Regulation of type 1 inositol 1,4,5-trisphosphate-gated calcium channels by InsP3 and calcium: Simulation of single channel kinetics based on ligand binding and electrophysiological analysis.

Cytosolic calcium acts as both a coagonist and an inhibitor of the type 1 inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel, resulting in a bell-shaped Ca dependence of channel activity (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature. 351:751-754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science. 252: 443-446; Iino, M. 1990. J. Gen. Physiol. 95:1103-1122). The ability of Ca to inhibit channel activity, however, varies dramatically depending on InsP3 concentration (Combettes, L., Z. Hannaert-Merah, J.F. Coquil, C. Rousseau, M. Claret, S. Swillens, and P. Champeil. 1994. J. Biol. Chem. 269:17561-17571; Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529-538). In the present report, we have extended the characterization of the effect of cytosolic Ca on both InsP3 binding and InsP3-gated channel kinetics, and incorporated these data into a mathematical model capable of simulating channel kinetics. We found that cytosolic Ca increased the Kd of InsP3 binding approximately 3.5-fold, but did not influence the maximal number of binding sites. The ability of Ca to decrease InsP3 binding is consistent with the rightward shift in the bell-shaped Ca dependence of InsP3-gated Ca channel activity. High InsP3 concentrations are able to overcome the Ca-dependent inhibition of channel activity, apparently due to a low affinity InsP3 binding site (Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529-538). Constants from binding analyses and channel activity determinations were used to develop a mathematical model that fits the complex Ca-dependent regulation of the type 1 InsP3-gated Ca channel. This model accurately simulated both steady state data (channel open probability and InsP3 binding) and kinetic data (channel activity and open time distributions), and yielded testable predictions with regard to the regulation of this intracellular Ca channel. Information gained from these analyses, and our current molecular model of this Ca channel, will be important for understanding the basis and regulation of intracellular Ca waves and oscillations in intact cells.     

Concerted mechanism of Swe1/Wee1 regulation by multiple kinases in budding yeast

In eukaryotes, entry into mitosis is induced by cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. In budding yeast, Swe1 (Wee1 ortholog) is targeted to the bud neck through Hsl1 (Nim1-related kinase) and its adaptor Hsl7, and is hyperphosphorylated prior to ubiquitin-mediated degradation. Here, we show that Hsl1 and Hsl7 are required for proper localization of Cdc5 (Polo-like kinase homolog) to the bud neck and Cdc5-dependent Swe1 phosphorylation. Mitotic cyclin (Clb2)-bound Cdc28 (Cdk1 homolog) directly phosphorylated Swe1 and this modification served as a priming step to promote subsequent Cdc5-dependent Swe1 hyperphosphorylation and degradation. Clb2-Cdc28 also facilitated Cdc5 localization to the bud neck through the enhanced interaction between the Clb2-Cdc28-phosphorylated Swe1 and the polo-box domain of Cdc5. We propose that the concerted action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates is an evolutionarily conserved mechanism that is crucial for effectively triggering mitotic entry and other critical mitotic events.     

Upregulated lncRNA-UCA1 contributes to metastasis of bile duct carcinoma through regulation of miR-122/CLIC1 and activation of the ERK/MAPK signaling pathway

In the present study, we aimed to identify specific lncRNAs and miRNAs, as well as mRNAs, involved in bile duct carcinoma (BDC) and to further explore the way in which lncRNA UCA1 regulates cell metastasis ability. Differentially expressed RNAs were screened out from the TCGA database. In in vitro experiments, qRT-PCR was used to measure lncRNA UCA1, miR-122 and CLIC1 expression. We performed a dual luciferase assay to validate the target relationships among UCA1, CLIC1 and miR-122. The cell migration ability was measured by a wound healing assay, and Transwell assays were applied to detect cell invasive ability. Western blot analysis was employed to detect the expression of related proteins in the MAPK signaling pathway. According to the bioinformatics analysis, lncRNA UCA1 and CLIC1 were both significantly upregulated in BDC, while the expression of miR-122 declined compared with the normal group. The target relationship among UCA1, CLIC1 and miR-122 was verified. UCA1 promoted BDC cell migration and invasiveness, while miR-122 inhibited their progression. CLIC1 served as the downstream target gene of miR-122 and had opposite effects. The ERK/MAPK signaling pathway was activated after upregulating UCA1. LncRNA-UCA1 promoted the metastasis of BDC cells by regulating the expression of miR-122 and its downstream gene mRNA CLIC1 and promoted the activation of the ERK/MAPK pathway, which expanded the horizons of targeted therapy of cholangiocarcinoma.     

紫檀芪调节Keap-1/Nrf2/HO-1信号通路对非酒精性脂肪肝大鼠氧化应激和细胞凋亡的影响

摘要 目的：研究紫檀芪调节Kelch样ECH关联蛋白1（Keap-1）/核因子E2相关因子2（Nrf2）/血红素加氧酶-1（HO-1）信号通路对非酒精性脂肪肝（NAFLD）大鼠氧化应激和细胞凋亡的影响。方法：将60只SD大鼠随机分为对照组、模型组、紫檀芪低剂量组（30 mg/kg）、紫檀芪高剂量组（60 mg/kg）、紫檀芪（60 mg/kg）+N-（4-（2,3-二氢-1-（2''-甲基苯甲酰）-1H-吲哚-5-基）-5-甲基-2-噻唑基）-1,3-苯并二氧唑-5-乙酰胺（ML385）（30 mg/kg）组,每组12只。模型组与药物干预组大鼠以高脂饲料饲养诱导NAFLD模型,对照组大鼠以普通饲料饲养,各组连续喂养12周。以紫檀芪和ML385分组处理14 d后（对照组以等剂量生理盐水处理）,检测各组大鼠脂代谢指标[三酰甘油（TG）、总胆固醇（TC）及游离脂肪酸（FFA）水平]、肝指数、肝功能指标[谷丙转氨酶（ALT）及谷草转氨酶（AST）]水平、血清白细胞介素（IL）-17、IL-6、IL-10、氧化应激指标[丙二醛（MDA）、超氧化物歧化酶（SOD）及过氧化氢酶（CAT）]水平;原位末端标记法（TUNEL）染色检测各组大鼠肝细胞凋亡率;蛋白免疫印迹法检测各组大鼠肝组织凋亡相关蛋白及Keap-1/Nrf2/HO-1通路相关蛋白表达。结果：与对照组相比,模型组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平显著降低（P＜0.05）,TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1及Bax表达水平显著升高（P＜0.05）。与模型组相比,紫檀芪低、高剂量组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平均升高（P＜0.05）,TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1、Bax表达水平均降低（P＜0.05）;与紫檀芪低剂量组相比,紫檀芪高剂量组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平升高（P＜0.05）,TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1及Bax表达水平降低（P＜0.05）;与紫檀芪高剂量组相比,紫檀芪+ML385组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平降低（P＜0.05）,TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Bax表达水平升高（P＜0.05）。结论：紫檀芪可能通过激活Keap-1/Nrf2/HO-1信号通路,改善NAFLD大鼠脂代谢水平,调节炎症反应及氧化应激,减轻肝组织脂肪变性及细胞凋亡。     

Functional identification of an Arabidopsis snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast

Yeast Snf4 is a prototype of activating gamma-subunits of conserved Snf1/AMPK-related protein kinases (SnRKs) controlling glucose and stress signaling in eukaryotes. The catalytic subunits of Arabidopsis SnRKs, AKIN10 and AKIN11, interact with Snf4 and suppress the snf1 and snf4 mutations in yeast. By expression of an Arabidopsis cDNA library in yeast, heterologous multicopy snf4 suppressors were isolated. In addition to AKIN10 and AKIN11, the deficiency of yeast snf4 mutant to grown on non-fermentable carbon source was suppressed by Arabidopsis Myb30, CAAT-binding factor Hap3b, casein kinase I, zinc-finger factors AZF2 and ZAT10, as well as orthologs of hexose/UDP-hexose transporters, calmodulin, SMC1-cohesin and Snf4. Here we describe the characterization of AtSNF4, a functional Arabidopsis Snf4 ortholog, that interacts with yeast Snf1 and specifically binds to the C-terminal regulatory domain of Arabidopsis SnRKs AKIN10 and AKIN11.     

4-1BBL通过激活Wnt/β-catenin信号通路促进人胃癌细胞的增殖和迁移

4-1BB和4-1BB配体(4-1BBL),又被称为CD137和CD137配体,分别属于肿瘤坏死因子(TNF)受体和配体家族的成员。4-1BBL 与4-1BB相互作用可以激活T细胞免疫应答。因此,4-1BBL一直在抗肿瘤免疫应答中发挥经典的免疫共刺激分子作用。近期研究发现,4-1BBL在肿瘤细胞中另有其他的生物学功能,但4-1BBL在胃癌进展过程中的功能尚不明确。本文探讨了4-1BBL在人胃癌细胞中的生物学功能和分子作用机制。首先,通过检索TCGA和Kaplan Meier plotter数据库发现,4-1BBL在胃癌组织中的表达显著高于癌旁组织（P<0.001）,且4-1BBL的高表达与胃癌的不良预后正相关（P<0.05）。细胞生物学的结果显示,敲除4-1BBL明显抑制胃癌细胞的增殖（P<0.05）、侵袭和迁移（P<0.05）,促进胃癌细胞的凋亡（P<0.05）;另外,蛋白质免疫印迹结果表明,敲除4-1BBL可使β-联蛋白、c-Myc和细胞周期蛋白D1(cyclin D1)的蛋白质表达水平下降,抑制Wnt/β-catenin信号通路。相反,过表达4-1BBL则显著促进胃癌细胞增殖（P<0.05）、侵袭和迁移（P<0.05）,减少胃癌细胞的凋亡（P<0.05）;且过表达4-1BBL促进β-联蛋白(β-catenin)、c-Myc和细胞周期蛋白D1的蛋白质表达,激活Wnt/β-catenin信号通路。综上所述,4-1BBL可通过激活Wnt/β-catenin信号通路促进人胃癌细胞的增殖和迁移。     

Carbachol-induced accumulation of inositol phosphates and its modulation by excitatory amino acids in cortical slices of young and aged rats with down-regulation of muscarinic M-1 receptors

The effects of a subacute intoxication with diisopropyl fluorophosphate (DPF) on total muscarinic acetylcholine receptor sites (mAChRs) and M-1 AChRs were evaluated in the cerebral cortex of young (2–4 months) and aged (22–24 months) Fischer 344 rats. Since M-1 AChRs are coupled to the metabolism of phosphoinositides, carbachol-induced accumulation of inositol phosphates (IP) and its inhibition by glutamate and NMDA was also measured in the cortical slices. DFP treatment caused about 75% inhibition of cholinesterase and 35% down-regulation of mAChRs (measured as [³H]quinuclidinyl benzylate binding) in both young and aged rats. The down-regulation of M-1-ACHRs (measured as [³H]pirenzepine binding) was more pronounced in aged (30%) than in young (17%) DFP-treated rats. There was a significant increase in carbachol-induced IP accumulation in aged, with respect to young, untreated rats. DFP treatment caused a considerable decrease in such IP accumulation in aged but not in young rats. Glutamate and NMDA antagonized carbachol-induced IP accumulation in untreated young and aged rats (and the effects of NMDA were reversed by carboxy-piperazinyl-propyl phosphonic acid). In DFP-treated rats such antagonism was somewhat less pronounced. The data appear of interest in relation to the use of anticholinesterase compounds in the therapy of senile dementia of Alzheimer's type. They suggest that beside their primary action (increasing brain ACh levels) such compounds also act on post-receptor mechanisms and on the interactions between cholinergic and glutamatergic neurotransmitter systems.     

NMDA-receptor regulation of muscarinic-receptor stimulated inositol 1,4,5-trisphosphate production and protein kinase C activation in single cerebellar granule neurons

Inositol 1,4,5-trisphosphate (InsP(3)) production in single cerebellar granule neurons (CGNs) grown in culture was measured using the PH domain of phospholipase C delta1 tagged with enhanced green fluorescent protein (eGFP-PH(PLCdelta1)). These measurements were correlated with changes in intracellular free Ca2+ determined by single cell imaging. In control CGNs, intracellular Ca2+ stores appeared replete. However, the refilling state of these stores appeared dependent on the fluorophore used to measure Ca2+-release. Thus, methacholine (MCH), acting via muscarinic acetylcholine-receptors (mAchRs), mobilised intracellular Ca2+ in cells loaded with fluo-3 and fura-4f, but not fura-2. Confocal measurements of single CGNs expressing eGFP-PH(PLCdelta1) demonstrated that MCH stimulated a robust peak increase in InsP(3), which was followed by a sustained plateau phase of InsP(3) production. In contrast, glutamate-induced InsP(3) signals were weak or not detectable. MCH-stimulated InsP(3) production was reduced by chelation of intracellular Ca2+ with BAPTA, and emptying of intracellular stores with thapsigargin, indicated a positive feedback effect of Ca2+ mobilisation onto PLC activity. In CGNs, NMDA- and KCl-mediated Ca2+-entry significantly enhanced MCH-induced InsP(3) production. Furthermore, mAchR-mediated PLC activation appeared sensitive to the full dynamic range of intracellular Ca2+ increases stimulated by 100 microm NMDA. This dynamic regulation was also observed at the level of PKC activation indicated by an enhanced translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate (MARCKS) protein in cells stimulated with MCH. Thus, NMDA-mediated Ca2+ influx and PLC activation may represent a coincident-detection system whereby ionotropic and metabotropic signals combine to stimulate InsP(3) production and PKC-mediated phosphorylation events in CGNs.     

Retracted: 9za plays cytotoxic and proapoptotic roles and induces cytoprotective autophagy through the PDK1/Akt/mTOR axis in non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) is an aggressive subtype of pulmonary carcinomas with high mortality. However, chemotherapy drug resistance and high recurrence rates hinder the curative effect of platinum-based first-line chemotherapy, which makes it urgent to develop new antitumor drugs for NSCLC. 9za, a new candidate drug synthesized by our research group, has been verified with potent antilung cancer activity in preliminary experiments. However, the underlying molecular mechanism of 9za remains largely vague. This work revealed that 9za could play important cytotoxic and proapoptotic roles in NSCLC cells. Moreover, 9za could induce autophagy and promote autophagy flux. Interestingly, the cytotoxic and proapoptotic roles were significantly dependent on 9za-induced cytoprotective autophagy. That is, the coadministration of 9za with an autophagy inhibitor such as chloroquine or 3-methyladenine exhibited increased cytotoxic and proapoptotic effects compared with 9za treatment alone. In addition, 9za exposure suppressed the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), protein kinase B (Akt), mammalian targets of rapamycin (mTOR), p70 S6 kinase, and 4E binding protein 1 by a dose-dependent way, manifesting that the Akt/mTOR axis was implicated in 9za-induced autophagy. In addition, the overexpression of PDK1 resulted in increased phosphorylation of PDK1 and Akt and blocking of 9za-mediated autophagy. These data showed that the PDK1/Akt/mTOR pathway was involved in 9za-induced autophagy. Hence, this work provides a theoretical basis for exploiting 9za as a new antilung cancer candidate drug and hints that the combination of 9za with an autophagy inhibitor is a feasible alternative approach for the therapy of NSCLC.