Cloning and function analysis of promoter of DcCDPK8 from Dendrobium catenatum / 中国中药杂志

DcCDPK8 involved in abiotic stress such as low temperature and signal transduction of hormones ABA and MeJA,but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism,the DcCDPK8 gene promoter sequence was cloned by PCR from D. catenatum. Promoter sequence function was studied by fusion of 5 'terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element( LTR) between~(-1) 749 bp and-614 bp,two MeJA responsive elements between~(-1) 749 bp and-230 bp,and one ABA responsive elements between-614 bp and-230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS,which were transformed into tobacco leaves. The GUS activity under cold stress treatment was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. GUS activity under exogenous ABA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3,and GUS activity under exogenous MeJA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. It is speculated that the ABA response element( ARE) in the promoter sequences of DcCDPK8 is positive regulatory role in response to exogenous ABA,the MeJA cis-acting element plays a negative role in response to exogenous MeJA.

Renovación contextual de la distribución temporal del responder / Contextual Renewal of Temporal Patterns of Responding

Resumen Se ha reportado que en ciertos fenómenos (recuperación espontánea, readquisición, resurgencia y renovación contextual) es posible observar la recuperación de una respuesta previamente extinguida. La renovación contextual, se refiere a la reaparición de una respuesta extinguida que resulta de un cambio de contexto entre la fase de extinción y prueba. El propósito del presente trabajo consistió en evaluar la renovación contextual, atendiendo tanto a la distribución temporal del responder, como a la tasa de respuesta. Se encontró una recuperación de la tasa de respuesta, siendo un resultado consistente con los reportados en el área de renovación contextual. Además, se pudo identificar una renovación de la distribución temporal de respuesta. Los resultados se discuten reconociendo que la tasa y la distribución temporal constituyen dos dimensiones de la ejecución que son moduladas por los cambios de contexto.

Abstract It has been reported that in certain phenomena (spontaneous recovery, reacquisition, resurgence and contextual renewal) it is possible to observe the recovery of a previously extinguished response. Contextual renewal refers to the reappearance of an extinguished response that result from a change of context between the extinction and testing phase. The purpose of the present study was to evaluate contextual renewal, considering both the temporal distribution of the responses and the response rate. A recovery of the response rate was found, being a result consistent with those reported in the area of ​​contextual renewal. In addition, a renewal of the temporal distribution of the responses could be identified. The results are discussed by recognizing that rate and temporal distribution are two behavioral dimensions that are modulated by contextual changes.

Histone deacetylase inhibition attenuates hepatic steatosis in rats with experimental Cushing's syndrome

Cushing's syndrome (CS) is a collection of symptoms caused by prolonged exposure to excess cortisol. Chronically elevated glucocorticoid (GC) levels contribute to hepatic steatosis. We hypothesized that histone deacetylase inhibitors (HDACi) could attenuate hepatic steatosis through glucocorticoid receptor (GR) acetylation in experimental CS. To induce CS, we administered adrenocorticotropic hormone (ACTH; 40 ng/kg/day) to Sprague-Dawley rats by subcutaneous infusion with osmotic mini-pumps. We administered the HDACi, sodium valproate (VPA; 0.71% w/v), in the drinking water. Treatment with the HDACi decreased steatosis and the expression of lipogenic genes in the livers of CS rats. The enrichment of GR at the promoters of the lipogenic genes, such as acetyl-CoA carboxylase (Acc), fatty acid synthase (Fasn), and sterol regulatory element binding protein 1c (Srebp1c), was markedly decreased by VPA. Pan-HDACi and an HDAC class I-specific inhibitor, but not an HDAC class II a-specific inhibitor, attenuated dexamethasone (DEX)-induced lipogenesis in HepG2 cells. The transcriptional activity of Fasn was decreased by pretreatment with VPA. In addition, pretreatment with VPA decreased DEX-induced binding of GR to the glucocorticoid response element (GRE). Treatment with VPA increased the acetylation of GR in ACTH-infused rats and DEX-induced HepG2 cells. Taken together, these results indicate that HDAC inhibition attenuates hepatic steatosis hrough GR acetylation in experimental CS.

The Memory-Enhancing Effects of Liquiritigenin by Activation of NMDA Receptors and the CREB Signaling Pathway in Mice

Liquiritigenin (LQ) is a flavonoid that can be isolated from Glycyrrhiza radix. It is frequently used as a tranditional oriental medicine herbal treatment for swelling and injury and for detoxification. However, the effects of LQ on cognitive function have not been fully explored. In this study, we evaluated the memory-enhancing effects of LQ and the underlying mechanisms with a focus on the N-methyl-D-aspartic acid receptor (NMDAR) in mice. Learning and memory ability were evaluated with the Y-maze and passive avoidance tests following administration of LQ. In addition, the expression of NMDAR subunits 1, 2A, and 2B; postsynaptic density-95 (PSD-95); phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII); phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2); and phosphorylation of cAMP response element binding (CREB) proteins were examined by Western blot. In vivo, we found that treatment with LQ significantly improved memory performance in both behavioral tests. In vitro, LQ significantly increased NMDARs in the hippocampus. Furthermore, LQ significantly increased PSD-95 expression as well as CaMKII, ERK, and CREB phosphorylation in the hippocampus. Taken together, our results suggest that LQ has cognition enhancing activities and that these effects are mediated, in part, by activation of the NMDAR and CREB signaling pathways.

TGF-β induces Smad2 Phosphorylation, ARE Induction, and Trophoblast Differentiation

BACKGROUND: Transforming growth factor beta (TGF-β) signaling has been shown to control a large number of critical cellular actions such as cell death, differentiation, and development and has been implicated as a major regulator of placental function. SM10 cells are a mouse placental progenitor cell line, which has been previously shown to differentiate into nutrient transporting, labyrinthine-like cells upon treatment with TGF-β. However, the signal transduction pathway activated by TGF-β to induce SM10 progenitor differentiation has yet to be fully investigated. MATERIALS AND METHODS: In this study the SM10 labyrinthine progenitor cell line was used to investigate TGF-β induced differentiation. Activation of the TGF-β pathway and the ability of TGF-β to induce differentiation were investigated by light microscopy, luciferase assays, and Western blot analysis. RESULTS AND CONCLUSIONS: In this report, we show that three isoforms of TGF-β have the ability to terminally differentiate SM10 cells, whereas other predominant members of the TGF-β superfamily, Nodal and Activin A, do not. Additionally, we have determined that TGF-β induced Smad2 phosphorylation can be mediated via the ALK-5 receptor with subsequent transactivation of the Activin response element. Our studies identify an important regulatory signaling pathway in SM10 progenitor cells that is involved in labyrinthine trophoblast differentiation.

The Regulatory Effects of Trans-chalcone on Adipogenesis

It is noted that chalcone derivatives have characteristic diverse pharmacological properties, and that precise evidence has been growing that they could regulate a tumor necrosis factor-α (TNF-α) induced insulin resistance. The purpose of the present investigation is to elucidate the effects of the identified chalcone derivatives on adipogenesis, and to find the underlying mechanism of action in that case. Consequently, we first investigated whether the chalcone derivatives could affect the identified PPARγ-induced transcriptional activity on the proliferator-activated receptor response elements (PPRE) at target promoters, and find that trans-chalcone most significantly increased the PPARγ-induced transcriptional activity. Additionally, we confirmed that there were up-regulatory effects of trans-chalcone during the adipogenesis and lipid accumulation, and on the mRNA of adipogenic factors in 3T3-L1 cells. Next, we examined the effect of trans-chalcone on the inhibition induced by TNF-α on adipogenesis. To that end, we noted that the treatment with trans-chalcone attenuated the effect of TNF-α mediated secretion of various adipokines that are involved in insulin sensitivity. For this reason, we noted that this study clearly demonstrates that trans-chalcone enhanced adipogenesis, in part, by its potent effect on PPARγ activation and by its reverse effect on TNF-α.

Novos reguladores de resposta envolvidos na virulência de Pseudomonas aeruginosa / New response regulators involved in Pseudomonas aeruginosa virulence

Os sistemas de sinalização de dois componentes são sistemas prevalentes em bactérias, permitindo a adaptação a diferentes condições ambientais. O sistema de dois componentes classicamente possui uma proteína histidina quinase, o primeiro componente, capaz de reconhecer o estímulo ambiental e fosforilar o regulador de resposta, o segundo componente. Pseudomonas aeruginosa é uma proteobactéria ubíqua, capaz de infectar hospedeiros filogeneticamente distintos. Esse patógeno oportunista apresenta um dos maiores conjuntos de sistemas de dois componentes em bactérias, que permite que ela sobreviva numa grande gama de ambientes, incluindo humanos. P. aeruginosa UCBPP-PA14 apresenta pelo menos 64 histidina quinases e 76 reguladores de resposta codificados em seu genoma. Diversos sistemas de dois componentes já foram correlacionados com a virulência, sendo o sistema GacSA o exemplo melhor caracterizado. Há poucos estudos sistemáticos sobre o envolvimendo dos reguladores de resposta na virulência de P. aeruginosa e os sinais que induzem a ativação dos reguladores de resposta precisam ser encontrados. Para identificar novos reguladores de resposta envolvidos na patogenicidade, infecções in vitro em macrófagos e in vivo em Drosophila melanogaster foram realizadas neste trabalho. Os macrófagos foram infectados com cada mutante dos reguladores de resposta ou com a linhagem selvagem, e a produção da citocina pró-inflamatória TNF-α e o clearance bacteriano foram determinados. Alternativamente, as moscas foram infectadas utilizando-se a estratégia de feeding e a sobrevivência foi verificada. Utilizando-se essas abordagens, a identificação de diversos reguladores de resposta com papel na virulência foi alcançada, além de se corfirmar o papel de reguladores de resposta já estudados. Um dos novos genes envolvidos em virulência, PA14_26570 (nomeado neste trabalho de atvR), codifica um regulador de resposta atípico com substituição no aspartato fosforilável para glutamato, o que usualmente induz um estado sempre ativo. Um mutante não polar em atvR foi construído e macrófagos infectados com a linhagem ΔatvR confirmaram um maior clearance bacteriano e maior produção de TNF-α em comparação aos macrófagos infectados com a linhagem selvagem. Para comprovar a participação de AtvR durante a patogênese, um modelo de pneumonia aguda em camundongos foi utilizado. Camundongos infectados com a linhagem ΔatvR apresentaram uma maior sobrevivência em comparação aos camundongos infectados com a linhagem selvagem. Além disso, os camundongos infectados com ΔatvR apresentaram menor carga bacteriana, aumento no recrutamento de neutrófilos ativados e aumento na produção de citocinas pró-inflamatórias (TNF-α e IFN-γ). Utilizando-se uma abordagem transcritômica (RNA-Seq), foi determindo diversos genes são regulados positivamente na linhagem superexpressando AtvR em relação à linhagem controle. Dentre esses, os clusters de respiração anaeróbia nar, nir, nor e nos estão incluídos. Esse resultado foi confirmado por qRT-PCR e análises fenotípicas, em que a linhagem ΔatvR apresentou menor crescimento e expressão da nitrato redutase durante condições de hipóxia em comparação à linhagem selvagem. Em suma, neste trabalho foi demonstrado que diversos reguladores de resposta são importantes para a virulência de P. aeruginosa em macrófagos in vitro e in vivo em Drosophila, além de caracterizar o regulador de resposta atípico AtvR, que regula a respiração anaeróbica por desnitrificação, permitindo que P. aeruginosa possa infectar e colonizar o hospedeiro com maior eficiência

Two-component systems are widespread in bacteria, allowing the adaptation to environmental changes. A two-component system is classically composed by a sensor kinase that phosphorylates a cognate response regulator. Pseudomonas aeruginosa is a ubiquitous proteobacterium able to cause disease in several hosts. This opportunistic pathogen presents one of the largest sets of two-component systems known in bacteria, which certainly contributes to its ability to thrive in a wide range of environmental settings, including humans. P. aeruginosa UCBPP-PA14 genome codes for at least 64 sensor kinases and 76 response regulators. Some response regulators are already known to be related to virulence, with the GacSA system as the best characterized. There are no systematic studies about the involvement of P. aeruginosa response regulators in virulence. Moreover, the input signal that triggers the response regulator activation is yet to be uncovered for most systems. To find new response regulators involved in virulence, in vitro infections werecarried out using macrophages. Briefly, the macrophages were infected with each response regulator mutant or the wild-type strain, the pro-inflammatory cytokine production (TNF-α) and the bacterial clearance were evaluated. Using this approach, we identified several response regulators involved in virulence, and we also confirmed the involvement of known response regulators in this process. One of the novel virulence-related response regulators, PA14_26570 (named here as AtvR), is an atypical response regulator with a substitution in the phosphorylable aspartate to glutamate, that usually leads to an always-on state. A non-polar mutant was constructed, and macrophage infection with ΔatvR confirmed an increased bacterial clearance as well as a higher TNF-α production as compared to the wild-type strain. To ascertain the role of AtvR during the pathogenic process, an acute pneumonia model was used. Mice infected with ΔatvR showed an increased survival as compared to mice infected with the wildtype strain. In addition, ΔatvR infected mice showed reduced bacterial burden, increased neutrophil recruitment and activation, as well as increased pro-inflammatory cytokine production (TNF-α and IFN-γ). Also, using a transcriptomic approach (RNASeq), we showed that several genes were upregulated in the strain overexpressing AtvR. These genes include the anaerobic respiration clusters nar, nir, nor and nos. This result was confirmed by qRT-PCR and phenotypic analysis, in which ΔatvR showed reduced growth and nitrate reductase expression during hypoxic conditions as compared to the wild-type strain. In conclusion, we have demonstrated that several response regulators are important for P. aeruginosa virulence in vitro. In addition, we further characterized the atypical response regulator AtvR, which regulates anaerobic respiration via denitrification, allowing this bacterium to infect and colonize the host more efficiently

The role of hypoxia on the acquisition of epithelial-mesenchymal transition and cancer stemness: a possible link to epigenetic regulation

A hypoxic microenvironment leads to cancer progression and increases the metastatic potential of cancer cells within tumors via epithelial-mesenchymal transition (EMT) and cancer stemness acquisition. The hypoxic response pathway can occur under oxygen tensions of < 40 mmHg through hypoxia-inducible factors (HIFs), which are considered key mediators in the adaptation to hypoxia. Previous studies have shown that cellular responses to hypoxia are required for EMT and cancer stemness maintenance through HIF-1α and HIF-2α. The principal transcription factors of EMT include Twist, Snail, Slug, Sip1 (Smad interacting protein 1), and ZEB1 (zinc finger E-box-binding homeobox 1). HIFs bind to hypoxia response elements within the promoter region of these genes and also target cancer stem cell-associated genes and mediate transcriptional responses to hypoxia during stem cell differentiation. Acquisition of stemness characteristics in epithelial cells can be induced by activation of the EMT process. The mechanism of these phenotypic changes includes epigenetic alterations, such as DNA methylation, histone modification, chromatin remodeling, and microRNAs. Increased expression of EMT and pluripotent genes also play a role through demethylation of their promoters. In this review, we summarize the role of hypoxia on the acquisition of EMT and cancer stemness and the possible association with epigenetic regulation, as well as their therapeutic applications.

Reversion of BDNF, Akt and CREB in Hippocampus of Chronic Unpredictable Stress Induced Rats: Effects of Phytochemical, Bacopa Monnieri

OBJECTIVE: The aims of the present study were to explore the behavioural effects and to understand the possible mode of action of Bacopa monnieri extract (BME) on chronic unpredictable stress (CUS) induced depressive model and the biochemical alterations such as brain derived neurotrophic factor (BDNF), Akt, cyclic-AMP response element binding (CREB) protein level in the hippocampus of rats. METHODS: We examined the effects of chronic administration of BME on CUS exposed rats for 28 days. Behavioural changes were assessed by sucrose consumption and open field test to assess the effect of BME on CUS-induced depression. The mechanisms underlying antidepressant like action of BME was further evaluated by measuring levels of BDNF, Akt, and CREB in the hippocampus of rat brain and compared with the standard tricyclic antidepressant drug imipramine (20 mg/kg body weight). RESULTS: Exposure to CUS for 28 days produced depression-like behavior in rats, as indicated by significant decreases in sucrose consumption, locomotor activity including decreased BDNF, Akt and CREB levels in the hippocampus. Daily administration of BME at a dose of (80 mg/kg body weight) significantly reverses the behavioral alteration and restored the normal level of BDNF, total and phospho-Akt, total and phospho CREB in the hippocampus of CUS induced rats as compared to vehicle treated control rats. CONCLUSION: These findings suggest that BME ameliorates CUS induced behavioural depression in rats and that can be used as a potent therapeutic agent in treating depressive like behavior.

Lonchocarpine Increases Nrf2/ARE-Mediated Antioxidant Enzyme Expression by Modulating AMPK and MAPK Signaling in Brain Astrocytes

Lonchocarpine is a phenylpropanoid compound isolated from Abrus precatorius that has anti-bacterial, anti-inflammatory, antiproliferative, and antiepileptic activities. In the present study, we investigated the antioxidant effects of lonchocarpine in brain glial cells and analyzed its molecular mechanisms. We found that lonchocarpine suppressed reactive oxygen species (ROS) production and cell death in hydrogen peroxide-treated primary astrocytes. In addition, lonchocarpine increased the expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), and manganese superoxide dismutase (MnSOD), which are all under the control of Nrf2/antioxidant response element (ARE) signaling. Further, mechanistic studies showed that lonchocarpine increases the nuclear translocation and DNA binding of Nrf2 to ARE as well as ARE-mediated transcriptional activities. Moreover, lonchocarpine increased the phosphorylation of AMP-activated protein kinase (AMPK) and three types of mitogen-activated protein kinases (MAPKs). By treating astrocytes with each signaling pathway-specific inhibitor, AMPK, c-jun N-terminal protein kinase (JNK), and p38 MAPK were identified to be involved in lonchocarpine-induced HO-1 expression and ARE-mediated transcriptional activities. Therefore, lonchocarpine may be a potential therapeutic agent for neurodegenerative diseases that are associated with oxidative stress.

Integrated computational approach to the analysis of RNA-seq data reveals new transcriptional regulators of psoriasis

Psoriasis is a common inflammatory skin disease with complex etiology and chronic progression. To provide novel insights into the regulatory molecular mechanisms of the disease, we performed RNA sequencing analysis of 14 pairs of skin samples collected from patients with psoriasis. Subsequent pathway analysis and extraction of the transcriptional regulators governing psoriasis-associated pathways was executed using a combination of the MetaCore Interactome enrichment tool and the cisExpress algorithm, followed by comparison to a set of previously described psoriasis response elements. A comparative approach allowed us to identify 42 core transcriptional regulators of the disease associated with inflammation (NFκB, IRF9, JUN, FOS, SRF), the activity of T cells in psoriatic lesions (STAT6, FOXP3, NFATC2, GATA3, TCF7, RUNX1), the hyperproliferation and migration of keratinocytes (JUN, FOS, NFIB, TFAP2A, TFAP2C) and lipid metabolism (TFAP2, RARA, VDR). In addition to the core regulators, we identified 38 transcription factors previously not associated with the disease that can clarify the pathogenesis of psoriasis. To illustrate these findings, we analyzed the regulatory role of one of the identified transcription factors (TFs), FOXA1. Using ChIP-seq and RNA-seq data, we concluded that the atypical expression of the FOXA1 TF is an important player in the disease as it inhibits the maturation of naive T cells into the (CD4+FOXA1+CD47+CD69+PD-L1(hi)FOXP3−) regulatory T cell subpopulation, therefore contributing to the development of psoriatic skin lesions.

Effect of estrogen on iron metabolism in mammals / 生理学报

Estrogen is a steroid hormone produced mainly by the ovaries. It combines with the nuclear receptors to exert the biological effects influencing the metabolism of body. Elevated levels of estrogen are often associated with altered iron levels in mammals. Furthermore, the findings of estrogen response element (ERE) have demonstrated that estrogen affects iron metabolism directly in peripheral tissues. In this review, we will briefly summarize the effect of estrogen on iron metabolism in mammals, and discuss recent progress in the mechanisms of estrogen on some iron related proteins in order to provide guidance for clinical use of estrogen. Estrogen and iron metabolism are closely related, but the exact regulatory mechanisms still need further exploration.

miR-148a is a downstream effector of X-box-binding protein 1 that silences Wnt10b during adipogenesis of 3T3-L1 cells

Wnt10b, an endogenous inhibitor of adipogenesis, maintains preadipocytes in an undifferentiated state by suppressing adipogenic transcription factors. We have previously demonstrated that Wnt10b transcription during adipogenesis is negatively regulated by X-box-binding protein 1 (XBP1), an important transcription factor of the unfolded protein response. In this report, we demonstrate that XBP1s can directly induce the transcription of microRNA-148a, which in turn mediates the silencing of Wnt10b mRNA during adipogenic differentiation of 3T3-L1 cells. Stability of Wnt10b mRNA was found to be significantly increased by knockdown of XBP1s. Using computational algorithms, a set of microRNAs was predicted to bind Wnt10b mRNA, of which microRNA-148a was selected as a potential target for XBP1s. Our results revealed that microRNA-148a could bind to the 3′UTR of Wnt10b mRNA. Its ectopic expression significantly suppressed both Wnt10b expression and β-catenin activity. When we altered the expression of XBP1 in 3T3-L1 cells, microRNA-148a levels changed accordingly. A potential XBP1 response element was found in the promoter region of microRNA-148a, and XBP1s directly bound to this response element as shown by point mutation analysis and chromatin immunoprecipitation assay. In addition, a microRNA-148a mimic significantly restored adipogenic potential in XBP1-deficient 3T3-L1 cells. These findings provide the first evidence that XBP1s can regulate Wnt10b by a post-transcriptional mechanism through directly inducing microRNA-148a.

miR-148a is a downstream effector of X-box-binding protein 1 that silences Wnt10b during adipogenesis of 3T3-L1 cells

Wnt10b, an endogenous inhibitor of adipogenesis, maintains preadipocytes in an undifferentiated state by suppressing adipogenic transcription factors. We have previously demonstrated that Wnt10b transcription during adipogenesis is negatively regulated by X-box-binding protein 1 (XBP1), an important transcription factor of the unfolded protein response. In this report, we demonstrate that XBP1s can directly induce the transcription of microRNA-148a, which in turn mediates the silencing of Wnt10b mRNA during adipogenic differentiation of 3T3-L1 cells. Stability of Wnt10b mRNA was found to be significantly increased by knockdown of XBP1s. Using computational algorithms, a set of microRNAs was predicted to bind Wnt10b mRNA, of which microRNA-148a was selected as a potential target for XBP1s. Our results revealed that microRNA-148a could bind to the 3′UTR of Wnt10b mRNA. Its ectopic expression significantly suppressed both Wnt10b expression and β-catenin activity. When we altered the expression of XBP1 in 3T3-L1 cells, microRNA-148a levels changed accordingly. A potential XBP1 response element was found in the promoter region of microRNA-148a, and XBP1s directly bound to this response element as shown by point mutation analysis and chromatin immunoprecipitation assay. In addition, a microRNA-148a mimic significantly restored adipogenic potential in XBP1-deficient 3T3-L1 cells. These findings provide the first evidence that XBP1s can regulate Wnt10b by a post-transcriptional mechanism through directly inducing microRNA-148a.

Pigmentation Effect of Electromagnetic Fields at Various Intensities to Melanocytes

Melanogenesis is the biological process that results in the synthesis of skin pigment of melanin and it has various functions in living systems and is synthesized by the melanosome within the melanocytes. A variety of physical treatments are used to promote melanin production in the melanocytes for pigmentation control. The purpose of this study was to evaluate the intensity-dependent effect of extremely low-frequency electromagnetic fields (ELF-EMFs) on melanogenesis by melanocytes in vitro. Melanocytes were exposed to ELF-EMFs at a frequency of 50 Hz and at intensities in the range of 0.5–20 G over 4 days. The results of lactate dehydrogenase assay showed that there were no significant differences between cells exposed to 0.5 G or 2 G groups and the controls. The melanin contents increased 1.2–1.5-fold in cells exposed to ELF-EMFs and tyrosinase activity increased 1.3-fold in cells exposed to ELF-EMFs, relative to the controls. Also, exposure to ELF-EMFs was associated with activation in cyclic-AMP response element binding protein and microphthalmia-associated transcription factor (MITF) was up-regulated. Up-regulation of MITF induces the expression of melanogenesis-related markers, such as tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2. In conclusion, the present study showed that the exposure to ELF-EMFs at low intensities can stimulate melanogenesis in melanocyte, and these results may be used to a therapeutic devices for inducing repigmentation in vitiligo patients.

Role of cyclic adenosine monophosphate response element binding protein in ventricular pacing induced cardiac electrical remodeling in a canine model / 中华心血管病杂志

<p><b>OBJECTIVE</b>This project is designed to explore the potential role of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) in cardiac electrical remodeling induced by pacing at different ventricular positions in dogs.</p><p><b>METHODS</b>An animal model by implanting the pacemakers in beagles was established. According to the different pacing positions, the animals were divided into 4 groups:conditional control group (n=6), left ventricle pacing group (n=6), right ventricle pacing group (n=6) and bi-ventricle pacing group (n=6). Cardiac and electrical remodeling were observed by echocardiography, electrocardiogram and plasma BNP. Myocardial pathology and protein expression of extracellular regulated protein kinases1/2 (ERK1/2), P38 mitogen activated protein kinases (P38 MAPK) and CREB were examined at 4 weeks post pacing.</p><p><b>RESULTS</b>Cardiac structure and plasma BNP level were similar among 4 groups (all P>0.05). Electrocardiogram derived Tp-Te interval was significantly prolonged post pacing (92±11, 91±10, and 79±13 ms vs. 60±12 ms), and the Tp-Te interval in bi-ventricle pacing group was shorter than in left or right ventricle pacing group (P < 0.05). Western blot results showed that the expression of p-ERK1/2 in left ventricular myocardium of left ventricle pacing group, right ventricular myocardium of right ventricle pacing group and bi-ventricular myocardium of bi-ventricle pacing group was 2.7±0.4, 2.4±0.2, 1.7±0.1 and 1.9±0.2, respectively, the expression of p-P38 MAPK was 1.9±0.3, 1.7±0.2, 0.8±0.1 and 1.1±0.1, respectively, and the expression of p-CREB was 2.1±0.2, 2.0±0.2, 2.7±0.4 and 2.6±0.3, respectively. The p-ERK1/2 and p-P38 MAPK expression of bi-ventricle pacing group was lower,but the p-CREB expression was higher compared to the other pacing groups (P < 0.05).</p><p><b>CONCLUSIONS</b>Ventricular pacing could induce electrical remodeling evidenced by prolonged Tp-Te interval and increased phosphorylation of ERK1/2 and p38 MAPK and reduced phosphorylation of CREB. Compared with single ventricle pacing, bi-ventricle pacing could attenuate electrical remodeling in this model.</p>

Thymosin Beta-4, Actin-Sequestering Protein Regulates Vascular Endothelial Growth Factor Expression via Hypoxia-Inducible Nitric Oxide Production in HeLa Cervical Cancer Cells

Vascular endothelial growth factor (VEGF) is an important regulator of neovascularization. Hypoxia inducible nitric oxide (NO) enhanced the expression of VEGF and thymosin beta-4 (Tbeta4), actin sequestering protein. Here, we investigated whether NO-mediated VEGF expression could be regulated by Tbeta4 expression in HeLa cervical cancer cells. Hypoxia inducible NO production and VEGF expression were reduced by small interference (si) RNA of Tbeta4. Hypoxia response element (HRE)-luciferase activity and VEGF expression were increased by the treatment with N-(beta-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, which was inhibited by the inhibition of Tbeta4 expression with Tbeta4-siRNA. In hypoxic condition, HRE-luciferase activity and VEGF expression were inhibited by the treatment with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor to nitric oxide synthase (NOS), which is accompanied with a decrease in Tbeta4 expression. VEGF expression inhibited by L-NMMA treatment was restored by the transfection with pCMV-Tbeta4 plasmids for Tbeta4 overexpression. Taken together, these results suggest that Tbeta4 could be a regulator for the expression of VEGF via the maintenance of NOS activity.

Study on anti-oxidative effect of extracts from Cichorium endivia on HepG2 cells and its mechanism / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the protective effect of extracts from Cichorium endivia (CEE) in H2O2-induced HepG2 cell oxidative stress injury, and explore the antioxidant mechanism of CEE in HepG2 cells.</p><p><b>METHOD</b>The viability of H2O2-induced HepG2 cells and the intracellular ROS level were measured by MTT assay and DCFH-DA fluorescence staining assay. The antioxidant-response element (ARE)-Luciferase activity was tested in HepG2 cells stably transected by ARE reporter gene. The fluorescence quantitative RT-PCR was adopted to determine the mRNA expressions of genes containing ARE sequence in HepG2 cells.</p><p><b>RESULT</b>The cell viability reduced, while the ROS level increased after HepG2 cells were treated by H2O2. Different concentrations of CEE could be added to significantly improve the above results. After HepG2 cells transected by ARE reporter gene were treated with different concentrations of CEE, the intracellular ARE activity could increase in a concentration-dependent manner. In addition, the mRNA expressions of regulatory genesGCLC, GCLM and HMOX-1 containing ARE sequence in HepG2 cells were up-regulated in a concentration-dependent manner by CEE.</p><p><b>CONCLUSION</b>CEE inhibited the H2O2-injured HepG2 cells by reducing the ROS level. CEE's antioxidant mechanism for HepG2 cells may be closely related to the antioxidant defense system associated with its effect of activating Nrf2-ARE pathway in HepG2 cells.</p>

Establishment and application of screening methods for non-agonist PPARγ ligand / 药学学报

In-vitro assay methods were established to evaluate transactivation and binding activity of compounds on peroxisome proliferator-activated receptor y (PPARγ). Firstly, plasmids were constructed for transactivation assay of PPARγ response element (PPRE) triggered reporter gene expression, and for cell-based binding activity assay of the chimeric receptor, which was fused with PPARγ ligand binding domain (LBD) and yeast transcriptional activator Gal4. Secondly, by using PPARy competitive binding assay based on time resolved-fluorescence resonance energy transfer (TR-FRET), affinities of compounds and drugs to PPARγ were evaluated. In application of these above methods, the PPARγ activating potency and characteristics of different compounds were evaluated, and a novel benzeneselfonamide derivative, ZLJ01, was found to have comparable binding activity and affinity with the well-known PPARy agonist, but lack of PPRE mediated transactivation activity. In preliminary study on in-vitro hypoglycemic activity, ZLJ1 was found to promote insulin-stimulated glucose uptake by liver cells. Therefore, we believe that combining transactivation and binding activity as well as affinity evaluation, the system could be used to screen non-agonist PPARγ ligand as anovel PPARγ modulator

Selective Estrogen Receptor Modulation by Larrea nitida on MCF-7 Cell Proliferation and Immature Rat Uterus

Larrea nitida is a plant that belongs to the Zygophyllaceae family and is widely used in South America to treat inflammatory diseases, tumors and menstrual pain. However, its pharmacological activity remains unclear. In this study we evaluated the property of selective estrogen receptor modulator (SERM) of Larrea nitida extracts (LNE) as a phytoestrogen that can mimic, modulate or disrupt the actions of endogenous estrogens, depending on the tissue and relative amount of other SERMs. To investigate the property of SERM of LNE, we performed MCF-7 cell proliferation assays, estrogen response element (ERE)-luciferase reporter gene assay, human estrogen receptor (hER) binding assays and in vivo uterotrophic assay. To gain insight into the active principles, we performed a bioassay-guided analysis of LNE employing solvents of various polarities and using classical column chromatography, which yielded 16 fractions (LNs). LNE showed high binding affinities for hERalpha and hERbeta with IC50 values of 1.20x10(-7) g/ml and 1.00x10(-7) g/ml, respectively. LNE induced 17beta-estradiol (E2)-induced MCF-7 cell proliferation, however, it reduced the proliferation in the presence of E2. Furthermore, LNE had an atrophic effect in the uterus of immature rats through reducing the expression level of progesterone receptor (PR) proteins. LN08 and LN10 had more potent affinities for binding on hER alpha and beta than other fractions. Our results indicate that LNE had higher binding affinities for hERbeta than hERalpha, and showed SERM properties in MCF-7 breast cancer cells and the rat uterus. LNE may be useful for the treatment of estrogen-related conditions, such as female cancers and menopause.