Proteins ; Ectopic Gene Expression ; Humans ; In Vitro Techniques ; Lipoprotein Lipase ; Lipoproteins ; Luciferases ; Mesenchymal Stromal Cells ; Obesity ; PPAR gamma ; Real-Time Polymerase Chain Reaction ; Transcription Factors ; Transfection ; Up-Regulation

Somatic cell nuclear transfer (SCNT) has various applications in research, as well as in the medical field and animal husbandry. However, the efficiency of SCNT is low and the accurate mechanism of SCNT in murine embryo development is unreported. In general, the developmental rate of SCNT murine embryos is lower than in vivo counterparts. In previous studies, polo-like kinase 1 (Plk1) was reported to be a crucial element in cell division including centrosome maturation, cytokinesis, and spindle formation. In an initial series of experiments in this study, BI2536, a Plk1 inhibitor, was treated to in vivo-fertilized embryos and the embryos failed to develop beyond the 2-cell stage. This confirmed previous findings that Plk1 is crucial for the first mitotic division of murine embryos. Next, we investigated Plk1's localization and intensity by immunofluorescence analysis. In contrast to normally developed embryos, SCNT murine embryos that failed to develop exhibited two types of Plk1 expressions; a low Plk1 expression pattern and ectopic expression of Plk1. The results show that Plk1 has a critical role in SCNT murine embryos. In conclusion, this study demonstrated that the SCNT murine embryos fail to develop beyond the 2-cell stage, and the embryos show abnormal Plk1 expression patterns, which may one of the main causes of developmental failure of early SCNT murine embryos.
Animal Husbandry ; Cell Division ; Centrosome ; Cytokinesis ; Ectopic Gene Expression ; Embryonic Development ; Embryonic Structures ; Female ; Fluorescent Antibody Technique ; Nuclear Transfer Techniques ; Phosphotransferases ; Pregnancy

ectopic expression and this is associated with an increased activity of tumor stem cells in these patients. This leads to a high aggressiveness of the tumor and the development of metastatic disease. The aim was to evaluate the prognostic significance of the presence of amplifications of stem genes and their expression in patients with early breast cancer (BC).METHODS: The study included 28 patients with T₁N(x)M₀ BC. We used surgical specimens, including formalin-fixed paraffin-embedded archive materials, for 8 patients. A microarray analysis was performed on high-density DNA chips from CytoScanHDArray to assess the status of copy number aberration (CNA) of stem genes locus. Gene expression was assessed using RT-qPCR.RESULTS: CNA analysis of the studied tumors of patients without chemotherapy showed that 17/18 patients without metastases did not have two or more amplifications of chromosomal regions. Ten patients had visceral metastases. In 9/10 of these patients in the primary tumor there were two or more amplifications of the stem genes locus. Two or more amplifications of stem genes locus were found in 12 patients with stage I. Hematogenous metastases did not develop in all patients. Comparison of metastasis-free survival rates in groups of patients with 1 or without amplifications and with two or more amplifications showed statistically significant differences (P = 0.01).CONCLUSION: Our studies have shown that the presence of clones with two or more amplifications of stem gene in patients with BC T₁N(x)M₀ has a significant prognostic value and determines an unfavorable prognosis for distant metastasis.]]>
Archives ; Breast Neoplasms ; Breast ; Clone Cells ; Drug Therapy ; Ectopic Gene Expression ; Gene Expression ; Humans ; Microarray Analysis ; Neoplasm Metastasis ; Neoplastic Stem Cells ; Oligonucleotide Array Sequence Analysis ; Prognosis ; Survival Rate

BACKGROUND: Dysregulation of hepatic glucose production (HGP) contributes to the development of type 2 diabetes mellitus. Telmisartan, an angiotensin II type 1 receptor blocker (ARB), has various ancillary effects in addition to common blood pressure-lowering effects. The effects and mechanism of telmisartan on HGP have not been fully elucidated and, therefore, we investigated these phenomena in hyperglycemic HepG2 cells and high-fat diet (HFD)-fed mice.METHODS: Glucose production and glucose uptake were measured in HepG2 cells. Expression levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase α (G6Pase-α), and phosphorylation levels of insulin receptor substrate-1 (IRS-1) and protein kinase C ζ (PKCζ) were assessed by western blot analysis. Animal studies were performed using HFD-fed mice.RESULTS: Telmisartan dose-dependently increased HGP, and PEPCK expression was minimally increased at a 40 μM concentration without a change in G6Pase-α expression. In contrast, telmisartan increased phosphorylation of IRS-1 at Ser302 (p-IRS-1-Ser302) and decreased p-IRS-1-Tyr632 dose-dependently. Telmisartan dose-dependently increased p-PKCζ-Thr410 which is known to reduce insulin action by inducing IRS-1 serine phosphorylation. Ectopic expression of dominant-negative PKCζ significantly attenuated telmisartan-induced HGP and p-IRS-1-Ser302 and -inhibited p-IRS-1-Tyr632. Among ARBs, including losartan and fimasartan, only telmisartan changed IRS-1 phosphorylation and pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist, did not alter this effect. Finally, in the livers from HFD-fed mice, telmisartan increased p-IRS-1-Ser302 and decreased p-IRS-1-Tyr632, which was accompanied by an increase in p-PKCζ-Thr410.CONCLUSION: These results suggest that telmisartan increases HGP by inducing p-PKCζ-Thr410 that increases p-IRS-1-Ser302 and decreases p-IRS-1-Tyr632 in a PPARγ-independent manner.
Animals ; Blotting, Western ; Diabetes Mellitus, Type 2 ; Diet, High-Fat ; Ectopic Gene Expression ; Glucose ; Glucose-6-Phosphatase ; Hep G2 Cells ; Insulin Receptor Substrate Proteins ; Insulin ; Liver ; Losartan ; Mice ; Peroxisomes ; Phosphoenolpyruvate ; Phosphorylation ; Protein Kinase C ; Protein Kinases ; Receptor, Angiotensin, Type 1 ; Receptor, Insulin ; Serine

The small GTP-binding protein Rab25 is associated with tumor formation and progression. However, recent studies have shown discordant effects of Rab25 on cancer cell progression depending on cell lineage. In the present study, we elucidate the underlying mechanisms by which Rab25 induces cellular invasion. We demonstrate that Rab25 increases β1 integrin levels and subsequent activation of EGFR and upregulation of VEGF-A expression, leading to increased Snail expression, epithelial-to-mesenchymal transition and cancer cell invasiveness. Strikingly, we identify that Snail mediates Rab25-induced cancer cell invasiveness through fascin expression and that ectopic expression of Rab25 aggravates metastasis of ovarian cancer cells to the lung. We thus demonstrate a novel role of a β1 integrin/EGFR/VEGF-A/Snail signaling cascade in Rab25-induced cancer cell aggressiveness through induction of fascin expression, thus providing novel biomarkers and potential therapeutic targets for Rab25-expressing cancer cells.
Biomarkers ; Cell Lineage ; Ectopic Gene Expression ; GTP-Binding Proteins ; Lung ; Neoplasm Metastasis ; Ovarian Neoplasms ; Snails ; Up-Regulation ; Vascular Endothelial Growth Factor A

Olfactory receptors (ORs) in mammals are generally considered to function as chemosensors in the olfactory organs of animals. They are membrane proteins that traverse the cytoplasmic membrane seven times and work generally by coupling to heterotrimeric G protein. The OR is a G protein–coupled receptor that binds the guanine nucleotide-binding G(αolf) subunit and the Gβγ dimer to recognize a wide spectrum of organic compounds in accordance with its cognate ligand. Mammalian ORs were originally identified from the olfactory epithelium of rat. However, it has been recently reported that the expression of ORs is not limited to the olfactory organ. In recent decades, they have been found to be expressed in diverse organs or tissues and even tumors in mammals. In this review, the expression and expected function of olfactory receptors that exist throughout an organism's system are discussed.
Animals ; Cell Membrane ; Ectopic Gene Expression ; GTP-Binding Proteins ; Guanine ; Mammals* ; Membrane Proteins ; Olfactory Mucosa ; Rats

PURPOSE: T helper (Th) 17 cells play a critical role in the development of asthma, but the underlying mechanism of how interleukin (IL)-17 is regulated in allergic airway inflammation is poorly understood. In this study, we investigated the impact of Bcl11b on Th17 response in asthma. METHODS: Blood samples from patients with mild asthma (MA) and severe asthma (SA) were collected. Expression of Bcl11b, IL-4, IL-5, IL-13, IL-17A and transforming growth factor (TGF)-β1 were determined in CD4+ T cells and plasma by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Relative mRNA and protein levels of Bcl11b, IL-17A and genes involved in the TGF/Smad signaling pathway were examined by PCR, ELISA and western blot analysis in house dust mite (HDM)-challenged mice. Ectopic expression of Bcl11b in HDM-stimulated primary mouse splenocytes was achieved by nucleofection of Bcl11b expression plasmid. RESULTS: We found significantly decreased Bcl11b but increased IL-17A and TGF-β1 expression in patients with asthma and a strongly negative correlation between Bcl11b and these 2 cytokines in SA patients. Similar expression patterns of Bcl11b, IL-17A and TGF-β1 were also found in mice with HDM-induced allergic airway inflammation. We demonstrated further that Smad2/3 phosphorylation was increased in HDM-challenged mice and that ectopic expression of Bcl11b in HDM-stimulated primary mouse splenocytes reduced Smad2 phosphorylation and IL-17 expression. CONCLUSIONS: Our findings demonstrate a potential effect of Bc111b in controlling IL-17-mediated inflammation in asthma and suggest that Bc111b may be a useful therapeutic target for asthma.
Animals ; Asthma* ; Blotting, Western ; Cytokines ; Ectopic Gene Expression ; Enzyme-Linked Immunosorbent Assay ; Humans ; Inflammation ; Interleukin-13 ; Interleukin-17* ; Interleukin-4 ; Interleukin-5 ; Interleukins ; Mice ; Phosphorylation ; Plasma ; Plasmids ; Polymerase Chain Reaction ; Pyroglyphidae ; RNA, Messenger ; T-Lymphocytes ; Transforming Growth Factors

IL-9 is a known T cell growth factor with pleiotropic immunological functions, especially in parasite infection and colitis. However, its role in tumor growth is controversial. In this study, we generated tumor clones expressing the membrane-bound form of IL-9 (MB-IL-9) and investigated their influences on immune system. MB-IL-9 tumor clones showed reduced tumorigenicity but shortened survival accompanied with severe body weight loss in mice. MB-IL-9 expression on tumor cells had no effect on cell proliferation or major histocompatibility complex class I expression in vitro. MB-IL-9 tumor clones were effective in amplifying CD4⁺ and CD8⁺ T cells and increasing cytotoxic activity against CT26 cells in vivo. We also observed a prominent reduction in body weights and survival period of mice injected intraperitoneally with MB-IL-9 clones compared with control groups. Ratios of IL-17 to interferon (IFN)-γ in serum level and tumor mass were higher in mice implanted with MB-IL-9 tumor clones than those observed in mice implanted with control cells. These results indicate that the ectopic expression of the MB-IL-9 on tumor cells exerts an immune-stimulatory effect with toxicity. To exploit its benefits as a tumor vaccine, a strategy to control the toxicity of MB-IL-9 tumor clones should be developed.
Animals ; Body Weight ; Cell Proliferation ; Clone Cells ; Colitis ; Colon ; Ectopic Gene Expression ; Immune System ; In Vitro Techniques ; Interferons ; Interleukin-17 ; Interleukin-2 ; Interleukin-9 ; Major Histocompatibility Complex ; Mice ; Parasites ; T-Lymphocytes