Modulation Of mRNA Expression Research Articles

Structural basis for the specific recognition of 18S rRNA by APUM23.

PUF (Pumilio/fem-3 mRNA binding factor) proteins, a conserved family of RNA-binding proteins, recognize specific single-strand RNA targets in a specific modular way. Although plants have a greater number of PUF protein members than do animal and fungal systems, they have been the subject of fewer structural and functional investigations. The aim of this study was to elucidate the involvement of APUM23, a nucleolar PUF protein in the plant Arabidopsis, in pre-rRNA processing. APUM23 is distinct from classical PUF family proteins, which are located in the cytoplasm and bind to 3′UTRs of mRNA to modulate mRNA expression and localization. We found that the complete RNA target sequence of APUM23 comprises 11 nt in 18S rRNA at positions 1141–1151. The complex structure shows that APUM23 has 10 PUF repeats; it assembles into a C-shape, with an insertion located within the inner concave surface. We found several different RNA recognition features. A notable structural feature of APUM23 is an insertion in the third PUF repeat that participates in nucleotide recognition and maintains the correct conformation of the target RNA. Our findings elucidate the mechanism for APUM23’s-specific recognition of 18S rRNA.

Multiple Sclerosis Treatments Affect Monocyte-Derived Microvesicle Production.

Microvesicles (MVs) are released by immune cells especially of the myeloid lineage upon stimulation with ATP on its cognate receptor P2X7, both in physiological and pathological conditions. In multiple sclerosis (MS) the role of MVs remains little investigated. We aimed to compare the release of MVs in peripheral blood monocytes from MS patients with healthy donors (HDs) and to see how current MS treatment may affect such a production. We also assessed the treatment effect on M1 and M2 monocyte polarization and on the inflammasome components. Spectrophotometric quantification was performed to compare monocyte-derived MVs from 20 untreated relapsing-remitting MS patients and 20 HDs and to evaluate the effect of different treatments. Subgroups of nine interferon-beta and of five teriflunomide-treated MS patients were evaluated at baseline and after 2, 6, and 12 months of treatment. Six MS patients taking Fingolimod, after switching from a first-line therapy, were included in the study and analyzed only at 12 months of treatment. MVs analysis revealed that monocytes from MS patients produced vesicles in higher amounts than controls. All treatments reduced vesicle production but only teriflunomide was associated with a downregulation of purinergic P2X7 receptor and inflammasome components expression. The therapies modulated mRNA expression of both M1 and M2 monocyte markers. Our results, suggesting new molecular targets for drugs currently used in MS, may potentially provide useful novel evidence to approach the disease.

Dietary Wolfberry Extract Modifies Oxidative Stress by Controlling the Expression of Inflammatory mRNAs in Overweight and Hypercholesterolemic Subjects: A Randomized, Double-Blind, Placebo-Controlled Trial.

In the present study, we evaluated the antioxidative and anti-inflammatory effects of an aqueous extract of wolfberry fruit (WBE) in mild hypercholesterolemic and overweight subjects. This study was a double-blind randomized trial of two parallel groups of free-living subjects (n = 53). The participants consumed the contents of an 80 mL pouch containing 13.5 g WBE or placebo after one meal per day over an 8-week period. Following 8 weeks of WBE supplementation, we observed a slight but significant decrease in erythrocyte superoxide dismutase activity and an increase in catalase activity. Furthermore, to assess endogenous DNA damage in lymphocytes, the alkaline comet assay was performed, showing that the percentage of DNA in the tail was significantly decreased by 8-week WBE intake. Additionally, the proportion of significantly deregulated mRNAs related to oxidative or inflammatory stress was considerably higher in the WBE intake group. The present data indicate that WBE intake has antioxidative and anti-inflammatory effects in overweight and hypercholesterolemic subjects by modulating mRNA expression.

Modulation of mRNA expression and activities of xenobiotic metabolizing enzymes, CYP1A1, CYP1A2, CYP2E1, GPx and GSTP1 by the Salicornia freitagii extract in HT-29 human colon cancer cells

Phase I-II detoxification and antioxidant enzymes are responsible for the detoxification and elimination of activated carcinogens, acting as important biomarkers for chemoprevention. Among them, cytochrome P450s plays a prominent role in the metabolic activation of xenobiotics. The herb Salicornia freitagii (SF) (Amaranthaceae) is known for its anticancer, antioxidant, antidiabetic and antiinflammatory activities. In this study, we determined the bioactive phenolics in the SF methanol extract and investigated its antiproliferative potential in HT-29 human colon cancer cells. We also investigated the modulation of some phase I and II enzyme (CYP 1A1, 1A2, 2E1, GSTP1 and GPx) mRNA expression and enzymatic activities by the SF extract and its major bioactive phenolic compounds. LC/MS-MS analysis showed that the main phenolic compounds of the methanolic SF extract are vanillic acid (48 ?g/100g) and p-coumaric acid (10.8 ?g/100g). SF extract, vanillic acid and p-coumaric acid exhibited high antiproliferative activities in HT-29 cells, with IC50 values of 81.79?g/mL, 98.8 ?M and 221.6 ?M, respectively. The mRNA expression levels of CYP1A2 and CYP2E1 were decreased, while those of GSTP1 and GPx in HT-29 cells were increased after application of either the SF extract or vanillic acid. The SF extract by itself also increased the activities of GPx and GSTP1 enzymes 1.68- and 1.49-fold, respectively. Our data indicate that the SF extract and its major bioactive compound, vanillic acid, could exert a modulatory effect on the expression of enzymes that are involved in xenobiotic activation and detoxification pathways in the gastrointestinal tract. For this reason, SF can be considered as a natural source of chemopreventive agents.

ELEVEN WEEKS OF STRENGTH TRAINING DECREASED THE EXPRESSION OF IMMUNE-RELATED GENES IN OLDER SUBJECTS INDEPENDENT OF PROTEIN SUPPLEMENT TYPE; A RANDOMIZED CONTROLLED TRIAL

Objective: To investigate the effects of eleven weeks of strength training combined with two isocaloric protein supplements on mRNA expression levels in skeletal muscle and peripheral blood mononuclear blood cells (PBMCs). Design: A double blind randomized controlled study. Setting: The Norwegian School of Sports Sciences, Norway. Participants: Untrained, but otherwise healthy, men and women (n=20, ≥ 70 yrs). Intervention: Participants were randomly allocated to receive either milk protein or a native whey protein supplement (20 g protein, morning and afternoon) combined with a standardized strength training protocol (6-10 RM, 1-3 sets, 3 times/week) for eleven weeks. Measurements: The mRNA expression levels of immune-related genes were measured before and after the intervention period, using RT-qPCR. Cytokines were measured using ELISA. Results: PBMC mRNA expression of interleukin (IL) 6, IL8, chemokine (C-C motif) ligand (CCL) 3, and nuclear receptor subfamily (NR) 1 group H (H) member 3 decreased significantly after the intervention period, whereas the mRNA expression of toll-like receptor (TLR2) increased. In skeletal muscle, the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) and PPARGC1B decreased significantly, whereas the mRNA expression of CCL2, CCL5, TLR2, TLR4 and hypoxia inducible factor 1 alpha subunit (HIF1A) increased significantly after the intervention. We found no significant differences in circulating C-reactive protein and IL6 after the intervention period. The consumption of whey and milk proteins had similar effects on mRNA expression levels after strength training in skeletal muscle as well as PBMCs. Conclusion: Eleven weeks of strength training and protein supplementation reduced the PBMC expression levels of genes involved in the immune system as well as in metabolism, underlining the close interaction between these processes. The upregulation of other immune-related genes observed in PBMCs as well as in skeletal muscle needs further investigations, but may be related to protein supplementation and training adaptations. Different protein supplementation (milk or native whey) did not differentially modulate mRNA expression after the intervention period.

Effects of Antifouling Biocides on Molecular and Biochemical Defense System in the Gill of the Pacific Oyster Crassostrea gigas.

Antifouling biocides such as organotin compounds and their alternatives are potent toxicants in marine ecosystems. In this study, we employed several molecular and biochemical response systems of the Pacific oyster Crassostrea gigas to understand a potential mode of action of antifouling biocides (i.e. tributyltin (TBT), diuron and irgarol) after exposure to different concentrations (0.01, 0.1, and 1 μg L-1) for 96 h. As a result, all the three antifouling biocides strongly induced the antioxidant defense system. TBT reduced both enzymatic activity and mRNA expression of Na+/K+-ATPase and acetylcholinesterase (AChE). Lower levels of both Na+/K+-ATPase activity and AChE mRNA expression were observed in the diuron-exposed oysters compared to the control, while the irgarol treatment reduced only the transcriptional expression of AChE gene. We also analyzed transcript profile of heat shock protein (Hsp) superfamily in same experimental conditions. All antifouling biocides tested in this study significantly modulated mRNA expression of Hsp superfamily with strong induction of Hsp70 family. Taken together, overall results indicate that representative organotin TBT and alternatives have potential hazardous effects on the gill of C. gigas within relatively short time period. Our results also suggest that analyzing a series of molecular and biochemical parameters can be a way of understanding and uncovering the mode of action of emerging antifouling biocides. In particular, it was revealed that Pacific oysters have different sensitivities depend on the antifouling biocides.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) promotes survival and migration of acute myeloid leukemia cells through CD63/PI3K/Akt/p21 signaling.

We and others have shown that the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1), a member of the inflammatory network exerting pleiotropic effects in the bone marrow (BM) microenvironment, regulates the survival and proliferation of different cell types, including normal hematopoietic progenitor cells. Moreover, TIMP-1 has been shown to be involved in cancer progression. However, its role in leukemic microenvironment has not been addressed. Here, we investigated the activity of TIMP-1 on Acute Myelogenous Leukemia (AML) cell functions. First, we found that TIMP-1 levels were increased in the BM plasma of AML patients at diagnosis. In vitro, recombinant human (rh)TIMP-1 promoted the survival and cell cycle S-phase entry of AML cells. These kinetic effects were related to the downregulation of cyclin-dependent kinase inhibitor p21. rhTIMP-1 increases CXCL12-driven migration of leukemic cells through PI3K signaling. Interestingly, activation of CD63 receptor was required for TIMP-1's cytokine/chemokine activity. Of note, rhTIMP-1 stimulation modulated mRNA expression of Hypoxia Inducible Factor (HIF)-1α, downstream of PI3K/Akt activation. We then co-cultured AML cells with normal or leukemic mesenchymal stromal cells (MSCs) to investigate the interaction of TIMP-1 with cellular component(s) of BM microenvironment. Our results showed that the proliferation and migration of leukemic cells were greatly enhanced by rhTIMP-1 in presence of AML-MSCs as compared to normal MSCs. Thus, we demonstrated that TIMP-1 modulates leukemic blasts survival, migration and function via CD63/PI3K/Akt/p21 signaling. As a “bad actor” in a “bad soil”, we propose TIMP-1 as a potential novel therapeutic target in leukemic BM microenvironment.

Pharmacokinetics, toxicity, and cytochrome P450 modulatory activity of plumbagin.

BackgroundThe antimalarial activity of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naturally occurring naphthoquinone widely distributed in the Plumbaginaceae family has previously been demonstrated in vitro (good activity) and in vivo (weak activity). The aim of the study was to investigate the pharmacokinetic profile following a single oral dosing to explain inconsistency of results of the in vitro and in vivo antimalarial activities. In addition, toxicity profiles and potential of modulation of cytochrome P450 enzymes (CYP1A2 and CYP3A11) were also investigated.MethodsThe pharmacokinetics and toxicity of plumbagin were investigated in rats. The propensity of plumbagin to modulate the mRNA expression and activities of the two inducible forms of hepatic drug metabolizing enzyme cytochrome P450 (CYP450), i.e., CYP1A2 and CYP3A11, was investigated using microsomes prepared from mouse livers.ResultsAcute and subacute toxicity tests indicate low toxicity of plumbagin with maximum tolerated doses of 150 (single oral dose) and 25 (daily doses for 28 days) mg/kg body weight, respectively. The pharmacokinetic profile of plumbagin following a single oral dose of 100 mg/kg body weight suggests that delayed absorption and short residence time (median values of time to maximal concentration and elimination half-life = 9.63 and 5.0 h, respectively) in plasma. Plumbagin did not modulate mRNA expression and activities of CYP1A2 and CYP3A11.ConclusionsPlumbagin was well tolerated following oral dose administration in rats. Pharmacokinetic property of this compound may be a limiting factor that explains the weak antimalarial activity of plumbagin observed in animal models. Potential metabolic interaction with co-administered drugs that are metabolized by CYP1A2 or CYP3A11 are unlikely.

Activation of G protein-coupled estrogen receptor 1 mimics, but does not mediate, the anti-proliferative action of estradiol on pituitary lactotrophs in primary culture.

Estrogen binds to nuclear estrogen receptors (ERs) to modulate transcription of target genes in estrogen-responsive cells. However, recent studies have shown that estrogen also binds to cytoplasmic membrane ERs to modulate protein kinase signaling cascades, leading to non-genomic actions. We investigated whether either nuclear or membrane ERs, including G protein-coupled estrogen receptor 1 (Gper1), mediate the inhibitory action of estrogen on insulin-like growth factor-1 (IGF-1)-induced proliferation of pituitary lactotrophs in primary culture. The cytoplasmic membrane-impermeable bovine serum albumin-conjugated estradiol (BSA-E2) at 1 nM, an equimolar concentration at which 17β-estradiol (E2) exerts anti-proliferative effects, did not inhibit IGF-1-induced lactotroph proliferation. In contrast, diethylstilbestrol, which is known to selectively activate nuclear ERs but not membrane ERs, inhibited IGF-1-induced proliferation and modulated mRNA expression of estrogen-responsive genes to a similar degree as E2. Activation of Gper1 by its agonist G-1 inhibited IGF-1-induced proliferation in a dose-dependent manner, but it had little effect on modulation of mRNA expression of estrogen-responsive genes. However, blockade of Gper1 by its antagonist G-15 did not affect the inhibitory action of E2 on IGF-1-induced proliferation. Here, we demonstrate that E2 inhibition of lactotroph proliferation is due to nuclear ER-mediated genomic action. Our results suggest that activation of Gper1 mimics, but does not mediate, the anti-proliferative action of E2 on lactotrophs.

Substrate stiffness modulates mRNA expression profiling in breast cancer cells.

Identifying effective targets induced by ECM stiffness is of critical importance for treating metastatic cancer diseases, which are followed by changes in the mechanical microenvironment in cancer cells. In this study, polyacrylamide hydrogel substrates with different stiffnesses were prepared and mRNA microarrays were performed to analyze the mRNA expression profiles in breast cancer cell line SK-BR-3 grown on different stiffness substrates. The results indicated that the expressions of 1831 genes were changed significantly in the SK-BR-3 cells on the different stiffness substrates. GO and KEGG pathway analyses of the differently expressed genes in five significant profiles annotated that the most significant pathways were cell cycle, ubiquitin mediated proteolysis RNA transport and pathways in cancer. Finally, the network of genes and gene interaction based on these differently expressed genes was established, and the phosphorylation of AKT and ERK, respectively the downstreams of the PI3K and Ras signal pathways, was further validated. The genes identified in this study may represent good therapeutic targets, and further study of these targets may help to increase our understanding of the mechanisms underlying the pathological processes and therapy for metastatic breast cancer disease.

Roles of 5-HT1A receptor in the expression of AMPA receptor and BDNF in developing mouse cortical neurons

The possible interactions between serotonergic and glutamatergic systems during neural development and under the pathogenesis of depression remain unclear. We now investigated roles of 5-HT1A receptor in the mRNA expression of AMPA receptor subunits (GluR1 and GluR2) and brain-derived neurotrophic factor (BDNF) using primary culture of cerebral cortex of mouse embryos. Neurons at embryonic day 18 were cultured for 3days or 14days and then treated with 5-HT1A receptor agonist (8-OH-DPAT) for 3h or 24h. In neurons cultured for 3 days, 8-OH-DPAT treatment for both 3h and 24h increased the mRNA levels of BDNF and GluR1, but not GluR2. In neurons cultured for 14 days, however, 8-OH-DPAT had no effects on these mRNA levels. Next, we examined in vivo roles of 5-HT1A receptor by administration of 8-OH-DPAT to newborn mice. Twenty-four hours after the oral administration of 8-OH-DPAT, the mRNA expression of BDNF was decreased in the frontal cortex, but had no effects on the mRNA expression of GluR1 and GluR2. Taken together, the present study suggests that 5-HT1A receptor activation modulates mRNA expression of AMPA receptor subunit and BDNF in cortical neurons, and the effects are different between in vitro and in vivo.

The effects of crocin (active contstituent of saffron) treatment on brain antioxidant enzyme mRNA levels in diabetic rats / Diyabetik ratlarda safranın aktif içeriği olan krosin tedavisinin beyindeki antioksidan enzimlerin mRNA seviyeleri üzerine etkisi

Abstract Objective: The aim of the present study is to evaluate the effect of crocin on mRNA expression of antioxidant enzymes, SOD, CAT and GPX in the brain of the STZ induced diabetic rats. Methods: Thirty animals randomized in three groups containing ten animals in each group as follows; control (non-diabetic rats), DM (STZ-induced untreated diabetic rats), DM+crocin (STZ-induced diabetic rats treated with crocin,). Crocin was given at a dose of 20 mg/kg bw/day by gavage for 21 days. Results: STZ injection caused a significant increase in mRNA expression of antioxidant enzymes, SOD, CAT and GPX when compared to control group. Crocin given to diabetic rats significantly decreased mRNA expression of antioxidant enzymes, SOD, CAT and GPX when compared to DM group. Conclusion: The present study demonstrates that crocin can modulate mRNA expression of antioxidant enzymes, SOD, CAT and GPX and oxidative stress in the brain of the STZ induced diabetic rats.

The Functional Haplotypes of CHRM3 Modulate mRNA Expression and Associate with Bladder Cancer among a Chinese Han Population in Kaohsiung City.

Bladder cancer is one of the major cancer types and both environmental factors and genetic background play important roles in its pathology. Kaohsiung is a high industrialized city in Taiwan, and here we focused on this region to evaluate the genetic effects on bladder cancer. Muscarinic acetylcholine receptor M3 (CHRM3) was reported as a key receptor in different cancer types. CHRM3 is located at 1q42-43 which was reported to associate with bladder cancer. Our study attempted to delineate whether genetic variants of CHRM3 contribute to bladder cancer in Chinese Han population in south Taiwan. Five selected SNPs (rs2165870, rs10802789, rs685550, rs7520974, and rs3738435) were genotyped for 30 bladder cancer patients and 60 control individuals and genetic association studies were performed. Five haplotypes (GTTAT, ATTGT, GCTAC, ACTAC, and ACCAC) were found significantly associated with low CHRM3 mRNA level and contributed to increased susceptibility of bladder cancer in Kaohsiung city after rigid 10000 consecutive permutation tests. To our knowledge, this is the first genetic association study that reveals the genetic contribution of CHRM3 gene in bladder cancer etiology.

A112: Vascular endothelial growth factor (VEGF) modulates functional activity of murine peritoneal macrophages in vitro

Recruitment of mononuclear phagocytes from the blood into tissues is considered to be a crucial process during inflammatory reactions, wound healing and tumor growth. Macrophages are known to reveal high plasticity and may change under the influence of microenvironment. The aim of the study was to evaluate the changes of macrophage functional activity under the influence of Vascular endothelial growth factor (VEGF) in vitro. This factor is known to be the main angiogenic factor but also possesses several immunomodulatory properties. Here we report that VEGF revealed a dose-dependent effects on cultured freshly isolated murine resident peritoneal macrophages: modulated iNOS mRNA expression, nitroxide and superoxide anion production, decreased 5′-nucleotidase (5′-N) activity, but had no influence on fluid-phase pinocytosis. Moreover, VEGF increased expression of its own mRNA via autocrine pathway as well as of VEGF protein expression. VEGF also induced up-regulation of extracellular matrix protein thrombospondin-1 (TSP-1) mRNA, which is considered as a part of macrophage activation phenotype. Production of cytokines and chemokines by macrophages was screened with the help of Multi- analyte ELISArray kits. It was found that incubation of macrophages in the presence of VEGF increased the production of angiogenic cytokines – TNF-α and IL-6 as well as several monocyte and leukocyte chemoattractants such as MCP-1, RANTES and MIP-1β. Therefore, we suggest that locally established VEGF gradient may influence inflammatory phenotype of tissue macrophages as well as potentiate their pro-angiogenic properties. This work was supported by Russian Foundation for Basic Research – Russia, Grant No. 15-04-06150 .

Abstract B22: Inhibition of c-MYC expression in oral squamous carcinoma cells contributes to the antiproliferative activity of the alkylphosphocholine erufosine

Abstract Erufosine is a third generation alkylphosphocholine, which has been shown to inhibit the proliferation of oral squamous cell carcinoma (OSCC) cells. It simultaneously induces apoptosis and autophagy by modulating the Akt–mTOR signaling pathway and is known to down regulate the expression of cell cycle related proteins, but the exact mechanism of action of this membrane seeking compound is still unknown. For investigating the mechanism of action in a systematic way, we studied the effects of erufosine in a panel of OSCC cell lines (Cal-27, FaDu and HN-5). The concentration effecting the 50% inhibition in cell proliferation (IC50) in response to erufosine was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using concentrations ranging from 0.6µM – 100µM at various time points (24h, 48h and 72h) after exposure. Based on these results, FaDu cells were exposed to erufosine at concentrations effecting IC25, IC50 and IC75, and then the mRNA of respectively treated cells was isolated and subjected to microarray analysis by Illumina Chip array. Selected genes with highly modulated mRNA expression were further investigated by quantitative RT-PCR and Western Blot. IC50 values in response to erufosine, following exposure for 24h, 48h and 72h exposure ranged from 20µM to 9.7µM for Cal-27 -, 27µM to 14µM for FaDu - , and 43µM to 37µM for HN-5 cells. Analysis of microarray data by the Ingenuity Pathways Analysis (IPA; Ingenuity® Systems, www.ingenuity.com) software revealed that 725 genes were modulated at 16h, averaged for all concentrations, with 47 genes being modulated more than 3 fold. The respective 24h gene expression analysis revealed that 1215 genes were significantly modulated, with 77 genes deregulated over 3 fold, whereas a total of 1305 genes were altered at the end of 48h, with 93 genes being deregulated over 3 fold. In a first approach, we concentrated on genes related to the c-MYC signaling pathway. The cyclin-dependent kinase inhibitor 1A (p21, Cip1) was found to be upregulated 9.2, 7.9 and 9.9 fold, averaged for all concentrations, at 16h, 24h and 48h, respectively. The Max dimerization protein 1 (MDX1) was upregulated 3.7 fold and 3.3 fold over the untreated control at 24h and 48h post treatment. The combined modulation of the two mRNA species hints at the involvement of c-MYC as a target of erufosine treatment. These results were further validated by qRT-PCR, which also confirmed the modulation of these genes across the OSCC cell panel. In line with this, the Western blots also showed a clear decrease in c-MYC protein levels and a corresponding increase in p21 in a concentration- and time-dependent manner. In summary, the known effect of erufosine on cell cycle related proteins as well as the increased mRNA (p21, MDX-1) and protein (p21) expression reported here is in accordance with the decreased expression of c-MYC at protein level. These findings support the hypothesis that c-MYC is an indirect target of erufosine in OSCC cells. Citation Format: Shariq S. Ansari, Martin R. Berger. Inhibition of c-MYC expression in oral squamous carcinoma cells contributes to the antiproliferative activity of the alkylphosphocholine erufosine. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B22.

Differential gene expression in two contrasting wheat cultivars under cadmium stress

The present study investigated differences in cadmium resistance of two wheat (Triticum aestivum L.) cultivars. The cvs. RAJ 4161 (Cd resistant) and PBW 343 (Cd sensitive) were treated with 200 mg(Cd) kg−1(soil) for 3, 5, 7, and 10 d. The effect of the Cd stress was estimated by measuring growth parameters, accumulation of cadmium, sulphur, and glutathione (GSH), and by expression of some defence genes [phytochelatin synthase (PCS), glutathione reductase (GR), and ascorbate peroxidase (APX)]. The Cd treatment resulted in a significant reduction in plant growth and in an increase in the accumulation of S and GSH. Further, the expressions of PCS, GR, and APX were also mostly enhanced. The PCS was upregulated significantly in roots of RAJ 4161 (0.6-fold) and downregulated (0.9-fold) in PBW 343 on day 3 of the Cd treatment. In RAJ 4161, the expressions of APX and GR recorded a maximum increase of 2.1- and 2.4-fold in roots and leaves, respectively, after 10 d of the stress. The results show that a different ability of RAJ 4161 and PBW 343 to modulate mRNA expression after the Cd treatment was related to their Cd tolerance.

Effects of chitosan on nitric oxide production and inducible nitric oxide synthase activity and mRNA expression in weaned piglets

The effects of chitosan on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity and gene expression in vivo or vitro were investigated in weaned piglets. In vivo, 180 weaned piglets were assigned to five dietary treatments with six replicates. The piglets were fed on a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. In vitro, the peripheral blood mononuclear cells (PBMCs) from a weaned piglet were cultured respectively with 0 (control), 40, 80, 160, and 320 µg chitosan/ml medium. Results showed that serum NO concentrations on days 14 and 28 and iNOS activity on day 28 were quadratically improved with increasing chitosan dose (P < 0.05). The iNOS mRNA expressions were linearly or quadratically enhanced in the duodenum on day 28, and were improved quadratically in the jejunum on days 14 and 28 and in the ileum on day 28 (P < 0.01). In vitro, the NO concentrations, iNOS activity, and mRNA expression in unstimulated PBMCs were quadratically enhanced by chitosan, but the improvement of NO concentrations and iNOS activity by chitosan were markedly inhibited by N-(3-[aminomethyl] benzyl) acetamidine (1400w) (P < 0.05). Moreover, the increase of NO concentrations, iNOS activity, and mRNA expression in PBMCs induced by lipopolysaccharide (LPS) were suppressed significantly by chitosan (P < 0.05). The results indicated that the NO concentrations, iNOS activity, and mRNA expression in piglets were increased by feeding chitosan in a dose-dependent manner. In addition, chitosan improved the NO production in unstimulated PBMCs but inhibited its production in LPS-induced cells, which exerted bidirectional regulatory effects on the NO production via modulated iNOS activity and mRNA expression.

MicroRNAs in chicken eggs are bioavailable in healthy adults and can modulate mRNA expression in peripheral blood mononuclear cells.

Previous research in our lab demonstrated the bioavailability of milk-borne microRNAs following milk consumption by healthy adults. An important question left unanswered from our previous work was if this phenomenon is unique to milk or if other foods could also be a source of exogenous microRNA. To explore this possibility, we tested the hypothesis that chicken egg microRNAs are bioavailable following consumption of hard boiled eggs by healthy adult humans. Plasma levels of miR-181a and miR-181b increased 9 hours after egg consumption to 146% and 304% above baseline levels, respectively (as per qRT-PCR). Abundance of miR-181b is 223% higher than miR-181a in eggs, suggesting that the increase in plasma microRNAs depended on the dose consumed. Importantly, the plasma concentrations of the chicken-specific microRNA, gga-miR-1451-5p, increased from below detection limit at hour zero to 1 fmol/L at hour nine. Expression of BCL2A1, an experimentally validated target of miR-181b, was 16% lower in human lymphocytes 9 hours after egg consumption. A bioinformatics analysis showed chicken egg microRNAs target at least 28 KEGG pathways. We conclude that chicken egg microRNAs are bioavailable and have the potential to influence endogenous gene expression in humans following consumption of physiologically relevant doses of eggs. Supported by USDA/NIFA, USDA/Egg Nutrition Center, NIH 1P20GM104320.

Cyclosporin A induces cardiac differentiation but inhibits hemato-endothelial differentiation of P19 cells.

Little is known about the mechanisms underlying the effects of Cyclosporin A (CsA) on the fate of stem cells, including cardiomyogenic differentiation. Therefore, we investigated the effects and the molecular mechanisms behind the actions of CsA on cell lineage determination of P19 cells. CsA induced cardiomyocyte-specific differentiation of P19 cells, with the highest efficiency at a concentration of 0.32 μM during embryoid body (EB) formation via activation of the Wnt signaling pathway molecules, Wnt3a, Wnt5a, and Wnt8a, and the cardiac mesoderm markers, Mixl1, Mesp1, and Mesp2. Interestingly, cotreatment of P19 cells with CsA plus dimethyl sulfoxide (DMSO) during EB formation significantly increases cardiac differentiation. In contrast, mRNA expression levels of hematopoietic and endothelial lineage markers, including Flk1 and Er71, were severely reduced in CsA-treated P19 cells. Furthermore, expression of Flk1 protein and the percentage of Flk1+ cells were severely reduced in 0.32 μM CsA-treated P19 cells compared to control cells. CsA significantly modulated mRNA expression levels of the cell cycle molecules, p53 and Cyclins D1, D2, and E2 in P19 cells during EB formation. Moreover, CsA significantly increased cell death and reduced cell number in P19 cells during EB formation. These results demonstrate that CsA induces cardiac differentiation but inhibits hemato-endothelial differentiation via activation of the Wnt signaling pathway, followed by modulation of cell lineage-determining genes in P19 cells during EB formation.

Interaction between microRNAs and OCT4

OCT4基因是POU转录因子家族中的一员，它能与含八聚体基序（ATGCAAAT）的DNA结合。OCT4是一个关键的转录因子，在未分化胚胎干细胞中参与维持多能性和自我更新性，在许多种癌症包括肺癌、生殖细胞肿瘤、乳腺癌、宫颈癌、前列腺癌、胃癌、肝癌和卵巢癌中过表达。MicroRNAs（miRNAs）是一种小的非编码RNA，通过和靶基因mRNA碱基配对来调控mRNA表达，降解mRNA或阻碍蛋白合成。一些miRNAs被证实在癌细胞中调控干细胞因子如OCT4、NANOG、SOX2和KLF4，进而调控癌细胞的增殖、凋亡、分化、抗药性和免疫性。