雌激素受体α在大鼠垂体的表达及其调控的初步探讨

目的 :观察雌激素受体α(ERα)免疫反应阳性细胞在大鼠垂体的分布 ,并初步探讨其调控因素。方法 :应用免疫组织化学和原代细胞培养方法结合图像分析。结果 :①正常大鼠垂体前叶和后叶有丰富的ERα阳性细胞 ,ERα在出生 3d即有表达 ,一月龄大鼠己达到成年大鼠水平 ;②卵巢切除大鼠ERα表达减少 ,补充雌激素后其表达有所增高 ,但仍末达到正常水平 ;③动情期大鼠垂体前叶ERα表达最高 ,动情后期逐渐减少 ,动情间期达最低值 ;④白细胞介素 2可促进ERα表达 ,但雌激素在离体情况下未能促进其表达 ;⑤Tamoxifen可能通过阻断ERα表达而导致垂体细胞死亡。结论 :ERα作为信号转导通路的重要环节 ,在垂体细胞应答。

DDR2胞外区的可溶性表达、纯化和功能检测

盘状结构域受体2(discoidindomainreceptor2,DDR2)一种受体型酪氨酸蛋白激酶,其配体为纤维型胶原,胶原诱导的DDR2磷酸化可上调细胞基质金属蛋白酶1的过表达。DDR2在人体内广泛分布并与肿瘤的转移相关。为进一步研究DDR2在肿瘤转移中的作用和DDR2在细胞内的信号通路,对DDR2的胞外区进行了原核融合表达并纯化可溶性部分,获得纯度约85%的纯化蛋白,竞争结合抑制实验证明,纯化的融合蛋白可以特异性阻断II型胶原与天然DDR2受体的结合。

Cloning of Brassica napus EIN3 Gene and Its Expression Induced by Sclerotinia sclerotiorum

[Objective] The study was to investigate roles of Brassica napus EINB in （ BnEIN3 ） resistance to Sclerotinia sclerotiorum. [ Methods] Genomic PCR and RT-PCR were carded out to isolate genomic DNA and cDNA sequences of BnEIN3 from oilseed rape, based on the highly conserved region of EIN3 gene from Arabidopsis thaliana and the homologous sequences of oilseed rape ESTs. Expression levels of BnEIN3 were detected in three varieties of oilseed rape inoculated with S. sclerotiorum by real-time quantitative PCR.[Results] A 1 947 bp DNA fragment was obtained from oilseed rape. The fragment shared 82% identity to A. thaliana EIIV3, encoded 614 amino acids containing an EIN3 domain, and was named as BnEIN3. Real-time PCR results showed that expression patterns of BnEIN3 were drastically different in different varieties. In highly resistant oilseed rape variety D083, BnEIN3 expression level was significantly increased 72 h after S. sclerotiorum inoculation whereas in middle resistant and susceptible varieties Zhongshuang 9 and 84039, BnEIN3 expression was suppressed. [ Conclusion ] BnEIIV3 may play an important role in oilseed rape resistance to S. sclerotiorum.

The Mediation of Defense Responses of Ginseng Cells to an Elicitor from Cell Walls of Colletotrichum lagerarium by Plasma Membrane NAD(P)H Oxidases

NAD(P)H oxidases were detected in suspension cultured cells of ginseng (Panax ginseng C. A. Meyer). The activities of these enzymes were induced by an elicitor (Cle) extracted from cell walls of Col-letotrichum lagerarium. In addition, Cle induced an oxidative burst and enhanced the synthesis of saponin, activity of phenylalanine ammonialyase (PAL) , accumulation of chalcone synthase (CHS) and the transcription of a hydroxyproline-rich glycoprotein gene ( hrgp ) . Pre-treatments with DPI and quinacrine (two inhibitors of mammalian neutrophil plasma membrane NADPH oxidase) for 30 min prior to Cle addition blocked the NAD(P)H oxidase activity induced by Cle. These inhibitors also inhibited the release of H2C2, the synthesis of saponin, PAL activity and CHS accumulation. Our data revealed homology between plasma membrane NAD(P)H oxidases of mammalian neutrophil cells and ginseng suspension cells. They also indicated that deactivated NAD(P)H oxidases catalysed the release of H2O2 and that H2O2 was functioning as a second messenger stimulating PAL activity, saponin synthesis and hrgp transcription. Elevations of Ca2 + and protein phos-phorylation/dephosphorylation were required for this defense process. We propose that NAD(P)H oxidases mediate the processes of Cle-induced defense responses in ginseng suspensions, and postulate the existence of a signalling cascade including extracellular Cle stimulation, activation of plasma membrane NAD(P)H oxidases, release of H2O2, and the intracellular responses of metabolism and gene transcription in ginseng suspension cells.

Multifaceted nature of membrane microdomains in colorectal cancer

Membrane microdomains or lipid rafts are known to be highly dynamic and to act as selective signal transduction mediators that facilitate interactions between the cell's external and internal environments.Lipid rafts play an important mediating role in the biology of cancer:they have been found in almost all existing experimental cancer models,including colorectal cancer (CRC),and play key regulatory roles in cell migration,metastasis,cell survival and tumor progression.This paper explores the current state of knowledge in this field by highlighting some of the pioneering and recent lipid raft studies performed on different CRC cell lines and human tissue samples.From this literature review,it becomes clear that membrane microdomains appear to be implicated in all key intracellular signaling pathways for lipid metabolism,drug resistance,cell adhesion,cell death,cell proliferation and many other processes in CRC.All signal transduction pathways seem to originate directly from those peculiar lipid islands,thereby orchestrating the colon cancer cells' state and fate.As confirmed by recent animal and preclinical studies in different CRC models,continuing to unravel the structure and function of lipid rafts-including their associated complex signaling pathways-will likely bring us one step closer to better monitoring and treating of colon cancer patients.

Oridonin induces apoptosis in gastric cancer through Apaf-1,cytochrome c and caspase-3 signaling pathway

AIM:To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.METHODS:The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide assay.After treatment with 10 μg/mL oridonin for 24 h and 48 h,the cells were stained with acridine orange/ethidium bromide.The morphologic changes were observed under an inverted fluorescence microscope.DNA fragmen-tation(a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay.After treated with oridonin(0,1.25,2.5,5 and 10 μg/mL),HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis,and oridonin-induced apoptosis in HGC-27 cells was detected.After treatment with oridonin for 24 h,the effects of oridonin on expression of Apaf-1,Bcl-2,Bax,caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction(RT-PCR) and Western blotting.RESULTS:Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose-and time-dependent manner.The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h(1.25,2.5,5 and 10 μg/mL) were 1.78% ± 0.36%,4.96% ± 1.59%,10.35% ± 2.76% and 41.6% ± 4.29%,respectively,which showed a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%,21.57% ± 3.75%,30.31% ± 4.91% and 61.19% ± 5.81%,with a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%,31.86% ± 3.86%,48.30% ± 4.16% and 81.80% ± 6.72%,with a significant difference(P < 0.05).Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining.After treatment with oridonin,the cells became round,shrank,and developed small buds around the nuclear membrane while forming apoptotic bodies.Lactate dehydrogenase(LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h,LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4%(P < 0.001).However,the change in the release of LDH caused by necrosis was insignificant,suggesting thatthe major cause of oridonin-induced HGC-27 cell death was apoptosis.Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls(P < 0.05).And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%,12.8% ± 2.53%,28.5% ± 4.23% and 49.6% ± 3.76%,which were in a dose-dependent manner(P < 0.05).After treatment for 24 h,DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dosedependent manner.RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3(0.917 ± 0.103 vs 0.357 ± 0.019,P < 0.05),cytochrome c(1.429 ± 0.111 vs 1.002 ± 0.014,P < 0.05),Apaf-1(0.688 ± 0.101 vs 0.242 ± 0.037,P < 0.05) and Bax(0.856 ± 0.101 vs 0.278 ± 0.027,P < 0.05)(P < 0.05),whereas down-regulated in Bcl-2(0.085 ± 0.012 vs 0.175 ± 0.030,P < 0.05).Western blotting analysis also confirmed this result.CONCLUSION:Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1,caspase-3 and cytochrome c,which are highly dependent upon the mitochondrial pathway.

Functional study of p38 mitogen-activated protein kinase based on cell-penetrating peptide delivery system

Objective p38 Mitogen-activated protein kinase （MAPK） is a crossing center of various pathways. In this study, protein transduction system based on human immunodeficiency virus （HIV）-1 transactivator of transcription （TAT）, which is an efficient delivery peptide of the foreign proteins into cells, was employed to study p38 MAPK functions in eukaryotic cells. Methods p38 And its dominant negative form, p38AF, were constructed into pET-His-TAT vector correctly to verify that the recombinant plasmids were well-founded through restriction enzyme digestion and DNA sequencing. The two proteins, His-TAT-p38 and His-TAT-p38AF, were expressed and purified in Escherichia coli by SDS-PAGE. Then they were incubated with ECV304 cells respectively and readily transduced into cells in a time-dependent and dose-dependent manner. The cells were stimulated by sorbitol. Activating transcription factor （ATF） 2 phosphorylation level was checked using Western blot to assess the activity of endogenous p38. Results Compared with controls, it was found that His-TAT-p38 increased the level ofATF2 phosphorylation in sorbitol-stimulated ECV304 cells, while His-TAT-p38AF inhibited it, indicating p38 MAPK protein delivery system based on TAT was constructed successfully. TAT-p38 and its dominant negative form possessed high biological activity after transduction into ECV304 cells by TAT protein delivery system. The results showed that p38AF fused with TAT could inhibit the transduction of endogenous p38 signal pathway in part, and other pathway might regulate p38 phosphorylation. Conclusions Our study provides a novel pathway to inhibit p38 signal pathway and establish a new method to study p38 function.

The role of phosphatidylinositol signaling pathway in regulating serotonin-induced oocyte maturation in Mercenaria mercenaria

Serotonin (5-HT) has been found to stimulate meiotic maturation of oocytes in many molluscs. During maturation, several signaling pathways are involved, especially the phosphatidylinositol and cAMP pathways. In order to examine the possible role of the phosphatidylinositol signaling pathway in regulating oocyte maturation in Mercenaria mercenaria, the effects of the activator/inhibitor of phospholipase (PLC) and protein kinase C (PKC) on serotonin-induced maturation were studied. Results show that high-concentrations of neomycin (inhibitor of PLC) blocked oocyte maturation, while 9, 10-dimethyl-1, 2-benzanthracene (DMBA, activator of PLC) promoted oocyte maturation in the presence of serotonin. It was also found that in the presence of serotonin, phorbol 12-myristate 13-acetate (PMA, activator of PKC) inhibited oocyte maturation, while sphingosine (inhibitor of PKC) stimulated oocyte maturation. These results indicate that serotonin-induced oocyte maturation requires the activation of the phosphatidylinositol pathway. Decrease of PLC inhibited serotonin-induced oocyte maturation, whereas a decrease of PKC stimulated the maturation. Thus, our study indicates that serotonin promotes maturation of M. mercenaria oocytes through PLC stimulated increase in calcium ion concentration via inositol-1, 4, 5-trisphosphate (IP3) but not PKC.

Screening and regulatory network analysis of survival-related genes of patients with colorectal cancer

The purpose of this study was to screen key survival-related genes from patients with colorectal cancer and explore signal transduction network of the involved genes.In a previous study,survival-related genes of patients with colorectal cancer were selected by colorectal cancer-related expression data GSE17538 using the Significance Analysis of Microarrays(SAM3.01)software,and 235 genes related to the survival of patients with colorectal cancer were obtained.Therefore,the following screening and analysis were conducted on these 235 genes in this study.First,the enrichment analysis of transcription factor binding sites was conducted on the 235 genes.Genes with more than seven transcription factor binding sites were screened.Then,these genes and upregulated genes in colorectal cancer were intersected.Finally,survival analysis and regulatory network analysis were conducted on the screened genes.This allowed clarification of the relationship between these genes and the survival of patients with colorectal cancer and the signaling network involving these genes in the cell signal transduction network of colorectal cancer.Through the above analysis,six upregulated genes in colorectal cancer related to the survival of colorectal cancer patients and highly regulated by transcription factors were selected,namely STX2,PODXL,KLK6,GRB10,EHBP1 and CREB5.These genes are involved in signal regulatory networks related to colorectal cancer metastasis-related signaling pathways.Therefore,the survival of patients with colorectal cancer is closely correlated with colorectal cancer metastasis.The six survival-related genes affect the survival of patients by regulating colorectal cancer metastasis-associated signaling pathways.

Host cellular signaling induced by influenza virus

A wide range of host cellular signal transduction pathways can be stimulated by influenza virus infection. Some of these signal transduction pathways induce the host cell's innate immune response against influenza virus, while others are essential for efficient influenza virus replication. This review examines the cellular signaling induced by influenza virus infection in host cells, including host pattern recognition receptor (PRR)-related signaling, protein kinase C (PKC), Raf/MEK/ERK and phosphatidylinositol-3-kinase (PI3K)/Akt signaling, and the corresponding effects on the host cell and/or virus, such as recognition of virus by the host cell, viral absorption and entry, viral ribonucleoprotein (vRNP) export, translation control of cellular and viral proteins, and virus-induced cell apoptosis. Research into influenza virus-induced cell signaling promotes a clearer understanding of influenza virus-host interactions and assists in the identification of novel antiviral targets and antiviral strategies.

Qingnaoyizhi decoction suppresses the formation of glial fibrillary acidic protein-positive cells in cultured neural stem cells by inhibiting the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway

OBJECTIVE: Inactivation of the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signaling axis plays a crucial role in determining the fate of neural stem cells(NSCs).Qingnaoyizhi decoction(QNYZD) has been used for the treatment of vascular dementia and has shown to improve synaptic remodeling. The aim of this study was to evaluate the effect of cerebrospinal fluid(CSF) containing QNYZD(CSF-QNYZD) on the differentiation of cultured NSCs and the involvement of the JAK2/STAT3 pathway.METHODS: The protein expression levels of glial fibrillary acidic protein(GFAP), tubulin, drosophila mothers against decapentaplegic protein(SMAD-1), STAT3, and phosphorylated-STAT3 were detected by western immunoblot analysis in the groups: control, CSF, JAK/STAT inhibitor(AG490),CSF-QNYZD, and CSF-XDZ(CSF-Xidezhen). The differentiation of NSCs was determined by immunofluorescence staining. The proliferation of NSCs was measured using the Cell Counting Kit-8 proliferation assay.RESULTS: Compared with the control group,CSF-QNYZD and AG490 significantly increased the number and expression of tubulin-positive cells, reduced the number and expression of GFAP-positive cells, and down-regulated the expression of p-STAT3. However, CSF-QNYZD also decreased the expression of SMAD-1 and STAT3.CONCLUSION: Enhanced neuronal differentiation may be associated with the down-regulation of glial differentiation instead of promoting proliferationin treated NSCs. Furthermore, QNYZD may play a direct role in suppressing the formation of GFAP-positive cells and enhancing neuronal differentiation by inhibiting JAK2/STAT3 activation. Overall, these results provide insights into the possible mechanism underlying QNYZD-mediated neurogenesis.

O-linked β-N-acetylglucosamine modification and its biological functions

The covalent attachment of O-linked β-N- acetylglucosamine （O-GIcNAc） to Ser/Thr residues of proteins acts as not only a posttranslational modification but also a nutritional sensor in nucleus and cytoplasm, which directly regulates the expression of genes and multiple crucial signal transduction pathways. Dynamic O- GlcNAcylation at Ser/Thr residues is catalyzed by two key enzymes, O-GIcNAc transferase （OGT） and O-GlcNAcase, which are responsible for addition and removal of the O- GlcNAc modification, respectively. O-GlcNAc modifica- tion plays important roles in cellular signaling in animals, especially in human diseases. Two orthologs of OGT in plants, SECRET AGENT and SPINDLY, have been reported to be involved in diverse plant processes. However, compared with the functional mechanisms revealed in animals, the consequences of protein O-GlcNAc modifi- cation in plants is largely unknown, and the relationship between O-GlcNAcylation and cellular processes needs to be explored. In this review, we summarized the recent advances on O-GlcNAc modification and its biological functions in animals and plants, and prospect of more special functions of O-GlcNAc will be revealed in plants.

Phototransduction in Drosophila

The Drosophila visual transduction is the fastest known G protein-coupled signaling cascade and has been served as a model for understanding the molecular mechanisms of other G protein-coupled signaling cascades. Numbers of components in visual transduction machinery have been identified. Based on the functional characterization of these genes, a model for Drosophila phototransduction has been outlined, including rhodopsin activation, phosphoinoside signaling, and the opening of TRP and TRPL channels. Recently, the characterization of mutants, showing slow termination, revealed the physiological significance and the mechanism of rapid termination of light response.

Potential antitumor mechanisms of phenothiazine drugs

In this study,three kinds of phenothiazine drugs were analyzed to explore their potential antitumor mechanisms.First,target proteins that could interact with chlorpromazine,fluphenazine and trifluoperazine were predicted.Then,the target proteins of the three drugs were intersected.Cell signaling pathway enrichment and related disease enrichment were conducted for the intersected proteins to extract the enrichment categories associated with tumors.By regulation network analysis of the protein interactions,the mechanisms of action of these target proteins in tumor tissue were clarified,thus confirming the potential antitumor mechanisms of the phenothiazine drugs.The final results of cell signaling pathway enrichment and related disease enrichment showed that the categories with the highest score were all found in tumors.Target proteins belonging to the tumor category included signaling pathway members such as Wnt,MAPK and retinoic acid receptor.Moreover,another target protein,MAPK8,could indirectly act on target proteins CDK2,IGF1R,GSK3B,RARA,FGFR2 and MAPK10,thereby affecting tumor cell division and proliferation.Therefore,phenothiazine drugs may have potential antitumor effects,and tumorassociated target proteins play important roles in the process of cell signaling transduction cascades.