Pregnane X receptor-agonists down-regulate hepatic ATP-binding cassette transporter A1 and scavenger receptor class B type I

Pregnane X receptor (PXR) is the molecular target for a wide variety of endogenous and xenobiotic compounds. It regulates the expression of genes central to the detoxification (cytochrome P-450 enzymes) and excretion (xenobiotic transporters) of potentially harmful compounds. The aim of the present investigation was to determine the role of PXR in regulation of high-density lipoprotein (HDL) cholesterol metabolism by studying its impact on ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression in hepatocytes. ABCA1 and SR-BI are major factors in the exchange of cholesterol between cells and HDL. Expression analyses were performed using Western blotting and quantitative real time RT-PCR. Luciferase reporter gene assays were used to measure promoter activities. Total cholesterol was measured enzymatically after lipid extraction (Folch's method). The expression of ABCA1 and SR-BI was inhibited by the PXR activators rifampicin and lithocholic acid (LCA) in HepG2 cells and pregnenolone 16alpha-carbonitrile (PCN) in primary rat hepatocytes. Thus, PXR appears to be a regulator of hepatic cholesterol transport by inhibiting genes central to cholesterol uptake (SR-BI) and efflux (ABCA1).     

Roles of aldo-keto reductases 1B10 and 1C3 and ATP-binding cassette transporter in docetaxel tolerance

Docetaxel (DTX) is widely used for treatment of inveterate lung and prostate cancers, but its continuous administration elicits the hyposensitivity. Here, we established the DTX-resistant variants of human lung cancer A549 and androgen-independent prostate cancer Du145 cells and found that the resistance development provoked aberrant up-regulations of aldo-keto reductase (AKR) 1B10 and AKR1C3 in A549 and Du145 cells, respectively. In addition, the sensitivity to the DTX toxicity was significantly decreased and increased by overexpression and knockdown of the two AKR isoforms, respectively. Furthermore, the resistant cells exhibited a decreased level of reactive 4-hydroxy-2-nonenal formed during DTX treatment, and the decrease was alleviated by adding the AKR inhibitors, inferring that the two AKRs confer the chemoresistance through elevating the antioxidant properties. The development of DTX resistance was also associated with enhanced expression of an ATP-binding cassette (ABC) transporter ABCB1 among the ABC transporter isoforms. The combined treatment with inhibitors of the two AKRs and ABCB1 additively sensitized the resistant cells to DTX. Intriguingly, the AKR1B10 inhibitor also suppressed the lung cancer cross-resistance against cisplatin. The results suggest that combined treatment with AKRs (1B10 and 1C3) and ABCB1 inhibitors exerts overcoming effect against the cancer resistance to DTX and cisplatin, and can be used as the adjuvant therapy.     

Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos.

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-1 beta induces autophagy of mouse preimplantation embryos and improves blastocyst quality

Autophagy is one of the basic cellular mechanism during preimplantation development of mammalian embryos, and it plays crucial role in several physiological processes. It is induced by interleukin (IL)-1β in mammalian cells. Our present study shows that IL-1β is important for autophagy activation in embryo development. Our in vitro culture system analysis shows effect of IL-1β in medium on the development of mouse embryos and it was found to be concentration dependent. A preimplantation embryo culture using medium containing IL-1β did not improve cleavage and blastocyst development rates of mouse embryos; however, blastocyst quality was significantly improved by increasing total cell number, especially in supplementary 20 ng/mL IL-1β. Furthermore, autophagy activation mainly occurs in 2 to 4 cell embryo and blastocyst, 20 ng/mL IL-1β into culture medium can effectively enhance levels of messenger RNA and protein of autophagy-related-factors in 2 to 4 cell embryos and blastocyst, while these factors reduce in VGX-1027 (IL-1β inhibitor) groups that also reduce the quality of blastocyst. Effects of IL-1β on the development of embryo reduced in 20 ng/mL IL-1β supplemented group when 5 mM 3-methyladenine (3-MA) was also added, which used to inhibit autophagy activation in endogenous PtdIns3Ks signal pathway. Our current results show that exogenous IL-1β can effectively induce autophagy in mouse embryos at stages of 2 to 8 cell and blastocyst, that also help to improve the quality of blastocyst.     

Effect of cryopreservation by slow cooling and vitrification on viral contamination of IVF embryos experimentally exposed to bovine viral diarrhea virus and bovine herpesvirus-1

The objective was to determine the effect of cryopreservation by conventional slow controlled cooling (0.5 °C/min) and by vitrification on the presence of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) infectivity associated with frozen-thawed Day 7 bovine embryos. In this study, Day 7 embryos generated by in vitro fertilization (IVF) were exposed in vitro for 1.5 h to BVDV (N = 393) and BHV-1 (N = 242) and subsequently tested before and after cryopreservation for the presence of infectivity. Exposure of embryos to viral agents resulted in 72% of them infected prior to cryopreservation. Stepwise exposure of embryos to cryoprotectants, as well as their removal, substantially reduced the proportion of contaminated embryos (46% vs. 72%, P < 0.05). Overall, both freezing methods reduced the percentage of infectious embryos compared with that of embryos similarly exposed to viruses but not cryopreserved (31% vs. 72%, respectively; P < 0.001). The percentage of embryos with infectious viruses was not significantly higher after vitrification than after slow cooling (38% vs. 22%). In addition, after cryopreservation, a higher percentage (P < 0.002) of embryos exposed to BHV-1 (42%) remained infectious than did embryos exposed to BVDV (24%). In conclusion, cryopreservation reduced the proportion of infected embryos but did not render all of them free from infectious pathogens.     

siRNA干扰TCAB1对人主动脉平滑肌增殖的影响及机制

目的:探究TCAB1沉默对人主动脉平滑肌细胞(HASMC)增殖的影响及可能机制。方法:采用RNAi技术设计并合成靶向沉默TCAB1基因表达的三对特异性si RNA序列(si TCAB1-331、si TCAB1-619、si TCAB-749)和一对阴性对照序列(NC),使用lipo2000将si TCAB1、NC转染HASMC,分为3个组:干扰组(si TCAB1)、空白对照组(BC)、阴性对照组(NC),转染24小时倒置荧光显微镜观察细胞转染情况;通过RT-qPCR和Western blot从3个干扰靶点中筛选效果最好的干扰靶点。进一步转染si TCAB1-749后,MTS检测HASMC 24、48、72 h的增殖能力,48小时用RT-qPCR和Western blot检测CyclinD1表达量变化,流式细胞术检测HASMC的细胞周期变化。结果:RT-qPCR和WB结果显示si TCAB1-749为最好的干扰靶点;转染24、48、72 h后,si TCAB1-749组增殖水平明显低于NC组、BC组(P0.05)。流式结果显示:si TCAB1-749组处于G1期细胞比率有所增加,处于S期细胞比率减少(P0.05),且si TCAB1-749组细胞周期蛋白cyclinD1表达也下降(P0.05)。结论:沉默TCAB1能抑制HASMC的增殖,其机制可能与阻碍细胞周期蛋白cyclinD1有关。     

Effect of carboxyl-reduced heparin on the growth inhibition of bovine pulmonary artery smooth muscle cells

Heparin (HP) inhibits the proliferation of bovine pulmonary artery smooth muscle cells (BPASMC’s), among other cell types in vitro. In order to develop a potential therapeutic agent to reverse vascular remodeling, we are involved in deciphering the relationship between the native HP structure and its antiproliferative potency. We have previously reported the influence of the molecular size and the effects of various O-sulfo and N-acetyl groups of HP on growth-inhibitory activity. In this study, to understand the influence of carboxyl groups in the HP structure required for endogenous activity, a chemically modified derivative of native HP was prepared by converting the carboxyl groups of hexuronic acid residues in HP to primary hydroxyl groups. This modification procedure involves the treatment of HP with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide followed by reduction with NaBH₄ to yield carboxyl-reduced heparin (CR-HP). When compared to the antiproliferative potency of native HP on cultured BPASMC’s at three dose levels (1, 10, and 100 μg/mL), the CR-HP showed significantly less potency at all the doses. These results suggest that hexuronic acid residues in both major and variable sequences in HP are essential for the antiproliferative properties of native HP.     

CKI p15INK4B高表达对人黑色素瘤细胞cyclinDl和c-myc表达的影响及生长特性的改变

通过 DNA体外重组和转染技术 ,将已构建好的含有 p1 5基因的质粒 p XJ- 41 - p1 5转染 p1 5缺失的人黑色素瘤细胞 A 375,经 G 41 8筛选出阳性单克隆 ,并经 PCR、Western等检测 ,证明建立了 p1 5稳定高表达的细胞模型 .生长曲线和 FCM实验表明 ,与对照组细胞相比 ,实验组细胞增殖能力减弱 ,G1期细胞增加 ,S期细胞减少 .同时 ,细胞血清依赖性有所恢复 ,在软琼脂的集落形成能力下降 ,显示出细胞部分恶性表型逆转 .进一步免疫印迹实验表明 ,癌基因 c- myc的蛋白表达水平明显下降 .Cyclin D1的表达受到抑制 ,而 CDK4的表达则基本不变 .以上结果显示出 ,p1 5基因高表达能够抑制人黑色素瘤细胞的增殖及部分恶性表型 ,负调细胞周期进程 ,而 c- Myc蛋白和 G1期引擎分子 Cyclin D1表达的被抑制可能是 p1 5负调细胞增殖的分子机理之一     

Transcriptional regulation of ATP-binding cassette transporter A1 expression by a novel signaling pathway

ATP-binding cassette transporter A1 (ABCA1) is a membrane-bound protein that regulates the efflux of cholesterol derived from internalized lipoproteins. Using a mouse macrophage cell line, this report studied the impact of low-density lipoproteins (LDL) on ABCA1 expression and the signaling pathway responsible for lipoprotein-induced ABCA1 expression. Our data demonstrated that treatment of macrophages with LDL increased ABCA1 mRNA and protein levels 4.3- and 3.5-fold, respectively. LDL also induced an ～2-fold increase in macrophage surface expression of ABCA1 and a 14-fold-increase in apolipoprotein AI-mediated cholesterol efflux. In addition, LDL significantly increased the level of phosphorylated specificity protein 1 (Sp1) and the amount of Sp1 bound to the ABCA1 promoter without alteration in total Sp1 protein level. Mutation of the Sp1 binding site in the ABCA1 promoter and inhibition of Sp1 DNA binding with mithramycin A suppressed the ABCA1 promoter activity and reduced the ABCA1 expression level induced by LDL. LDL treatment also elevated protein kinase C-ζ (PKC-ζ) phosphorylation and induced PKC-ζ binding with Sp1. Inhibition of PKC-ζ with kinase inhibitors or overexpression of kinase-dead PKC-ζ attenuated Sp1 phosphorylation and ABCA1 expression induced by LDL. These results demonstrate for the first time that activation of the PKCζ-Sp1 signaling cascade is a mechanism for regulation of LDL-induced ABCA1 expression.     

Effects of cellular, chemical, and pharmacological chaperones on the rescue of a trafficking-defective mutant of the ATP-binding cassette transporter proteins ABCB1/ABCB4

The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG(2) cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.     

CKIp15~(INK4B)高表达对人黑色素瘤细胞cyclinD1和c-myc表达的影响及生长特性的改变

通过 DNA体外重组和转染技术 ,将已构建好的含有 p1 5基因的质粒 p XJ- 41 - p1 5转染 p1 5缺失的人黑色素瘤细胞 A 375,经 G 41 8筛选出阳性单克隆 ,并经 PCR、Western等检测 ,证明建立了 p1 5稳定高表达的细胞模型 .生长曲线和 FCM实验表明 ,与对照组细胞相比 ,实验组细胞增殖能力减弱 ,G1期细胞增加 ,S期细胞减少 .同时 ,细胞血清依赖性有所恢复 ,在软琼脂的集落形成能力下降 ,显示出细胞部分恶性表型逆转 .进一步免疫印迹实验表明 ,癌基因 c- myc的蛋白表达水平明显下降 .Cyclin D1的表达受到抑制 ,而 CDK4的表达则基本不变 .以上结果显示出 ,p1 5基因高表达能够抑制人黑色素瘤细胞的增殖及部分恶性表型 ,负调细胞周期进程 ,而 c- Myc蛋白和 G1期引擎分子 Cyclin D1表达的被抑制可能是 p1 5负调细胞增殖的分子机理之一     

Differential regulation of cyclin B1 degradation between the first and second meiotic divisions of bovine oocytes

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.     

Identification of the tree shrew ATP-binding cassette transporter A1 (ABCA1) and its expression in tissues

ATP-binding cassette transporter A1 (ABCA1) modulates plasma levels of high density lipoprotein (HDL), a cardiovascular protecting factor. Tree shrew was considered to be an animal protected from atherosclerosis characterized by high proportion of HDL in plasma. The cDNA clones and expression of tree shrew ABCA1 was identified using SMART-RACE and Real-Time PCR techniques respectively. The nucleotide sequence of tree shrew ABCA1 covered 7,762 bp, including a 6,786 bp coding region which encoded a 2,261 amino acids protein with the high identity to human ABCA1 (95%). Tree shrew ABCA1 was expressed in various tissues, the highest in lung, followed by liver, kidney, spleen and cardiac muscle in turn from high to medium expression levels. This pattern was partially different from that of human ABCA1 which was low in kidney and cardiac muscle. This work could shed new light on its role of ABCA1 in the distinctive HDL metabolism in tree shrew.     

Meta-analysis on association between the ATP-binding cassette transporter A1 gene (ABCA1) and Alzheimer's disease

Purpose

In the past decade, a number of case–control studies have been carried out to investigate the relationship between ABCA1 polymorphisms and Alzheimer's disease (AD). However, these studies have yielded contradictory results. To investigate this inconsistency, a meta-analysis was performed.

Methods

Databases including PubMed, Web of Science, EMBASE and CNKI were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association.

Results

A total of 13 case–control studies, involving 6214 patients and 6034 controls for ABCA1 polymorphisms were included. In a combined analysis, the summary per-allele odds ratio for AD of the 219 K was 1.03 (95% CI: 0.93–1.14, p = 0.56). A meta-analysis of studies on the 883 M and 1587 K variant showed no significant overall association with AD, yielding a per-allele odds ratio of 1.10 (95% CI: 0.96–1.26, p = 0.16), and 1.09 (95% CI: 0.97–1.24, p = 0.16) respectively. Similar results were also found for heterozygous and homozygous. In the subgroup analysis by ethnicity, sample size, APOE status and onset type, no significant associations were found in almost all genetic models.

Conclusions

In summary, there was no significant association detected between ABCA1 R219K, I883M and R1587K polymorphisms and risk for AD.     

BRCA1相关蛋白1对肾癌增殖和侵袭的实验研究

目的:研究BRCA1相关蛋白(BRCA1-associated protein-1,BAP1)对肾癌细胞增殖和侵袭的影响。方法:通过含有不同BAP1和HAT1载体的慢病毒感染后用嘌呤霉素筛选的方法在肾癌细胞系中构建BAP1稳定低表达、HAT1稳定高表达、HAT1稳定低表达以及BAP1低表达且HAT1低表达的慢病毒细胞稳转株。通过WB和PCR验证BAP1在肾癌细胞系中对于HAT1的调控。使用CCK-8细胞增殖和TRANSWELL侵袭实验检测对于肾癌细胞增殖和侵袭能力的影响。利用免疫组化和临床回访数据分析其临床意义。结果:肾癌细胞系中BAP1表达降低后HAT1表达升高。BAP1低表达部分通过了HAT1表达升高促进肾癌的增殖和侵袭能力。在肾癌组织中BAP1的表达与HAT1表达具有相关性(P0.05)。结论:BAP1敲低的肾癌细胞中HAT1特异性的高表达可能是导致肾癌细胞增殖和侵袭能力增强的原因。BAP1与HAT1在肾癌组织中的表达具有显著相关性。