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Sample records for p-gp protein expression

  1. P-gp Protein Expression and Transport Activity in Rodent Seizure Models and Human Epilepsy.

    PubMed

    Hartz, Anika M S; Pekcec, Anton; Soldner, Emma L B; Zhong, Yu; Schlichtiger, Juli; Bauer, Bjoern

    2017-03-02

    A cure for epilepsy is currently not available, and seizure genesis, seizure recurrence, and resistance to antiseizure drugs remain serious clinical problems. Studies show that the blood-brain barrier is altered in animal models of epilepsy and in epileptic patients. In this regard, seizures increase expression of blood-brain barrier efflux transporters such as P-glycoprotein (P-gp), which is thought to reduce brain uptake of antiseizure drugs, and thus, contribute to antiseizure drug resistance. The goal of the current study was to assess the viability of combining in vivo and ex vivo preparations of isolated brain capillaries from animal models of seizures and epilepsy as well as from patients with epilepsy to study P-gp at the blood-brain barrier. Exposing isolated rat brain capillaries to glutamate ex vivo upregulated P-gp expression to levels that were similar to those in capillaries isolated from rats that had status epilepticus or chronic epilepsy. Moreover, the fold-increase in P-gp protein expression seen in animal models is consistent with the fold-increase in P-gp observed in human brain capillaries isolated from patients with epilepsy compared to age-matched control individuals. Overall, the in vivo/ex vivo approach presented here allows detailed analysis of the mechanisms underlying seizure-induced changes of P-gp expression and transport activity at the blood-brain barrier. This approach can be extended to other blood-brain barrier proteins that might contribute to drug-resistant epilepsy or other CNS disorders as well.

  2. Down-regulation of P-gp expression and function after Mulberroside A treatment: potential role of protein kinase C and NF-kappa B.

    PubMed

    Li, Yuhua; Huang, Ling; Zeng, Xuezhen; Zhong, Guoping; Ying, Mengjia; Huang, Min; Bi, Huichang

    2014-04-25

    P-Glycoprotein (P-gp) plays a major role in drug-drug and herb-drug interactions. Mulberroside A (Mul A) is one of the main bioactive constituents of Sangbaipi, the dried root-bark of Morus alba L. (white mulberry), which is officially listed in the Chinese Pharmacopoeia. In the present study, we investigated the effect of Mul A treatment on mRNA expression and protein expression of P-gp in the Caco-2 cells by real-time qPCR and Western blot analysis. The effect of Mul A treatment on the function of P-gp in vitro and in vivo was assessed by Rho123 transport assay and a pharmacokinetic study. The potential roles of protein kinase C (PKC) and nuclear factor kappa B (NF-κB) in the expression regulation of P-gp after Mul A treatment were also investigated. The results revealed that Mul A treatment significantly decreased the mRNA and protein expression of P-gp in Caco-2 cells after treatment with Mul A (5-20 μM). Furthermore, Mul A treatment displayed apparently inhibitory effect on the function of P-gp both in vitro and in vivo. In addition, activation of PKC activity and NF-κB nuclear translocation were observed in the presence of Mul A, which suggested that PKC and NF-κB might play crucial roles in Mul A-induced suppression of P-gp. Our study demonstrated that Mul A treatment could down-regulate P-gp expression and function accompanied by the activation of PKC and NF-κB, and this should be taken into consideration in potential herb-drug interactions when Mul A or M. alba are co-administered with other drugs transported by P-gp.

  3. Effect of Phoneutria nigriventer venom on the expression of junctional protein and P-gp efflux pump function in the blood-brain barrier.

    PubMed

    Rapôso, Catarina; Odorissi, Paulo Alexandre Miranda; Oliveira, Alexandre L R; Aoyama, Hiroshi; Ferreira, Carmen Verissima; Verinaud, Liana; Fontana, Karina; Ruela-de-Sousa, Roberta R; da Cruz-Höfling, Maria Alice

    2012-09-01

    Phoneutria nigriventer spider venom (PNV) contains Ca(2+), K(+) and Na(+) channel-acting peptides that affect neurotransmitter release and causes excitotoxicity in PNS and CNS. It has been demonstrated that PNV causes blood-brain barrier (BBB) breakdown of hippocampal microvessels time-dependently through enhanced microtubule-mediated vesicular transport. Herein, it is hypothesized that PNV can cause BBB breakdown in the hippocampus and cerebellum time-dependently through other molecular mechanisms. The BBB integrity was assessed through the analysis of expression of Poly-glycoprotein (P-gp) efflux transporter protein, laminin from basement membrane and endothelial tight junctional and adhesion junctional (TJ/AJ) proteins. Phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) expression, which are known to have a role in the phosphorylation of junctional proteins and BBB opening, were also investigated. Astrocytes P-gp activity was determined by flow cytometry. The study demonstrated temporary decreased expression of laminin, TJ and AJ proteins (ZO1//occludin//claudin-5//beta-catenin) and P-gp (more prominently in hippocampus), which was completely or partially resolved between 2 and 5 h (and more quickly for cerebellum). PNV inhibited P-gp activity in astrocytes. PP2A phosphorylation, which inhibits the enzyme activity, was increased in both regions (15-45 min); however the phosphorylation level returned to baseline after 2 h. In conclusion, PNV disrupts paracellular transport in the BBB and possesses substrates for the active P-gp efflux transporter located in the BBB complex. Further studies into cellular mechanisms of astrocyte/endothelial interactions, using PNV as tool, may identify how astrocytes regulate the BBB, a characteristic that may be useful for the temporary opening of the BBB.

  4. Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines.

    PubMed

    Feinshtein, Valeria; Erez, Offer; Ben-Zvi, Zvi; Erez, Noam; Eshkoli, Tamar; Sheizaf, Boaz; Sheiner, Eyal; Huleihel, Mahmud; Holcberg, Gershon

    2013-01-01

    Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD), a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) expression, and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp) for comparison). Study design. Following the establishment of the basal expression of these transporters in the membrane fraction of all three cell lines, P-gp and BCRP protein and mRNA levels were determined following chronic (24-72 h) exposure to CBD, by Western Blot and qPCR. CBD impact on P-gp efflux function was examined by uptake of specific P-gp fluorescent substrates (calcein-AM, DiOC2(3) and rhodamine123(rh123)). Cyclosporine A (CsA) served as a positive control. Results. Chronic exposure to CBD resulted in significant changes in the protein and mRNA levels of both transporters. While P-gp was down-regulated, BCRP levels were up-regulated in the choriocarcinoma cell lines. CBD had a remarkably different influence on P-gp and BCRP expression in MCF7/P-gp cells, demonstrating that these are cell type specific effects. P-gp dependent efflux (of calcein, DiOC2(3) and rh123) was inhibited upon short-term exposure to CBD. Conclusions. Our study shows that CBD might alter P-gp and BCRP expression in the human placenta, and inhibit P-gp efflux function. We conclude that marijuana use during pregnancy may reduce placental protective functions and change its morphological and physiological characteristics.

  5. Mini-P-gp and P-gp Co-Expression in Brown Trout Erythrocytes: A Prospective Blood Biomarker of Aquatic Pollution

    PubMed Central

    Valton, Emeline; Amblard, Christian; Desmolles, François; Combourieu, Bruno; Penault-Llorca, Frédérique; Bamdad, Mahchid

    2015-01-01

    In aquatic organisms, such as fish, blood is continually exposed to aquatic contaminants. Multidrug Resistance (MDR) proteins are ubiquitous detoxification membrane pumps, which recognize various xenobiotics. Moreover, their expression is induced by a large class of drugs and pollutants. We have highlighted the co-expression of a mini P-gp of 75 kDa and a P-gp of 140 kDa in the primary culture of brown trout erythrocytes and in the erythrocytes of wild brown trout collected from three rivers in the Auvergne region of France. In vitro experiments showed that benzo[a]pyrene, a highly toxic pollutant model, induced the co-expression of mini-P-gp and P-gp in trout erythrocytes in a dose-dependent manner and relay type response. Similarly, in the erythrocytes of wild brown trout collected from rivers contaminated by a mixture of PAH and other multi-residues of pesticides, mini-P-gp and P-gp were able to modulate their expression, according to the nature of the pollutants. The differential and complementary responses of mini-P-gp and P-gp in trout erythrocytes suggest the existence in blood cells of a real protective network against xenobiotics/drugs. This property could be exploited to develop a blood biomarker of river pollution. PMID:26854141

  6. Chitosan Influences the Expression of P-gp and Metabolism of Norfloxacin in Grass Carp.

    PubMed

    Hu, Kun; Xie, Xinyan; Zhao, Yi-Ni; Li, Yi; Ruan, Jiming; Li, Hao-Ran; Jin, Tianyi; Yang, Xian-Le

    2015-06-01

    The aim of this study was to investigate the relationship between the administration of chitosan (CTS), expression of permeability glycoprotein (P-gp), and the metabolism of norfloxacin (NOR) in Grass Carp Ctenopharyngodon idella. Fish were administrated with a single dose of either NOR, CTS, 1:5 NOR-CTS or 1:10 NOR-CTS. The P-gp expression was analyzed by immunohistochemistry and real time-PCR. The concentration of NOR was determined using HPLC. The mRNA and protein expression of P-gp in the fish intestine was significantly enhanced following a single dosage of 40 mg/kg NOR, and peak expression occurred at 3 h after drug administration (P < 0.05). A single dosage of both 1:5 NOR-CTS and 1:10 NOR-CTS reduced the intestinal P-gp expression to levels significantly lower than that from NOR alone (P < 0.05), but significantly higher than that from the control (P < 0.05). Interestingly, CTS alone also led to a slight decrease in P-gp expression. In addition, pharmacokinetic assays revealed a marked increase in area under the curve (AUC) of NOR with 1:5 and 1:10 NOR-CTS, by approximately 1.5-fold and threefold, respectively. Finally, the relative bioavailability of NOR after a single oral dosage of 1:5 and 1:10 NOR-CTS was enhanced to 148.02% and 304.98%, respectively. In this study, we demonstrated that the transmembrane glycoprotein P-gp regulates NOR metabolism in the intestine of Grass Carp, suggesting that NOR may be a direct substrate of P-gp. More importantly, we showed that CTS can inhibit P-gp expression in a dose-dependent manner and improve the relative bioavailability of NOR in this species.

  7. Increased brain uptake of venlafaxine loaded solid lipid nanoparticles by overcoming the efflux function and expression of P-gp.

    PubMed

    Zhou, Yan; Zhang, Guoqiang; Rao, Zhi; Yang, Yang; Zhou, Qian; Qin, Hongyan; Wei, Yuhui; Wu, Xin'an

    2015-07-01

    Venlafaxine (VLX) could be pumped out of the brain by P-glycoprotein (P-gp). Moreover, the expression of P-gp distributed in blood-brain barrier could be significantly induced by VLX. Thus, P-gp could be considered as the nature barrier for delivering of VLX to the brain. The aim of this study was to investigate whether the efflux function and increased expression of P-gp could be reversed by utilizing solid lipid nanoparticles (SLN). VLX solid lipid nanoparticles (VLX - SLN) were prepared and evaluated. Pharmacokinetics and brain distribution of VLX in different formulations were conducted after oral or intravenous administration. P-gp efflux function to VLX was evaluated by the brain uptake amount of VLX, while P-gp expression was investigated by Western blotting. Results indicated that the entrapment, mean size and zata potential of VLX - SLN was 74.9 ± 3.0 %, 186.3 ± 69.26 nm and -22.8 ± 7.78 mv, respectively. After vein injection of VLX formulations, the brain uptake amount of VLX from VLX - SLN was significantly higher than that of VLX solution, VLX solution with empty SLN (VLX+ empty SLN) and VLX solution with Verapamil (VLX + Ver), respectively. Furthermore, the protein mass of P-gp in VLX - SLN treated group was the lowest among all the investigated groups. These results indicated that SLN could overcome P-gp and achieve brain target by intravenous administration.

  8. AZT and emodin exhibit synergistic growth-inhibitory effects on K562/ADM cells by inducing S phase cell cycle arrest and suppressing MDR1 mRNA/p-gp protein expression.

    PubMed

    Chen, Peng; Liu, Yingxia; Sun, Yanqing; Chen, Che; Qi, Yongmei; Zhang, Yingmei

    2013-12-01

    Previous studies have demonstrated that both 3'-azido-3'-deoxythymidine (AZT) and emodin, a traditional chemotherapy agent, can inhibit the growth of many types of cancer cells. This study aimed to evaluate the effect of AZT and emodin on adriamycin-resistant human chronic myelogenous leukemia (K562/ADM) cells, determine the expression of multidrug resistance 1 (MDR1) mRNA/p-glycoprotein (p-gp) protein, a protein known to induce resistance to anticancer agents, and to elucidate the underlying molecular mechanisms. K562/ADM cells were treated with AZT (10-160 μM) or emodin (5-80 μM) for 24, 48 and 72 h and cell viability was measured using the MTT assay. The effect of AZT (16.5, 33 and 66 μM) and emodin (6.1, 17.6 and 33.2 μM) on K562/ADM cell cycle distribution was determined by flow cytometry, and MDR1 mRNA/p-gp protein expression was determined by real time RT-PCR and western blotting. The growth suppression of emodin was dramatically enhanced by AZT in K562/ADM cells. The IC50 of AZT and emodin was lower than that of emodin alone. All examined combinations of AZT and emodin yielded a synergetic effect (CI < 1). Furthermore, AZT and emodin altered the cell cycle distribution and led to an accumulation of cells in S phase. Meanwhile, the expression of MDR1 mRNA/p-gp protein was markedly decreased. These results show a synergistic growth-inhibitory effect of AZT and emodin in K562/ADM cells, which is achieved through S phase arrest. MDR1 might ultimately be responsible for these phenomena.

  9. In vitro and in vivo evaluation of the effects of piperine on P-gp function and expression

    SciTech Connect

    Han Yi; Chin Tan, Theresa May; Lim, Lee-Yong

    2008-08-01

    Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 {mu}M, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [{sup 3}H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [{sup 3}H]-digoxin polarized transport attained at 50 {mu}M of piperine. In contrast, prolonged (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 {mu}M) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by a higher degree of [{sup 3}H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. Peroral administration of piperine at the dose of 112 {mu}g/kg body weight/day to male Wistar rats for 14 consecutive days also led to increased intestinal P-gp levels. However, there was a concomitant reduction in the rodent liver P-gp although the kidney P-gp level was unaffected. Our data suggest that caution should be exercised when piperine is to be co-administered with drugs that are P-gp substrates, particularly for patients whose diet relies heavily on pepper.

  10. [Effect of 18alpha-glycyrrhizic acid and 18beta-glycyrrhizic acid on P-gp function and expression in Caco-2 cells].

    PubMed

    Yan, Miao; Li, Lanfang; Li, Huande; Fang, Pingfei; Xu, Ping; Zheng, Mei; Xu, Danhua

    2012-01-01

    The aim of the present study was to evaluate the modulating effect of glycyrrhizic acid C-18 epimers, 18alpha-glycyrrhizic acid (alpha-GL) and 18beta-glycyrrhizic acid (beta-GL) on both P-glycoprotein (P-gp) activity and expression in Caco-2 cell. The effects of P-gp activity were analyzed by rhodamine (Rhd 123) accumulation test, and those of P-gp expression were analyzed by flow cytometry and real-time PCR. At middle and high concentrations (10, 60 micromol x L(-1)), alpha-GL inhibited the function of P-gp and with on dose dependent while beta-GL induced the function of P-gp at three test concentrations with no dose dependent too. At middle and high concentrations (10, 60 micromol x L(-1)), alpha-GL down-regulated the expression of MDR1 mRNA. At high concentrations (60 micromol x L(-1)), beta-GL up-regulated the expression of MDR1 mRNA; At high concentrations (60 micromol x L(-1)), beta-GL induced the expression of P-gp protein while alpha-GL has no effect on the expression of P-gp protein at three test concentrations. The effects of alpha-GL and beta-GL on the expression of MDR1 mRNA and CYP3A mRNA showed the same trend. The character that epimers of GL act on CYP3A and P-gp show similar stereo selectivity whether relate to PXR need further study.

  11. Mulberroside A suppresses PXR-mediated transactivation and gene expression of P-gp in LS174T cells.

    PubMed

    Li, Yuhua; Huang, Ling; Sun, Jiahong; Wei, Xiaohua; Wen, Jinhua; Zhong, Guoping; Huang, Min; Bi, Huichang

    2016-12-05

    Mulberroside A (Mul A) is the main bioactive constituents of Sangbaipi, which is officially listed in the Chinese Pharmacopoeia. The pregnane X receptor (PXR) has been recognized as the critical mediator of human P-glycoprotein (P-gp) gene transactivation. In this study, the effect of Mul A on PXR-mediated transactivation and gene expression of P-gp was investigated. It was found that Mul A significantly suppressed PXR-mediated P-gp luciferase activity induced by rifampicin (Rif). Furthermore, Rif induced an elevation of P-gp expression and transport activity, which was apparently suppressed by Mul A. However, Mul A did not suppress the P-gp luciferase activity, P-gp expression, and function in the absence of Rif. These findings suggest that Mul A suppresses PXR-mediated transactivation and P-gp expression induced by Rif. This should be taken into consideration to predict any potential herb-drug interactions when Mul A or Sangbaipi are co-administered with Rif or other PXR agonist drugs.

  12. Dual-phase 99mTc-MIBI imaging and the expressions of P-gp, GST-π, and MRP1 in hyperparathyroidism.

    PubMed

    Xue, Jianjun; Liu, Yan; Yang, Danrong; Yu, Yan; Geng, Qianqian; Ji, Ting; Yang, Lulu; Wang, Qi; Wang, Yuanbo; Lu, Xueni; Yang, Aimin

    2017-08-11

    The aim of this study was to further elucidate the mechanisms of dual-phase technetium-99m methoxyisobutylisonitrile (Tc-MIBI) parathyroid imaging by exploring the association between early uptake results (EUR), delayed uptake results (DUR), and the retention index (RI) in dual-phase Tc-MIBI parathyroid imaging and P glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and glutathione S-transferase-π (GST-π) expression in hyperparathyroidism (HPT). Preoperative dual-phase (early and delayed) Tc-MIBI imaging was performed on 74 patients undergoing parathyroidectomy for HPT. EUR, DUR, and RI were calculated. P-gp, MRP1, and GST-π expressions were assessed using immunohistochemistry in resected tissue from HPT and control patients. The association between P-gp, MRP1, and GST-π expressions and EUR, DUR, and RI in HPT was evaluated. The positive rate of dual-phase T c-MIBI imaging was 91.89% (68/74) and the false-negative rate was 8.11% (6/74). P-gp and GST-π expressions were higher in tissues resected from control compared with HPT patients (47.37 and 81.5%, P<0.05); there was no difference in MRP1. EUR were associated with P-gp and GST-π expressions, and DUR were associated with MRP1 expression. There was a significant difference in MRP1 expression between RI greater than or equal to 0 and RI less than 0. There was no relationship between the sensitivity of dual-phase Tc-MIBI imaging and P-gp, MRP1, and GST-π expressions in resected parathyroid tissue. The six false-negative HPT cases consisted of three P-gp (-)/MRP1 (-) tissues, three P-gp (-)/GST-π (-) tissues, and four MRP1 (-)/GST-π (-) tissues. As P-gp and GST-π expressions were higher in tissues resected from control compared with HPT patients, Tc-MIBI may wash out faster from normal parathyroid tissue surrounding the lesion compared with the lesion itself, facilitating detection.This is an open-access article distributed under the terms of the Creative Commons Attribution

  13. Expression of HIF-1α and P-gp in non-small cell lung cancer and the relationship with HPV infection.

    PubMed

    Lu, Yimin; Yu, Le-Qun; Zhu, Lixia; Zhao, Nian; Zhou, Xing-Ju; Lu, Xudong

    2016-08-01

    The aim of the study was to study the expression of hypoxia-inducible factor-1α (HIF-1α) and P-glycoprotein (P-gp) and analyze its correlation with human papillomavirus (HPV) infection. From January, 2012 to May, 2014, 72 cases of non-small cell lung cancer (NSCLC) pathologic tissue samples were selected from the study group. Fifty-four lung benign lesions were selected to serve as the control group. HIF-1α and P-gp expression levels were detected using immunohistochemistry. PCR was used to detect the expression of HPV genome employing specific primers for HPV 16 and 18 types. The results showed that there was 47.2 and 63.9% positive HIF-1α and P-gp expression in the study group. No P-gp or HIF-1α expression was detected in the control group. The results established a positive correlation between the expression of HIF-1α and P-gp. In the study group, the expression and differentiation degree of HIF-1α was related to lymphatic metastasis. The HIF-1α expression in the well-differentiated samples was lower than that in the moderate or poorly differentiated samples. HIF-1α expression in patients with lymphatic metastasis was higher than in patients without metastasis. The expression rate of P-gp in adenocarcinoma was higher than that in squamous carcinoma. The detection rate of HPV DNA was 45.83 and 3.70% in the study and control groups, respectively. The HPV infection and differentiation degree had relevance to lymphatic metastasis in the study group. The HPV DNA detection rate in the well-differentiated samples was lower than that in the moderate or poorly differentiated samples. The HPV DNA detection rate in patients with lymphatic metastasis was higher than that in patients with no lymphatic metastasis. There was a close link between HIF-1α, P-gp expression and NSCLC occurrence, and the development of multidrug resistance. In conclusion, the detection of HIF-1α and P-gp expression can effectively predict drug resistance during chemotherapy in NSCLC, and

  14. Actin filament-associated protein 1 (AFAP-1) is a key mediator in inflammatory signaling-induced rapid attenuation of intrinsic P-gp function in human brain capillary endothelial cells.

    PubMed

    Hoshi, Yutaro; Uchida, Yasuo; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

    2017-04-01

    The purpose of this study was to identify regulatory molecule(s) involved in the inflammatory signaling-induced decrease in P-glycoprotein (P-gp) efflux function at the blood-brain barrier (BBB) that may occur in brain diseases. We confirmed that in vivo P-gp efflux activity at the BBB was decreased without any change in P-gp protein expression level in a mouse model of acute inflammation induced by 3 mg/kg lipopolysaccharide. In a human BBB model cell line (human brain capillary endothelial cells; hCMEC/D3), 1-h treatment with 10 ng/mL tumor necrosis factor-α (TNF-α; an inflammatory mediator) rapidly reduced P-gp efflux activity, but had no effect on P-gp protein expression level. To clarify the non-transcriptional mechanism that causes the decrease in intrinsic efflux activity of P-gp in acute inflammation, we applied comprehensive quantitative phosphoproteomics to compare hCMEC/D3 cells treated with TNF-α and vehicle (control). Actin filament-associated protein-1 (AFAP-1), MAPK1, and transcription factor AP-1 (AP-1) were significantly phosphorylated in TNF-α-treated cells, and were selected as candidate proteins. In validation experiments, knockdown of AFAP-1 expression blocked the reduction in P-gp efflux activity by TNF-α treatment, whereas inhibition of MAPK function or knockdown of AP-1 expression did not. Quantitative targeted absolute proteomics revealed that the reduction in P-gp activity by TNF-α did not require any change in P-gp protein expression levels in the plasma membrane. Our results demonstrate that AFAP-1 is a key mediator in the inflammatory signaling-induced, translocation-independent rapid attenuation of P-gp efflux activity in human brain capillary endothelial cells. © 2017 International Society for Neurochemistry.

  15. P-gp expression levels in the erythrocytes of brown trout: a new tool for aquatic sentinel biomarker development.

    PubMed

    Valton, Emeline; Wawrzyniak, Ivan; Amblard, Christian; Combourieu, Bruno; Bayle, Marie-Laure; Desmolles, François; Kwiatkowski, Fabrice; Penault-Llorca, Frédérique; Bamdad, Mahchid

    2017-09-01

    P-glycoprotein (P-gp) is a ubiquitous membrane detoxification pump involved in cellular defence against xenobiotics. Blood is a hub for the trade and transport of physiological molecules and xenobiotics. Our recent studies have highlighted the expression of a 140-kDa P-gp in brown trout erythrocytes in primary cell culture and its dose-dependent response to Benzo[a]pyrene pollutant. The purpose of this study was focused on using P-gp expression in brown trout erythrocytes as a biomarker for detecting the degree of river pollution. abcb1 gene and P-gp expression level were analysed by reverse transcriptase-PCR and Western blot, in the erythrocytes of brown trouts. The latter were collected in upstream and downstream of four rivers in which 17 polycyclic aromatic hydrocarbons and 348 varieties of pesticides micro-residues were analysed by liquid chromatography and mass spectrometry. The abcb1 gene and the 140-kDa P-gp were not expressed in trout erythrocytes from uncontaminated river. In contrast, they are clearly expressed in contaminated rivers, in correlation with the river pollution degree and the nature of the pollutants. This biological tool may offer considerable advantages since it provides an effective response to the increasing need for an early biomarker.

  16. Transport Inhibition of Digoxin Using Several Common P-gp Expressing Cell Lines Is Not Necessarily Reporting Only on Inhibitor Binding to P-gp

    PubMed Central

    Lumen, Annie Albin; Li, Libin; Li, Jiben; Ahmed, Zeba; Meng, Zhou; Owen, Albert; Ellens, Harma; Hidalgo, Ismael J.; Bentz, Joe

    2013-01-01

    We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable

  17. Validation of a P-Glycoprotein (P-gp) Humanized Mouse Model by Integrating Selective Absolute Quantification of Human MDR1, Mouse Mdr1a and Mdr1b Protein Expressions with In Vivo Functional Analysis for Blood-Brain Barrier Transport

    PubMed Central

    Sadiq, Muhammad Waqas; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Hammarlund-Udenaes, Margareta

    2015-01-01

    It is essential to establish a useful validation method for newly generated humanized mouse models. The novel approach of combining our established species-specific protein quantification method combined with in vivo functional studies is evaluated to validate a humanized mouse model of P-gp/MDR1 efflux transporter. The P-gp substrates digoxin, verapamil and docetaxel were administered to male FVB Mdr1a/1b(+/+) (FVB WT), FVB Mdr1a/1b(-/-) (Mdr1a/1b(-/-)), C57BL/6 Mdr1a/1b(+/+) (C57BL/6 WT) and humanized C57BL (hMDR1) mice. Brain-to-plasma total concentration ratios (Kp) were measured. Quantitative targeted absolute proteomic (QTAP) analysis was used to selectively quantify the protein expression levels of hMDR1, Mdr1a and Mdr1b in the isolated brain capillaries. The protein expressions of other transporters, receptors and claudin-5 were also quantified. The Kp for digoxin, verapamil, and docetaxel were 20, 30 and 4 times higher in the Mdr1a/1b(-/-) mice than in the FVB WT controls, as expected. The Kp for digoxin, verapamil and docetaxel were 2, 16 and 2-times higher in the hMDR1 compared to the C57BL/6 WT mice. The hMDR1 mice had 63- and 9.1-fold lower expressions of the hMDR1 and Mdr1a proteins than the corresponding expression of Mdr1a in C57BL/6 WT mice, respectively. The protein expression levels of other molecules were almost consistent between C57BL/6 WT and hMDR1 mice. The P-gp function at the BBB in the hMDR1 mice was smaller than that in WT mice due to lower protein expression levels of hMDR1 and Mdr1a. The combination of QTAP and in vivo functional analyses was successfully applied to validate the humanized animal model and evaluates its suitability for further studies. PMID:25932627

  18. Validation of a P-Glycoprotein (P-gp) Humanized Mouse Model by Integrating Selective Absolute Quantification of Human MDR1, Mouse Mdr1a and Mdr1b Protein Expressions with In Vivo Functional Analysis for Blood-Brain Barrier Transport.

    PubMed

    Sadiq, Muhammad Waqas; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Hammarlund-Udenaes, Margareta

    2015-01-01

    It is essential to establish a useful validation method for newly generated humanized mouse models. The novel approach of combining our established species-specific protein quantification method combined with in vivo functional studies is evaluated to validate a humanized mouse model of P-gp/MDR1 efflux transporter. The P-gp substrates digoxin, verapamil and docetaxel were administered to male FVB Mdr1a/1b(+/+) (FVB WT), FVB Mdr1a/1b(-/-) (Mdr1a/1b(-/-)), C57BL/6 Mdr1a/1b(+/+) (C57BL/6 WT) and humanized C57BL (hMDR1) mice. Brain-to-plasma total concentration ratios (Kp) were measured. Quantitative targeted absolute proteomic (QTAP) analysis was used to selectively quantify the protein expression levels of hMDR1, Mdr1a and Mdr1b in the isolated brain capillaries. The protein expressions of other transporters, receptors and claudin-5 were also quantified. The Kp for digoxin, verapamil, and docetaxel were 20, 30 and 4 times higher in the Mdr1a/1b(-/-) mice than in the FVB WT controls, as expected. The Kp for digoxin, verapamil and docetaxel were 2, 16 and 2-times higher in the hMDR1 compared to the C57BL/6 WT mice. The hMDR1 mice had 63- and 9.1-fold lower expressions of the hMDR1 and Mdr1a proteins than the corresponding expression of Mdr1a in C57BL/6 WT mice, respectively. The protein expression levels of other molecules were almost consistent between C57BL/6 WT and hMDR1 mice. The P-gp function at the BBB in the hMDR1 mice was smaller than that in WT mice due to lower protein expression levels of hMDR1 and Mdr1a. The combination of QTAP and in vivo functional analyses was successfully applied to validate the humanized animal model and evaluates its suitability for further studies.

  19. The process behind the expression of mdr-1/P-gp and mrp/MRP in human leukemia/lymphoma.

    PubMed

    Hirose, Masao

    2009-04-01

    There is a controversy over the link between phenotypes of multidrug resistance (MDR) and clinical outcome in leukemia/lymphoma patients. This may be because the process behind the induction and loss of expression of genotypes and phenotypes by which MDR develops and the role of MDR in fresh cells of human leukemia/lymphoma are not clearly defined. P-glycoprotein (P-gp) increased and decreased along with mdr-1 expression in three cell lines out of five vincristine (VCR)-resistant cell lines. MRP appeared with increased mrp expression in the other two cell lines. After the drug was removed from the culture system, mdr-1/P-gp changed in parallel with the level of VCR resistance, although mrp and MRP did not. It was concluded that P-gp is directly derived from mdr-1 and that mdr-1/P-gp supports the VCR-resistance but mrp/MRP is not directly linked to the VCR-resistance. These results should contribute to a better understanding of MDR phenomenon in cancer.

  20. Chrysosplenetin inhibits artemisinin efflux in P-gp-over-expressing Caco-2 cells and reverses P-gp/MDR1 mRNA up-regulated expression induced by artemisinin in mouse small intestine.

    PubMed

    Ma, Liping; Wei, Shijie; Yang, Bei; Ma, Wei; Wu, Xiuli; Ji, Hongyan; Sui, Hong; Chen, Jing

    2017-12-01

    CYP3A4 and P-gp together form a highly efficient barrier for orally absorbed drugs and always share the same substrates. Our previous work revealed that chrysosplenetin (CHR) significantly augmented the rat plasma level and anti-malarial efficacy of artemisinin (ART), partially due to the uncompetitive inhibition effect of CHR on rat CYP3A. But the impact of CHR on P-gp is still unknown. The present study investigates whether CHR interferes with P-gp-mediated efflux of ART and elucidates the underlying mechanism. P-gp-over-expressing Caco-2 cells were treated with ART (10 μM) or ART-CHR (1:2, 10:20 μM) in ART bidirectional transport experiment. ART concentration was determined by UHPLC-MS/MS method. Healthy male ICR mice were randomly divided into nine groups (n = 6) including negative control (0.5% CMC-Na solution, 13 mL/kg), ART alone (40 mg/kg), verapamil (positive control, 40 mg/kg), ART-verapamil (1:1, 40:40 mg/kg), CHR alone (80 mg/kg), ART-CHR (1:0.1, 40:4 mg/kg), ART-CHR (1:1, 40:40 mg/kg), ART-CHR (1:2, 40:80 mg/kg) and ART-CHR (1:4, 40:160 mg/kg). The drugs were administrated intragastrically for seven consecutive days. MDR1 and P-gp expression levels in mice small intestine were examined by performing RT-PCR and western blot analysis. ABC coupling ATPase activity was also determined. After combined with CHR (1:2), Papp (AP-BL) and Papp (BL-AP) of ART changed to 4.29 × 10 (-) (8) (increased 1.79-fold) and 2.85 × 10 (-) (8 )cm/s (decreased 1.24-fold) from 2.40 × 10 (-) (8) and 3.54 × 10 (-) (8 )cm/s, respectively. Efflux ratio (PBA/PAB) declined 2.21-fold (p < 0.01) versus to ART alone. ART significantly up-regulated both MDR1 mRNA and P-gp levels compared with vehicle, while CHR in combination ratio of 0:1, 0.1:1, 1:1, 2:1 and 4:1 with ART, reversed them to normal levels as well as negative control (p < 0.05). The ATPase activities in ART-CHR 1:4 and CHR alone groups achieved a

  1. Expression of P-gp, MRP, LRP, GST-π and TopoIIα and intrinsic resistance in human lung cancer cell lines.

    PubMed

    Wang, Jiarui; Zhang, Jinhui; Zhang, Lichuan; Zhao, Long; Fan, Sufang; Yang, Zhonghai; Gao, Fei; Kong, Ying; Xiao, Gary Guishan; Wang, Qi

    2011-11-01

    This study aimed to determine the relationship between the endogenous levels of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione-s-transferase-π (GST‑π) and topoisomerase IIα (TopoIIα) and intrinsic drug resistance in four human lung cancer cell lines, SK-MES-1, SPCA-1, NCI-H-460 and NCI-H-446, of different histological types. The expression of P-gp, MRP, LRP, GST-π and TopoIIα was measured by immunofluorescence, Western blotting and RT-PCR. Drug resistance to cisplatin, doxorubicin and VP-16 was determined using MTT assays. The correlation between expression of the resistance-related proteins and their roles in the resistance to drugs in these cancer cell lines was analyzed. We found that the endogenous levels of P-gp, MRP, LRP, GST-π and TopoIIα in the four cell lines varied. The level of GST-π in the SK-MES-1 cells was the highest, whereas the level of P-gp in the SPCA-1 cells was the lowest. The chemoresistance to cisplatin, doxorubicin and VP-16 in the four cell lines was different. The SPCA-1 cell line was most resistance to cisplatin; SK-MES-1 was most resistance to VP-16; whereas SK-MES-1 was most sensitive to doxorubicin. There was a positive correlation between GST-π expression and resistance to cisplatin, between TopoIIα expression and resistance to VP-16; and a negative correlation was noted between TopoIIα expression and resistance to doxorubicin. In summary, the endogenous expression of P-gp, MRP, LRP, GST-π and TopoIIα was different in the four human lung cancer cell lines of different histological types, and this variance may be associated with the variation in chemosensitivity to cisplatin, doxorubicin and VP-16. Among the related proteins, GST-π may be useful for the prediction of the intrinsic resistance to cisplatin, whereas TopoIIα may be useful to predict resistance to doxorubicin and VP-16 in human lung cancer cell lines.

  2. Serum serotonin reduced the expression of hepatic transporter Mrp2 and P-gp via regulating nuclear receptor CAR in PI-IBS rats.

    PubMed

    Shao, Yun-Yun; Huang, Jing; Ma, Yan-Rong; Han, Miao; Ma, Kang; Qin, Hong-Yan; Rao, Zhi; Wu, Xin-An

    2015-08-01

    Hepatic transporters and drug metabolizing enzymes (DMEs) play important roles in the pharmacological effects and (or) side-effects of many drugs, and are regulated by several mediators, including neurotransmitters. This work aimed to investigate whether serum levels of 5-hydroxytryptamine (5-HT) affected the expression of hepatic transporters or DMEs. The expression of hepatic transporters was assessed using the Western-blot technique in a 2,4,6-trinitrobenzenesulfonic-acid-induced rat model of post-infectious irritable bowel syndrome (PI-IBS), in which serum levels of 5-HT were significantly elevated. To further clarify the underlying mechanism, the 5-HT precursor 5-hydroxytryptophan (5-HTP) and the 5-HT depleting agent parachlorophenylalanine (pCPA) were applied to adjust serum levels of 5-HT. Serum levels of 5-HT were measured using LC-MS/MS; the expression of hepatic transporters, DMEs, and nuclear receptors were examined by Western-blot technique. Our results showed that in PI-IBS rats the expression of multidrug resistance protein 2 (Mrp2) was significantly decreased, while colonic enterochromaffin cell density and serum levels of 5-HT were all significantly increased. Moreover, 5-HTP treatment significantly increased serum levels of 5-HT and decreased the expression of Mrp2 and glycoprotein P (P-gp), whereas treatment with pCPA markedly decreased serum levels of 5-HT and increased the expression of Mrp2 and P-gp. Our results indicated that serum 5-HT regulates the expression of Mrp2 and P-gp, and the underlying mechanism may be related to the altered expression of the nuclear receptor constitutive androstane receptor (CAR).

  3. Nanoparticle-mediated drug delivery to tumor neovasculature to combat P-gp expressing multidrug resistant cancer.

    PubMed

    Bai, Fan; Wang, Chao; Lu, Qin; Zhao, Mei; Ban, Fu-Qiang; Yu, De-Hong; Guan, Ying-Yun; Luan, Xin; Liu, Ya-Rong; Chen, Hong-Zhuan; Fang, Chao

    2013-08-01

    Anticancer drug resistance is a common intractable obstacle in clinical cancer chemotherapy. Here, we hypothesize that antiangiogenic cancer therapy through the targeted delivery of antiangiogenic agents to the tumor endothelial cells (EC), not the resistant cancer cells, may have the potential of combating multidrug resistant cancer. The K237 peptide-conjugated paclitaxel loaded nanoparticles (K237-PTX-NP), which can target KDR receptors highly expressed in the tumor vasculature, were fabricated for this investigation and the human colorectal adenocarcinoma HCT-15 with naturally expressed P-gp on the cell surface was adopted as the resistant tumor model. The human umbilical vein endothelial cells (HUVEC, a classical cell model mimicking tumor EC) were much more sensitive, in the cytotoxicity and apoptosis test, to K237-PTX-NP than Taxol and non-targeted PTX-NP. The enhanced antiangiogenic feature of K237-PTX-NP can be ascribed to the active internalization mediated by the interaction of K237 and KDR specifically highly expressed on the HUVEC, and the significantly extended intracellular drug retention. The tumor vessel targeting of K237-PTX-NP led to increased nanoparticle accumulation in HCT-15 tumors, and more importantly, induced significant apoptosis of tumor vascular EC and necrosis of tumor tissues. Low dose paclitaxel formulated in K237-PTX-NP (1 mg/kg) achieved significant anticancer efficacy of inhibiting the growth of HCT-15 tumors, but the same efficacy could be only obtained with 8 fold dose paclitaxel (8 mg/kg) in Taxol plus XR9576, a potent P-gp inhibitor. The anticancer efficacy of K237-PTX-NP was well related with the improved antiangiogenic effect shown in the dramatically decreased intratumoral microvessel density and pronouncedly increased apoptotic tumor cells, and such approach did not lead to obvious toxicity in the mice. These results suggest that the nanoparticles targeting drug to tumor neovasculature may be a promising strategy for the

  4. Prognostic significance of miR-23b in combination with P-gp, MRP and LRP/MVP expression in non-small cell lung cancer.

    PubMed

    Janikova, M; Zizkova, V; Skarda, J; Kharaishvili, G; Radova, L; Kolar, Z

    2016-01-01

    Recently, miR-23b has emerged as a promising new cancer biomarker but its role in lung cancer has not been established yet. Patients still do not respond well to available treatments, probably due to expression of multidrug resistance (MDR) proteins, such as P-gp, MRP and LRP/MVP. The aim of this study was to determine the role of miR-23b in non-small cell lung cancer (NSCLC) and its relationship to the patient outcome together with MDR transporter proteins. We immunohistochemically evaluated expression of P-gp, MRP and LRP/MVP and quantified the relative levels of miR-23b in 62 NSCLC patients´ samples. The prognostic significance of miR-23b and MDR proteins was tested by Kaplan-Meier and Cox-regression analysis. Our results showed that miR-23b is mostly downregulated in NSCLC samples (57/62) and that its upregulation in tumors is connected with longer progression-free survival (PFS; P = 0.065) and overall survival (OS; P = 0.048). The Cox proportional hazard model revealed that the risk of death or relapse in NSCLC patients with miR-23b downregulation increases together with LRP/MVP expression and both risks decrease with miR-23b upregulation (HRPFS = 4.342, PPFS = 0.022; HROS = 4.408, POS = 0.015). Our findings indicate that miR-23b, especially in combination with LRP/MVP expression, might serve as a suitable prognostic biomarker for NSCLC patients.

  5. Estradiol and progesterone-mediated regulation of P-gp in P-gp overexpressing cells (NCI-ADR-RES) and placental cells (JAR).

    PubMed

    Coles, Lisa D; Lee, Insong J; Voulalas, Pamela J; Eddington, Natalie D

    2009-01-01

    The effect of progesterone and estrogen treatment on the expression and function of P-glycoprotein (P-gp) was evaluated in JAR cells and a P-gp overexpressing cell line, NCI-ADR-RES. Western blot analysis and real-time Q-PCR were used to evaluate P-gp protein and MDR1 mRNA expression respectively in the cells following incubation with progesterone (P4) and/or beta-estradiol (E2). Cellular uptake studies of the P-gp substrates, saquinavir and paclitaxel, were performed to evaluate function. Treatment with either E2 or P4 resulted in a significant increase in P-gp protein levels in the NCI-ADR-RES cells at concentrations of or greater than 100 nM or 10 nM, respectively. JAR cells also had increased levels of P-gp with 100 nM of P4 but were much more sensitive to E2 showing increased P-gp at a concentration of 1 nM. Furthermore, E2 or P4 treatment resulted in a significant decrease in cellular uptake of the P-gp substrates tested in these cells lines. Based on mRNA quantitation, a transient increase (2-fold) in MDR1 levels was observed at 8 h postincubation with either E2 or P4, while MDR1 levels remained high in the JAR cells treated with E2 for 72 h postincubation. The addition of actinomycin D, a transcription inhibitor negated the increase in P-gp by P4 and E2. P4 and E2 increase P-gp expression and function in NCI-ADR-RES and JAR cells with the ERalpha-expressing cells (JAR) much more sensitive to E2. Furthermore, transcriptional regulation by E2 and P4 likely contributes to the modulation of P-gp levels.

  6. Multidrug resistance protein P-gp interaction with nanoparticles (fullerenes and carbon nanotube) to assess their drug delivery potential: a theoretical molecular docking study.

    PubMed

    Shityakov, Sergey; Förster, Carola

    2013-01-01

    P-glycoprotein (P-gp)-mediated efflux system plays an important role to maintain chemical balance in mammalian cells for endogenous and exogenous chemical compounds. However, despite the extensive characterisation of P-gp potential interaction with drug-like molecules, the interaction of carbon nanoparticles with this type of protein molecule is poorly understood. Thus, carbon nanoparticles were analysed, such as buckminsterfullerenes (C20, C60, C70), capped armchair single-walled carbon nanotube (SWCNT or C168), and P-gp interactions using different molecular docking techniques, such as gradient optimisation algorithm (ADVina), Lamarckian genetic algorithm (FastDock), and shape-based approach (PatchDock) to estimate the binding affinities between these structures. The theoretical results represented in this work show that fullerenes might be P-gp binders because of low levels of Gibbs free energy of binding (ΔG) and potential of mean force (PMF) values. Furthermore, the SWCNT binding is energetically unfavourable, leading to a total decrease in binding affinity by elevation of the residual area (Ares), which also affects the π-π stacking mechanisms. Further, the obtained data could potentially call experimental studies using carbon nanostructures, such as SWCNT for development of drug delivery vehicles, to administer and assess drug-like chemical compounds to the target cells since organisms probably did not develop molecular sensing elements to detect these types of carbon molecules.

  7. Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

    PubMed

    Braun, Clemens; Sakamoto, Atsushi; Fuchs, Holger; Ishiguro, Naoki; Suzuki, Shinobu; Cui, Yunhai; Klinder, Klaus; Watanabe, Michitoshi; Terasaki, Tetsuya; Sauer, Achim

    2017-10-02

    Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. Kp,uu,brain and Kp,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, Kp,uu,brain for the BCRP substrates was low. In contrast, Kp,uu,CSF for both BCRP substrates was close to unity, resulting in Kp,uu,CSF/Kp,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the

  8. Pseudolaric acid B circumvents multidrug resistance phenotype in human gastric cancer SGC7901/ADR cells by downregulating Cox-2 and P-gp expression.

    PubMed

    Yu, Fei; Li, Kai; Chen, Suning; Liu, Yunpeng; Li, Yan

    2015-01-01

    Multidrug resistance (MDR) is a challenging issue in the treatment of gastric cancer. Pseudolaric acid B is a new diterpene acid compound isolated from pseudolarix, which has been found to have anti-tumor activities in recent studies. The purpose of the present study was to evaluate the effects of pseudolaric acid B in an MDR gastric cancer cell line and elucidate the possible underlying mechanisms of action. SGC7901/ADR, a P-glycoprotein (P-gp)-overexpressing cell line, was used to evaluate the efficacy of pseudolaric acid B against MDR phenotypes. The effects of pseudolaric acid B and chemotherapeutic agents on cell proliferation and apoptosis were assessed using the MTT assay and flow cytometry, respectively. Immunocytochemistry and Western blot were used to detect the possible relevant molecules in order to elucidate the underlying mechanism of action. The results showed that pseudolaric acid B inhibited cell proliferation and induced apoptosis in SGC7901/ADR cells. A low dose of pseudolaric acid B (0.5 µmol/L) augmented the inhibitory effects of chemotherapeutic agents on proliferation (p < 0.05). The expression of P-gp and cyclooxygenase 2 (Cox-2) was downregulated with pseudolaric acid B treatment. The present results showed that pseudolaric acid B inhibited cell proliferation, induced apoptosis, circumvented MDR, and increased the sensitivity of chemotherapeutic agents in vitro by downregulating the expression of P-gp and Cox-2.

  9. Characterization of human colorectal cancer MDR1/P-gp Fab antibody.

    PubMed

    Zhang, Xuemei; Xiao, Gary Guishan; Gao, Ying

    2013-01-01

    In this study, the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, were isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21 and human colorectal cancer tissue homogenate, resulting in identification of an optimal recombinant Fab clone (Number 29). Further characterization by recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29 by Isopropyl β-D-1-thiogalactopyranoside (IPTG). After purified by HiTrap Protein L, the specificity of the Fab antibody to P-gp21 was also confirmed. Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope (ALKDKKELEGSGKIAT) comprising residues 883-898 within the transmembrane (TM) domain of human P-gp, but also the binding ability with it was verified. The clinical implication of our results for development of personalized therapy of colorectal cancer will be further studied.

  10. HOXB4 knockdown reverses multidrug resistance of human myelogenous leukemia K562/ADM cells by downregulating P-gp, MRP1 and BCRP expression via PI3K/Akt signaling pathway.

    PubMed

    Wang, Hong; Jia, Xiu-Hong; Chen, Jie-Ru; Yi, Ying-Jie; Wang, Jian-Yong; Li, You-Jie; Xie, Shu-Yang

    2016-12-01

    Multidrug resistance (MDR) plays a pivotal role in human chronic myelogenous leukemia (CML) chemotherapy failure. MDR is mainly associated with the overexpression of drug efflux transporters of the ATP-binding cassette (ABC) proteins. Phosphoinositide 3-kinase (PI3K)/Akt signaling cascade is involved in the MDR phenotype and is correlated with multidrug resistance 1 (MDR1)/P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) expression in many human malignancies. Homeobox (HOX) B4, a member of the HOX gene family, has been reported to be correlated with occurrence, development, poor prognosis and drug resistance of human leukemia. In the present study, HOXB4 expression was analyzed in K562 cell line and its MDR subline K562/ADM. Compared with K562 cells, drug-resistant K562/ADM cells demonstrated evidently higher HOXB4 expression. In addition, we firstly investigated the reversal effect of HOXB4 deletion on K562/ADM cells and the underlying mechanism. The Cell Counting kit-8 (CCK-8) and flow cytometry assays showed that knockdown of HOXB4 enhanced chemosensitivity and decreased drug efflux in K562/ADM cells. Moreover, HOXB4 knockout led to downregulation of P-gp, MRP1 and BCRP expression and PI3K/Akt signaling activity, suggesting that repression of HOXB4 might be a key point to reverse MDR of K562/ADM cells.

  11. Potentiating effect of the flavonolignan (-)-hydnocarpin in combination with vincristine in a sensitive and P-gp-expressing acute lymphoblastic leukemia cell line.

    PubMed

    Bueno Pérez, Lynette; Pan, Li; Sass, Ellen; Gupta, Sneha V; Lehman, Amy; Kinghorn, A Douglas; Lucas, David M

    2013-11-01

    The potentiating action of the flavonolignan, (-)-hydnocarpin, in combination with vincristine was evaluated in the 697 acute lymphoblastic leukemia cell line and a P-gp-expressing variant, 697-R. Vincristine at 3 nM caused nearly complete growth inhibition in 697 cells versus a 17% growth inhibition in 697-R cells. When combined with (-)-hydnocarpin at concentrations of 10 and 5 μM, vincristine-mediated growth inhibition in the 697-R cells increased significantly over the sum of the individual agents to 72% (p ≤ 0.0001) and 41% (p = 0.0256), respectively. Vincristine at 1.5 nM (66% growth inhibition) and 0.75 nM (39% growth inhibition) combined with (-)-hydnocarpin at 10 μM (42% growth inhibition) in the 697 cells caused a significant increase in growth inhibition to 83% (p = 0.03) and to 61% (p < 0.0001), respectively, when compared to vincristine treatment as a single agent. To investigate the mechanism for the vincristine re-sensitization caused by (-)-hydnocarpin, the P-gp inhibitory effect of (-)-hydnocarpin was evaluated.

  12. Tariquidar sensitizes multiple myeloma cells to proteasome inhibitors via reduction of hypoxia-induced P-gp-mediated drug resistance.

    PubMed

    Muz, Barbara; Kusdono, Hubert D; Azab, Feda; de la Puente, Pilar; Federico, Cinzia; Fiala, Mark; Vij, Ravi; Salama, Noha N; Azab, Abdel Kareem

    2017-12-01

    Multiple myeloma (MM) presents a poor prognosis and high lethality of patients due to development of drug resistance. P-glycoprotein (P-gp), a drug-efflux transporter, is upregulated in MM patients post-chemotherapy and is involved in the development of drug resistance since many anti-myeloma drugs (including proteasome inhibitors) are P-gp substrates. Hypoxia develops in the bone marrow niche during MM progression and has long been linked to chemoresistance. Additionally, hypoxia-inducible transcription factor (HIF-1α) was demonstrated to directly regulate P-gp expression. We found that in MM patients P-gp expression positively correlated with the hypoxic marker, HIF-1α. Hypoxia increased P-gp protein expression and its efflux capabilities in MM cells in vitro using flow cytometry. We reported herein that hypoxia-mediated resistance to carfilzomib and bortezomib in MM cells is due to P-gp activity and was reversed by tariquidar, a P-gp inhibitor. These results suggest combining proteasome inhibitors with P-gp inhibition for future clinical studies.

  13. [Effect of different heating methods combined with neferine on the expressions of γH2AX and mdr-1/P-gp in MCF-7/Adr breast cancer cells].

    PubMed

    Huang, Chenghui; Cao, Peiguo; Xie, Zhaoxia; Zhu, Hong

    2011-04-01

    To determine the effect of different heating Methods combined with neferine(Nef) on the proliferation and expressions of γH2AX and mdr-1/P-gp in MCF-7/Adr breast cancer cells. MTT assay was used to determine block heating, water submerged heating, medium heating, and oven heating combined with 10 μg/mL Nef on adriamycin cultured MCF-7/Adr cell proliferation. The mdr-1mRNA expression was detected by real-time quantitative PCR. γH2AX and P-gp expressions were detected by Western blot. The absorbance values of MCF-7/Adr cells in different heating groups at 42 degree and 45 degree were significantly decreased, the mdr-1/P-gp expression was decreased, and γH2AX expression was upregulated compared with those of the 37 degree control group (all P<0.01). The absorbance values (P<0.01) and mdr-1/P-gp expression(P<0.05) were significantly lower and γH2AX expression(P<0.05) was significantly higher in the hyperthermia combined with 10 μg/mL Nef group than those of 10 μg/mL Nef group and hyperthermia group in MCF-7/Adr cells. The water submerged heating group had the lowest P-gp expression and the highest γH2AX expression among different heating groups at 42 degree and 45 degree in MCF-7/Adr cells (P<0.05). Hyperthermia can increase the cell toxicity of adriamycin to multidrug resistant breast cancer cells. Hyperthermia significantly damages DNA of MCF-7/Adr cells and the higher temperature, the worse effect. Multidrug resistant breast cancer cells may respond differently to the different heating methods. Combined treatment of hyperthermia with Nef can increase the sensitivity in adriamycin chemotherapy.

  14. Enhancement of placental antioxidative function and P-gp expression by sodium ferulate mediated its protective effect on rat IUGR induced by prenatal tobacco/alcohol exposure.

    PubMed

    Li, Yan; Yan, You-E; Wang, Hui

    2011-11-01

    This study was aimed to explore the therapeutic effect of sodium ferulate (SF) on rats with intrauterine growth retardation (IUGR), and then to clarify the corresponding mechanism. Pregnant rats were divided into normal group, tobacco/alcohol exposure group, and tobacco/alcohol+SF groups. Fetal developmental indices, placental weight, histological alteration, oxidative and antioxidative-function (e.g. MDA, SOD, CAT) and Mdr1 levels were assayed. Results showed exposure to tobacco/alcohol resulted in reduced fetal developmental indices and placental histological alteration, as well as the increased MDA content, decreased SOD and CAT activities and decreased Mdr1a level. After SF treatment, fetal developmental indices, and placental weight, histological alteration, oxidative and antioxidative-function and mdr1a levels were reversed. Our study indicated SF may be effective in reversing IUGR production, and its underlying mechanism may be due to enhanced placental antioxidative function and P-gp expression, which may be related to IUGR formation by tobacco/alcohol exposure.

  15. Inhibition of the Human ABC Efflux Transporters P-gp and ...

    EPA Pesticide Factsheets

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity.

  16. A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics.

    PubMed

    Saaby, Lasse; Brodin, Birger

    2017-09-01

    Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate kinetic parameters, such as Km and Vmax, for carrier-mediated transport, whereas half-maximal inhibitor concentration (IC50) and the disassociation constant for an inhibitor/P-gp complex (Ki) have been determined to estimate P-gp inhibition. This review addresses in vitro methods commonly used to study P-gp transport kinetics and aims at providing a critical evaluation of the application of steady-state Michaelis-Menten analysis of kinetic parameters for substrate/P-gp interactions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  17. Natural Products as Alternative Choices for P-Glycoprotein (P-gp) Inhibition.

    PubMed

    Dewanjee, Saikat; Dua, Tarun K; Bhattacharjee, Niloy; Das, Anup; Gangopadhyay, Moumita; Khanra, Ritu; Joardar, Swarnalata; Riaz, Muhammad; Feo, Vincenzo De; Zia-Ul-Haq, Muhammad

    2017-05-25

    Multidrug resistance (MDR) is regarded as one of the bottlenecks of successful clinical treatment for numerous chemotherapeutic agents. Multiple key regulators are alleged to be responsible for MDR and making the treatment regimens ineffective. In this review, we discuss MDR in relation to P-glycoprotein (P-gp) and its down-regulation by natural bioactive molecules. P-gp, a unique ATP-dependent membrane transport protein, is one of those key regulators which are present in the lining of the colon, endothelial cells of the blood brain barrier (BBB), bile duct, adrenal gland, kidney tubules, small intestine, pancreatic ducts and in many other tissues like heart, lungs, spleen, skeletal muscles, etc. Due to its diverse tissue distribution, P-gp is a novel protective barrier to stop the intake of xenobiotics into the human body. Over-expression of P-gp leads to decreased intracellular accretion of many chemotherapeutic agents thus assisting in the development of MDR. Eventually, the effectiveness of these drugs is decreased. P-gp inhibitors act by altering intracellular ATP levels which are the source of energy and/or by affecting membrane contours to increase permeability. However, the use of synthetic inhibitors is known to cause serious toxicities. For this reason, the search for more potent and less toxic P-gp inhibitors of natural origin is underway. The present review aims to recapitulate the research findings on bioactive constituents of natural origin with P-gp inhibition characteristics. Natural bioactive constituents with P-gp modulating effects offer great potential for semi-synthetic modification to produce new scaffolds which could serve as valuable investigative tools to recognize the function of complex ABC transporters apart from evading the systemic toxicities shown by synthetic counterparts. Despite the many published scientific findings encompassing P-gp inhibitors, however, this article stand alones because it provides a vivid picture to the readers

  18. Co-administration strategy to enhance brain accumulation of vandetanib by modulating P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) mediated efflux with m-TOR inhibitors

    PubMed Central

    Minocha, Mukul; Khurana, Varun; Qin, Bin; Pal, Dhananjay; Mitra, Ashim K

    2012-01-01

    The objectives of this study were (i) to characterize the interaction of vandetanib with P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) in vitro and in vivo (ii) to study the modulation of P-gp and BCRP mediated efflux of vandetanib with specific transport inhibitors and m-TOR inhibitors, everolimus and temsirolimus. Cellular accumulation and bi-directional transport studies in MDCKII cell monolayers were conducted to delineate the role of efflux transporters on disposition of vandetanib. Brain distribution studies were conducted in male FVB wild-type mice with vandetanib administered intravenously either alone or in the presence of specific inhibitors and m-TOR inhibitors. In vitro studies suggested that vandetanib is a high affinity substrate of Bcrp1 but is not transported by P-gp. Interestingly, in vivo brain distribution studies in FVB wild type mice indicated that vandetanib penetration into the brain is restricted by both Bcrp1 and P-gp mediated active efflux at the blood brain barrier (BBB). Co-administration of elacridar, a dual P-gp/BCRP inhibitor increased the brain to plasma concentration ratio of vandetanib upto 5 fold. Of the two m-TOR pathway inhibitors examined; everolimus showed potent effect on modulating vandetanib brain penetration whereas no significant affect on vandetanib brain uptake was observed following temsirolimus co-administration. This finding could be clinically relevant as everolimus can provide synergistic pharmacological effect in addition to primary role of vandetanib efflux modulation at BBB for the treatment of brain tumors. PMID:22633931

  19. ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice.

    PubMed

    Brzozowska, Natalia; Li, Kong M; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S; Arnold, Jonathon C

    2016-01-01

    Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-∕-)), Bcrp knockout (Abcg2 (-∕-)), combined P-gp/Bcrp knockout (Abcb1a/b (-∕-) Abcg2 (-∕-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.

  20. ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice

    PubMed Central

    Brzozowska, Natalia; Li, Kong M.; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S.

    2016-01-01

    Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b−∕−), Bcrp knockout (Abcg2−∕−), combined P-gp/Bcrp knockout (Abcb1a/b−∕−Abcg2−∕−) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders. PMID:27257556

  1. Effects of norfloxacin on hepatic genes expression of P450 isoforms (CYP1A and CYP3A), GST and P-glycoprotein (P-gp) in Swordtail fish (Xiphophorus Helleri).

    PubMed

    Liang, Ximei; Wang, Lan; Ou, Ruikang; Nie, Xiangping; Yang, YuFeng; Wang, Fang; Li, Kaibin

    2015-10-01

    The presence of antibiotics including norfloxacin in the aquatic environment may cause adverse effects in non-target organisms. But the toxic mechanisms of fluoroquinolone to fish species are still not completely elucidated. Thus, it is essential to investigate the response of fish to the exposure of fluoroquinolone at molecular or cellular level for better and earlier prediction of these environmental pollutants toxicity. The sub-chronic toxic effects of norfloxacin (NOR) on swordtail fish (Xiphophoru s helleri) were investigated by measuring mRNA expression of cytochrome P450 1A (CYP1A), cytochrome P450 3A (CYP3A), glutathione S-transferase (GST) and P-glycoprotein (P-gp) and their corresponding enzyme activities (including ethoxyresorufin O-deethylase, erythromycin N-demethylase and GST. Results showed that NOR significantly affected the expression of CYP1A, CYP3A, GST and P-gp genes in swordtails. The gene expressions were more responsive to NOR exposure than their corresponding enzyme activities. Moreover, sexual differences were found in gene expression and enzyme activities of swordtails exposed to NOR. Females displayed more dramatic changes than males. The study further demonstrated that the combined biochemical and molecular parameters were considered as useful biomarkers to improve our understanding of potential ecotoxicological risks of NOR exposure to aquatic organisms.

  2. Effect of Subchronic Intravenous Morphine Infusion and Naloxone-Precipitated Morphine Withdrawal on P-gp and Bcrp at the Rat Blood-Brain Barrier.

    PubMed

    Chaves, Catarina; Gómez-Zepeda, David; Auvity, Sylvain; Menet, Marie-Claude; Crété, Dominique; Labat, Laurence; Remião, Fernando; Cisternino, Salvatore; Declèves, Xavier

    2016-01-01

    Chronic morphine regimen increases P-glycoprotein (P-gp) and breast cancer-resistance protein (Bcrp) expressions at the rat blood–brain barrier (BBB) but what drives this effect is poorly understood. The objective of this study is to assess subchronic continuous morphine infusion and naloxone-precipitated morphine withdrawal effects on P-gp/Bcrp contents and activities at the rat BBB. Rats were treated either with (i) a continuous i.v. morphine for 120 h, (ii) escalating morphine dosing (10-40 mg/kg, i.p., 5 days), (iii) a chronic morphine regimen (10 mg/kg s.c., 5 days) followed by a withdrawal period (2 days) and treatment for 3 additional days. Animal behavior was assessed after naloxone-precipitated withdrawal (1 mg/kg, s.c.). P-gp/Bcrp expressions and activities were determined in brain microvessels by qRT-PCR, Western blot, UHPLC–MS/MS, and in situ brain perfusion of P-gp or Bcrp substrates. Results show continuous i.v. morphine did not change P-gp/Bcrp protein levels in rat brain microvessels, whereas naloxone-precipitated withdrawal after escalating or chronic morphine dose regimen increased Mdr1a and Bcrp mRNA levels by 1.4-fold and 2.4-fold, respectively. Conversely, P-gp/Bcrp protein expressions remained unchanged after naloxone administration, and brain uptake of [3H]-verapamil (P-gp) and [3H]-mitoxantrone (Bcrp) was not altered. The study concludes subchronic morphine infusion and naloxone-precipitated morphine withdrawal have poor effect on P-gp/Bcrp levels at the rat BBB.

  3. The consequence of regional gradients of P-gp and CYP3A4 for drug-drug interactions by P-gp inhibitors and the P-gp/CYP3A4 interplay in the human intestine ex vivo.

    PubMed

    Li, Ming; de Graaf, Inge A M; van de Steeg, Evita; de Jager, Marina H; Groothuis, Geny M M

    2017-04-01

    Intestinal P-gp and CYP3A4 work coordinately to reduce the intracellular concentration of drugs, and drug-drug interactions (DDIs) based on this interplay are of clinical importance and require pre-clinical investigation. Using precision-cut intestinal slices (PCIS) of human jejunum, ileum and colon, we investigated the P-gp/CYP3A4 interplay and related DDIs with P-gp inhibitors at the different regions of the human intestine with quinidine (Qi), dual substrate of P-gp and CYP3A4, as probe. All the P-gp inhibitors increased the intracellular concentrations of Qi by 2.1-2.6 fold in jejunum, 2.6-3.8 fold in ileum but only 1.2-1.3 fold in colon, in line with the different P-gp expression in these intestinal regions. The selective P-gp inhibitors (CP100356 and PSC833) enhanced 3-hydroxy-quinidine (3OH-Qi) in jejunum and ileum, while dual inhibitors of P-gp and CYP3A4 (verapamil and ketoconazole) decreased the 3OH-Qi production, despite of the increased intracellular Qi concentration, due to inhibition of CYP3A4. The outcome of DDIs based on P-gp/CYP3A4 interplay, shown as remarkable changes in the intracellular concentration of both the parent drug and the metabolite, varied among the intestinal regions, probably due to the different expression of P-gp and CYP3A4, and were different from those found in rat PCIS, which may have important implications for the disposition and toxicity of drugs and their metabolites.

  4. Reversal of P-gp-mediated multidrug resistance in colon cancer by cinobufagin.

    PubMed

    Yuan, Zeting; Shi, Xiaojing; Qiu, Yanyan; Jia, Tingting; Yuan, Xia; Zou, Yu; Liu, Cheng; Yu, Hui; Yuan, Yuxia; He, Xue; Xu, Ke; Yin, Peihao

    2017-03-01

    Cinobufagin (CBF) is isolated from the skin and posterior auricular glands of the Asiatic toad (Bufo gargarizans). This study investigated the reversal effect of CBF on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in colon cancer. The effect of CBF on the cytotoxicity of anticancer drugs in P-gp overexpressing LoVo/ADR, HCT116/L, Cao-2/ADR cells and their parental cells was determined using CCK-8 assay. Apoptosis of anti-cancer drugs and accumulation of doxorubicin (DOX) and Rhodamine 123 (Rho123) in P-gp overexpressing cells were evaluated by flow cytometry. Results indicated that CBF significantly enhanced the sensitivity of P-gp substrate drugs on P-gp overexpressing cells, but had no effect on their parental cells. CBF enhanced the effect of DOX against P-gp-overexpressing LoVo/ADR cell xenografts in nude mice. Moreover, CBF also increased cell apoptosis of chemotherapy agents and intracellular accumulation of DOX and Rho123 in the MDR cells. Further research on the mechanisms revealed non-competitive inhibition of P-gp ATPase activity, but without altering the expression of P-gp. These findings demonstrated that CBF could be further developed into a safe and potent P-gp modulator for combination use with anticancer drugs in cancer chemotherapy.

  5. A Multi-System Approach Assessing the Interaction of Anticonvulsants with P-gp

    PubMed Central

    Dickens, David; Yusof, Siti R.; Abbott, N. Joan; Weksler, Babette; Romero, Ignacio A.; Couraud, Pierre-Olivier; Alfirevic, Ana; Pirmohamed, Munir; Owen, Andrew

    2013-01-01

    30% of epilepsy patients receiving antiepileptic drugs (AEDs) are not fully controlled by therapy. The drug transporter hypothesis for refractory epilepsy proposes that P-gp is over expressed at the epileptic focus with a role of P-gp in extruding AEDs from the brain. However, there is controversy regarding whether all AEDs are substrates for this transporter. Our aim was to investigate transport of phenytoin, lamotrigine and carbamazepine by using seven in-vitro transport models. Uptake assays in CEM/VBL cell lines, oocytes expressing human P-gp and an immortalised human brain endothelial cell line (hCMEC/D3) were carried out. Concentration equilibrium transport assays were performed in Caco-2, MDCKII ±P-gp and LLC-PK1±P-gp in the absence or presence of tariquidar, an inhibitor of P-gp. Finally, primary porcine brain endothelial cells were used to determine the apparent permeability (Papp) of the three AEDs in the absence or presence of P-gp inhibitors. We detected weak transport of phenytoin in two of the transport systems (MDCK and LLC-PK1 cells transfected with human P-gp) but not in the remaining five. No P-gp interaction was observed for lamotrigine or carbamazepine in any of the seven validated in-vitro transport models. Neither lamotrigine nor carbamazepine was a substrate for P-gp in any of the model systems tested. Our data suggest that P-gp is unlikely to contribute to the pathogenesis of refractory epilepsy through transport of carbamazepine or lamotrigine. PMID:23741405

  6. Mitotane enhances doxorubicin cytotoxic activity by inhibiting P-gp in human adrenocortical carcinoma cells.

    PubMed

    Gagliano, Teresa; Gentilin, Erica; Benfini, Katiuscia; Di Pasquale, Carmelina; Tassinari, Martina; Falletta, Simona; Feo, Carlo; Tagliati, Federico; Uberti, Ettore Degli; Zatelli, Maria Chiara

    2014-12-01

    Mitotane is currently employed as adjuvant therapy as well as in the medical treatment of adrenocortical carcinoma (ACC), alone or in combination with chemotherapeutic agents. It was previously demonstrated that mitotane potentiates chemotherapeutic drugs cytotoxicity in cancer cells displaying chemoresistance due to P-glycoprotein (P-gp), an efflux pump involved in cancer multidrug resistance. The majority of ACC expresses high levels of P-gp and is highly chemoresistent. The aim of our study was to explore in vitro whether mitotane, at concentrations lower than those currently reached in vivo, may sensitize ACC cells to the cytotoxic effects of doxorubicin and whether this effect is due to a direct action on P-gp. NCI-H295 and SW13 cell lines as well as 4 adrenocortical neoplasia primary cultures were treated with mitotane and doxorubicin, and cell viability was measured by MTT assay. P-gp activity was measured by calcein and P-gp-Glo assays. P-gp expression was evaluated by Western blot. We found that very low mitotane concentrations sensitize ACC cells to the cytotoxic effects of doxorubicin, depending on P-gp expression. In addition, mitotane directly inhibits P-gp detoxifying function, allowing doxorubicin cytotoxic activity. These data provide the basis for the greater efficacy of combination therapy (mitotane plus chemotherapeutic drugs) on ACC patients. Shedding light on mitotane mechanisms of action could result in an improved design of drug therapy for patients with ACC.

  7. P-gp activity and inhibition in the different regions of human intestine ex vivo.

    PubMed

    Li, Ming; de Graaf, Inge A M; de Jager, Marina H; Groothuis, Geny M M

    2017-03-01

    Although intestinal P-glycoprotein (P-gp) has been extensively studied in vitro and in animals, its activity and the consequences of P-gp inhibition for drug disposition and toxicity in humans are still difficult to accurately extrapolate from these studies. Moreover, existing in vitro models do not take into consideration that the intestine is heterogeneous with respect to P-gp expression. Recently, we reported rat precision-cut intestinal slices (PCIS) as a physiological ex vivo model to study the regional gradient of P-gp activity and inhibition. Here we extended the application of PCIS to the human intestine. For this purpose rhodamine 123 (R123) accumulation in the presence or absence of the P-gp inhibitors verapamil, cyclosporine A, quinidine, ketoconazole, PSC833 and CP100356 was measured in PCIS of human duodenum, jejunum, ileum and colon. R123 accumulation in the presence of the P-gp inhibitors appeared to be most enhanced in the ileum compared to the other regions. Moreover, the regional differences in accumulation are in line with published differences in abundance of P-gp. The rank order of the potency of the P-gp inhibitors, reflected by their IC50 , was comparable to that in rat PCIS. However, the increase in accumulation of the P-gp substrate R123 by the inhibitors was larger in human ileum PCIS than in rat PCIS, indicating species difference in P-gp abundance. These data show that human PCIS are an appropriate ex vivo model to study the activity of intestinal P-gp and predict the inhibitory effect of drugs and of transporter-mediated drug-drug interactions in the human intestine. Copyright © 2016 John Wiley & Sons, Ltd.

  8. P-gp/ABCB1 exerts differential impacts on brain and fetal exposure to norbuprenorphine.

    PubMed

    Liao, Michael Z; Gao, Chunying; Shireman, Laura M; Phillips, Brian; Risler, Linda J; Neradugomma, Naveen K; Choudhari, Prachi; Prasad, Bhagwat; Shen, Danny D; Mao, Qingcheng

    2017-01-19

    fetal exposure to norbuprenorphine, but plays a significant role in restricting its brain distribution. The differential impacts of P-gp on norbuprenorphine distribution into the brain and fetus are likely, at least in part, due to the differences in amounts of P-gp protein expressed in the blood-brain and blood-placental barriers. BCRP is not as important as P-gp in determining both the systemic and tissue exposure to norbuprenorphine. Finally, fetal AUCs of the metabolite norbuprenorphine-β-d-glucuronide were 3-7 times greater than maternal plasma AUCs, while the maternal brain AUCs were <50% of maternal plasma AUCs, suggesting that a reversible pool of conjugated metabolite in the fetus may contribute to the high fetal exposure to norbuprenorphine.

  9. Protection promoted by pGP3 or pGP4 against Chlamydia muridarum is mediated by CD4(+) cells in C57BL/6N mice.

    PubMed

    Mosolygó, Tímea; Szabó, Agnes M; Balogh, Emese P; Faludi, Ildikó; Virók, Dezső P; Endrész, Valéria; Samu, Alíz; Krenács, Tibor; Burián, Katalin

    2014-09-08

    Urogenital tract infection with Chlamydia trachomatis is a leading cause of sexually transmitted infections. There is currently no commercially available vaccine against C. trachomatis. The highly conserved plasmid of chlamydiae has been considered to be a virulence factor and the plasmid proteins have important roles in the Chlamydia-specific immune response. This study was designed to evaluate the efficacy of vaccination with plasmid proteins in the prevention of C. muridarum lung infection in a mouse model. C57BL/6N mice were immunised 3 times subcutaneously with recombinant pGP3 or pGP4 and infected with C. muridarum. Immunisation of the mice with recombinant pGP3 or pGP4 protein caused a significantly lower chlamydial burden in the lungs of the infected mice; the lower IFN-γ level indicated a reduced extent of inflammation. In vitro or in vivo neutralisation of C. muridarum with sera obtained from immunised mice did not reduce the number of viable C. muridarum in the lungs of mice. However, adoptive transfer of the CD4(+) spleen cells isolated from the immunised mice resulted in a significantly reduced bacterial burden. Our results indicate that it is not the pGP3- and pGP4-specific antibodies, but the CD4(+) cells that are responsible for the protective effect of the immune response to plasmid proteins.

  10. Protein phosphatase complex PP5/PPP2R3C dephosphorylates P-glycoprotein/ABCB1 and down-regulates the expression and function.

    PubMed

    Katayama, Kazuhiro; Yamaguchi, Miho; Noguchi, Kohji; Sugimoto, Yoshikazu

    2014-04-01

    P-glycoprotein (P-gp)/ABCB1 is a key molecule of multidrug resistance in cancer. Protein phosphatase (PP) 2A, regulatory subunit B, gamma (PPP2R3C), which is a regulatory subunit of PP2A and PP5, was identified as a binding candidate to P-gp. Immunoprecipitation-western blotting revealed that PP5 and PPP2R3C were coprecipitated with P-gp, while PP2A was not. PP5/PPP2R3C dephosphorylated protein kinase A/protein kinase C-phosphorylation of P-gp. Knockdown of PP5 and/or PPP2R3C increased P-gp expression and lowered the sensitivity to vincristine and doxorubicin. Consequently, our results indicate that PP5/PPP2R3C negatively regulates P-gp expression and function.

  11. The putative P-gp inhibitor telmisartan does not affect the transcellular permeability and cellular uptake of the calcium channel antagonist verapamil in the P-glycoprotein expressing cell line MDCK II MDR1

    PubMed Central

    Saaby, Lasse; Tfelt-Hansen, Peer; Brodin, Birger

    2015-01-01

    Verapamil is used in high doses for the treatment of cluster headache. Verapamil has been described as a P-glycoprotein (P-gp, ABCB1) substrate. We wished to evaluate in vitro whether co administration of a P-gp inhibitor with verapamil could be a feasible strategy for increasing CNS uptake of verapamil. Fluxes of radiolabelled verapamil across MDCK II MDR1 monolayers were measured in the absence and presence of the putative P-gp inhibitor telmisartan (a clinically approved drug compound). Verapamil displayed a vectorial basolateral-to-apical transepithelial efflux across the MDCK II MDR1 monolayers with a permeability of 5.7 × 10−5 cm sec−1 compared to an apical to basolateral permeability of 1.3 × 10−5 cm sec-1. The efflux could be inhibited with the P-gp inhibitor zosuquidar. Zosuquidar (0.4 μmol/L) reduced the efflux ratio (PB-A/PA-B) for verapamil 4.6–1.6. The presence of telmisartan, however, only caused a slight reduction in P-gp-mediated verapamil transport to an efflux ratio of 3.4. Overall, the results of the present in vitro approach indicate, that clinical use of telmisartan as a P-gp inhibitor may not be an effective strategy for increasing brain uptake of verapamil by co-administration with telmisartan. PMID:26171231

  12. Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure.

    PubMed

    Marchitti, Satori A; Mazur, Christopher S; Dillingham, Caleb M; Rawat, Swati; Sharma, Anshika; Zastre, Jason; Kenneke, John F

    2017-01-01

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [((3)H)-paclitaxel and ((3)H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed.

  13. Lobular Distribution and Variability in Hepatic ATP Binding Cassette Protein B1 (ABCB1, P-gp): Ontogenetic Differences and Potential for Toxicity

    PubMed Central

    Abanda, Ngu Njei; Riches, Zoe; Collier, Abby C.

    2017-01-01

    The ATP Binding Cassette B1 (ABCB1) transporter has critical roles in endo- and xenobiotic efficacy and toxicity. To understand population variability in hepatic transport we determined ABCB1 mRNA and protein levels in total liver lysates sampled from 8 pre-defined sites (n = 24, 18–69 years), and in S9 from randomly acquired samples (n = 87, 7 days–87 years). ABCB1 levels did not differ significantly throughout individual livers and showed 4.4-fold protein variation between subjects. Neither mRNA nor protein levels varied with sex, ethnicity, obesity or triglycerides in lysates or S9 (that showed the same relationships), but protein levels were lower in pediatric S9 (p < 0.0001), with 76% of adult ABCB1 present at birth and predicted to mature in 5 years. Pediatric total liver lysates were not available. In summary, opportunistic collection for studying human hepatic ABCB1 is acceptable. Additionally, ABCB1 may be lower in children, indicating differential potential for toxicity and response to therapy in this special population. PMID:28218636

  14. CacyBP/SIP enhances multidrug resistance of pancreatic cancer cells by regulation of P-gp and Bcl-2.

    PubMed

    Chen, Xiong; Zheng, Peichan; Xue, Zengfu; Li, Jie; Wang, Wenwu; Chen, Xi; Xie, Fangwei; Yu, Zongyang; Ouyang, Xuenong

    2013-07-01

    Our former report indicates that calcyclin-binding protein or Siah-1-interacting protein (CacyBP/SIP) is over-expressed in the SGC7901/ADR cell line. However, the potential role of CacyBP/SIP in the development of multidrug resistance (MDR) of pancreatic cancer is still uncertain. In this paper, we investigated the role of CacyBP/SIP in MDR of pancreatic cancer cells and its possible underlying mechanisms, and found that CacyBP/SIP was over-expressed in the Gemcitabine induced MDR pancreatic cancer cell PC-3/Gem compared with its parental cell PC-3. Up-regulation of CacyBP/SIP expression could enhance resistance of chemotherapy drugs on PC-3 cells and inhibit Adriamycin-induced apoptosis accompanied by decreased accumulation of intracellular Adriamycin. Furthermore, CacyBP/SIP could significantly up-regulate the expression of P-gp, Bcl-2, and the transcription of the MDR1 gene. In addition, the decrease of CacyBP/SIP expression using RNA interference or P-gp inhibitor could partially reverse CacyBP/SIP-mediated MDR. In brief, our study demonstrated that CacyBP/SIP could enhance the MDR phenotype of pancreatic cancer cells by increasing the expression of P-gp and Bcl-2, thus inhibiting apoptosis of pancreatic cancer cell.

  15. Esters of the Marine-Derived Triterpene Sipholenol A Reverse P-GP-Mediated Drug Resistance

    PubMed Central

    Zhang, Yongchao; Zhang, Yun-Kai; Wang, Yi-Jun; Vispute, Saurabh G.; Jain, Sandeep; Chen, Yangmin; Li, Jessalyn; Youssef, Diaa T. A.; El Sayed, Khalid A.; Chen, Zhe-Sheng

    2015-01-01

    Our previous studies showed that several sipholane triterpenes, sipholenol A, sipholenone E, sipholenol L and siphonellinol D, have potent reversal effect for multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp/ABCB1). Through comparison of cytotoxicity towards sensitive and multi-drug resistant cell lines, we identified that the semisynthetic esters sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate potently reversed P-gp-mediated MDR but had no effect on MRP1/ABCC1 and BCRP/ABCG2-mediated MDR. The results from [3H]-paclitaxel accumulation and efflux studies suggested that these two triterpenoids were able to increase the intracellular accumulation of paclitaxel by inhibiting its active efflux. In addition, western blot analysis revealed that these two compounds did not alter the expression levels of P-gp when treated up to 72 h. These sipholenol derivatives also stimulated the ATPase activity of P-gp membranes, which suggested that they might be substrates of P-gp. Moreover, in silico molecular docking studies revealed the virtual binding modes of these two compounds into human homology model of P-gp. In conclusion, sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate efficiently inhibit the P-gp and may represent potential reversal agents for the treatment of multidrug resistant cancers. PMID:25874923

  16. Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

    PubMed

    Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Li, You-Jie; Wang, Hong; Xie, Shu-Yang

    2016-05-01

    Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML.

  17. Multifunctional PLGA Nanobubbles as Theranostic Agents: Combining Doxorubicin and P-gp siRNA Co-Delivery Into Human Breast Cancer Cells and Ultrasound Cellular Imaging.

    PubMed

    Yang, Hong; Deng, Liwei; Li, Tingting; Shen, Xue; Yan, Jie; Zuo, Liangming; Wu, Chunhui; Liu, Yiyao

    2015-12-01

    Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. One of the effective approaches to overcome MDR is to use nanoparticle-mediated the gene silence of chemotherapeutic export proteins by RNA interference to increase drug accumulation in drug resistant cancer cells. In this work, a new co-delivery system, DOX-PLGA/PEI/P-gp shRNA nanobubbles (NBs) around 327 nm, to overcome doxorubicin (DOX) resistance in MCF-7 human breast cancer was designed and developed. Positively charged polyethylenimine (PEI) were modified onto the surface of DOX-PLGA NBs through DCC/NHS crosslinking, and could efficiently condense P-gp shRNA into DOX-PLGA/PEI NBs at vector/shRNA weight ratios of 70:1 and above. An in vitro release profile demonstrated an efficient DOX release (more than 80%) from DOX-PLGA/PEI NBs at pH 4.4, suggesting a pH-responsive drug release for the multifunctionalized NBs. Cellular experimental results further showed that DOX-PLGA/PEI/P-gp shRNA NBs could facilitate cellular uptake of DOX into cells and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The IC50 of DOX-PLGA NBs against MCF-7/ADR cells was 2-fold lower than that of free DOX. The increased cellular uptake and nuclear accumulation of DOX delivered by DOX-PLGA/PEI/P-gp shRNA NBs in MCF-7/ADR cells was confirmed by fluorescence microscopy and fluorescence spectrophotometry, and might be owning to the down-regulation of P-gp and reduced the efflux of DOX. The cellular uptake mechanism of DOX-PLGA/PEI/P-gp shRNA NBs indicated that the macropinocytosis was one of the pathways for the uptake of NBs by MCF-7/ADR cells, which was also an energy-dependent process. Furthermore, the in vitro cellular ultrasound imaging suggested that the employment of the DOX-PLGA/PEI/P-gp shRNA NBs could efficiently enhance ultrasound imaging of cancer cells. These results demonstrated

  18. BZML, a novel colchicine binding site inhibitor, overcomes multidrug resistance in A549/Taxol cells by inhibiting P-gp function and inducing mitotic catastrophe.

    PubMed

    Bai, Zhaoshi; Gao, Meiqi; Zhang, Huijuan; Guan, Qi; Xu, Jingwen; Li, Yao; Qi, Huan; Li, Zhengqiang; Zuo, Daiying; Zhang, Weige; Wu, Yingliang

    2017-08-28

    Multidrug resistance (MDR) interferes with the efficiency of chemotherapy. Therefore, developing novel anti-cancer agents that can overcome MDR is necessary. Here, we screened a series of colchicine binding site inhibitors (CBSIs) and found that 5-(3, 4, 5-trimethoxybenzoyl)-4-methyl-2-(p-tolyl) imidazol (BZML) displayed potent cytotoxic activity against both A549 and A549/Taxol cells. We further explored the underlying mechanisms and found that BZML caused mitosis phase arrest by inhibiting tubulin polymerization in A549 and A549/Taxol cells. Importantly, BZML was a poor substrate for P-glycoprotein (P-gp) and inhibited P-gp function by decreasing P-gp expression at the protein and mRNA levels. Cell morphology changes and the expression of cycle- or apoptosis-related proteins indicated that BZML mainly drove A549/Taxol cells to die by mitotic catastrophe (MC), a p53-independent apoptotic-like cell death, whereas induced A549 cells to die by apoptosis. Taken together, our data suggest that BZML is a novel colchicine binding site inhibitor and overcomes MDR in A549/Taxol cells by inhibiting P-gp function and inducing MC. Our study also offers a new strategy to solve the problem of apoptosis-resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Snail-Induced Epithelial-to-Mesenchymal Transition Enhances P-gp-Mediated Multi Drug Resistance in HCC827 Cells.

    PubMed

    Tomono, Takumi; Yano, Kentaro; Ogihara, Takuo

    2017-03-17

    Overexpression and/or activation of P-glycoprotein (P-gp), which mediates efflux transport of various anti-cancer drugs in cancer cells, are associated with multi-drug resistance (MDR). On the other hand, malignant cancer cells frequently undergo epithelial-to-mesenchymal transition (EMT), thereby acquiring high migratory mobility and invasive ability. Snail is a transcriptional factor that represses multiple other factors, and its overexpression is a trigger of EMT. Since both P-gp and Snail are involved in malignant evolution of cancer, in this work, we evaluated whether or not EMT induced by overexpression of Snail influences P-gp expression and/or activity. Snail-overexpressing cells showed downregulation of epithelial markers, E-cadherin, occludin and claudin-1, and upregulation of mesenchymal markers, vimenin and ZEB1. Although Western blot analysis showed that P-gp expression levels were similar in Mock and Snail-overexpressing cells, the results of P-gp functional assays with P-gp substrates rhodamine123 and paclitaxel indicated that P-gp is activated in Snail-overexpressing cells. Indeed, Snail-overexpressing cells showed greater viability than Mock cells in the presence of paclitaxel. We observed caveolin-1 dephosphorylation and decreased GRB2 expression in Snail-overexpressing cells. These findings suggest a novel pathway leading to cancer MDR, in which Snail induces EMT concomitantly with a decrease of GRB2-mediated caveolin-1 phosphorylation, resulting in activation of P-gp.

  20. The JAK2 inhibitors CEP-33779 and NVP-BSK805 have high P-gp inhibitory activity and sensitize drug-resistant cancer cells to vincristine.

    PubMed

    Cheon, Ji Hyun; Kim, Kyeong Seok; Yadav, Dharmendra Kumar; Kim, Mihyun; Kim, Hyung Sik; Yoon, Sungpil

    2017-09-02

    P-glycoprotein (P-gp) is overexpressed in cancer cells in order to pump out chemotherapeutic drugs, and is one of the major mechanisms responsible for multidrug resistance (MDR). It is important to identify P-gp inhibitors with low toxicity to normal cells in order to increase the efficacy of anti-cancer drugs. Previously, a JAK2 inhibitor CEP-33779 demonstrated inhibitory actions against P-gp and an ability to sensitize drug-resistant cancer cells to treatment. In the present study, we tested another JAK2 inhibitor NVP-BSK805 for P-gp inhibitory activity. In molecular docking simulation modeling, NVP-BSK805 showed higher binding affinity docking scores against a P-gp member (ABCB1) than CEP-33779 did. Furthermore, we found that lower doses of NVP-BSK805 are required to inhibit P-gp in comparison with that of CEP-33779 or verapamil (an established P-gp inhibitor) in KBV20C cells, suggesting that NVP-BSK805 has higher specificity. NVP-BSK805, CEP-33779, and verapamil demonstrated similar abilities to sensitize KBV20C cells to vincristine (VIC) treatment. Our results suggested that the JAK2 inhibitors were able to inhibit P-gp pump-action via a direct binding mechanism, similar to verapamil. However, JAK2 inhibitor-induced sensitization was not observed in VIC-treated sensitive KB parent cells, suggesting that these effects are specific to resistant cancer cells. FACS, western-blot, and annexin V analyses were used to further investigate the mechanism of action of JAK2 inhibitors in VIC-treated KBV20C cells. Both CEP-33779 and NVP-BSK805 induced the sensitization of KBV20C cells to VIC treatment via the same mechanisms; they each caused a reduction in cell viability, increased G2 arrest, and upregulated expression of the DNA damaging protein pH2AX when used as co-treatments with VIC. These findings indicate that inhibition of JAK2 may be a promising target in the treatment of cancers that are resistant to anti-mitotic drugs. Copyright © 2017 Elsevier Inc. All rights

  1. BCRP and P-gp relay overexpression in triple negative basal-like breast cancer cell line: a prospective role in resistance to Olaparib.

    PubMed

    Dufour, Robin; Daumar, Pierre; Mounetou, Emmanuelle; Aubel, Corinne; Kwiatkowski, Fabrice; Abrial, Catherine; Vatoux, Catherine; Penault-Llorca, Frédérique; Bamdad, Mahchid

    2015-08-03

    The triple negative basal-like (TNBL) breast carcinoma is an aggressive and unfavorable prognosis disease. Inhibitors of poly(ADP-ribose) polymerase such as Olaparib could represent a promising targeted therapy but their sensitivity against Multidrug Resistance proteins (MDR), which causes resistance, is not well defined. Thus, our work focused on the analysis of P-gp and BCRP coexpression in the SUM1315 TNBL human cell line, in correlation with Olaparib intracellular concentration. Western blot analyses showed a clear coexpression of P-gp and BCRP in SUM1315 cells. A low cytotoxic Olaparib treatment clearly led to an increased expression of both BCRP and P-gp in these cells. Indeed, after 1.5 h of treatment, BCRP expression was increased with a 1.8 fold increase rate. Then, P-gp took over from 3 h to 15 h with an average increase rate of 1.8 fold, and finally returned to control value at 24 h. HPLC-UV analyses showed that, in the same treatment conditions, the intracellular Olaparib concentration increased from 1 h to 3 h and remained relatively stable until 24 h. Results suggest that the resistance mechanism induced by Olaparib in TNBL SUM1315 cell line may be overpassed if a cytotoxic and stable intracellular level of the drug can be maintained.

  2. BCRP and P-gp relay overexpression in triple negative basal-like breast cancer cell line: a prospective role in resistance to Olaparib

    PubMed Central

    Dufour, Robin; Daumar, Pierre; Mounetou, Emmanuelle; Aubel, Corinne; Kwiatkowski, Fabrice; Abrial, Catherine; Vatoux, Catherine; Penault-Llorca, Frédérique; Bamdad, Mahchid

    2015-01-01

    The triple negative basal-like (TNBL) breast carcinoma is an aggressive and unfavorable prognosis disease. Inhibitors of poly(ADP-ribose) polymerase such as Olaparib could represent a promising targeted therapy but their sensitivity against Multidrug Resistance proteins (MDR), which causes resistance, is not well defined. Thus, our work focused on the analysis of P-gp and BCRP coexpression in the SUM1315 TNBL human cell line, in correlation with Olaparib intracellular concentration. Western blot analyses showed a clear coexpression of P-gp and BCRP in SUM1315 cells. A low cytotoxic Olaparib treatment clearly led to an increased expression of both BCRP and P-gp in these cells. Indeed, after 1.5 h of treatment, BCRP expression was increased with a 1.8 fold increase rate. Then, P-gp took over from 3 h to 15 h with an average increase rate of 1.8 fold, and finally returned to control value at 24 h. HPLC-UV analyses showed that, in the same treatment conditions, the intracellular Olaparib concentration increased from 1 h to 3 h and remained relatively stable until 24 h. Results suggest that the resistance mechanism induced by Olaparib in TNBL SUM1315 cell line may be overpassed if a cytotoxic and stable intracellular level of the drug can be maintained. PMID:26234720

  3. A New Class of Safe, Potent, and Specific P-gp Modulator: Flavonoid Dimer FD18 Reverses P-gp-Mediated Multidrug Resistance in Human Breast Xenograft in Vivo.

    PubMed

    Yan, Clare S W; Wong, Iris L K; Chan, Kin-Fai; Kan, Jason W Y; Chong, Tsz Cheung; Law, Man Chun; Zhao, Yunzhe; Chan, Shun Wan; Chan, Tak Hang; Chow, Larry M C

    2015-10-05

    Flavonoid dimer FD18 is a new class of dimeric P-gp modulator that can reverse cancer drug resistance. FD18 is a potent (EC50 = 148 nM for paclitaxel), safe (selective index = 574), and selective P-glycoprotein (P-gp) modulator. FD18 can modulate multidrug resistance toward paclitaxel, vinblastine, vincristine, doxorubicin, daunorubicin, and mitoxantrone in human breast cancer LCC6MDR in vitro. FD18 (1 μM) can revert chemosensitivity of LCC6MDR back to parental LCC6 level. FD18 was 11- to 46-fold more potent than verapamil. FD18 (1 μM) can increase accumulation of doxorubicin by 2.7-fold, daunorubicin (2.1-fold), and rhodamine 123 (5.2-fold) in LCC6MDR. FD18 inhibited P-gp-mediated doxorubicin efflux and has no effect on influx. FD18 at 1 μM did not affect the protein expression level of P-gp. Pharmacokinetics studies indicated that intraperitoneal administration of 45 mg/kg FD18 was enough to maintain a plasma level above EC50 (148 nM) for more than 600 min. Toxicity studies with FD18 (90 mg/kg, i.p. for 12 times in 22 days) with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) revealed no obvious toxicity or death in mice. In vivo efficacy studies indicated that FD18 (45 mg/kg, i.p. for 12 times in 22 days) together with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) resulted in a 46% reduction in LCC6MDR xenograft volume (n = 11; 648 ± 84 mm(3)) compared to paclitaxel control (n = 8; 1201 ± 118 mm(3)). There were no animal deaths or significant drop in body weight and vital organ wet weight. FD18 can increase paclitaxel accumulation in LCC6MDR xenograft by 1.8- to 2.2-fold. The present study suggests that FD18 represents a new class of safe and potent P-gp modulator in vivo.

  4. The application of P-gp inhibiting phospholipids as novel oral bioavailability enhancers - An in vitro and in vivo comparison.

    PubMed

    Weinheimer, Manuel; Fricker, Gert; Burhenne, Jürgen; Mylius, Patricia; Schubert, Rolf

    2016-08-30

    The efflux transporter P-glycoprotein (P-gp) significantly modulates drug transport across the intestinal mucosa, strongly reducing the systemic absorption of various active pharmaceutical ingredients. P-gp inhibitors could serve as helpful tools to enhance the oral bioavailability of those substances. As a membrane-associated protein P-gp is surrounded and influenced by phospholipids. Some synthetic phospholipids have been found to strongly reduce P-gp's activity. In this study two representative phospholipids, 1,2-dioctanoyl-sn-glycero-3-phosphocholine (8:0 PC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine (10:0 PC), were compared with Tween® 80 and Cremophor® EL, both commonly used surfactants with P-gp inhibitory properties. Their influence on the cellular transport of the P-gp substrate rhodamine 123 (RH123) was examined using Caco-2 cell layers. In addition, fluorescence anisotropy measurements were performed in order to investigate their effect on membrane fluidity. Finally, we compared the phospholipids with Tween® 80 and the competitive P-gp inhibitor verapamil in an in vivo study, testing their effects on the oral bioavailability of the P-gp substrate drug ritonavir. Both phospholipids not only led to the strongest absorption of RH123, but a permeability enhancing effect was detected in addition to the P-gp inhibition. Their effects on membrane fluidity were not consistent with their P-gp inhibiting effects, and therefore suggested a more complex mode of action. Both phospholipids significantly increased the area under the ritonavir plasma level curve (AUC) within 150min by more than tenfold, but were inferior to Tween® 80, which showed superior solubilizing effects. Finally, these phospholipids represent a novel substance class showing a high permeabilization potential for P-gp substrates. Because of their physiological structure and intestinal degradability, good tolerability without systemic absorption is expected. Formulating P-gp substrates with

  5. P-glycoprotein expression in lamina propria lymphocytes of duodenal biopsy samples in dogs with chronic idiopathic enteropathies.

    PubMed

    Allenspach, K; Bergman, P J; Sauter, S; Gröne, A; Doherr, M G; Gaschen, F

    2006-01-01

    P-glycoprotein (p-gp) is a transmembrane protein functioning as a drug-efflux pump in the intestinal epithelium. Human patients with inflammatory bowel disease (IBD) who fail to respond to treatment with steroids express high levels of p-gp in lamina propria lymphocytes. The purpose of this study was to investigate p-gp expression in duodenal biopsy samples of dogs with chronic enteropathies and to evaluate the expression of p-gp after treatment with a known inducer of p-gp (prednisolone). Duodenal biopsy samples from 48 dogs were evaluated immunohistochemically with the mouse monoclonal antibody C219 for expression of p-gp in lamina propria lymphocytes. Biopsy samples were available from 15 dogs after treatment with prednisolone and 16 dogs after dietary therapy alone ("elimination diet"). Treatment with prednisolone resulted in an increase in p-gp expression (P=0.005). In contrast, dietary treatment alone produced no significant change in p-gp expression (P=0.59). A low p-gp score before initiation of steroid treatment was significantly associated with a positive response to treatment (P=0.01). These results indicate that lamina propria lymphocyte expression of p-gp is upregulated after prednisolone treatment in dogs with IBD, and that mucosal expression of p-gp may be of value in predicting the response to therapy.

  6. A Potent and Selective P-gp Modulator for Altering Multidrug Resistance Due to Pump Overexpression.

    PubMed

    Guglielmo, Stefano; Contino, Marialessandra; Lazzarato, Loretta; Perrone, Maria Grazia; Blangetti, Marco; Fruttero, Roberta; Colabufo, Nicola Antonio

    2016-02-17

    P-glycoprotein (P-gp) is a membrane protein responsible for the active transport of several endogenous and exogenous substances. It constitutes a defense mechanism and, at the same time, it severely compromises the success rate of antitumor chemotherapy. In this study a small library of alkyl/oxyalkyl derivatives of MC70 [4'-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-ylmethyl)biphenyl-4-ol], a well-known P-gp inhibitor, was synthesized through straightforward functionalization of the phenolic group present in the structure of MC70. All compounds were characterized for their effect on P-gp, proving capable of blocking P-gp-mediated calcein-AM efflux with micromolar potency, following their ability to act as high-affinity substrates of this transporter. Excitingly, compound 4 [6,7-dimethoxy-2-((4'-butoxybiphen-4-yl)methyl)-1,2,3,4-tetrahydroisoquinoline] exhibited low nanomolar potency (5.2 nm) and had a peculiar activity profile, acting both as a positive allosteric modulator and as a substrate of the transporter. A new and more efficient synthesis of MC70 is also described.

  7. Differential effect of P-gp and MRP2 on cellular translocation of gemifloxacin.

    PubMed

    Vadlapatla, Ramya Krishna; Vadlapudi, Aswani Dutt; Kwatra, Deep; Pal, Dhananjay; Mitra, Ashim K

    2011-11-25

    Fluoroquinolones are broad spectrum antibiotics widely indicated in the treatment of both human and animal diseases. The primary objective of this study was to assess short and long term affinities of gemifloxacin towards efflux transporters (P-gp, MRP2) and nuclear hormone receptor (PXR). Uptake and dose dependent inhibition studies were performed with [(14)C] erythromycin (0.25 μCi/ml) on MDCKII-MDR1 and MDCKII-MRP2 cells. Cellular accumulation of calcein-AM was further determined to confirm the affinity of gemifloxacin towards P-gp and MRP2. Transport studies were conducted to determine bi-directional permeability and to assess efflux ratio of gemifloxacin. LS-180 cells were treated with three different concentrations of gemifloxacin for 72 h and real-time PCR analysis was performed to study the quantitative gene expression levels of PXR, MDR1 and MRP2. Further, [(14)C] erythromycin uptake was also performed on LS-180 treated cells to better delineate the functional activity of efflux transporters. Results from our study suggest that gemifloxacin may be a substrate of both the efflux transporters studied. This compound inhibited both P-gp and MRP2 mediated efflux of [(14)C] erythromycin in a dose dependent manner with IC(50) values of 123 ± 2 μM and 16 ± 2 μM, respectively. The efflux ratio of [(14)C] erythromycin lowered from 3.56 to 1.63 on MDCKII-MDR1 cells and 4.93 to 1.26 on MDCKII-MRP2 cells. This significant reduction in efflux ratio further confirmed the substrate specificity of gemifloxacin towards P-gp and MRP2. Long term exposure significantly induced the expression of PXR (18 fold), MDR1 (6 fold) and MRP2 (6 fold). A decrease (20%) in [(14)C] erythromycin uptake further confirmed the elevated functional activity of P-gp and MRP2. In conclusion, our studies demonstrated that gemifloxacin is effluxed by both P-gp and MRP2. Long term exposure induced their gene expression and functional activity. This substrate specificity of gemifloxacin towards

  8. Differential effect of P-gp and MRP2 on cellular translocation of gemifloxacin

    PubMed Central

    Vadlapatla, Ramya Krishna; Vadlapudi, Aswani Dutt; Kwatra, Deep; Pal, Dhananjay; Mitra, Ashim K.

    2011-01-01

    Fluoroquinolones are broad spectrum antibiotics widely indicated in the treatment of both human and animal diseases. The primary objective of this study was to assess short and long term affinity of gemifloxacin towards efflux transporters (P-gp, MRP2) and nuclear hormone receptor (PXR). Uptake and dose dependent inhibition studies were performed with [14C] erythromycin (0.25μCi/ml) on MDCKII-MDR1 and MDCKII-MRP2 cells. Cellular accumulation of calcein-AM was further determined to confirm the affinity of gemifloxacin towards P-gp and MRP2. Transport studies were conducted to determine bi-directional permeability and to assess efflux ratio of gemifloxacin. LS-180 cells were treated with three different concentrations of gemifloxacin for 72hrs and real-time PCR analysis was performed to study the quantitative gene expression levels of PXR, MDR1 and MRP2. Further, [14C] erythromycin uptake was also performed on LS-180 treated cells to better delineate the functional activity of efflux transporters. Results from our study suggest that gemifloxacin may be a substrate of both the efflux transporters studied. This compound inhibited both P-gp and MRP2 mediated efflux of [14C] erythromycin in a dose dependent manner with IC50 values of 123 ± 2μM and 16 ± 2μM, respectively. The efflux ratio of [14C] erythromycin lowered from 3.56 to 1.63 on MDCKII-MDR1 cells and 4.93 to 1.26 on MDCKII-MRP2 cells. This significant reduction in efflux ratio further confirmed the substrate specificity of gemifloxacin towards P-gp and MRP2. Long term exposure significantly induced the expression of PXR (18 fold), MDR1 (6 fold) and MRP2 (6 fold). A decrease (20%) in [14C] erythromycin uptake further confirmed the elevated functional activity of P-gp and MRP2. In conclusion, our studies demonstrated that gemifloxacin is effluxed by both P-gp and MRP2. Long term exposure induced their gene expression and functional activity. This substrate specificity of gemifloxacin towards these efflux

  9. Both P-gp and MRP2 mediate transport of Lopinavir, a protease inhibitor

    PubMed Central

    Agarwal, Sheetal; Pal, Dhananjay; Mitra, Ashim K.

    2011-01-01

    Polarized epithelial non-human (canine) cell lines stably transfected with human or murine complementary DNA (cDNA) encoding for various efflux transporters (P-gp/MDR1, MRP1, MRP2, and Bcrp1) were used to study transepithelial transport of Lopinavir (LVR) and compare results with the MDCKII-Wild type cells. These transmembrane proteins cause multidrug resistance by decreasing the total intracellular accumulation of drugs. Lopinavir efflux was directional and was completely inhibited by MK-571, a selective MRP family inhibitor in the MDCKII-MRP2 cell line. Similarly, LVR efflux was also inhibited by P-gp inhibitors P-gp 4008 and GF120918 in the MDCKII-MDR1 cell line. The efflux ratios (Efflux rate/ Influx rate) of LVR in the absence of any efflux inhibitors in the MDCK-Wild type, MDCKII-MDR1, MDCKII-MRP1 and MDCKII-MRP2 cell monolayers were 1.32, 4.91, 1.26 and 2.89 respectively. The MDCKII-MDR1 and MDCKII-MRP2 cells have significantly increased LVR efflux ratio relative to the parental cells due to the apically directed transport by MDR1 and MRP2 respectively. The efflux ratios in MRP2 and MDR1 transfected cell lines were close to unity in the presence of MK-571 and P-gp 4008 respectively; indicating that LVR efflux by MRP2 and P-gp was completely inhibited by their selective inhibitors. MDCKII-MRP1 cells did not exhibit a significant reduction in the LVR efflux relative to the parental cells, indicating that LVR is not a good substrate for MRP1. Transport studies across MDCKII-Bcrp1 cells indicated that LVR is not transported by Bcrp1 and is not a substrate for this efflux protein. In conclusion, this study presents direct evidence that LVR is effluxed by both P-gp and MRP2 which may contribute to its poor oral bioavailability and limited penetration into the CNS. PMID:17451894

  10. Fructose-induced metabolic syndrome decreases protein expression and activity of intestinal P-glycoprotein.

    PubMed

    Novak, Analía; Godoy, Yanina Cynthia; Martinez, Sonia Amalia; Ghanem, Carolina Inés; Celuch, Stella Maris

    2015-06-01

    Metabolic syndrome (MetS) is a health disorder that increases the risk for cardiovascular complications such as heart disease and type 2 diabetes. Some drugs used in patients with MetS are substrates of intestinal P-glycoprotein (P-gp), one of the most important efflux pumps that limit the absorption of xenobiotics. Thus, their bioavailability could be affected by changes in this transporter. Because one of the major causes of MetS in humans is excessive sugar intake, the aim of this study was to evaluate the effect of a fructose-rich diet on intestinal P-gp activity and protein expression in male Sprague-Dawley rats. Fructose-drinking animals received standard chow and 15% (w/v) fructose in the drinking water over 8 wk; control rats were fed on standard chow and tap water. Ileal protein expression of P-gp was 50% lower in fructose-drinking rats than in control animals. This reduction was confirmed by immunofluorescence microscopy. These results correlated well with the decrease of about 50% in the transport rate of the substrate rhodamine 123 in everted intestinal sacs. Finally, an increase of 62% in the intestinal absorption of digoxin, a P-gp substrate used as therapeutic drug, was observed in vivo, in fructose-drinking animals. The present study demonstrated that MetS-like conditions generated by enhanced fructose intake in rats decreased the protein expression and activity of ileal P-gp, thus increasing the bioavailability of P-gp substrates. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover.

    PubMed

    Orr, Mona W; Donaldson, Gregory P; Severin, Geoffrey B; Wang, Jingxin; Sintim, Herman O; Waters, Christopher M; Lee, Vincent T

    2015-09-08

    The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A from Pseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆orn strains of P. aeruginosa PA14 for pGpG stability. The lysates from ∆orn showed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆orn strain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP-regulated pel promoter. Additionally, the c-di-GMP-governed auto-aggregation and biofilm phenotypes were elevated in the ∆orn strain in a pel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆orn strain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.

  12. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover

    PubMed Central

    Orr, Mona W.; Donaldson, Gregory P.; Severin, Geoffrey B.; Wang, Jingxin; Sintim, Herman O.; Waters, Christopher M.; Lee, Vincent T.

    2015-01-01

    The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A from Pseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆orn strains of P. aeruginosa PA14 for pGpG stability. The lysates from ∆orn showed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆orn strain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP–regulated pel promoter. Additionally, the c-di-GMP–governed auto-aggregation and biofilm phenotypes were elevated in the ∆orn strain in a pel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆orn strain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway. PMID:26305945

  13. Cbl-b inhibits P-gp transporter function by preventing its translocation into caveolae in multiple drug-resistant gastric and breast cancers.

    PubMed

    Zhang, Ye; Qu, Xiujuan; Teng, Yuee; Li, Zhi; Xu, Ling; Liu, Jing; Ma, Yanju; Fan, Yibo; Li, Ce; Liu, Shizhou; Wang, Zhenning; Hu, Xuejun; Zhang, Jingdong; Liu, Yunpeng

    2015-03-30

    The transport function of P-glycoprotein (P-gp) requires its efficient localization to caveolae, a subset of lipid rafts, and disruption of caveolae suppresses P-gp transport function. However, the regulatory molecules involved in the translocation of P-gp into caveolae remain unknown. In the present study, we showed that c-Src dependent Caveolin-1 phosphorylation promoted the translocation of P-gp into caveolae, resulting in multidrug resistance in adriamycin resistant gastric cancer SGC7901/Adr and breast cancer MCF-7/Adr cells. In a negative feedback loop, the translocation of Cbl-b from the nucleus to the cytoplasm prevented the localization of P-gp to caveolae resulting in the reversal of MDR through the ubiquitination and degradation of c-Src. Clinical data showed a significant positive relationship between Cbl-b expression and survival in P-gp positive breast cancer patients who received anthracycline-based chemotherapy. Our findings identified a new regulatory mechanism of P-gp transport function in multiple drug-resistant gastric and breast cancers.

  14. Exposure of LS-180 cells to drugs of diverse physicochemical and therapeutic properties up-regulates P-glycoprotein expression and activity.

    PubMed

    Abuznait, Alaa H; Patrick, Shawn G; Kaddoumi, Amal

    2011-01-01

    Drug transporters are increasingly recognized as important determinants of variability in drug disposition and therapeutic response, both in pre-clinical and clinical stages of drug development process. The role P-glycoprotein (P-gp) plays in drug interactions via its inhibition is well established. However, much less knowledge is available about drugs effect on P-gp up-regulation. The objective of this work was to in vitro investigate and rank commonly used drugs according to their potencies to up-regulate P-gp activity utilizing the same experimental conditions. The in vitro potencies of several drugs of diverse physicochemical and therapeutic properties including rifampicin, dexamethasone, caffeine, verapamil, pentylenetetrazole, hyperforin, and β-estradiol over broad concentration range to up-regulate P-gp expression and activity were examined. For dose-response studies, LS-180 cells were treated with different concentrations of the selected drugs followed by P-gp protein and gene expressions analyses. P-gp functionality was determined by uptake studies with rhodamine 123 as a P-gp substrate, followed by Emax/EC50 evaluation. The results demonstrated a dose-dependent increase in P-gp expression and activity following treatments. At 50 uM concentration (hyperforin, 0.1 uM), examined drugs increased P-gp protein and gene expressions by up to 5.5 and 6.2-fold, respectively, while enhanced P-gp activity by 1.8-4-fold. The rank order of these drugs potencies to up-regulate P-gp activity was as following: hyperforin > dexamethasone ~ beta-estradiol > caffeine > rifampicin ~ pentylenetetrazole > verapamil. These drugs have the potential to be involved in drug interactions when administered with other drugs that are P-gp substrates. Further studies are needed to in vivo evaluate these drugs and verify the consequences of such induction on P-gp activity for in vitro-in vivo correlation purposes.

  15. Brain Accumulation of Ponatinib and Its Active Metabolite, N-Desmethyl Ponatinib, Is Limited by P-Glycoprotein (P-GP/ABCB1) and Breast Cancer Resistance Protein (BCRP/ABCG2).

    PubMed

    Kort, Anita; van Hoppe, Stéphanie; Sparidans, Rolf W; Wagenaar, Els; Beijnen, Jos H; Schinkel, Alfred H

    2017-10-02

    Ponatinib is an oral BCR-ABL1 inhibitor for treatment of advanced leukemic diseases that carry the Philadelphia chromosome, specifically containing the T315I mutation yielding resistance to previously approved BCR-ABL1 inhibitors. Using in vitro transport assays and knockout mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 transport ponatinib and whether they, or the drug-metabolizing enzyme CYP3A, affect the oral availability and brain accumulation of ponatinib and its active N-desmethyl metabolite (DMP). In vitro, mouse Abcg2 and human ABCB1 modestly transported ponatinib. In mice, both Abcb1 and Abcg2 markedly restricted brain accumulation of ponatinib and DMP, but not ponatinib oral availability. Abcg2 deficiency increased DMP plasma levels ∼3-fold. Cyp3a deficiency increased the ponatinib plasma AUC 1.4-fold. Our results suggest that pharmacological inhibition of ABCG2 and ABCB1 during ponatinib therapy might benefit patients with brain (micro)metastases positioned behind an intact blood-brain barrier, or with substantial expression of these transporters in the malignant cells. CYP3A inhibitors might increase ponatinib oral availability, enhancing efficacy but possibly also toxicity of this drug.

  16. Insulin resistance contributes to multidrug resistance in HepG2 cells via activation of the PERK signaling pathway and upregulation of Bcl-2 and P-gp.

    PubMed

    Liu, Xinyue; Li, Linjing; Li, Jing; Cheng, Yan; Chen, Jing; Shen, Minghui; Zhang, Shangdi; Wei, Hulai

    2016-05-01

    Liver tumorigenesis frequently causes insulin resistance which may be used as an independent risk factor for evaluation of survival and post-surgery relapse of liver cancer patients. In the present study, HepG2/IR, an insulin resistant HepG2 cell line, was established by exposing HepG2 cells to 0.5 µmol/l of insulin for 72 h, and comparison of HepG2/IR with the parental HepG2 cells indicated that the HepG2/IR cells showed significantly enhanced resistance to the most frequently used chemotherapeutics for solid tumors, such as cisplatin, 5-fluorouracil, vincristine and mitomycin. Flow cytometric analysis of cisplatin-treated HepG2/IR cells showed a significantly decreased hypodiploid peak and a significantly downregulated expression level of pro-apoptotic protein caspase-3 compared with the parental HepG2 cells. Our data further showed swollen endoplasmic reticulum (ER) in the cisplatin-treated HepG2/IR cells with significantly increased levels of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like ER kinase (p-PERK) and P-glycoprotein (P-gp). There was also an upregulated expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) whereas no significant change was observed for CCAAT-enhancer-binding protein homologous protein (CHOP), which is known to be induced by ER stress and to mediate apoptosis. Our results demonstrated that insulin resistance in HepG2 cells promoted a protective unfolded protein response and upregulated the expression of ER chaperone protein GRP78, which resulted in the phosphorylation of PERK kinase to activate the PERK-mediated ER stress signal transduction pathway and the upregulation of Bcl-2 and P-gp, leading to the inhibition of the caspase-3-dependent apoptosis pathway and to the survival of liver tumor cells.

  17. Enhanced brain accumulation of pazopanib by modulating P-gp and Bcrp1 mediated efflux with canertinib or erlotinib

    PubMed Central

    Minocha, Mukul; Khurana, Varun; Qin, Bin; Pal, Dhananjay; Mitra, Ashim K.

    2013-01-01

    Primary objective of this investigation was to delineate the differential impact of efflux transporters P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) on brain disposition and plasma pharmacokinetics of pazopanib. In addition, this research investigated whether inhibition of these efflux transporters with clinically relevant efflux modulators canertinib or erlotinib could be a viable strategy for improving pazopanib brain delivery. In vitro assays with MDCKII cell monolayers suggested that pazopanib is a high affinity substrate for Bcrp1 and a moderate substrate for P-gp. Co-incubation with specific transport inhibitors restored cell accumulation and completely abolished the directionality of pazopanib flux. Brain and plasma pharmacokinetic studies were conducted in FVB wild type mice in the absence and presence of specific transport inhibitors. Drug levels in plasma and brain were determined using a validated high performance liquid chromatography method using vandetanib as an internal standard. In vivo studies indicated that specific inhibition of either P-gp (by zosuquidar or LY335979) or Bcrp1 (by Ko143) alone did not significantly alter pazopanib brain accumulation. However, dual P-gp/Bcrp1 inhibition by elacridar (GF120918), significantly enhanced pazopanib brain penetration by ~5-fold without altering its plasma concentrations. Thus, even though Bcrp1 showed higher affinity towards pazopanib in vitro, in vivo at the mouse BBB both P-gp and Bcrp1 act in concert to limit brain accumulation of pazopanib. Furthermore, erlotinib and canertinib as clinically relevant efflux modulators efficiently abrogated directionality in pazopanib efflux in vitro and their co-administration resulted in 2–2.5-fold increase in pazopanib brain accumulation in vivo. Further pre-clinical and clinical investigations are warranted as erlotinib or canertinib may have a synergistic pharmacological effect in addition to their primary role of pazopanib

  18. An electrically tight in vitro blood-brain barrier model displays net brain-to-blood efflux of substrates for the ABC transporters, P-gp, Bcrp and Mrp-1.

    PubMed

    Helms, Hans Christian; Hersom, Maria; Kuhlmann, Louise Borella; Badolo, Lasina; Nielsen, Carsten Uhd; Brodin, Birger

    2014-09-01

    Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate if a bovine endothelial/rat astrocyte in vitro BBB co-culture model displayed polarized transport of known efflux transporter substrates. The co-culture model displayed low mannitol permeabilities of 0.95 ± 0.1 · 10(-6) cm·s(-1) and high transendothelial electrical resistances of 1,177 ± 101 Ω·cm(2). Bidirectional transport studies with (3)H-digoxin, (3)H-estrone-3-sulphate and (3)H-etoposide revealed polarized transport favouring the brain-to-blood direction for all substrates. Steady state efflux ratios of 2.5 ± 0.2 for digoxin, 4.4 ± 0.5 for estrone-3-sulphate and 2.4 ± 0.1 for etoposide were observed. These were reduced to 1.1 ± 0.08, 1.4 ± 0.2 and 1.5 ± 0.1, by addition of verapamil (digoxin), Ko143 (estrone-3-sulphate) or zosuquidar + reversan (etoposide), respectively. Brain-to-blood permeability of all substrates was investigated in the presence of the efflux transporter inhibitors verapamil, Ko143, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport.

  19. Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells.

    PubMed

    Jumnongprakhon, Pichaya; Sivasinprasasn, Sivanan; Govitrapong, Piyarat; Tocharus, Chainarong; Tocharus, Jiraporn

    2017-02-20

    Melatonin has been known as a neuroprotective agent for the central nervous system (CNS) and the blood-brain barrier (BBB), which is the primary structure that comes into contact with several neurotoxins including methamphetamine (METH). Previous studies have reported that the activation of melatonin receptors (MT1/2) by melatonin could protect against METH-induced toxicity in brain endothelial cells via several mechanisms. However, its effects on the P-glycoprotein (P-gp) transporter, the active efflux pump involved in cell homeostasis, are still unclear. Thus, this study investigated the role of melatonin and its receptors on the METH-impaired P-gp transporter in primary rat brain microvascular endothelial cells (BMVECs). The results showed that METH impaired the function of the P-gp transporter, significantly decreasing the efflux of Rho123 and P-gp expression, which caused a significant increase in the intracellular accumulation of Rho123, and these responses were reversed by the interaction of melatonin with its receptors. Blockade of the P-gp transporter by verapamil caused oxidative stress, apoptosis, and cell integrity impairment after METH treatment, and these effects could be reversed by melatonin. Our results, together with previous findings, suggest that the interaction of melatonin with its receptors protects against the effects of the METH-impaired P-gp transporter and that the protective role in METH-induced toxicity was at least partially mediated by the regulation of the P-gp transporter. Thus, melatonin and its receptors (MT1/2) are essential for protecting against BBB impairment caused by METH.

  20. Inhibitory effect of phospholipids on P-glycoprotein: cellular studies in Caco-2, MDCKII mdr1 and MDCKII wildtype cells and P-gp ATPase activity measurements.

    PubMed

    Simon, S; Schubert, R

    2012-09-01

    Phospholipids are widely used excipients for pharmaceutical formulations, such as for preparing biphasic systems or to solubilize or encapsulate poorly soluble drugs. The present study investigates a new property of this class of substance: its ability to inhibit the efflux transporter Pglycoprotein (P-gp). P-gp is expressed in the intestinal epithelium, thereby significantly impairing the systemic absorption of various pharmaceutically active substances. The phospholipid screening performed in this study involved derivatives with different headgroups and fatty acid residues and a number of experimental parameters. For in vitro studies we carried out transport experiments and calcein accumulation assays in Caco-2- and MDCKII mdr1 and wildtype cell lines. The three compounds which displayed significant P-gp inhibition in both assays and in Caco-2 as well as in MDCKII mdr1, consisted of phosphatidylcholine (PC) and either two saturated fatty acid residues of eight (8:0 PC) or ten carbon atoms (10:0 PC), or of two unsaturated docosahexaeonic acid residues (cis-22:6 PC).Supported by P-gp ATPase activity measurements, 8:0 and 10:0 PC were assumed to function as direct P-gp inhibitors interacting with the transporter probably in their monomeric state, whereas a different, as yet unknown mechanism of action applied for cis-22:6 PC.Because of their proven ability to significantly inhibit P-gp in vitro, these phospholipids shall further be elucidated in vivo, whether they may truly serve to increase the bioavailability of orally applied drugs with a P-gp substrate character.

  1. MiR-129 regulates cisplatin-resistance in human gastric cancer cells by targeting P-gp.

    PubMed

    Lu, Chaojing; Shan, Zhengxiang; Li, Chunguang; Yang, Lixin

    2017-02-01

    Development of multiple drug resistance (MDR) to chemotherapy is the major reason for the failure of gastric cancer (GC) treatment. P-glycoprotein (P-gp), which is encoded by MDR gene 1, as one of the mechanisms responsible for MDR. Mounting evidence has demonstrated that the drug-induced dysregulation of microRNAs (miRNAs) function may mediate MDR in cancer cells. However, the underling mechanisms of miRNA-mediated MDR in GC remain unclear. Here, we found that miR-129 was downregulated in cisplatin-resistant GC tissues/cells. Our results also showed that overexpression of miR-129 decreased cisplatin-resistance in cisplatin-resistant GC cells, and miR-129 knockdown reduced chemosensitivity to cisplatin in cisplatin-sensitive GC cells. Furthermore, miR-129 activated the intrinsic apoptotic pathway via upregulating caspase-9 and caspase-3. Most importantly, we further confirmed that P-gp is the functional target of miR-129 by regulating cisplatin-resistance in GC cells. These results suggested that miR-129 reversed cisplatin-resistance through inhibiting the P-gp expression in GC cells.

  2. MDR1/P-gp and VEGF synergistically enhance the invasion of Hep-2 cells with multidrug resistance induced by taxol.

    PubMed

    Li, Li; Jiang, Alice C; Dong, Pin; Wang, Haibo; Xu, Wei; Xu, Chengzhi

    2009-05-01

    Tumor invasion/metastasis and multidrug resistance (MDR) are the main causes of treatment failure and high mortality in all kinds of cancer patients. The relationship between the two factors is still unclear. The aim of this study is to investigate the association between MDR and invasion, especially the role of multidrug resistance 1/P-glycoprotein (MDR1/P-gp) and vascular endothelial growth factor (VEGF) during the invasion. Multidrug resistance 1 (MDR1) and VEGF receptor 2 (VEGFR-2) were detected with real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting at the levels of messenger RNA (mRNA) and protein, respectively. RNA interference was applied to inhibit the expression of MDR1. The invasive assays were performed with the CHEMICON cell invasion assay kit. The MDR cell line induced by Taxol (Hep-2T cell) was more invasive than its parent cell line (Hep-2 cell), which was at least in part mediated through the overexpressed MDR1/P-pg. MDR1-targeted RNA interference could effectively inhibit the expression of MDR1 and obviously decrease the invasive ability. Synergistic enhancing effects existed between MDR1/P-gp and VEGF on the invasion of Hep-2T cells. The expression of VEGFR-2 was elevated in Hep-2T cells. SU1498 could significantly decrease the invasion of Hep-2T cells. MDR1-targeted RNA interference and SU1498 had synergistic decreasing effect on the invasion of Hep-2T cells. MDR1/P-pg may be a risk predictor for the invasion of laryngeal cancer. MDR1 knock down and VEGFR-2 inhibitor may be two promising treatment regiments for advanced laryngeal carcinoma patients with MDR and invasion/metastasis.

  3. Enhancement effect of P-gp inhibitors on the intestinal absorption and antiproliferative activity of bestatin.

    PubMed

    Huo, Xiaokui; Liu, Qi; Wang, Changyuan; Meng, Qiang; Sun, Huijun; Peng, Jinyong; Ma, Xiaochi; Liu, Kexin

    2013-11-20

    Bestatin is an immunomodulator with antitumor activity. This study was performed to investigate the effect of P-gp on the intestinal absorption and antiproliferative activity of bestatin. Our results showed that P-gp inhibitors significantly increased rat intestinal absorption of bestatin in vivo and in vitro. The net efflux ratio of bestatin was 2.2 across mock-/MDR1-MDCK cell monolayers and was decreased by P-gp inhibitors, indicating bestatin was a substrate of P-gp. Furthermore, the IC50 values of bestatin on U937 and K562 cells were decreased dramatically and the intracellular concentrations of bestatin were increased by incubation of cells with verapamil or Cyclosporin A. K562/ADR cells exhibited a higher IC50 value and a lower intracellular level of bestatin. The bestatin level in K562/ADR cells was partially restored by incubation with doxorubicin. However, P-gp and APN mRNA levels were not changed by bestatin. These results suggested that the intestinal absorption and accumulation in cancer cells for bestatin were limited by P-gp-mediated efflux. Additional attention should be paid to the alternative exposure of bestatin when bestatin was coadministered with drugs as P-gp substrates in clinic.

  4. Assessing the Impact of Lithium Chloride on the Expression of P-Glycoprotein at the Blood-Brain Barrier.

    PubMed

    Newman, Stephanie A; Pan, Yijun; Short, Jennifer L; Nicolazzo, Joseph A

    2017-01-16

    In addition to extruding drugs from the brain, P-glycoprotein (P-gp) at the blood-brain barrier (BBB) facilitates the brain-to-blood clearance of beta-amyloid (Aβ) and is down-regulated in Alzheimer's disease. Studies suggest that the mood-stabilizing drug lithium exerts a protective effect against Alzheimer's disease. Although the mechanisms underlying this effect are not fully understood, evidence suggests that lithium chloride (LiCl) increases P-gp expression in vitro, albeit at concentrations substantially outside the therapeutic window. Therefore, we investigated the effects of pharmacologically-relevant concentrations of LiCl on P-gp expression using in vitro and in vivo approaches. Swiss outbred mice administered LiCl (300 mg/kg/day, 21 days) showed no change in brain microvascular P-gp protein expression. Furthermore, P-gp transcript and protein levels were unaltered by LiCl (1.25-5 mM, 24 h) in human immortalized brain endothelial cells, while both gene and protein expression were significantly enhanced by the P-gp up-regulator, SR12813 by 1.5-fold and 2.0-fold, respectively. P-gp efflux function was also unaffected by LiCl in vitro, by measuring accumulation of the fluorescent P-gp substrate rhodamine-123. This suggests therefore that LiCl is unlikely to affect the BBB efflux of Aβ or other P-gp substrates at pharmacologically-relevant concentrations, suggesting that the Aβ-lowering effects of LiCl are unrelated to elevated BBB P-gp expression.

  5. Effects of rhinacanthin-C on function and expression of drug efflux transporters in Caco-2 cells.

    PubMed

    Wongwanakul, Ratjika; Vardhanabhuti, Nontima; Siripong, Pongpun; Jianmongkol, Suree

    2013-09-01

    Rhinacanthin-C is a bioactive naphthoquinone ester found in Rhinacanthus nasutus Kurz (Acanthaceae). This compound has potential therapeutic value as an anticancer and antiviral agent. The purposes of this study were to determine the effects of this compound on the function and the expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2), using the in vitro model of Caco-2 cells. The activities of P-gp and MRP2 were determined by following the intracellular accumulation of calcein and 5(6)-carboxy-2',7'-dichlorofluorescein in the uptake assays with fluorescence spectroscopy. The expression of P-gp after prolonged exposure was evaluated by flow cytometry with the use of a fluorescein isothiocyanate-conjugated anti-human P-gp antibody. Our results showed that the inhibitory effect of rhinacanthin-C was more potent toward P-gp than MRP2, and was reversible. Short-term exposure of Caco-2 cells with rhinacanthin-C (100 μM) resulted in increase in P-gp expression without any significant change in its function. Extended exposure of Caco-2 cells to the naphthoquinone at the highest non-cytotoxic concentration (0.625 μM) for 7 days had no effect on the expression and the function of P-gp. These findings suggested that rhinacanthin-C might raise the problem of herb-drug interaction when co-administered with other P-gp substrates.

  6. Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression.

    PubMed

    Yuan, Wei-Qi; Zhang, Rong-Rong; Wang, Jun; Ma, Yan; Li, Wen-Xue; Jiang, Ren-Wang; Cai, Shao-Hui

    2016-05-24

    Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0μM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants.

  7. Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression

    PubMed Central

    Wang, Jun; Ma, Yan; Li, Wen-Xue; Jiang, Ren-Wang; Cai, Shao-Hui

    2016-01-01

    Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0μM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants. PMID:27129170

  8. Induction of drug efflux protein expression by venlafaxine but not desvenlafaxine.

    PubMed

    Bachmeier, Corbin J; Beaulieu-Abdelahad, David; Ganey, Nowell J; Mullan, Michael J; Levin, Gary M

    2011-05-01

    Venlafaxine and its metabolite desvenlafaxine are serotonin-norepinephrine reuptake inhibitors currently prescribed for the treatment of depression. Previously, it was reported that venlafaxine is an inducer of MDR1, the gene responsible for P-glycoprotein (P-gp). The present study expanded upon these findings by examining the effect of venlafaxine and desvenlafaxine on the expression of both P-gp and the breast cancer resistance protein (BCRP) in human brain endothelial cells (HBMEC), an in vitro model of the blood-brain barrier (BBB). The HBMEC were treated for 1 h with various concentrations (500 nM to 50 µM) of venlafaxine and desvenlafaxine. Western blot analysis revealed treatment with venlafaxine significantly induced the expression of P-gp (2-fold) and BCRP (1.75-fold) in a dose-dependent manner, while treatment with desvenlafaxine had no effect on drug efflux transporter expression. To determine the functional significance of this effect, the permeability of a known drug efflux probe, rhodamine 123, across the BBB model and Caco-2 cells, a model of intestinal absorption, were examined. Treatment with venlafaxine (1-50 µM) for 1 h significantly reduced the apical-to-basolateral permeability of R123 across the BBB model (30%) and Caco-2 cell monolayers (25%), indicative of increased drug efflux transporter expression at the apical membrane. Conversely, desvenlafaxine had no effect on R123 permeability in either cellular model. These studies indicate that venlafaxine, but not desvenlafaxine is an inducer of drug efflux transporter expression, which consequently increases the potential for clinical drug-drug interactions. Therefore, based on these preliminary results, caution should be taken when prescribing venlafaxine with other P-gp substrates. Copyright © 2011 John Wiley & Sons, Ltd.

  9. Induction of P-glycoprotein expression and activity by Aconitum alkaloids: Implication for clinical drug–drug interactions

    PubMed Central

    Wu, Jinjun; Lin, Na; Li, Fangyuan; Zhang, Guiyu; He, Shugui; Zhu, Yuanfeng; Ou, Rilan; Li, Na; Liu, Shuqiang; Feng, Lizhi; Liu, Liang; Liu, Zhongqiu; Lu, Linlin

    2016-01-01

    The Aconitum species, which mainly contain bioactive Aconitum alkaloids, are frequently administered concomitantly with other herbal medicines or chemical drugs in clinics. The potential risk of drug–drug interactions (DDIs) arising from co-administration of Aconitum alkaloids and other drugs against specific targets such as P-glycoprotein (P-gp) must be evaluated. This study focused on the effects of three representative Aconitum alkaloids: aconitine (AC), benzoylaconine (BAC), and aconine, on the expression and activity of P-gp. We observed that Aconitum alkaloids increased P-gp expression in LS174T and Caco-2 cells in the order AC > BAC > aconine. Nuclear receptors were involved in the induction of P-gp. AC and BAC increased the P-gp transport activity. Strikingly, intracellular ATP levels and mitochondrial mass also increased. Furthermore, exposure to AC decreased the toxicity of vincristine and doxorubicin towards the cells. In vivo, AC significantly up-regulated the P-gp protein levels in the jejunum, ileum, and colon of FVB mice, and protected them against acute AC toxicity. Taken together, the findings of our in vitro and in vivo experiments indicate that AC can induce P-gp expression, and that co-administration of AC with P-gp substrate drugs may cause DDIs. Our findings have important implications for Aconitum therapy in clinics. PMID:27139035

  10. Inhibitory effects of herbal constituents on P-glycoprotein in vitro and in vivo: Herb–drug interactions mediated via P-gp

    SciTech Connect

    Li, Xue Hu, Jinping Wang, Baolian Sheng, Li Liu, Zhihao Yang, Shuang Li, Yan

    2014-03-01

    Modulation of drug transporters via herbal medicines which have been widely used in combination with conventional prescription drugs may result in herb–drug interactions in clinical practice. The present study was designed to investigate the inhibitory effects of 50 major herbal constituents on P-glycoprotein (P-gp) in vitro and in vivo as well as related inhibitory mechanisms. Among these herbal medicines, four constituents, including emodin, 18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and 20(S)-ginsenoside F{sub 1} [20(S)-GF{sub 1}] exhibited significant inhibition (> 50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC{sub 50} = 9.42 μM), followed by 18β-GA (IC{sub 50} = 21.78 μM), 20(S)-GF{sub 1} (IC{sub 50} = 76.08 μM) and DAG (IC{sub 50} = 77.80 μM). P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with K{sub m} and V{sub max} values of 48.61, 29.09 μM and 71.29, 38.45 nmol/min/mg protein, respectively. However, 18β-GA and 20(S)-GF{sub 1} exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Molecular docking analysis (CDOCKER) further elucidated the mechanism for structure–inhibition relationships of herbal constituents with P-gp. When digoxin was co-administered to male SD rats with emodin or 18β-GA, the AUC{sub 0−t} and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18β-GA, DAG, 20(S)-GF{sub 1} and Rh{sub 1} at 10 μM significantly inhibited CYP3A4/5 activity, while emodin activated the metabolism of midazolam in human liver microsomes. In conclusion, four herbal constituents demonstrated inhibition of P-gp to specific extents in vitro and in vivo. Taken together, our findings provided the basis for the reliable assessment of the potential risks of herb–drug interactions in humans. - Highlights: • Emodin, 18

  11. Nasal delivery of P-gp substrates to the brain through the nose-brain pathway.

    PubMed

    Shingaki, Tomotaka; Hidalgo, Ismael J; Furubayashi, Tomoyuki; Sakane, Toshiyasu; Katsumi, Hidemasa; Yamamoto, Akira; Yamashita, Shinji

    2011-06-01

    The objective of this study was to evaluate in rats the potential utility of the nasal route to enhance central nervous system (CNS) delivery of drugs recognized by P-glycoprotein (P-gp). Well-known P-gp substrates verapamil and talinolol were perfused nasally or infused intravenously, and when plasma concentrations following intravenous infusion and nasal perfusion showed similar profiles. The concentration of verapamil in the brain after nasal perfusion was twice that after intravenous infusion. Although talinolol in the brain and the cerebrospinal fluid after i.v. infusion were below the detection limit, it was detected after nasal perfusion. When rats were treated with cyclosporin A, brain concentrations of verapamil after both administration modes were increased significantly, while those of talinolol were not significantly changed. Since the permeability of talinolol is low, talinolol in the brain which was transported directly from the nasal cavity has little chance of transport by P-gp localized in the apical membrane of cerebral microvessel endothelial cells. The potential for drug delivery utilizing the nose-CNS route was confirmed for P-gp substrates. The advantage of nasal delivery over i.v. delivery of talinolol to the brain was more significant than that of verapamil, suggesting that nasal administration is more useful strategy for the brain delivery of low-permeability P-gp substrates than the use of P-gp inhibitors.

  12. Chronic inflammation up-regulates P-gp in peripheral mononuclear blood cells via the STAT3/Nf-κb pathway in 2,4,6-trinitrobenzene sulfonic acid-induced colitis mice

    PubMed Central

    Liu, Jiali; Zhou, Fang; Chen, Qianying; Kang, An; Lu, Meng; Liu, Wenyue; Zang, Xiaojie; Wang, Guangji; Zhang, Jingwei

    2015-01-01

    Patients with inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis, often suffer drug intolerance. This resistance can be divided into intrinsic resistance and acquired resistance. Although there is agreement on acquired resistance, studies regarding intrinsic resistance have demonstrated inconsistencies, especially for Crohn’s disease. For this reason, an animal model of Crohn’s disease was induced with 2,4,6-trinitrobenzene sulfonic acid solution (TNBS), and intrinsic resistance was analyzed by measuring the function and expression of P-glycoprotein (P-gp) in peripheral mononuclear blood cells (PMBC), followed by mechanistic studies. The results revealed reduced retention of cyclosporine A in PMBC over-expressing P-gp in a TNBS-treated group and enhanced secretion of the cytokines IL-1β, IL-6, IL-17, and TNF-α as well as LPS in plasma. These cytokines and LPS can induce P-gp expression through the STAT3/Nf-κb pathway, contributing to a decrease of cyclosporine A retention, which can be reversed by the application of a P-gp inhibitor. Our results demonstrated that the sustained chronic inflammation could induce the intrinsic resistance presented as P-gp over-expression in PBMC in Crohn’s disease through STAT3/Nf-κb pathway and this resistance might be reversed by combinational usage of P-gp inhibitors. PMID:26324318

  13. Doxorubicin delivery enhanced by electroporation to gastrointestinal adenocarcinoma cells with P-gp overexpression.

    PubMed

    Kulbacka, Julita; Daczewska, Małgorzata; Dubińska-Magiera, Magda; Choromańska, Anna; Rembiałkowska, Nina; Surowiak, Paweł; Kulbacki, Marek; Kotulska, Małgorzata; Saczko, Jolanta

    2014-12-01

    Electroporation (EP) can effectively support the penetration of macromolecules from the extracellular space into cells. Electropores induced by the influence of electromagnetic field generate additional paths of transport for macromolecules. The aim of this study was evaluation of the electroporation effect on doxorubicin transport efficiency to human colon (LoVo and LoVo/DX) and gastric (EPG85-257/P and EPG85-257/RDB) adenocarcinoma cells with overexpression of P-glycoprotein and murine macrophage cell line (P388/D1). In our EP experiments cells were placed into a cuvette with aluminum electrodes and pulsed with five square electric pulses of 1300 V/cm and duration of 50 μs each. Cells were also treated with low doxorubicin concentration ([DOX]=1.7 μM). The ultrastructure (TEM) and changes of P-glycoprotein expression of tumor cells subjected to electric field were monitored. The mitochondrial cell function and trypan blue staining were evaluated after 24h. Our results indicate the most pronounced effect of EP with DOX and disturbed ultrastructure in resistant gastric and colon cells with decrease of P-gp expression. Electroporation may be an attractive delivery method of cytostatic drugs in chemotherapy, enabling reduction of drug dose, exposure time and side effects.

  14. Targeting autophagy augments the activity of DHA-E3 to overcome p-gp mediated multi-drug resistance.

    PubMed

    Xi, Guangmin; Wang, Ming; Sun, Bing; Shaikh, Abdul Sami; Liu, Yongqing; Wang, Wei; Lou, Hongxiang; Yuan, Huiqing

    2016-12-01

    Multidrug resistance (MDR) is a major obstacle for successful chemotherapy treatment. Searching for effective MDR modulators and combining them with anticancer drug therapies has been a promising strategy against clinical MDR. In our previous study, we have found that DHA-E3, a synthetic derivative of DHA, has the ability to modulate the function of P-glycoprotein (P-gp) and reverse MDR in cancer cells. In this study, we further evaluated the reversal effect of DHA-E3 on MDR and explored its mechanism of action in vitro. Our findings showed that DHA-E3 significantly potentiated the cytotoxicity of vincristine(VCR) and adriamycin(ADR) in the P-gp over-expressing KB/VCR and A02 cells. The mechanistic study found that DHA-E3 increased the intracellular accumulation of ADR and rhodamine-123 by directly inhibiting the drug-transport activity of P-gp. In the present study, we found that DHA-E3 not only reversed MDR, but also induced autophagy in MDR cancer cells. To determine whether DHA-E3-induced autophagy is an adaptive survival response or contributes to cell death, we manipulated autophagic activity using autophagy inhibitor 3-MA or siRNA targeting Beclin1. We found that the reversal activity of DHA-E3 was significantly exacerbated in the presence of 3-MA or blocking the expression of Beclin1. These results suggest that DHA-E3 is capable of reversing MDR, induction of autophagy represents a defense mechanism and inhibiting this process may be an effective strategy to augment the reversal activity of reversal agents. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Function of Aurora kinase A in Taxol-resistant breast cancer and its correlation with P-gp.

    PubMed

    Li, Yan; Tang, Ke; Zhang, Haijing; Zhang, Yi; Zhou, Wanqi; Chen, Xiaoguang

    2011-01-01

    Breast cancer is one of the most common malignant diseases among women. In early and metastatic breast cancer, Taxane (Taxol) is widely used as an adjuvant and neoadjuvant therapies. Although breast cancer is initially responsive to Taxol, inherent or developed resistance to Taxol often limits the efficacy of the drug. The oncogene Aurora kinase A is frequently up-regulated in human cancer, and is associated with sensitivity to chemotherapy in certain types of cancer. In the present study, we aimed to clarify the functional role of Aurora kinase A in breast cancer resistance to Taxol, and to determine the means to overcome this resistance. The correlation between the expression levels of Aurora kinase A and chemoresistance to Taxol in breast cancer cells, and resistance to Taxol in a xenograft model were demonstrated. MTT assay was performed to determine cell viability. Subsequently, the relationship of Aurora kinase A with the expression and functional role of P-gp was clarified, as well as its relationship with p-ERK2, which regulates the expression of P-gp. The expression of Aurora A was determined to be capable of enhancing the sensitivity of cells resistant to Taxol in vitro and in vivo using stable knockdown Aurora kinase A cells. We propose that this kinase may be used as a target for overcoming chemoresistance and enhancing the chemosensitivity of breast cancer to Taxol.

  16. Intracellular accumulation of Praziquantel in T lymphoblastoid cell lines, CEM (parental) and CEMVBL(P-gp-overexpressing).

    PubMed

    Kigen, Gabriel; Edwards, Geoffrey

    2016-08-14

    Praziquantel (PZQ) is an antihelminthic drug whose P-glycoprotein (P-gp) substrate specificity has not been conclusively characterized. We investigated its specificity by comparing its in vitro intracellular accumulation in CEM (parental), and CEMVBL cells which over express P-gp, a drug efflux transporter. Saquinavir (SQV), a known substrate of efflux transporters was used as control. A reversed phase liquid chromatography method was developed to simultaneously quantify PZQ and SQV in cell culture media involving involved a liquid - liquid extraction followed by ultra-high performance liquid chromatography using a Hypurity C18 column and ultraviolet detection set at a wavelength of 215 nm. The mobile phase consisted of ammonium formate, acetonitrile and methanol (57:38:5 v/v). Separation was facilitated via isocratic elution at a flow rate of 1.5 ml/min, with clozapine (CLZ) as internal standard. This was validated over the concentration range of 1.6 to 25.6 μM for all analytes. Intracellular accumulation of SQV in CEMVBL was significantly lower compared to that in CEM cells (0.1 ± 0.031 versus 0.52 ± 0.046, p = 0.03 [p <0.05]). Accumulation of PZQ in both cell lines cells were similar (0.05 ± 0.005 versus 0.04 ± 0.009, p = 0.4) suggesting that it is not a substrate of P-gp in CEM cells. In presence tariquidar, a known inhibitor of P-gp, the intracellular accumulation of SQV in CEMVBL cells increased (0.52 ± 0.068 versus 0.61 ± 0.102, p = 0.34 in CEM cells and 0.09 ± 0.015 versus 0.56 ± 0.089, p = 0.029 [p < 0.05] in CEMVBL cells). PZQ did not significantly affect the accumulation of SQV in either CEM (0.52 ± 0.068 versus 0.54 ± 0.061, p = 0.77), or in CEMVBL cells (0.09 ± 0.015 versus 0.1 ± 0.031, p = 0.89) cells compared to tariquidar, implying that PZQ is not an inhibitor of P-gp in CEMVBL cells. PZQ is neither a substrate nor an inhibitor of the efflux drug transporter

  17. Modulation of paclitaxel transport by flavonoid derivatives in human breast cancer cells. Is there a correlation between binding affinity to NBD of P-gp and modulation of transport?

    PubMed

    Václavíková, Radka; Boumendjel, Ahcene; Ehrlichová, Marie; Kovár, Jan; Gut, Ivan

    2006-07-01

    We have investigated the effect of 13 flavonoid derivatives on [(14)C]paclitaxel transport in two human breast cancer cell lines, the adriamycin-resistant NCI/ADR-RES and sensitive MDA-MB-435. For this study, we selected representatives of aurones, chalcones, flavones, flavonols, chromones, and isoflavones with known binding affinity toward nucleotide-binding domain (NBD2) of P-glycoprotein and for which no reported work is available regarding paclitaxel transport. Aurones CB-284, CB-285, CB-287, and ML-50 most effectively inhibited P-gp related transport in the resistant line in comparison with chalcones, flavones, flavonols, chromones, and isoflavone derivatives and accordingly increased the accumulation of [(14)C]paclitaxel and decreased its efflux. Those agents efficiently modulated paclitaxel transport in P-gp highly expressing resistant human breast cancer cells and they could increase the efficiency of chemotherapy in paclitaxel-resistant tumors. In contrast, the sensitive cell line responded reversely in that CB-284, CB-285, CB-287, and ML-50 significantly inhibited accumulation of [(14)C]paclitaxel and especially CB-287, which significantly stimulated its efflux. Some, but not all, of the data correlated with the binding of flavonoid derivatives to P-gp, and indicated that even in the P-gp highly expressing NCI/ADR-RES cells, the binding was not the only factor influencing the transport of [(14)C]paclitaxel. Opposite effects of flavonoid derivatives on the P-gp highly expressing and MDA-MB-435 non-expressing cell lines indicate that paclitaxel is not only transported by P-gp and let us assume that Mrp2 or ABCC5 seem to be good transport-candidates in these cells. The inhibition of paclitaxel accumulation and stimulation of its efflux are potentially unfavorable for drug therapy and since they could be due to modulation of drug transporters other than P-gp, their expression in tumors is of great significance for efficient chemotherapy.

  18. P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins.

    PubMed

    Huang, Lu; Wang, Jie; Chen, Wen-Chang; Li, Hong-Ye; Liu, Jie-Sheng; Tao Jiang; Yang, Wei-Dong

    2014-08-01

    Bivalves naturally exposed to toxic algae have mechanisms to prevent from harmful effects of diarrhetic shellfish poisoning (DSP) toxins. However, quite few studies have examined the mechanisms associated, and the information currently available is still insufficient. Multixenobiotic resistance (MXR) is ubiquitous in aquatic invertebrates and plays an important role in defense against xenobiotics. Here, to explore the roles of P-glycoprotein (P-gp) in the DSP toxins resistance in shellfish, complete cDNA of P-gp gene in the mussel Perna viridis was cloned and analyzed. The accumulation of okadaic acid (OA), a main component of DSP toxins, MXR activity and expression of P-gp in gills of P. viridis were detected after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins in the presence or absence of P-gp inhibitors PGP-4008, verapamil (VER) and cyclosporin A (CsA). The mussel P. viridis P-gp closely matches MDR/P-gp/ABCB protein from various organisms, having a typical sequence organization as full transporters from the ABCB family. After exposure to P. lima, OA accumulation, MXR activity and P-gp expression significantly increased in gills of P. viridis. The addition of P-gp-specific inhibitors PGP-4008 and VER decreased MXR activity induced by P. lima, but had no effect on the OA accumulation in gills of P. viridis. However, CsA, a broad-spectrum inhibitor of ABC transporter not only decreased MXR activity, but also increased OA accumulation in gills of P. viridis. Together with the ubiquitous presence of other ABC transporters such as MRP/ABCC in bivalves and potential compensatory mechanism in P-gp and MRP-mediated resistance, we speculated that besides P-gp, other ABC transporters, especially MRP might be involved in the resistance mechanisms to DSP toxins.

  19. Glycolytic pyruvate regulates P-Glycoprotein expression in multicellular tumor spheroids via modulation of the intracellular redox state.

    PubMed

    Wartenberg, Maria; Richter, Madeleine; Datchev, André; Günther, Sebastian; Milosevic, Nada; Bekhite, Mohamed M; Figulla, Hans-Reiner; Aran, Josep M; Pétriz, Jordi; Sauer, Heinrich

    2010-02-01

    ABC transporters like P-glycoprotein (P-gp/ABCB1) are membrane proteins responsible for the transport of toxic compounds out of non-malignant cells and tumor tissue. To investigate the effect of glycolysis and the tissue redox state on P-gp expression in multicellular tumor spheroids derived from prostate adenocarcinoma cells (DU-145), glioma cells (Gli36), and the human cervix carcinoma cell line KB-3-1 transfected with a P-gp-EGFP fusion gene that allows monitoring of P-gp expression in living cells. During cell culture of DU-145, Gli36, and KB-3-1 tumor spheroids P-gp expression was observed as well as increased lactate and decreased pyruvate levels and expression of glycolytic enzymes. Inhibition of glycolysis for 24 h by either iodoacetate (IA) or 2-deoxy-D-glucose (2-DDG) downregulated P-gp expression which was reversed upon coincubation with the radical scavenger ebselen as shown by semi-quantitative immunohistochemisty in DU-145 and Gli36 tumor spheroids, and by EGFP fluorescence in KB-3-1 tumor spheroids. Consequently endogenous ROS generation in DU-145 tumor spheroids was increased in the presence of either IA or 2-DDG, which was abolished upon coincubation with ebselen. Exogenous addition of pyruvate significantly reduced ROS generation, increased P-gp expression as well as efflux of the P-gp substrate doxorubicin. Doxorubicin transport was significantly blunted by 2-DDG and IA, indicating that inhibition of glycolysis reversed the multidrug resistance phenotype. In summary our data demonstrate that P-gp expression in tumor spheroids is closely related to the glycolytic metabolism of tumor cells and can be downregulated by glycolysis inhibitors via mechanisms that involve changes in the cellular redox state. (c) 2009 Wiley-Liss, Inc.

  20. P-glycoprotein and its inducible expression in three bivalve species after exposure to Prorocentrum lima.

    PubMed

    Huang, Lu; Liu, Su-Li; Zheng, Jian-Wei; Li, Hong-Ye; Liu, Jie-Sheng; Yang, Wei-Dong

    2015-12-01

    P-glycoprotein (P-gp or ABCB1) belongs to the family of ATP-binding cassette (ABC) transporters responsible for multixenobiotic resistance (MXR) in aquatic organisms. To provide more information of P-gp in shellfish, in this study, complete cDNA of P-gp in three bivalve species including Ruditapes philippinarum, Scapharca subcrenata and Tegillarca granosa were cloned and its expressions in gill, digestive gland, adductor muscle and mantle of the three bivalves were detected after exposure to Prorocentrum lima, a toxogenic dinoflagellate. The complete sequences of R. philippinarum, S. subcrenata and T. granosa P-gp showed high homology with MDR/P-gp/ABCB proteins from other species, having a typical sequence organization as full transporters from the ABCB family. Phylogenetic analyses revealed that the amino acid sequences of P-gp from S. subcrenata and T. granosa had a closest relationship, forming an independent branch, then grouping into the other branch with Mytilus californianus, Mytilus galloprovincialis and Crassostrea gigas. However, P-gp sequences from R. philippinarum were more similar to the homologs from the more distantly related Aplysia californica than to homologs from S. subcrenata and T. granosa, suggesting that bivalves P-gp might have different paralogs. P-glycoprotein expressed in all detected tissues but there were large differences between them. After exposure to P. lima, the expression of P-gp changed in the four tissues in varying degrees within the same species and between different species, but the changes in mRNA and protein level were not always synchronous. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Natural alkaloids as P-gp inhibitors for multidrug resistance reversal in cancer.

    PubMed

    Joshi, Prashant; Vishwakarma, Ram A; Bharate, Sandip B

    2017-09-29

    The biggest challenge associated with cancer chemotherapy is the development of cross multi-drug resistance to almost all anti-cancer agents upon chronic treatment. The major contributing factor for this resistance is efflux of the drugs by the p-glycoprotein pump. Over the years, inhibitors of this pump have been discovered to administer them in combination with chemotherapeutic agents. The clinical failure of first and second generation P-gp inhibitors (such as verapamil and cyclosporine analogs) has led to the discovery of third generation potent P-gp inhibitors (tariquidar, zosuquidar, laniquidar). Most of these inhibitors are nitrogenous compounds and recently a natural alkaloid CBT-01(®) (tetrandrine) has advanced to the clinical phase. CBT-01 demonstrated positive results in Phase-I study in combination with paclitaxel, which warranted conducting it's Phase II/III trial. Apart from this, there exist a large number of natural alkaloids possessing potent inhibition of P-gp efflux pump and other related pumps responsible for the development of resistance. Despite the extensive contribution of alkaloids in this area, has never been reviewed. The present review provides a comprehensive account on natural alkaloids possessing P-gp inhibition activity and their potential for multidrug resistance reversal in cancer. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Effects of Kampo medicines on CYP and P-gp activity in vitro.

    PubMed

    Ito, Kiyomi; Satoh, Toshiyuki; Watanabe, Yuka; Ikarashi, Nobutomo; Asano, Takayuki; Morita, Toshimi; Sugiyama, Kiyoshi

    2008-05-01

    The Kampo medicines are more and more often used in recent years, usually together with the Western drugs. The need for the investigation of drug interactions between Kampo medicines and Western drugs are, therefore, widely recognized. In the present study, the effects of 3 Kampo medicines (Rikkunshito, Yokukansan and Boiogito) on the activity of cytochrome P450 (CYP), a superfamily of drug-metabolizing enzymes, were investigated in an in vitro study using human CYP recombinants. Their effects on the P-glycoprotein (P-gp), one of the major drug transporters, were also evaluated by the ATPase assay using human P-gp membranes and verapamil as a substrate. The inhibition rate of Rikkunshito, Yokukansan and Boiogito on human CYP3A4, 2C9, 2C19, 2D6 and 2E1 was less than 50% at the concentrations below 0.1 mg/ml except for the inhibition of CYP2D6 by Boiogito. Furthermore, none of the Kampo medicines affected the ATPase activity at the concentrations lower than 0.1 mg/ml, either in the absence or presence of verapamil, indicating their low inhibitory potency against P-gp. These findings indicate that Rikkunshito, Yokukansan and Boiogito are unlikely to cause clinically relevant drug interactions involving the inhibition of major CYP isozymes and P-gp.

  3. Localization and mRNA expression of CYP3A and P-glycoprotein in human duodenum as a function of age.

    PubMed

    Fakhoury, May; Litalien, Catherine; Medard, Yves; Cavé, Hélène; Ezzahir, Nadia; Peuchmaur, Michel; Jacqz-Aigrain, Evelyne

    2005-11-01

    Cytochromes P450 3A (CYP3A) and P-glycoprotein (P-gp) are mainly located in enterocytes and hepatocytes. The CYP3A/P-gp system contributes to the first-pass metabolism of many drugs, resulting in a limited bioavailability. During the neonatal period, a shift between CYP3A7, the fetal form, and CYP3A4 occurs in the liver, but data on the expression of the CYP3A/P-gp complex in the intestine are very limited. A total of 59 normal duodenal biopsies from white children aged 1 month to 17 years were studied. Localization of the proteins by immunohistochemistry analysis was performed using a polyclonal antibody, Nuage anti-CYP3A, and a monoclonal antibody, C494 anti-P-gp. The mRNA quantification was performed using highly specific real-time reverse transcription-polymerase chain reaction. Villin mRNA quantification was used for normalization. CYP3A protein was detected in all enterocytes in the samples from patients over 6 months of age, whereas it was not in younger samples. P-gp protein was expressed at the apical and upper lateral surfaces of the enterocytes. CYP3A isoforms and P-gp mRNA levels were highly variable. CYP3A4 and CYP3A5 mRNA levels were high during the first year of life and decreased with age, whereas CYP3A7 was detected at a low level in 64% of samples, whatever the age. P-gp mRNA expression level was also highly variable. Our results showed that neonates and infants had a significant expression of CYP3A and P-gp mRNA in the intestine, suggesting a different maturation profile of CYP3A and P-gp with age in the liver and the intestine.

  4. Exposure of LS-180 Cells to Drugs of Diverse Physicochemical and Therapeutic Properties Up-regulates P-glycoprotein Expression and Activity

    PubMed Central

    Abuznait, Alaa H.; Patrick, Shawn G.; Kaddoumi, Amal

    2011-01-01

    Purpose Drug transporters are increasingly recognized as important determinants of variability in drug disposition and therapeutic response, both in pre-clinical and clinical stages of drug development process. The role P-glycoprotein (P-gp) plays in drug interactions via its inhibition is well established. However, much less knowledge is available about drugs effect on P-gp up-regulation. The objective of this work was to in vitro investigate and rank commonly used drugs according to their potencies to up-regulate P-gp activity utilizing the same experimental conditions. Methods The in vitro potencies of several drugs of diverse physicochemical and therapeutic properties including rifampicin, dexamethasone, caffeine, verapamil, pentylenetetrazole, hyperforin, and β-estradiol over broad concentration range to up-regulate P-gp expression and activity were examined. For dose-response studies, LS-180 cells were treated with different concentrations of the selected drugs followed by P-gp protein and gene expressions analyses. P-gp functionality was determined by uptake studies with rhodamine 123 as a P-gp substrate, followed by Emax/EC50 evaluation. Results The results demonstrated a dose-dependent increase in P-gp expression and activity following treatments. At 50 μM concentration (hyperforin, 0.1 μM), examined drugs increased P-gp protein and gene expressions by up to 5.5 and 6.2-fold, respectively, while enhanced P-gp activity by 1.8–4-fold. The rank order of these drugs potencies to up-regulate P-gp activity was as following: hyperforin ⋙ dexamethasone ≈ β-estradiol > caffeine > rifampicin ≈ pentylenetetrazole > verapamil. Conclusions These drugs have the potential to be involved in drug interactions when administered with other drugs that are P-gp substrates. Further studies are needed to in vivo evaluate these drugs and verify the consequences of such induction on P-gp activity for in vitro-in vivo correlation purposes. PMID:21733412

  5. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  6. [The effect of MDR1 (P-gp) and ABCG2 on the drug resistance in Hep 2 cells].

    PubMed

    Sun, Zhenfeng; Shen, Bin; Zhang, Jia; Su, Tiantian; Dong, Pin

    2015-06-01

    To study the effect of MDR1 (P-gp) and ABCG2 on the drug resistance in Hep 2 cells. Flow cytometry was used to detect the variations of the antitumor drugs accumulation and discharging, and activity variations when MDR1 and ABCG2 inhibitors were used in Hep-2. The accumulation and discharging of mitoxantrone was significantly higher than the control group when ABCG2 inhibitor FTC was used in Hep-2 (P<0. 05). In contrast, P-gp did not appear similar case; To the mitoxantrone and cisplatin, there was no statistical correlation about activity of Hep-2 between P-gp or ABCG2 antagonist and the control; To the doxorubicin, combining FTC and P-gp, the activity of Hep-2 was higher than the control and difference was significant (P<. 05), In contrast, FTC and P-gp did not appear similar case when used alone; To the 5-FU, when PGP used, the activity of Hep-2 was higher than that in the control and difference was significant (P<0. 05), In con- trast, FTC and FTC+P-gp did not appear similar case; To the paclitaxel, when P-gp or FTC+P-gp used, the activity of Hep-2 was higher than that in the control and difference was significant(P<0. 05). ABCG2 may lead to drug resistance mainly by changing the ability of cell in accumulating and discharging chemotherapy drugs. P-gp has other way. P-gp and ABCG2 play different roles in different drug resistance.

  7. Interaction of BDE-47 and its Hydroxylated Metabolite 6-OH-BDE-47 with the Human ABC Efflux Transporters P-gp and BCRP: Considerations for Human Exposure and Risk Assessment

    EPA Science Inventory

    ATP binding cassette (ABC) transporters, including P-glycoprotein (P-gp; also known as MDR1, ABCB1) and breast cancer resistance protein (BCRP; also known as ABCG2), are membrane-bound proteins that mediate the cellular efflux of xenobiotics as an important defense against chemic...

  8. Interaction of BDE-47 and its Hydroxylated Metabolite 6-OH-BDE-47 with the Human ABC Efflux Transporters P-gp and BCRP: Considerations for Human Exposure and Risk Assessment

    EPA Science Inventory

    ATP binding cassette (ABC) transporters, including P-glycoprotein (P-gp; also known as MDR1, ABCB1) and breast cancer resistance protein (BCRP; also known as ABCG2), are membrane-bound proteins that mediate the cellular efflux of xenobiotics as an important defense against chemic...

  9. P-glycoprotein (multi-xenobiotic resistance) and heat shock protein gene expression in the reef coral Montastraea franksi in response to environmental toxicants.

    PubMed

    Venn, Alexander A; Quinn, Jennifer; Jones, Ross; Bodnar, Andrea

    2009-07-26

    The deleterious impacts of marine pollutants on reef corals and their symbiotic algae are an important element of global coral reef decline. In the current study we examined the impacts of toxicants on the reef coral Montastraea franksi by analysing the expression of three stress-related genes belonging to the coral host, using Taqman real-time quantitative reverse transcription-PCR. Gene expression profiles of P-glycoprotein (or multi-xenobiotic resistance protein) (P-gp); heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) were examined following 4 and 8h exposures to the heavy metal copper (3, 10, 30 and 100 microgL(-1)) or the third generation oil dispersant Corexit9527 (1, 5, 10 and 50 ppm). Additionally, the expression of P-gp was examined following exposure to 0.5 and 5 microM concentrations of the chemotherapeutic drug vinblastine, a classic substrate of P-gp. The expression of P-gp increased significantly in corals treated with vinblastine and also increased following exposure to copper and Corexit9527. Hsp70, and to a lesser extent Hsp90 expression increased following exposure to copper and Corexit9527 indicating a general cellular stress response. Densities of symbiotic algae in the tissues of the corals did not change significantly during the experiments, nor was any loss or paling of coral tissues observed. These findings provide important insight into how corals defend themselves against pollution and complement ongoing initiatives developing molecular biomarkers of stress in reef-building corals.

  10. Expression and localization of p-glycoprotein, multidrug resistance protein 4, and breast cancer resistance protein in the female lower genital tract of human and pigtailed macaque.

    PubMed

    Zhou, Tian; Hu, Minlu; Pearlman, Andrew; Patton, Dorothy; Rohan, Lisa

    2014-11-01

    Antiretroviral drug absorption and disposition in cervicovaginal tissue is important for the effectiveness of vaginally or orally administered drug products in preexposure prophylaxis (PrEP) of HIV-1 sexual transmission to women. Therefore, it is imperative to understand critical determinants of cervicovaginal tissue pharmacokinetics. This study aimed to examine the mRNA expression and protein localization of three efflux transporters, P-glycoprotein (P-gp), multidrug resistance-associated protein 4 (MRP4), and breast cancer resistance protein (BCRP), in the lower genital tract of premenopausal women and pigtailed macaques. Along the human lower genital tract, the three transporters were moderately to highly expressed compared to colorectal tissue and liver, as revealed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). In a given genital tract segment, the transporter with the highest expression level was either BCRP or P-gp, while MRP4 was always expressed at the lowest level among the three transporters tested. The immunohistochemical staining showed that P-gp and MRP4 were localized in multiple cell types including epithelial cells and vascular endothelial cells. BCRP was predominantly localized in the vascular endothelial cells. Differences in transporter mRNA level and localization were observed among endocervix, ectocervix, and vagina. Compared to human tissues, the macaque cervicovaginal tissues displayed comparable expression and localization patterns of the three transporters, although subtle differences were observed between the two species. The role of these cervicovaginal transporters in drug absorption and disposition warrants further studies. The resemblance between human and pigtailed macaque in transporter expression and localization suggests the utility of the macaque model in the studies of human cervicovaginal transporters.

  11. Acetaminophen Modulates P-Glycoprotein Functional Expression at the Blood-Brain Barrier by a Constitutive Androstane Receptor–Dependent Mechanism

    PubMed Central

    Thompson, Brandon J.; Sanchez-Covarrubias, Lucy; Zhang, Yifeng; Laracuente, Mei-Li; Vanderah, Todd W.; Ronaldson, Patrick T.; Davis, Thomas P.

    2013-01-01

    Effective pharmacologic treatment of pain with opioids requires that these drugs attain efficacious concentrations in the central nervous system (CNS). A primary determinant of CNS drug permeation is P-glycoprotein (P-gp), an endogenous blood-brain barrier (BBB) efflux transporter that is involved in brain-to-blood transport of opioid analgesics (i.e., morphine). Recently, the nuclear receptor constitutive androstane receptor (CAR) has been identified as a regulator of P-gp functional expression at the BBB. This is critical to pharmacotherapy of pain/inflammation, as patients are often administered acetaminophen (APAP), a CAR-activating ligand, in conjunction with an opioid. Our objective was to investigate, in vivo, the role of CAR in regulation of P-gp at the BBB. Following APAP treatment, P-gp protein expression was increased up to 1.4–1.6-fold in a concentration-dependent manner. Additionally, APAP increased P-gp transport of BODIPY-verapamil in freshly isolated rat brain capillaries. This APAP-induced increase in P-gp expression and activity was attenuated in the presence of CAR pathway inhibitor okadaic acid or transcriptional inhibitor actinomycin D, suggesting P-gp regulation is CAR-dependent. Furthermore, morphine brain accumulation was enhanced by P-gp inhibitors in APAP-treated animals, suggesting P-gp–mediated transport. A warm-water (50°C) tail-flick assay revealed a significant decrease in morphine analgesia in animals treated with morphine 3 or 6 hours after APAP treatment, as compared with animals treated concurrently. Taken together, our data imply that inclusion of APAP in a pain treatment regimen activates CAR at the BBB and increases P-gp functional expression, a clinically significant drug-drug interaction that modulates opioid analgesic efficacy. PMID:24019224

  12. Cloning and heterologous expression of the ovine (Ovis aries) P-glycoprotein (Mdr1) in Madin-Darby canine kidney (MDCK) cells.

    PubMed

    Zahner, D; Alber, J; Petzinger, E

    2010-06-01

    P-glycoprotein (P-gp) plays a crucial role in the multidrug resistance of pathogenic helminths in sheep (Ovis aries) as well as in antiparasitic drug pharmacokinetics in the host. We cloned sheep P-gp cDNA and expressed it stably in Madin-Darby canine kidney (MDCK) cells. The open reading frame consists of 3858 nucleotides coding for a 1285 amino acids containing protein. The sequence shows high homology to the orthologs of other mammalian species, especially cattle. Both ruminant DNA sequences show a 9 bp insertion that is lacking in all other investigated sequences. Expressed in MDCK cells, the protein displays a size of 170 kDa on Western analysis. Transfection of MDCK cells with sheep P-gp resulted in 10- to 50-fold resistance to the cytotoxic P-gp substrates colchicin and daunorubicin, and in reduced digoxin accumulation.

  13. Susceptibility of juvenile and adult blood–brain barrier to endothelin-1: regulation of P-glycoprotein and breast cancer resistance protein expression and transport activity

    PubMed Central

    2012-01-01

    Background P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) play a critical role in keeping neurotoxic substances from entering the brain. We and others have previously reported an impact of inflammation on the regulation of adult blood–brain barrier (BBB) efflux transporters. However, studies in children have not been done. From the pediatric clinical perspective, it is important to understand how the central nervous system (CNS) and BBB drug efflux transporters differ in childhood from those of adults under normal and inflammatory conditions. Therefore, we examined and compared the regulation of P-gp and BCRP expression and transport activity in young and adult BBB and investigated the molecular mechanisms underlying inflammatory responses. Methods Rats at postnatal day (P) P21 and P84, corresponding to the juvenile and adult stages of human brain maturation, respectively, were treated with endothelin-1 (ET-1) given by the intracerebroventricular (icv) route. Twenty-four hours later, we measured P-gp and BCRP protein expression in isolated brain capillary by immunoblotting as well as by transport activity in vivo by measuring the unbound drug partitioning coefficient of the brain (Kp,uu,brain) of known efflux transporter substrates administered intravenously. Glial activation was measured by immunohistochemistry. The release of cytokines/chemokines (interleukins-1α, 1-β (IL-1β), -6 (IL-6), -10 (IL-10), monocyte chemoattractant protein (MCP-1/CCL2), fractalkine and tissue inhibitor of metalloproteinases-1 (TIMP-1)) were simultaneously measured in brain and serum samples using the Agilent Technology cytokine microarray. Results We found that juvenile and adult BBBs exhibited similar P-gp and BCRP transport activities in the normal physiological conditions. However, long-term exposure of the juvenile brain to low-dose of ET-1 did not change BBB P-gp transport activity but tended to decrease BCRP transport activity in the juvenile brain, while a

  14. Effect of venlafaxine and desvenlafaxine on drug efflux protein expression and biodistribution in vivo.

    PubMed

    Bachmeier, Corbin; Levin, Gary M; Beaulieu-Abdelahad, David; Reed, Jon; Mullan, Michael

    2013-10-01

    Venlafaxine, and to a lesser extent desvenlafaxine, has previously been shown to induce the expression of the drug efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in whole cells and alter the cellular permeability of a known drug efflux probe (rhodamine 123). To validate these in vitro findings, wild-type mice were treated for 4 days with 10 mg/kg venlafaxine or desvenlafaxine, and drug efflux transporter expression was examined in the brain, liver, and intestine. P-gp and BCRP expression was significantly upregulated in the intestine, following a treatment with venlafaxine (2.6- and 6.7-fold, respectively) or desvenlafaxine (2.3- and 4.8-fold, respectively). In addition, venlafaxine increased the BCRP expression in the brain (40%) and liver (60%), whereas desvenlafaxine had no effect on drug efflux transporter levels in these tissues. Using the same treatment paradigm, we observed a minimal impact of either drug on the brain disposition of the known drug efflux probe, topotecan. However, in the periphery, venlafaxine treatment significantly reduced the topotecan oral bioavailability by nearly 40%, whereas the impact of desvenlafaxine on topotecan plasma levels was more modest (23%). These studies demonstrate an effect of venlafaxine on the drug efflux transport activity and the potential for clinical drug-drug interactions. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

  15. Effect of 9-cis retinoic acid and all-trans retinoic acid in combination with verapamil on P-glycoprotein expression in L1210 cells.

    PubMed

    Breier, A; Stetka, J; Bohacova, V; Macejova, D; Brtko, J; Sulova, Z

    2014-01-01

    The development of the most common multidrug resistance (MDR) phenotype is associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells. In the current study, we used three L1210 cell variants: S cells - parental drug-sensitive cells; R cells - drug-resistant cells with P-gp overexpression due to selection with vincristine; T cells - drug-resistant cells with P-gp overexpression due to stable transfection with the pHaMDRwt plasmid, which encodes human full-length P-gp. Several authors have described the induction of P-gp expression/activity in malignant cell lines after treatment with all-trans retinoic acid (AtRA; ligand of retinoic acid nuclear receptors, RARs). An isomer of AtRA also exists, 9-cis retinoic acid, which is a ligand of both RARs and nuclear retinoid X receptors (RXRs). In a previous work, we described that the combined treatment of R cells with verapamil and AtRA induces the downregulation of P-gp expression/activity. In the current study, we studied the expression of RARs and RXRs in S, R and T cells and the effects of treatment with AtRA, 9cRA and verapamil on P-gp expression, cellular localization and efflux activity in R and T cells. We found that the overexpression of P-gp in L1210 cells is associated with several changes in the specific transcription of both subgroups of nuclear receptors, RARs and RXRs. We also demonstrated that treatment with AtRA, 9cRA and verapamil induces alterations in P-gp expression in R and T cells. Particularly, combined treatment of R cells with verapamil and AtRA induced downregulation of P-gp content/activity. In contrast, similar treatment of T cells induced slight increase of P-gp content without any changes in efflux activity of this protein. These findings indicate that active crosstalk between the RAR and RXR regulatory pathways and P-gp-mediated MDR could take place.

  16. P-glycoprotein function and expression during obstructive cholestasis in rats.

    PubMed

    Micuda, Stanislav; Brcakova, Eva; Fuksa, Leos; Cermanova, Jolana; Osterreicher, Jan; Hroch, Milos; Mokry, Jaroslav; Pejchal, Jaroslav; Martinkova, Jirina; Staud, Frantisek

    2008-05-01

    The present study was aimed at evaluation of in vivo biliary and renal excretion of rhodamine 123 (Rho123), a P-glycoprotein (P-gp) substrate, in rats during either acute or chronic cholestasis induced by bile duct obstruction (BDO). The Rho123 clearance study was performed either one (BDO1) or seven (BDO7) days after BDO. Bile flow was reconstituted, and bile and urine were collected after steady-state plasma concentration of Rho123 was attained. Tissue expression of P-gp was evaluated by quantitative immunohistochemistry, and immunoblotting. Significant up-regulation of the liver P-gp protein was observed in acute and chronic cholestasis. Primary periportal location of P-gp was enlarged also to pericentral areas. In the kidneys, immunohistochemistry showed pancellular increase in P-gp after 1 day of BDO, which subsided after 7 days of BDO. Nevertheless, biliary and renal clearances (CL(Bile) and CL(R)) of Rho123 did not reflect the induction of P-gp expression. While CL(Bile) was reduced one day after cholestasis and restored on the seventh day, the CL(R) was preserved in BDO1 group and reduced in BDO7 group without change in glomerular filtration rate. In parallel, biliary and renal clearances of conjugated bilirubin were significantly reduced in both cholestatic groups compared with controls. These findings suggest that extrahepatic cholestasis causes time-dependent changes in elimination of Rho123 which do not exactly reflect alteration of P-gp expression in the rat liver and kidney. These data may help to explain impaired elimination of P-gp substrates after short-term cholestasis that may commonly occur in clinical practice.

  17. Active brain targeting of a fluorescent P-gp substrate using polymeric magnetic nanocarrier system.

    PubMed

    Kirthivasan, B; Singh, D; Bommana, M M; Raut, S L; Squillante, E; Sadoqi, M

    2012-06-29

    Magnetic nanoparticles (NP) were developed for the active brain targeting of water-soluble P-glycoprotein (P-gp) substrate rhodamine 123 (Rh123). The NP matrix of poly(lactide-co-glycolide) (PLGA) and methoxy poly(ethyleneglycol)-poly(lactic acid) (M-PEG-PLA) was prepared by single emulsion solvent evaporation of polymers with oleic acid-coated magnetic nanoparticles (OAMNP) and Rh123. All formulations were characterized in terms of morphology, particle size, magnetic content and Rh123 encapsulation efficiency. The maximum encapsulation efficiency of Rh123 was 45 ± 3% and of OAMNP was 42 ± 4%. The brain targeting and biodistribution study was performed on Sprague Dawley rats (3 groups, n = 6). Rh123 (0.4 mg kg(-1)) was administered in saline form, NP containing Rh123, and NP containing Rh123 in the presence of a magnetic field (0.8 T). The fluorimetric analysis of brain homogenates revealed a significant uptake (p < 0.05) of Rh123 in the magnetically targeted group relative to controls. These results were supported by fluorescence microscopy. This study reveals the ability of magnetically targeted nanoparticles to deliver substances to the brain, the permeation of which would otherwise be inhibited by the P-gp system.

  18. Active brain targeting of a fluorescent P-gp substrate using polymeric magnetic nanocarrier system

    NASA Astrophysics Data System (ADS)

    Kirthivasan, B.; Singh, D.; Bommana, M. M.; Raut, S. L.; Squillante, E.; Sadoqi, M.

    2012-06-01

    Magnetic nanoparticles (NP) were developed for the active brain targeting of water-soluble P-glycoprotein (P-gp) substrate rhodamine 123 (Rh123). The NP matrix of poly(lactide-co-glycolide) (PLGA) and methoxy poly(ethyleneglycol)-poly(lactic acid) (M-PEG-PLA) was prepared by single emulsion solvent evaporation of polymers with oleic acid-coated magnetic nanoparticles (OAMNP) and Rh123. All formulations were characterized in terms of morphology, particle size, magnetic content and Rh123 encapsulation efficiency. The maximum encapsulation efficiency of Rh123 was 45 ± 3% and of OAMNP was 42 ± 4%. The brain targeting and biodistribution study was performed on Sprague Dawley rats (3 groups, n = 6). Rh123 (0.4 mg kg-1) was administered in saline form, NP containing Rh123, and NP containing Rh123 in the presence of a magnetic field (0.8 T). The fluorimetric analysis of brain homogenates revealed a significant uptake (p < 0.05) of Rh123 in the magnetically targeted group relative to controls. These results were supported by fluorescence microscopy. This study reveals the ability of magnetically targeted nanoparticles to deliver substances to the brain, the permeation of which would otherwise be inhibited by the P-gp system.

  19. Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

    PubMed

    Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

    2016-05-01

    P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP.

  20. Persistent expression and function of P-glycoprotein on peripheral blood lymphocytes identifies corticosteroid resistance in patients with systemic lupus erythematosus.

    PubMed

    Kansal, Amit; Tripathi, Deepak; Rai, Mohit K; Agarwal, Vikas

    2016-02-01

    Corticosteroids (CS) are the mainstay of treatment in systemic lupus erythematosus (SLE) patients. However, some patients have poor response to CS treatment. Among the multiple mechanisms of CS resistance, overexpression of P-glycoprotein (P-gp) on peripheral blood lymphocytes (PBL) may be one of them as this result in efflux of CS from lymphocytes. Thus, we evaluated the role of P-gp protein on PBLs in patients with SLE in its response to CS therapy. SLE patients (n = 42) (fulfilling ACR revised criteria) who were naïve to CS and immunosuppressive drugs were enrolled. Disease activity was assessed using SLE disease activity index (SLEDAI) and expression, and function of P-gp was evaluated by flow cytometry at baseline and after 3 months of therapy with CS. At 3 months, patients with SLEDAI >4 and SLEDAI ≤4 were grouped as nonresponders and responders, respectively. P-gp expression was significantly increased on PBLs of SLE patients as compared to healthy controls (p < 0.001). P-gp expression and function correlated with SLEDAI (r = 0.49, p = 0.005; and r = 0.49, p = 0.001, respectively). P-gp expression and function were not different in responders and nonresponders at baseline. However, at 3 months of CS therapy, P-gp expression and function decreased in responders (p < 0.001 and p < 0.005, respectively), whereas in nonresponders, it remained unchanged. Persistent overexpression and activity of P-gp are associated with poor response to CS in CS naïve patients of SLE.

  1. P-glycoprotein expression and localization in the rat uterus throughout gestation and labor.

    PubMed

    Huang, Qi-Tao; Shynlova, Oksana; Kibschull, Mark; Zhong, Mei; Yu, Yan-Hong; Matthews, Stephen G; Lye, Stephen J

    2016-09-01

    Uterine tissues contain the efflux transporter P-glycoprotein (P-gp, encoded by Abcb1a/1b gene), but little is known about how it changes through gestation. Our aim was to investigate the expression profile and cellular localization of P-gp in the pregnant, laboring and post-partum (PP) rat uterus. We propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial Abcb1a/1b/P-gp. Samples from bilaterally and unilaterally pregnant rats were collected throughout gestation, during labor, and PP (n=4-6/gestational day). RNA and protein were isolated and subjected to quantitative PCR and immunoblotting; P-gp transcript and protein were localized by in situ hybridization and immunohistochemistry. Expression of Abcb1a/1b gene and membrane P-gp protein in uterine tissue (1) increased throughout gestation, peaked at term (GD19-21) and dropped during labor (GD23L); and (2) was upregulated only in gravid but not in empty horn of unilaterally pregnant rats. (3) The drop of Abcb1a/1b mRNA on GD23 was prevented by artificial maintenance of elevated progesterone (P4) levels in late gestation; (4) injection of the P4 receptor antagonist RU486 on GD19 caused a significant decrease in Abcb1 mRNA levels. (5) In situ hybridization and immunohistochemistry indicated that Abcb1/P-gp is absent from myometrium throughout gestation; (6) was expressed exclusively by uterine microvascular endothelium (at early gestation) and luminal epithelium (at mid and late gestation), but was undetectable during labor. In conclusion, ABC transporter protein P-gp in pregnant uterus is hormonally and mechanically regulated. However, its substrate(s) and precise function in these tissues during pregnancy remains to be determined.

  2. Inhibitory effect of clemastine on P-glycoprotein expression and function: an in vitro and in situ study

    PubMed Central

    Abbasi, Mehran Mesgari; Valizadeh, Hadi; Hamishekar, Hamed; Mohammadnejad, Leila; Zakeri-Milani, Parvin

    2016-01-01

    Objective(s): Transporters have an important role in pharmacokinetics of drugs. Inhibition or induction of drug transporters activity can affect drug absorption, safety, and efficacy. P-glycoprotein (P-gp) is the most important membrane transporter that is responsible for active efflux of drugs. It is important to understand which drugs are substrates, inhibitors, or inducers of P-gp to minimize or avoid unwanted interactions. The aim of this study was to investigate the effects of clemastine on the expression and function of P-gp. Materials and Methods: The effect of clemastine on P-gp function and expression was evaluated in vitro byrhodamine-123 (Rho123) efflux assay in Caco-2 cells and Western blot analysis. Rat in situ single pass intestinal permeability model was used to investigate the clemastine effect on digoxin Peff, as a known P-gp substrate. Digoxin levels in intestinal perfusates were assayed by high performance liquid chromatography (HPLC) method. Results: The Caco-2 intracellular accumulation of Rho123 in clemastine and verapamil treated cells was 90.8 ± 9.8 and 420.6±25.4 pg/mg protein, respectively which was significantly higher than that in control cells (50.2±6.0; P<0.05). Immunoblotting results indicated that clemastine decreased expression of P-gp in Caco-2 cells in vitro. More over effective intestinal permeability (Peff) of digoxin in the presence of clemastine, was significantly increased compare to control group. Conclusion: Findings of our study suggested dose dependent P-gp inhibition activity for clemastine in vitro and in situ. Therefore co-administration of clemastine with P-gp substrates may result in unwanted interactions and side effects. PMID:27279987

  3. Anti-AIDS agents 89. Identification of DCX derivatives as anti-HIV and chemosensitizing dual function agents to overcome P-gp-mediated drug resistance for AIDS therapy

    PubMed Central

    Zhou, Ting; Ohkoshi, Emika; Shi, Qian; Bastow, Kenneth F.; Lee, Kuo-Hsiung

    2012-01-01

    In this study, 19 dicamphanoyl-dihydropyranochromone (DCP) and dicamphanoyl-dihydropyranoxanthone (DCX) derivatives, previously discovered as novel anti-HIV agents, were evaluated for their potential to reverse multi-drug resistance (MDR) in a cancer cell line over-expressing P-glycoprotein (P-gp). Seven compounds fully reversed resistance to vincristine (VCR) at 4 μM, a 20-fold enhancement compared to the first generation chemosensitizer, verapamil (4 μM). The mechanism of action of DCPs and DCXs was also resolved, since the most active compounds (3, 4, and 7) significantly increased intracellular drug accumulation due, in part, to inhibiting the P-gp mediated drug efflux from cells. We conclude that DCPs (3 and 4) and DCXs (7, 11, and 17) can exhibit polypharmacologic behavior by acting as dual inhibitors of HIV replication and chemoresistance mediated by P-gp. As such, they may be useful in combination therapy to overcome P-gp-associated drug resistance for AIDS treatment. PMID:22465634

  4. Design of PEG-grafted-PLA nanoparticles as oral permeability enhancer for P-gp substrate drug model Famotidine.

    PubMed

    Mokhtar, Mohamed; Gosselin, Patrick M; Lacasse, François; Hildgen, Patrice

    2017-02-02

    Bioavailability of oral drugs can be limited by an intestinal excretion process mediated by P-glycoprotein (P-gp). Polyethylene glycol (PEG) is a known P-gp inhibitor. Dispersion of Famotidine (a P-gp substrate) within PEGylated nanoparticles (NPs) was used to improve its oral bioavailability. In this work, we evaluated the potential impact of NPs prepared from a grafted copolymer of polylactic acid and PEG on P-gp function by studying in vitro permeability of Famotidine across Caco-2 cells. Copolymers of PEG grafted on polylactic acid a (PLA) backbone (PLA-g-PEG) were synthesized with 1 mol% and 5 mol% PEG vs. lactic acid monomer using PEG 750 and 2000 Da. The polymers were used to prepare Famotidine-loaded NPs and tested in vitro on Caco-2 cells. Significant decrease in basolateral-to-apical transport of Famotidine was observed when Famotidine was encapsulated in NPs prepared from PLA-g-PEG5%. NPs prepared from PLA-g-PEG5% are promising to improve oral bioavailability of P-gp substrates.

  5. Autoinduction of Protein Expression

    PubMed Central

    Fox, Brian G.; Blommel, Paul G.

    2017-01-01

    This unit contains protocols for the use of lactose-derived autoinduction in Escherichia coli. The protocols allow for reproducible expression trials to be undertaken with minimal user intervention. A basic protocol covers production of unlabeled proteins for functional studies. Alternate protocols for selenomethionine labeling for X-ray structural studies, and multi-well plate growth for screening and optimization are also included. PMID:19365792

  6. MiR-466b-1-3p regulates P-glycoprotein expression in rat cerebral microvascular endothelial cells.

    PubMed

    Yang, Xiaobo; Ren, Weimin; Shao, Yiye; Chen, Yinghui

    2017-04-03

    Epilepsy is one of the most common neurological disorders, and approximately one-third of epilepsy cases are resistant to treatment with anti-epileptic drug (AED). P-glycoprotein (P-gp) is a multi-drug transporter that is thought to play a pivotal role in multiple drug resistance (MDR) in epilepsy. The regulatory mechanism of P-gp remains largely unknown; however, recent studies have demonstrated that microRNAs (miRNAs) may regulate the chemo-resistance mediated by P-gp. This study investigated the effect of specific miRNAs that regulate P-gp expression in rat cerebral microvascular endothelial cells (RCMECs). Primary cultures of RCMECs were treated with phenobarbital (PB) at various concentrations to induce P-gp overexpression. MiRNA microarrays were used to investigate the expression profiles of miRNAs in the resistant RCMECs induced by PB and corresponding non-resistant cells. Our data demonstrated decreased miR-466b-1-3p expression in the resistant cells compared with the non-resistant cells. Moreover, the recombinant RNA of 466b-1-3p (mimic) and the artificial antisense RNA of miR-466b-1-3p (inhibitor) were constructed and transfected into resistant RCMECs. The expression and function of P-gp were measured by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry using rhodamine efflux. The mRNA and protein levels of P-gp increased as the concentration of PB increased, whereas miR-466b-1-3p levels decreased with increasing PB concentrations (P<0.05). The miR-466b-1-3p mimic down-regulated P-gp expression, whereas the miR-466b-1-3p inhibitor up-regulated P-gp expression (P<0.05). These findings demonstrate that miR-466b-1-3p may regulate PB-induced P-gp expression in RCMECs.

  7. Protein expression in liposomes.

    PubMed

    Oberholzer, T; Nierhaus, K H; Luisi, P L

    1999-08-02

    Compartmentalization is one of the key steps in the evolution of cellular structures and, so far, only few attempts have been made to model this kind of "compartmentalized chemistry" using liposomes. The present work shows that even such complex reactions as the ribosomal synthesis of polypeptides can be carried out in liposomes. A method is described for incorporating into 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC) liposomes the ribosomal complex together with the other components necessary for protein expression. Synthesis of poly(Phe) in the liposomes is monitored by trichloroacetic acid of the (14)C-labelled products. Control experiments carried out in the absence of one of the ribosomal subunits show by contrast no significant polypeptide expression. This methodology opens up the possibility of using liposomes as minimal cell bioreactors with growing degree of synthetic complexity, which may be relevant for the field of origin of life as well as for biotechnological applications. Copyright 1999 Academic Press.

  8. Piperine, a piperidine alkaloid from Piper nigrum re-sensitizes P-gp, MRP1 and BCRP dependent multidrug resistant cancer cells.

    PubMed

    Li, Sen; Lei, Yu; Jia, Yingjie; Li, Na; Wink, Michael; Ma, Yonggang

    2011-12-15

    Over-expression of P-gp, MRP1 and BCRP in tumor cells is one of the important mechanisms leading to multidrug resistance (MDR), which impairs the efficacy of chemotherapy. P-gp, MRP1 and BCRP are ABC (ATP-Binding Cassette) transporters, which can expel a variety of lipophilic anti-cancer drugs and protect tumor cells. During a screening of MDR reversal agents among alkaloids of various structural types, a piperidine alkaloid, piperine (a main piperidine alkaloid in Piper nigurm) was identified as an inhibitor. Piperine can potentiate the cytotoxicity of anti-cancer drugs in resistant sublines, such as MCF-7/DOX and A-549/DDP, which were derived from MCF-7 and A-549 cell lines. At a concentration of 50 μM piperine could reverse the resistance to doxorubicin 32.16 and 14.14 folds, respectively. It also re-sensitized cells to mitoxantrone 6.98 folds. In addition, long-term treatment of cells by piperine inhibits transcription of the corresponding ABC transporter genes. These results suggest that piperine can reverse MDR by multiple mechanisms and it may be a promising lead compound for future studies.

  9. Abraxane, the Nanoparticle Formulation of Paclitaxel Can Induce Drug Resistance by Up-Regulation of P-gp

    PubMed Central

    Bu, Xiangli; Ma, Huailei; Gong, He; Liu, Juan; Fang, Xiangdong; Hu, Zhiyuan; Fang, Qiaojun

    2015-01-01

    P-glycoprotein (P-gp) can actively pump paclitaxel (PTX) out of cells and induces drug resistance. Abraxane, a nanoparticle (NP) formulation of PTX, has multiple clinical advantages over the single molecule form. However, it is still unclear whether Abraxane overcomes the common small molecule drug resistance problem mediated by P-gp. Here we were able to establish an Abraxane-resistant cell line from the lung adenocarcinoma cell line A549. We compared the transcriptome of A549/Abr resistant cell line to that of its parental cell line using RNA-Seq technology. Several pathways were found to be up or down regulated. Specifically, the most significantly up-regulated gene was ABCB1, which translates into P-glycoprotein. We verified the overexpression of P-glycoprotein and confirmed its function by reversing the drug resistance with P-gp inhibitor Verapamil. The results suggest that efflux pathway plays an important role in the Abraxane-resistant cell line we established. However, the relevance of this P-gp mediated Abraxane resistance in tumors of lung cancer patients remains unknown. PMID:26182353

  10. P-glycoprotein (P-gp)-mediated efflux limits intestinal absorption of the Hsp90 inhibitor SNX-2112 in rats.

    PubMed

    Liu, Hongming; Sun, Hua; Wu, Zhufeng; Zhang, Xingwang; Wu, Baojian

    2014-08-01

    1. The promising anticancer agent SNX-2112 (a novel Hsp90 inhibitor) is poorly bioavailable after oral administration. Here, we aim to determine the role of P-glycoprotein (P-gp) in the intestinal absorption of SNX-2112. 2. We found that SNX-2112 significantly stimulated P-gp ATPase activity in in vitro ATPase assay with a small EC50 (the half-maximal effective concentration) value of 0.32 µM. 3. In the single-pass perfused rat intestine model, absorption of SNX-2112 was not favored in the small intestine with a [Formula: see text] (the wall permeability) value of 0.38-0.64. By contrast, the compound was well absorbed in the colon with a [Formula: see text] value of 1.19. The P-gp inhibitors cyclosporine and elacridar (i.e. GF120918A) markedly enhanced SNX-2112 absorption in all four intestinal segments (i.e. duodenum, jejunum, ileum and colon) and the fold change ranged from 3.1 to 14.1. Pharmacokinetic study revealed that cyclosporine increased the systemic exposure of SNX-2112 by a 2.5-fold after oral administration. 4. This is the first report that P-gp-mediated efflux is a limiting factor for intestinal absorption of SNX-2112 in rats.

  11. Inhibition of p38 mitogen-activated protein kinase signaling reduces multidrug transporter activity and anti-epileptic drug resistance in refractory epileptic rats.

    PubMed

    Shao, Yiye; Wang, Cuicui; Hong, Zhen; Chen, Yinghui

    2016-03-01

    It is widely recognized that P-glycoprotein (P-gp) mediates drug resistance in refractory epilepsy. However, the molecular mechanism underlying the up-regulation of P-gp expression remains unclear. Our previous studies have demonstrated that p38 mitogen-activated protein kinase (MAPK) regulates P-gp expression in cultured K562 cells. However, a lack of in vivo research leaves unanswered questions regarding whether p38MAPK regulates P-gp expression or drug resistance in refractory epilepsy. This in vivo study examined the effects of p38MAPK on the expression of P-gp and mdr1 in the rat brain and quantified antiepileptic drug (AED) concentrations in the hippocampal extracellular fluid. In addition, the role of p38MAPK in electrical and behavioral activity in a rat epilepsy model was studied. The results indicated that p38MAPK inhibition by SB202190 reduced P-gp expression, while increasing AED concentration in the hippocampal extracellular fluid in refractory epileptic rats. SB202190 also reduced the resistance to AEDs in drug-resistant rats and significantly reduced the severity of seizure activity. These results suggest that p38MAPK could participate in drug resistance in refractory epilepsy through the regulation of P-gp. We show that the specific inhibitor of p38MAPK could down-regulate the expression of multidrug transporter (P-glycoprotein) in blood-brain barrier, increase the concentration of antiepileptic drugs in the hippocampal extracellular fluid and reduce anti-epileptic drug resistance in refractory epileptic rats. We propose that the p38MAPK signaling pathway participates in drug resistance in refractory epilepsy through the regulation of P-glycoprotein expression. © 2015 International Society for Neurochemistry.

  12. Inhibition of P-glycoprotein, multidrug resistance-associated protein 2 and cytochrome P450 3A4 improves the oral absorption of octreotide in rats with portal hypertension.

    PubMed

    Sun, Xiao-Yu; Duan, Zhi-Jun; Liu, Zhen; Tang, Shun-Xiong; Li, Yang; He, Shou-Cheng; Wang, Qiu-Ming; Chang, Qing-Yong

    2016-12-01

    The aim of the present study was to increase the intestinal transport of octreotide (OCT) by targeting the first-pass impact to identify a potential method for decreasing portal vein pressure (PVP) using oral OCT. Thus, the bioavailability of intestinally absorbed OCT was evaluated in normal rats and rats with portal hypertension (PH) that had been administered P-glycoprotein/multidrug resistance-associated protein 2/cytochrome P450 3A4 (P-gp/MRP2/CYP3A4) inhibitors. The mRNA and protein expression levels of P-gp, MRP2 and CYP3A4 were evaluated in normal and PH rats with or without OCT and the inhibitors using RT-PCR, western blot and immunohistochemical analyses. The potential effects of the inhibitor administration on PVP were also examined. The results suggest that P-gp, MRP2 and CYP3A4 play important roles in prohibiting the enteral absorption of OCT, particularly under a PH environment. Moreover, inhibitors of P-gp, MRP2 and CYP3A4 decrease the first-pass effects of OCT and effectively reduce PVP under PH conditions. Therefore, the present results suggest P-gp, MRP2 and CYP3A4 are key factors in the intestinal absorption of OCT. The inhibition of P-gp, MRP2 and CYP3A4 can markedly decrease the first-pass effects of OCT, and their use may facilitate the use of orally administered OCT.

  13. Inhibition of P-glycoprotein, multidrug resistance-associated protein 2 and cytochrome P450 3A4 improves the oral absorption of octreotide in rats with portal hypertension

    PubMed Central

    Sun, Xiao-Yu; Duan, Zhi-Jun; Liu, Zhen; Tang, Shun-Xiong; Li, Yang; He, Shou-Cheng; Wang, Qiu-Ming; Chang, Qing-Yong

    2016-01-01

    The aim of the present study was to increase the intestinal transport of octreotide (OCT) by targeting the first-pass impact to identify a potential method for decreasing portal vein pressure (PVP) using oral OCT. Thus, the bioavailability of intestinally absorbed OCT was evaluated in normal rats and rats with portal hypertension (PH) that had been administered P-glycoprotein/multidrug resistance-associated protein 2/cytochrome P450 3A4 (P-gp/MRP2/CYP3A4) inhibitors. The mRNA and protein expression levels of P-gp, MRP2 and CYP3A4 were evaluated in normal and PH rats with or without OCT and the inhibitors using RT-PCR, western blot and immunohistochemical analyses. The potential effects of the inhibitor administration on PVP were also examined. The results suggest that P-gp, MRP2 and CYP3A4 play important roles in prohibiting the enteral absorption of OCT, particularly under a PH environment. Moreover, inhibitors of P-gp, MRP2 and CYP3A4 decrease the first-pass effects of OCT and effectively reduce PVP under PH conditions. Therefore, the present results suggest P-gp, MRP2 and CYP3A4 are key factors in the intestinal absorption of OCT. The inhibition of P-gp, MRP2 and CYP3A4 can markedly decrease the first-pass effects of OCT, and their use may facilitate the use of orally administered OCT. PMID:28105103

  14. P-glycoprotein, lung resistance-related protein and multidrug resistance-associated protein in de novo adult acute lymphoblastic leukaemia.

    PubMed

    Damiani, Daniela; Michelutti, Angela; Michieli, Mariagrazia; Masolini, Paola; Stocchi, Raffaella; Geromin, Antonella; Ermacora, Anna; Russo, Domenico; Fanin, Renato; Baccarani, Michele

    2002-03-01

    P-glycoprotein (P-gp), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP) expression, and blast cell intracellular daunorubicin accumulation (IDA) were evaluated in 95 previously untreated cases of adult acute lymphoblastic leukaemia (ALL) using flow cytometry. Forty-five out of 95 (47%) patients were P-gp positive (+), 12/66 (18%) were LRP+ and 11/66 (17%) were MRP+. Eighteen out of 66 (28%) patients showed a simultaneous multidrug resistance (MDR)-related protein expression higher than controls for more than one protein, while 24/66 (36%) cases did not overexpress any protein. Twenty-one out of 24 (87%) cases overexpressing at least one MDR-related protein had a defect in accumulating daunorubicin into their blast cells, while only 4/24 (16%) cases who did not overexpress any protein had similar features. The complete remission rates were similar in MDR-positive and -negative (-) patients but relapses within 6 months were more frequent in P-gp+ cases, and therefore the disease-free survival duration was shorter in P-gp+ than in P-gp- patients (P = 0.01). The number of MRP+ and/or LRP+ cases was too small to be able to draw any conclusion on their role in affecting or predicting therapy outcome. In conclusion, P-gp overexpression associated with a defect in daunorubicin accumulation is a frequent feature in adult ALL at onset and seems to be related to poorer therapy outcome and, consequently, a shorter disease-free survival. LRP and MRP overexpression seems to be a rare event and no conclusion can be drawn on its prognostic role.

  15. Leptospira Protein Expression During Infection

    USDA-ARS?s Scientific Manuscript database

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  16. Involvement of PtdIns(4,5)P2 in the regulatory mechanism of small intestinal P-glycoprotein expression.

    PubMed

    Kobori, Takuro; Harada, Shinichi; Nakamoto, Kazuo; Tokuyama, Shogo

    2014-02-01

    Previously, we reported that repeated oral administration of etoposide (ETP) activates the ezrin/radixin/moesin (ERM) scaffold proteins for P-glycoprotein (P-gp) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK) signaling, leading to increased ileal P-gp expression. Recent studies indicate that phosphatidyl inositol 4,5-bisphosphate [PtdIns(4,5)P2] regulates the plasma-membrane localization of certain proteins, and its synthase, the type I phosphatidyl inositol 4-phosphate 5-kinase (PI4P5K), is largely controlled by RhoA/ROCK. Here, we examined whether PtdIns(4,5)P2 and PI4P5K are involved in the increased expression of ileal P-gp following the ERM activation by ETP treatment. Male ddY mice (4-week-old) were treated with ETP (10 mg/kg/day, per os, p.o.) for 5 days. Protein-expression levels were measured by either western blot or dot blot analysis and molecular interactions were assessed using immunoprecipitation assays. ETP treatment significantly increased PI4P5K, ERM, and P-gp expression in the ileal membrane. This effect was suppressed following the coadministration of ETP with rosuvastatin (a RhoA inhibitor) or fasudil (a ROCK inhibitor). Notably, the PtdIns(4,5)P2 expression in the ileal membrane, as well as both P-gp and ERM levels coimmunoprecipitated with anti-PtdIns(4,5)P2 antibody, were increased by ETP treatment. PtdIns(4,5)P2 and PI4P5K may contribute to the increase in ileal P-gp expression observed following the ETP treatment, possibly through ERM activation via the RhoA/ROCK pathway.

  17. Interactions of Pluronic Block Copolymers on P-gp Efflux Activity: Experience With HIV-1 Protease Inhibitors

    PubMed Central

    SHAIK, NAVEED; PAN, GUOYU; ELMQUIST, WILLIAM F.

    2016-01-01

    The objective was to examine the influence of Pluronic block-copolymers on the interaction between the drug efflux transporter, P-glycoprotein and HIV-1 protease inhibitors (PIs). The ATPase assay determined the effect of various Pluronics on PI-stimulated P-gp ATPase activity. Cellular accumulation studies were conducted using MDCKII and LLC-PK1 cells transfected with human MDR1 to assess Pluronic modulation of PI efflux. Pluronic P85 inhibited both basal and nelfinavir-stimulated P-gp ATPase activity, while Pluronic F127 had no effect. In cell accumulation studies, Pluronic P85 restored the accumulation of nelfinavir in MDCKII-MDR1 cells while Pluronic F127 and F88 had no effect. Pluronic P85 increased saquinavir accumulation in wild-type and MDR1-transfected cells in both the MDCKII and LLC-PK1 cell models, suggesting inhibition of multiple transporters, including MRPs. In conclusion, this study provides evidence that a block-copolymer, Pluronic P85, effectively inhibits the interaction of P-gp with nelfinavir and saquinavir. These data indicate that effective inhibition of HIV-1 PI efflux by Pluronic P85 may influence the distribution of antiretroviral agents to sites protected by efflux mechanisms, such as the blood–brain barrier, and possibly increase the brain exposure of these drugs resulting in suppression of viral replication and reduction in the incidence of drug resistant mutants. PMID:18393290

  18. 4,5-Di-substituted benzyl-imidazol-2-substituted amines as the structure template for the design and synthesis of reversal agents against P-gp-mediated multidrug resistance breast cancer cells.

    PubMed

    Zhang, Nan; Zhang, Zhaohui; Wong, Iris L K; Wan, Shengbiao; Chow, Larry M C; Jiang, Tao

    2014-08-18

    Over-expression of P-glycoprotein (P-gp), a primary multidrug transporter which is located in plasma membranes, plays a major role in the multidrug resistance (MDR) of cytotoxic chemotherapy. Naamidines are a class of marine imidazole alkaloids isolated from Leucetta and Clathrina sponges, possessing a Y-shaped scaffold. Based on the results previously obtained from the third-generation MDR modulator ONT-093 and other modulators developed in our group, we designed and synthesized a series of novel 4,5-di-substituted benzyl-1-methyl-1H-imidazol-2-substituted amines using the Naamidine scaffold as the structure template. Subsequently, their reversing activity for Taxol resistance has been evaluated in P-gp-mediated multidrug resistance breast cancer cell line MDA435/LCC6MDR. Compounds 12c with a Y-shaped scaffold, and compound 17c which is 'X-shaped' scaffold and possesses a 4-diethylamino group at aryl ring B, turned out to be the most potent P-gp modulators. It appears that compounds 12c and 17c at 1 μM concentration can sensitize LCC6MDR cells toward Taxol by 26.4 and 24.5 folds, with an EC50 212.5 and 210.5 nM, respectively. These two compounds are about 5-6 folds more potent than verapamil (RF = 4.5). Moreover, compounds 12c and 17c did not exhibit obvious cytotoxicity in either cancer cell lines or normal mouse fibroblast cell lines. This study has demonstrated that the synthetic Naamidine analogues can be potentially employed as effective, safe modulators for the P-gp-mediated drug resistance cancer cells.

  19. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  20. Effect of phorbol 12-myristate 13-acetate on function and gene expression of P-glycoprotein in adriamycin-resistant K562/ADM cells.

    PubMed

    Li, Yuhua; Bi, Huichang; Zhong, Guoping; Huang, Ling; Li, Gelin; Xia, Yanzhe; Chen, Xiao; Huang, Min

    2013-01-01

    Multidrug resistance (MDR) is a critical issue during chemotherapy of cancers. Phorbol 12-myristate 13-acetate (PMA), a diester of phorbol, is a typical activator of protein kinase C (PKC). In the present study, we investigated the effect of PMA on MDR and P-glycoprotein (P-gp) gene expression in K562/ADM cells. 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay was used to assess adriamycin (Adr)-induced cytotoxicity towards K562/ADM cells in the absence or presence of PMA. The intracellular accumulation of Adr was measured by determining the mean fluorescence intensity. The effect of PMA on P-gp activity was investigated by rhodamine-123 accumulation and efflux experiment. Protein expression and mRNA expression of P-gp in K562/ADM cells were determined by Western blot analysis and real-time qPCR, respectively. Adr-induced cytotoxicity towards K562/ADM cells was significantly decreased by PMA at 5 μmol/l. Furthermore, intracellular Adr-associated mean fluorescence intensity was attenuated by 53.8% 1 h after exposure to PMA at 5 μmol/l compared with the control group (p < 0.05). A dose-dependent decrease of intracellular rhodamine-123 and increase of efflux activity of P-gp were also observed in K562/ADM cells incubation with PMA. In addition, P-gp mRNA and protein expression were significantly induced by PMA. Activation of PKC pathway by PMA can significantly induce expression and activity of P-gp, and thus decrease intracellular Adr level and strengthen MDR in K562/ADM cells. © 2013 S. Karger AG, Basel.

  1. Nanolipoparticles-mediated MDR1 siRNA delivery reduces doxorubicin resistance in breast cancer cells and silences MDR1 expression in xenograft model of human breast cancer

    PubMed Central

    Nourbakhsh, Mahnaz; Jaafari, Mahmoud Reza; Lage, Hermann; Abnous, Khalil; mosaffa, Fatemeh; Badiee, Ali; Behravan, Javad

    2015-01-01

    Objective(s): P-glycoprotein (P-gp) is an efflux protein, the overexpression of which has been associated with multidrug resistance in various cancers. Although siRNA delivery to reverse P-gp expression may be promising for sensitizing of tumor cells to cytotoxic drugs, the therapeutic use of siRNA requires effective carriers that can deliver siRNA intracellularly with minimal toxicity on target cells. We investigated a special class of PEGylated lipid-based nanoparticles (NP), named nanolipoparticles (NLPs), for siRNA-mediated P-gp downregulation. Materials and Methods: NLPs were prepared based on low detergent dialysis method. After characterization, we evaluated the effect of NLPs on siRNA delivery, and P-gp downregulation compared to oligofectamine™ (OFA) in vitro and in vivo. Results: Our results showed a significant decrease in P-gp expression and subsequent enhancement of chemosensitivity to doxorubicin in vitro. Although the effectiveness of NLPs for in vitro siRNA delivery compared to OFA was limited, the results of in vivo studies showed noticeable effectiveness of NLPs for systemic siRNA delivery. siRNA delivery using NLPs could downregulate MDR1 in tumor cells more than 80%, while OFA had a reverse effect on MDR1 expression in vivo. Conclusion: The results indicated that the prepared NLPs could be suitable siRNA delivery systems for tumor therapy. PMID:26019802

  2. Influence of overexpression of efflux proteins on the function and gene expression of endogenous peptide transporters in MDR-transfected MDCKII cell lines

    PubMed Central

    Wang, Zhiying; Pal, Dhananjay; Patel, Ashaben; Kwatra, Deep; Mitra, Ashim K.

    2013-01-01

    The objective of this study is to delineate whether overexpression of human efflux transporters (P-gp, MRP2, and BCRP) in transfected MDCK cells affect the functional activities, and gene and protein expression of endogenous influx peptide transporter system (PepT). Real-time PCR, immunoblotting, uptake and permeability studies of [3H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [3H]Gly-Sar accumulation was significantly lower in transfected MDCKII cell lines compared to wild-type cells. Transport efficiency of apical peptide transporters was markedly reduced to around 25%, 30%, and 40% in P-gp-, MRP2-, and BCRP-overexpressed MDCK cell lines, respectively. With ascending cell-passage, transport efficiency was enhanced. A significantly higher Gly-Sar permeability was observed across parental cell-monolayers over transfected cells at all pHs. Levels of mRNA for both canine PepT1 and PepT2 were substantially reduced when efflux transporters overexpressed but enhanced when mRNA-levels of efflux genes diminished with ascending cell-passage of transfected cells. An inverse correlation was evident between endogenous PepT and exogenous efflux transporters in transfected MDCKII cells. Results of protein expression also supported these findings. Overexpression of MDR genes can affect endogenous PepT function which might be due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes. PMID:23262422

  3. N-alkylated isatins evade P-gp mediated efflux and retain potency in MDR cancer cell lines.

    PubMed

    Vine, Kara L; Belfiore, Lisa; Jones, Luke; Locke, Julie M; Wade, Samantha; Minaei, Elahe; Ranson, Marie

    2016-01-01

    The search for novel anticancer therapeutics with the ability to overcome multi-drug resistance (MDR) mechanisms is of high priority. A class of molecules that show potential in overcoming MDR are the N-alkylated isatins. In particular 5,7-dibromo-N-alkylisatins are potent microtubule destabilizing agents that act to depolymerize microtubules, induce apoptosis and inhibit primary tumor growth in vivo. In this study we evaluated the ability of four dibrominated N-alkylisatin derivatives and the parent compound, 5,7-dibromoisatin, to circumvent MDR. All of the isatin-based compounds examined retained potency against the MDR cell lines; U937VbR and MES-SA/Dx5 and displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell lines. We show that one mechanism by which the isatin-based compounds overcome MDR is by circumventing P-glycoprotein (P-gp) mediated drug efflux. Thus, as the isatin-based compounds are not susceptible to extrusion from P-gp overexpressing tumor cells, they represent a promising alternative strategy as a stand-alone or combination therapy for treating MDR cancer.

  4. Pregnane X Receptor Not Nuclear Factor-kappa B Up-regulates P-glycoprotein Expression in the Brain of Chronic Epileptic Rats Induced by Kainic Acid.

    PubMed

    Yu, Nian; Zhang, Yan-Fang; Zhang, Kang; Cheng, Yong-Fei; Ma, Hai-Yan; Di, Qing

    2017-03-16

    Drug-resistance epilepsy (DRE) is attributed to the brain P-glycoprotein (P-gp) overexpression. We previously reported that nuclear factor-kappa B (NF-κB) played a critical role in regulating P-gp expression at the brain of the acute seizure rats. This study was extended further to investigate the interaction effect of NF-κB and pregnane X receptor (PXR) on P-gp expression at the brain of chronic epileptic rats treated with carbamazepine (CBZ). The chronic epileptic models were induced by the micro-injection of kainic acid (KA) into rats' hippocampus. Subsequently, the successful models were treated with different intervention agents of CBZ; PMA(a non-specific PXR activity inhibitor) or PDTC(a specific NF-κB activity inhibitor) respectively. The expression levels of P-gp and its encoded gene mdr1a/b were significantly up-regulated on the brain of KA-induced chronic epilepsy rats or the epilepsy rats treated with CBZ for 1 week, meanwhile with a high expression of PXR. The treatment of PMA dramatically reduced both PXR and P-gp expressions at the protein and mRNA levels in the chronic epilepsy brain. By compared to the epilepsy model group, the P-gp expression was not markedly attenuated by the inhibition of NF-κB activity with PDTC treatment, nevertheless with a decrease of NF-κB expression in this intervention group. Higher levels of proinflammatory cytokines(IL-1β, IL-6, TNF-α) were found both in the brain tissue and the serum in the epilepsy rats of each group. There was a declined trend of the pro-inflammatory cytokines expression of the PDTC treatment group but with no statistical significance. This study demonstrates for the first time that P-gp up-regulation is due to increase PXR expression in the chronic phase of epilepsy, differently from that NF-κB signaling may induce the P-gp expression in the acute seizure phase. Our results offer insights into the mechanism underlying the development of DRE using or not using CBZ treatment.

  5. Extracts of Immature Orange (Aurantii fructus immaturus) and Citrus Unshiu Peel (Citri unshiu pericarpium) Induce P-Glycoprotein and Cytochrome P450 3A4 Expression via Upregulation of Pregnane X Receptor

    PubMed Central

    Okada, Naoto; Murakami, Aki; Urushizaki, Shiori; Matsuda, Misa; Kawazoe, Kazuyoshi; Ishizawa, Keisuke

    2017-01-01

    P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4) are expressed in the intestine and are associated with drug absorption and metabolism. Pregnane X receptor (PXR) is the key molecule that regulates the expression of P-gp and CYP3A4. Given that PXR activity is regulated by a variety of compounds, it is possible that unknown PXR activators exist among known medicines. Kampo is a Japanese traditional medicine composed of various natural compounds. In particular, immature orange [Aurantii fructus immaturus (IO)] and citrus unshiu peel [Citri unshiu pericarpium (CP)] are common ingredients of kampo. A previous study reported that kampo containing IO or CP decreased the blood concentration of concomitant drugs via upregulation of CYP3A4 although the mechanism was unclear. Some flavonoids are indicated to alter P-gp and CYP3A4 activity via changes in PXR activity. Because IO and CP include various flavonoids, we speculated that the activity of P-gp and CYP3A4 in the intestine may be altered via changes in PXR activity when IO or CP is administered. We tested this hypothesis by using LS180 intestinal epithelial cells. The ethanol extract of IO contained narirutin and naringin, and that of CP contained narirutin and hesperidin. Ethanol extracts of IO and CP induced P-gp, CYP3A4, and PXR expression. The increase of P-gp and CYP3A4 expression by the IO and CP ethanol extracts was inhibited by ketoconazole, an inhibitor of PXR activation. The ethanol extract of IO and CP decreased the intracellular concentration of digoxin, a P-gp substrate, and this decrease was inhibited by cyclosporine A, a P-gp inhibitor. In contrast, CP, but not IO, stimulated the metabolism of testosterone, a CYP3A4 substrate, and this was inhibited by a CYP3A4 inhibitor. These findings indicate that the ethanol extract of IO and CP increased P-gp and CYP3A4 expression via induction of PXR protein. Moreover, this induction decreased the intracellular substrate concentration. PMID:28270768

  6. Extracts of Immature Orange (Aurantii fructus immaturus) and Citrus Unshiu Peel (Citri unshiu pericarpium) Induce P-Glycoprotein and Cytochrome P450 3A4 Expression via Upregulation of Pregnane X Receptor.

    PubMed

    Okada, Naoto; Murakami, Aki; Urushizaki, Shiori; Matsuda, Misa; Kawazoe, Kazuyoshi; Ishizawa, Keisuke

    2017-01-01

    P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4) are expressed in the intestine and are associated with drug absorption and metabolism. Pregnane X receptor (PXR) is the key molecule that regulates the expression of P-gp and CYP3A4. Given that PXR activity is regulated by a variety of compounds, it is possible that unknown PXR activators exist among known medicines. Kampo is a Japanese traditional medicine composed of various natural compounds. In particular, immature orange [Aurantii fructus immaturus (IO)] and citrus unshiu peel [Citri unshiu pericarpium (CP)] are common ingredients of kampo. A previous study reported that kampo containing IO or CP decreased the blood concentration of concomitant drugs via upregulation of CYP3A4 although the mechanism was unclear. Some flavonoids are indicated to alter P-gp and CYP3A4 activity via changes in PXR activity. Because IO and CP include various flavonoids, we speculated that the activity of P-gp and CYP3A4 in the intestine may be altered via changes in PXR activity when IO or CP is administered. We tested this hypothesis by using LS180 intestinal epithelial cells. The ethanol extract of IO contained narirutin and naringin, and that of CP contained narirutin and hesperidin. Ethanol extracts of IO and CP induced P-gp, CYP3A4, and PXR expression. The increase of P-gp and CYP3A4 expression by the IO and CP ethanol extracts was inhibited by ketoconazole, an inhibitor of PXR activation. The ethanol extract of IO and CP decreased the intracellular concentration of digoxin, a P-gp substrate, and this decrease was inhibited by cyclosporine A, a P-gp inhibitor. In contrast, CP, but not IO, stimulated the metabolism of testosterone, a CYP3A4 substrate, and this was inhibited by a CYP3A4 inhibitor. These findings indicate that the ethanol extract of IO and CP increased P-gp and CYP3A4 expression via induction of PXR protein. Moreover, this induction decreased the intracellular substrate concentration.

  7. Cyclosporine a augments P-glycoprotein expression in the regenerating rat liver.

    PubMed

    Daoudaki, Maria; Fouzas, Ioannis; Stapf, Verena; Ekmekcioglu, Cem; Imvrios, George; Andoniadis, Antonios; Demetriadou, Aphrodite; Thalhammer, Theresia

    2003-03-01

    In the liver, the multidrug resistance (MDR) protein P-glycoprotein (P-gp) is physiologically expressed at the bile canalicular membrane, where it participates in the biliary excretion of various lipophilic drugs and xenobiotics. Previous studies showed that the immunosuppressive agent cyclosporine A (CsA) modulates P-gp and exerts a hepatotrophic influence in the regenerating liver. Hepatocytes isolated from regenerating rat liver, after 2/3 partial hepatectomy (PH 2/3), were used as an in vivo experimental model of cells with high proliferating activity in order to investigate whether CsA influences cellular levels of P-gp in those cells. Male Wistar rats were treated with CsA (20 mg/kg body weight) for 4 d preoperatively and 1 d postoperatively, and regenerating hepatocytes were isolated by collagenase perfusion 12, 24 and 48 h after PH 2/3. Flow cytometry and Western blotting studies with the monoclonal antibodies C494 and C219 showed that after PH 2/3, cellular levels of P-gp were initially suppressed, 12 h after PH 2/3, by 23%, but were significantly elevated thereafter, 24 and 48 h after PH 2/3 by 28% and 73%, respectively. In CsA pretreated animals, P-gp levels were increased even in normal hepatocytes by 34%, and an additional augmentation was seen in hepatocytes from 24 and 48 h regenerating livers (60% and 56%, respectively). In summary, we demonstrate for the first time that CsA has an additive effect on the expression of P-glycoprotein during liver regeneration in the rat. Therefore, induction of P-gp might also be considered in patients receiving CsA after liver transplantation for hepatocellular carcinoma and chemotherapy as an adjuvant treatment for the prevention of tumor recurrence.

  8. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  9. Modulation of CYPs, P-gp, and PXR by Eschscholzia californica (California Poppy) and Its Alkaloids.

    PubMed

    Manda, Vamshi K; Ibrahim, Mohamed A; Dale, Olivia R; Kumarihamy, Mallika; Cutler, Stephen J; Khan, Ikhlas A; Walker, Larry A; Muhammad, Ilias; Khan, Shabana I

    2016-04-01

    Eschscholzia californica, a native US plant, is traditionally used as a sedative, analgesic, and anxiolytic herb. With the rapid rise in the use of herbal supplements together with over-the-counter and prescription drugs, the risk for potential herb-drug interactions is also increasing. Most of the clinically relevant pharmacokinetic drug interactions occur due to modulation of cytochrome P450 enzymes (CYPs), P-glycoprotein, and the pregnane X receptor by concomitantly used herbs. This study aimed to determine the effects of an EtOH extract, aqueous extract (tea), basic CHCl3 fractions, and isolated major alkaloids, namely protopine (1), escholtzine (2), allocryptopine (3), and californidine (4), of E. californica on the activity of cytochrome P450s, P-glycoprotein and the pregnane X receptor. The EtOH extract and fractions showed strong time-dependent inhibition of CYP 3A4, CYP 2C9, and CYP 2C19, and reversible inhibition of CYP 2D6. Among the alkaloids, escholtzine (2) and allocryptopine (3) exhibited time-dependent inhibition of CYP 3A4, CYP 2C9, and CYP 2C19 (IC50 shift ratio > 2), while protopine (1) and allocryptopine (3) showed reversible inhibition of CYP 2D6 enzyme. A significant activation of the pregnane X receptor (> 2-fold) was observed with the EtOH extract, basic CHCl3 fraction, and alkaloids (except protopine), which resulted into an increased expression of mRNA and the activity of CYP 3A4 and CYP 1A2. The expression of P-glycoprotein was unaffected. However, aqueous extract (tea) and its main alkaloid californidine (4) did not affect cytochrome P450s, P-glycoprotein, or the pregnane X receptor. This data suggests that EtOH extract of E. californica and its major alkaloids have a potential of causing interactions with drugs that are metabolized by cytochrome P450s, while the tea seems to be safer.

  10. [Effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expression of membrane transport proteins in K562/A02 cell xenografts].

    PubMed

    Li, Dong-Yun; Zheng, Zhi; Hou, Li; Jiang, Miao; Dong, Qing; Tian, Shao-Dan; Ma, Wei; Chen, Ju; Wang, Jing; Chen, Xin-Yi

    2010-02-01

    This study was purposed to investigate the effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expressions of P-gp, MRP, LRP in K562/A02 cell xenografts. Tumor xenograft model were established by injecting the multidrug resistant cell line K562/A02 in the axillary flank of BALB/c-nu-nu mice. CZBG-intragastric administration and doxorubicin-intraperitoneal injection in combination were given to the BALB/c-nu nude mice. The tumor xenografts were made into slice after the dissection, and the expression of P-gp, MRP, LRP in K562/A02 tumor xenografts in mice were investigated by immunohistochemical technique. The integral optical density (IOD) of P-gp, MRP, LRP in K562/A02 tumor xenografts were measured by Image Pro Plus 6.0. The results showed that as compared with the doxorubicin alone, the combination of the doxorubicin and CZBG with high, middle and low dosage could decrease IOD of P-gp, MRP in K562/A02 tumor xenografts with statistical significance (p < 0.05). The LRP expression in K562/A02 tumor xenografts was not observed in five groups. It is concluded that the combination of CZBG with doxorubicin decreases the expressions of P-gp, MRP in K562/A02 tumor xenografts of mice.

  11. B4GALT family mediates the multidrug resistance of human leukemia cells by regulating the hedgehog pathway and the expression of p-glycoprotein and multidrug resistance-associated protein 1

    PubMed Central

    Zhou, H; Ma, H; Wei, W; Ji, D; Song, X; Sun, J; Zhang, J; Jia, L

    2013-01-01

    β-1, 4-Galactosyltransferase gene (B4GALT) family consists of seven members, which encode corresponding enzymes known as type II membrane-bound glycoproteins. These enzymes catalyze the biosynthesis of different glycoconjugates and saccharide structures, and have been recognized to be involved in various diseases. In this study, we sought to determine the expressional profiles of B4GALT family in four pairs of parental and chemoresistant human leukemia cell lines and in bone marrow mononuclear cells (BMMC) of leukemia patients with multidrug resistance (MDR). The results revealed that B4GALT1 and B4GALT5 were highly expressed in four MDR cells and patients, altered levels of B4GALT1 and B4GALT5 were responsible for changed drug-resistant phenotype of HL60 and HL60/adriamycin-resistant cells. Further data showed that manipulation of these two gene expression led to increased or decreased activity of hedgehog (Hh) signaling and proportionally mutative expression of p-glycoprotein (P-gp) and MDR-associated protein 1 (MRP1) that are both known to be related to MDR. Thus, we propose that B4GALT1 and B4GALT5, two members of B4GALT gene family, are involved in the development of MDR of human leukemia cells, probably by regulating the activity of Hh signaling and the expression of P-gp and MRP1. PMID:23744354

  12. Data Mining for Expressivity of Recombinant Protein Expression

    NASA Astrophysics Data System (ADS)

    Kira, Satoshi; Isoai, Atsushi; Yamamura, Masayuki

    We analyzed the expressivity of recombinant proteins by using data mining methods. The expression technique of recombinant protein is a key step towards elucidating the functions of genes discovered through genomic sequence projects. We have studied the productive efficiency of recombinant proteins in fission yeast, Schizosaccharomyces pombe (S.pombe), by mining the expression results. We gathered 57 proteins whose expression levels were known roughly in the host. Correlation analysis, principal component analysis and decision tree analysis were applied to these expression data. Analysis featuring codon usage and amino acid composition clarified that the amino acid composition affected to the expression levels of a recombinant protein strongly than the effect of codon usage. Furthermore, analysis of amino acid composition showed that protein solubility and the metabolism cost of amino acids correlated with a protein expressivity. Codon usage was often interesting in the field of recombinant expressions. However, our analysis found the weak correlation codon features with expressivities. These results indicated that ready-made indices of codon bias were irrelevant ones for modeling the expressivities of recombinant proteins. Our data driven approach was an easy and powerful method to improve recombinant protein expression, and this approach should be concentrated attention with the huge amount of expression data accumulating through the post-genome era.

  13. In Vitro-In Vivo Extrapolation Scaling Factors for Intestinal P-Glycoprotein and Breast Cancer Resistance Protein: Part I: A Cross-Laboratory Comparison of Transporter-Protein Abundances and Relative Expression Factors in Human Intestine and Caco-2 Cells.

    PubMed

    Harwood, Matthew D; Achour, Brahim; Neuhoff, Sibylle; Russell, Matthew R; Carlson, Gordon; Warhurst, Geoffrey

    2016-03-01

    Over the last 5 years the quantification of transporter-protein absolute abundances has dramatically increased in parallel to the expanded use of in vitro-in vivo extrapolation (IVIVE) and physiologically based pharmacokinetics (PBPK)-linked models, for decision-making in pharmaceutical company drug development pipelines and regulatory submissions. Although several research groups have developed laboratory-specific proteomic workflows, it is unclear if the large range of reported variability is founded on true interindividual variability or experimental variability resulting from sample preparation or the proteomic methodology used. To assess the potential for methodological bias on end-point abundance quantification, two independent laboratories, the University of Manchester (UoM) and Bertin Pharma (BPh), employing different proteomic workflows, quantified the absolute abundances of Na/K-ATPase, P-gp, and breast cancer resistance protein (BCRP) in the same set of biologic samples from human intestinal and Caco-2 cell membranes. Across all samples, P-gp abundances were significantly correlated (P = 0.04, Rs = 0.72) with a 2.4-fold higher abundance (P = 0.001) generated at UoM compared with BPh. There was a systematically higher BCRP abundance in Caco-2 cell samples quantified by BPh compared with UoM, but not in human intestinal samples. Consequently, a similar intestinal relative expression factor (REF), derived from distal jejunum and Caco-2 monolayer samples, between laboratories was found for P-gp. However, a 2-fold higher intestinal REF was generated by UoM (2.22) versus BPh (1.11). We demonstrate that differences in absolute protein abundance are evident between laboratories and they probably result from laboratory-specific methodologies relating to peptide choice.

  14. Dose-dependent exposure and metabolism of GNE-892, a β-secretase inhibitor, in monkeys: contributions by P450, AO, and P-gp.

    PubMed

    Takahashi, Ryan; Ma, Shuguang; Yue, Qin; Kim-Kang, Heasook; Yi, Yijun; Lyssikatos, Joseph P; Regal, Kelly; Hunt, Kevin W; Kallan, Nicholas C; Siu, Michael; Hop, Cornelis E C A; Liu, Xingrong; Khojasteh, S Cyrus

    2015-06-01

    (R)-2-Amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one (GNE-892) is an orally administered inhibitor of β-secretase 1 (β-site amyloid precursor protein cleaving enzyme 1, BACE1) that was developed as an intervention therapy against Alzheimer's disease. A clinical microdosing strategy was being considered for de-risking the potential pharmacokinetic liabilities of GNE-892. We tested whether dose-proportionality was observed in cynomolgus monkey as proof-of-concept for a human microdosing study. With cryopreserved monkey hepatocytes, concentration-dependency for substrate turnover and the relative contribution of P450- versus AO-mediated metabolism were observed. Characterization of the kinetics of these metabolic pathways demonstrated differences in the affinities of P450 and AO for GNE-892, which supported the metabolic profiles that had been obtained. To test if this metabolic shift occurred in vivo, mass balance studies in monkeys were conducted at doses of 0.085 and 15 mg/kg. Plasma exposure of GNE-892 following oral administration was more than 20-fold greater than dose proportional at the high-dose. P-gp-mediated efflux was unable to explain the discrepancy. The profiles of metabolites in circulation and excreta were indicative that oxidative metabolism limited the exposure to unchanged GNE-892 at the low dose. Further, the in vivo data supported the concentration-dependent metabolic shift between P450 and AO. In conclusion, microdosing of GNE-892 was not predictive of pharmacokinetics at a more pharmacologically relevant dose due to saturable absorption and metabolism. Therefore, it is important to consider ADME liabilities and their potential concentration-dependency when deciding upon a clinical microdosing strategy.

  15. High Levels of Expression of P-glycoprotein/Multidrug Resistance Protein Result in Resistance to Vintafolide.

    PubMed

    Guertin, Amy D; O'Neil, Jennifer; Stoeck, Alexander; Reddy, Joseph A; Cristescu, Razvan; Haines, Brian B; Hinton, Marlene C; Dorton, Ryan; Bloomfield, Alicia; Nelson, Melissa; Vetzel, Marilynn; Lejnine, Serguei; Nebozhyn, Michael; Zhang, Theresa; Loboda, Andrey; Picard, Kristen L; Schmidt, Emmett V; Dussault, Isabelle; Leamon, Christopher P

    2016-08-01

    Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR.

  16. Curcumin-carboxymethyl chitosan (CNC) conjugate and CNC/LHR mixed polymeric micelles as new approaches to improve the oral absorption of P-gp substrate drugs.

    PubMed

    Ni, Jiang; Tian, Fengchun; Dahmani, Fatima Zohra; Yang, Hui; Yue, Deren; He, Shuwang; Zhou, Jianping; Yao, Jing

    2016-11-01

    The low oral bioavailability of numerous drugs has been mostly attributed to the significant effect of P-gp-mediated efflux on intestinal drug transport. Herein, we developed mixed polymeric micelles (MPMs) comprised of curcumin-carboxymethyl chitosan (CNC) conjugate, as a potential inhibitor of P-gp-mediated efflux and gastrointestinal absorption enhancer, and low-molecular-weight heparin-all-trans-retinoid acid (LHR) conjugate, as loading material, with the aim to improve the oral absorption of P-gp substrate drugs. CNC conjugate was synthesized by chemical bonding of curcumin (Cur) and carboxymethyl chitosan (CMCS) taking advantage of the inhibition of intestinal P-gp-mediated secretion by Cur and the intestinal absorption enhancement by CMCS. The chemical structure of CNC conjugate was characterized by (1)H NMR with a degree of substitution of Cur of 4.52-10.20%. More importantly, CNC conjugate markedly improved the stability of Cur in physiological pH. Cyclosporine A-loaded CNC/LHR MPMs (CsA-CNC/LHR MPMs) were prepared by dialysis method, with high drug loading 25.45% and nanoscaled particle size (∼200 nm). In situ single-pass perfusion studies in rats showed that both CsA + CNC mixture and CsA-CNC/LHR MPMs achieved significantly higher Ka and Peff than CsA suspension in the duodenum and jejunum segments (p <  0.01), which was comparable to verapamil coperfusion effect. Similarly, CsA + CNC mixture and CsA-CNC/LHR MPMs significantly increased the oral bioavailability of CsA as compared to CsA suspension. These results suggest that CNC conjugate might be considered as a promising gastrointestinal absorption enhancer, while CNC/LHR MPMs had the potential to improve the oral absorption of P-gp substrate drugs.

  17. Interaction of pyridostigmine bromide and N,N-diethyl-m-toluamide alone and in combination with P-glycoprotein expressed in Escherichia coli leaky mutant.

    PubMed

    El-Masry, Eman M; Abou-Donia, Mohamed B

    2006-05-01

    P-glycoprotein (P-gp), the most extensively studied ATP-binding transporter, functions as a biological barrier by extruding toxic substances and xenobiotics out of the cell. This study was carried out to determine the effect of N,N-diethyl-m-toluamide (DEET) and pyridostigmine bromide (PB), alone and in combination, on P-gp expression using Escherichia coli leaky mutant transformed with Mdr1 gene (pT5-7/mdr1), which codes for P-gp or lactose permease (pT5-7/lacY) as negative control. Also, daunomycin (a known P-gp sustrate) was used as a positive control and reserpine (a known P-gp inhibitor) served as a negative control. An in vitro cell-resistant assay was used to monitor the potential of test compounds to interact with P-gp. Following exposure of the cells to pyridostigmine bromide or daunomycin, P-gp conferred significant resistance against both compounds, while reserpine and DEET significantly inhibited the glycoprotein. Cells were grown in the presence of noncytotoxic concentrations of daunomycin, pyridostigmine bromide, reserpine, or DEET, and membrane fractions were examined by Western immunoblotting for expression of P-gp. Daunomycin induced P-gp expression quantitatively more than pyridostigmine bromide, while reserpine and DEET significantly inhibited P-gp expression in cells harboring mdr1. Photoaffinity labeling experiment performed with the P-gp ligand [125I]iodoarylazidoprazosin demonstrated that compounds that induced or inhibited P-gp transport activity also bound to P-gp. DEET was also found to be a potent inhibitor of P-gp-mediated ATPase activity, whereas pyridostigmine bromide increased P-gp ATPase activity. Cells expressing P-gp or lac permease were exposed to pyridostigmine bromide and DEET, alone and in combination. Noncytotoxic concentrations of DEET significantly inhibited P-gp-mediated resistance against pyridostigmine bromide, resulting in a reduction of the number of effective drug interactions with biological targets. An explanation of

  18. Novel binding interactions of the DNA fragment d(pGpG) cross-linked by the antitumor active compound tetrakis(mu-carboxylato)dirhodium(II,II).

    PubMed

    Chifotides, Helen T; Koshlap, Karl M; Pérez, Lisa M; Dunbar, Kim R

    2003-09-03

    Insight into the N7/O6 equatorial binding interactions of the antitumor active complex Rh(2)(OAc)(4)(H(2)O)(2) (OAc(-) = CH(3)CO(2)(-)) with the nucleotide 5'-GMP and the DNA fragment d(pGpG) has been obtained by one- (1D) and two-dimensional (2D) NMR spectroscopy. The lack of N7 protonation at low pH values and the significant increase in the acidity of N1-H (pK(a) approximately 5.6 as compared to 8.5 for N7 only bound platinum adducts), indicated by the pH dependence study of the H8 (1)H NMR resonance for the HT (head-to-tail) isomer of Rh(2)(OAc)(2)(5'-GMP)(2), are consistent with bidentate N7/O6 binding of the guanine. The H8 (1)H NMR resonance of the HH (head-to-head) Rh(2)(OAc)(2)(5'-GMP)(2) isomer, as well as the 5'-G and 3'-G H8 resonances of the Rh(2)(OAc)(2) [d(pGpG)] adduct exhibit pH-independent titration curves, attributable to the added effect of the 5'-phosphate group deprotonation at a pH value similar to that of the N1 site. The enhancement in the acidity of N1-H, with respect to N7 only bound metal adducts, afforded by the O6 binding of the bases to the rhodium centers, has been corroborated by monitoring the pH dependence of the purine C6 and C2 (13)C NMR resonances for Rh(2)(OAc)(2)(5'-GMP)(2) and Rh(2)(OAc)(2) [d(pGpG)]. The latter studies resulted in pK(a) values in good agreement with those derived from the pH-dependent (1)H NMR titrations of the H8 resonances. Comparison of the (13)C NMR resonances of C6 and C2 for the dirhodium adducts Rh(2)(OAc)(2)(5'-GMP)(2) and Rh(2)(OAc)(2) [d(pGpG)] with the corresponding resonances of the unbound ligands at pH 8.0, showed substantial downfield shifts of Deltadelta approximately 11.0 and 6.0 ppm, respectively. The HH arrangement of the bases in the Rh(2)(OAc)(2) [d(pGpG)] adduct is evidenced by intense H8/H8 ROE cross-peaks in the 2D ROESY NMR spectrum. The presence of the terminal 5'-phosphate group in d(pGpG) results in stabilization of one left-handed Rh(2)(OAc)(2) [d(pGpG)] HH1 L conformer, due to

  19. Amphiphilic carboxymethyl chitosan-quercetin conjugate with P-gp inhibitory properties for oral delivery of paclitaxel.

    PubMed

    Wang, Xiaoying; Chen, Yihang; Dahmani, Fatima Zohra; Yin, Lifang; Zhou, Jianping; Yao, Jing

    2014-08-01

    An amphiphilic carboxymethyl chitosan-quercetin (CQ) conjugate was designed and synthesized for oral delivery of paclitaxel (PTX) to improve its oral bioavailability by increasing its water solubility and bypassing the P-gp drug efflux pumps. CQ conjugate had low critical micelle concentration (55.14 μg/mL), and could self assemble in aqueous condition to form polymeric micelles (PMs). PTX-loaded CQ PMs displayed a particle size of 185.8 ± 4.6 nm and polydispersity index (PDI) of 0.134 ± 0.056. The drug-loading content (DL) and entrapment efficiency (EE) were 33.62 ± 1.34% and 85.63 ± 1.26%, respectively. Moreover, PTX-loaded CQ PMs displayed similar sustained-release profile in simulated gastrointestinal fluids (pH 1.2/pH 6.8) and PBS (pH 7.4). In situ intestinal absorption experiment showed that PTX-loaded CQ PMs significantly improved the effective permeability of PTX as compared to verapamil (P < 0.01). Likewise, PTX-loaded CQ PMs significantly enhanced the oral bioavailability of PTX, resulting in strong antitumor efficacy against tumor xenograft models with better safety profile as compared to Taxol(®) and Taxol(®) with verapamil. Overall, the results implicate that CQ PMs are promising vehicles for the oral delivery of water-insoluble anticancer drugs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  1. CAN a P-gp modulator assist in the control of methotrexate concentrations in the rat brain? -inhibitory effects of rhodamine 123, a specific substrate for P-gp, on methotrexate excretion from the rat brain and its optimal route of administration.

    PubMed

    Ogushi, Naofumi; Sasaki, Kazuaki; Shimoda, Minoru

    2017-02-14

    Although methotrexate (MTX) is mainly transported by reduced folate carrier, P-gp and MRP1 may also be involved in its transport. In our previous study, a potent P-gp and MRP1 modulator, Cyclosporine A, potentiated MTX concentration in rat brain. Since it is important for MTX therapy for brain tumor to clarify which transporter is dominant, we herein determined whether the specific P-gp substrate, rhodamine123 (Rho123), potentiates the transport and retention of MTX in the brain. Rho123 was injected intravenously or intrathecally into rats immediately after injection of MTX. 6 or 12 hr after the MTX injection, brains were isolated just after the sampling of cerebrospinal fluid (CSF). Blood was also collected intermittently. MTX concentrations were determined in plasma, CSF and the brain using high-performance liquid chromatography with UV detection. When MTX was intravenously injected, Rho123 didn't affect MTX concentrations in the brain. However, Rho123 resulted in significantly higher MTX concentrations in the brain at 12 hr after injection when MTX was intrathecally injected. It is suggested that Rho123 inhibits the excretion of MTX from the brain, but does not potentiate its distribution from the blood into the brain. This reveals that P-gp can be one of the major transporters of MTX in rat brain. Therefore, treatments with P-gp modulators may contribute to intrathecal MTX therapy for brain tumor. Since plasma concentration-time curves of MTX were not affected by Rho123, treatments with P-gp modulators may not potentiate the adverse effects of MTX.

  2. CAN a P-gp modulator assist in the control of methotrexate concentrations in the rat brain? −inhibitory effects of rhodamine 123, a specific substrate for P-gp, on methotrexate excretion from the rat brain and its optimal route of administration

    PubMed Central

    OGUSHI, Naofumi; SASAKI, Kazuaki; SHIMODA, Minoru

    2016-01-01

    Although methotrexate (MTX) is mainly transported by reduced folate carrier, P-gp and MRP1 may also be involved in its transport. In our previous study, a potent P-gp and MRP1 modulator, Cyclosporine A, potentiated MTX concentration in rat brain. Since it is important for MTX therapy for brain tumor to clarify which transporter is dominant, we herein determined whether the specific P-gp substrate, rhodamine123 (Rho123), potentiates the transport and retention of MTX in the brain. Rho123 was injected intravenously or intrathecally into rats immediately after injection of MTX. 6 or 12 hr after the MTX injection, brains were isolated just after the sampling of cerebrospinal fluid (CSF). Blood was also collected intermittently. MTX concentrations were determined in plasma, CSF and the brain using high-performance liquid chromatography with UV detection. When MTX was intravenously injected, Rho123 didn’t affect MTX concentrations in the brain. However, Rho123 resulted in significantly higher MTX concentrations in the brain at 12 hr after injection when MTX was intrathecally injected. It is suggested that Rho123 inhibits the excretion of MTX from the brain, but does not potentiate its distribution from the blood into the brain. This reveals that P-gp can be one of the major transporters of MTX in rat brain. Therefore, treatments with P-gp modulators may contribute to intrathecal MTX therapy for brain tumor. Since plasma concentration-time curves of MTX were not affected by Rho123, treatments with P-gp modulators may not potentiate the adverse effects of MTX. PMID:27916761

  3. Influence of overexpression of efflux proteins on the function and gene expression of endogenous peptide transporters in MDR-transfected MDCKII cell lines.

    PubMed

    Wang, Zhiying; Pal, Dhananjay; Patel, Ashaben; Kwatra, Deep; Mitra, Ashim K

    2013-01-30

    The objective of this study is to delineate whether overexpression of human efflux transporters (P-gp, MRP2, and BCRP) in transfected MDCK cells affect the functional activities, and gene and protein expression of endogenous influx peptide transporter system (PepT). Real-time PCR, immunoblotting, uptake and permeability studies of [(3)H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [(3)H]Gly-Sar accumulation was significantly lower in transfected MDCKII cell lines compared to wild-type cells. Transport efficiency of apical peptide transporters was markedly reduced to around 25%, 30%, and 40% in P-gp-, MRP2-, and BCRP-overexpressed MDCK cell lines, respectively. With ascending cell-passage, transport efficiency was enhanced. A significantly higher Gly-Sar permeability was observed across parental cell-monolayers over transfected cells at all pHs. Levels of mRNA for both canine PepT1 and PepT2 were substantially reduced when efflux transporters overexpressed but enhanced when mRNA-levels of efflux genes diminished with ascending cell-passage of transfected cells. An inverse correlation was evident between endogenous PepT and exogenous efflux transporters in transfected MDCKII cells. Results of protein expression also supported these findings. Overexpression of MDR genes can affect endogenous PepT function which might be due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Excretion of the dipeptidyl peptidase-4 inhibitor linagliptin in rats is primarily by biliary excretion and P-gp-mediated efflux.

    PubMed

    Fuchs, Holger; Runge, Frank; Held, Heinz-Dieter

    2012-04-11

    Linagliptin is a selective, competitive dipeptidyl peptidase-4 (DPP-4) inhibitor, recently approved in the USA, Japan and Europe for the treatment of type 2 diabetes. It has non-linear pharmacokinetics and, unlike other DPP-4 inhibitors, a largely non-renal excretion route. It was hypothesised that P-glycoprotein (P-gp)-mediated intestinal transport could influence linagliptin bioavailability, and might contribute to its elimination. Two studies evaluated the role of P-gp-mediated transport in the bioavailability and intestinal secretion of linagliptin in rats. In the bioavailability study, male Wistar rats received single oral doses of linagliptin, 1 or 15 mg/kg, plus either the P-gp inhibitor, zosuquidar trihydrochloride, or vehicle. For the intestinal secretion study, rats underwent bile duct cannulation, and urine, faeces, and bile were collected. At the end of the study, gut content was sampled. Inhibition of intestinal P-gp increased the bioavailability of orally administered linagliptin, indicating that this transport system plays a role in limiting the uptake of linagliptin from the intestine. This effect was dependent on linagliptin dose, and could play a role in its non-linear pharmacokinetics after oral dosing. Systemically available linagliptin was mainly excreted unchanged via bile (49% of i.v. dose), but some (12%) was also excreted directly into the gut independently of biliary excretion. Thus, direct excretion of linagliptin into the gut may be an alternative excretion route in the presence of liver and renal impairment. The primarily non-renal route of excretion is likely to be of benefit to patients with type 2 diabetes, who have a high prevalence of renal insufficiency.

  5. Protein structure protection commits gene expression patterns.

    PubMed

    Chen, Jianping; Liang, Han; Fernández, Ariel

    2008-01-01

    Gene co-expressions often determine module-defining spatial and temporal concurrences of proteins. Yet, little effort has been devoted to tracing coordinating signals for expression correlations to the three-dimensional structures of gene products. We performed a global structure-based analysis of the yeast and human proteomes and contrasted this information against their respective transcriptome organizations obtained from comprehensive microarray data. We show that protein vulnerability quantifies dosage sensitivity for metabolic adaptation phases and tissue-specific patterns of mRNA expression, determining the extent of co-expression similarity of binding partners. The role of protein intrinsic disorder in transcriptome organization is also delineated by interrelating vulnerability, disorder propensity and co-expression patterns. Extremely vulnerable human proteins are shown to be subject to severe post-transcriptional regulation of their expression through significant micro-RNA targeting, making mRNA levels poor surrogates for protein-expression levels. By contrast, in yeast the expression of extremely under-wrapped proteins is likely regulated through protein aggregation. Thus, the 85 most vulnerable proteins in yeast include the five confirmed prions, while in human, the genes encoding extremely vulnerable proteins are predicted to be targeted by microRNAs. Hence, in both vastly different organisms protein vulnerability emerges as a structure-encoded signal for post-transcriptional regulation. Vulnerability of protein structure and the concurrent need to maintain structural integrity are shown to quantify dosage sensitivity, compelling gene expression patterns across tissue types and temporal adaptation phases in a quantifiable manner. Extremely vulnerable proteins impose additional constraints on gene expression: They are subject to high levels of regulation at the post-transcriptional level.

  6. Direct in vivo evidence on the mechanism by which nanoparticles facilitate the absorption of a water insoluble, P-gp substrate.

    PubMed

    Soundararajan, Ramesh; Sasaki, Kenji; Godfrey, Lisa; Odunze, Uchechukwu; Fereira, Nancy; Schätzlein, Andreas; Uchegbu, Ijeoma

    2016-11-30

    Here we examine the mechanisms by which nanoparticles enable the oral absorption of water-insoluble, P-glycoprotein efflux pump (P-gp) substrates, without recourse to P-gp inhibitors. Both 200nm paclitaxel N-(2-phenoxyacetyl)-6-O-glycolchitosan (GCPh) nanoparticles (GCPh-PTX) and a simulated Taxol formulation, facilitate drug dissolution in biorelevant media, unlike paclitaxel nanocrystals. Verapamil (40mgkg(-1)) increased the oral absorption from low dose Taxol (6 or 10mgkg(-1)) by 100%, whereas the oral absorption from high dose Taxol (20mgkg(-1)) or low dose GCPh-PTX (6 or 10mgkg(-1)) was largely unchanged by verapamil. There was virtually no absorption from control paclitaxel nanocrystals (20mgkg(-1)). Imaging of ex-vivo rat ileum samples showed that fluorescently labelled GCPh nanoparticles are mucoadhesive and are taken up by ileum epithelial cells. GCPh nanoparticles were also found to open Caco-2 cell tight junctions. In conclusion, mucoadhesive, drug solubilising GCPh nanoparticles enable the oral absorption of paclitaxel via the saturation of the P-gp pump (by high local drug concentrations) and by particle uptake and tight junction opening mechanisms.

  7. Synergistic effect of folate-mediated targeting and verapamil-mediated P-gp inhibition with paclitaxel -polymer micelles to overcome multi-drug resistance.

    PubMed

    Wang, Feihu; Zhang, Dianrui; Zhang, Qiang; Chen, Yuxuan; Zheng, Dandan; Hao, Leilei; Duan, Cunxian; Jia, Lejiao; Liu, Guangpu; Liu, Yue

    2011-12-01

    Multidrug resistance (MDR) in tumor cells is a significant obstacle for successful cancer chemotherapy. Overexpression of drug efflux transporters such as P-glycoprotein (P-gp) is a key factor contributing to the development of tumor drug resistance. Verapamil (VRP), a P-gp inhibitor, has been reported to be able to reverse completely the resistance caused by P-gp. For optimal synergy, the drug and inhibitor combination may need to be temporally colocalized in the tumor cells. Herein, we investigated the effectiveness of simultaneous and targeted delivery of anticancer drug, paclitaxel (PTX), along with VRP, using DOMC-FA micelles to overcome tumor drug resistance. The floate-functionalized dual agent loaded micelles resulted in the similar cytotoxicity to PTX-loaded micelles/free VRP combination and co-administration of two single-agent loaded micelles, which was higher than that of PTX-loaded micelles. Enhanced therapeutic efficacy of dual agent micelles could be ascribe to increased accumulation of PTX in drug-resistant tumor cells. We suggest that the synergistic effect of folate receptor-mediated internalization and VRP-mediated overcoming MDR could be beneficial in treatment of MDR solid tumors by targeting delivery of micellar PTX into tumor cells. As a result, the difunctional micelle systems is a very promising approach to overcome tumor drug resistance. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  8. Glucagon-like peptide 2 prevents down-regulation of intestinal multidrug resistance-associated protein 2 and P-glycoprotein in endotoxemic rats.

    PubMed

    Arana, Maite Rocío; Tocchetti, Guillermo Nicolás; Zecchinati, Felipe; Londero, Ana Sofía; Dominguez, Camila; Perdomo, Virginia; Rigalli, Juan Pablo; Villanueva, Silvina Stella Maris; Mottino, Aldo Domingo

    2017-08-23

    Multidrug resistance-associated protein 2 (Mrp2, ABCC2) and P-glycoprotein (P-gp, ABCB1) constitute essential components of the intestinal biochemical barrier that prevent incorporation of food contaminants, drugs or toxic metabolites into the blood stream. Endotoxemia induced in rats by administration of bacterial lipopolysaccharide (LPS) results in elevated intestinal permeability and toxicity of xenobiotics in part associated with down-regulation of expression and activity of Mrp2 and P-gp. We evaluated the protective effect of glucagon-like peptide 2 (GLP-2), a peptide hormone with enterotrophic properties, on Mrp2 and P-gp alterations induced by single i.p. injection of LPS (5mg/kg b.wt.) to rats. Two different protocols of GLP-2 administration, namely prevention and reversion, were examined. The prevention protocol consisted of 7s.c. injections of GLP-2 (125μg/kg b.wt.) administered every 12h, starting 60h before LPS administration. The reversion protocol consisted of 2 doses of GLP-2, starting 3h after LPS injection. Intestinal samples were collected 24h after LPS administration and expression (protein and mRNA) and activity of Mrp2 were evaluated in proximal jejunum whereas those of P-gp were studied in ileum. GLP-2 completely neutralized down-regulation of expression of Mrp2 and P-gp and loss of their respective activities induced by LPS under prevention protocol. GLP-2 was also able to prevent internalization of both transporters from the apical membrane of the enterocyte to intracellular compartments, as detected by confocal microscopy. LPS induced an increase in IL-1β and oxidized glutathione tissue levels, which were also counterbalanced by GLP-2 administration. In contrast, the reversion protocol failed to attenuate Mrp2 and P-gp down-regulation induced by LPS. We conclude that GLP-2 can prevent down-regulation of intestinal expression and activity of Mrp2 and P-gp in endotoxemic rats and that IL-1β and oxidative stress constitute potential targets

  9. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  10. Investigation on modulation of human P-gp by multiple doses of Radix Astragali extract granules using fexofenadine as a phenotyping probe.

    PubMed

    Zhou, Quan; Ye, Zhen; Ruan, Zourong; Zeng, Su

    2013-04-19

    Herb-drug interactions may potentially affect drug efficacy and/or the likelihood of adverse drug reactions. Radix Astragali (RA) extract formulation is usually prescribed for long-term use for patients with immunodeficiency, diabetes, nephropathy or cardiovascular diseases. Its use in combination with P-glycoprotein (P-gp) substrates is possible in clinical practice. Currently there is little knowledge about whether concomitant use of RA extract has an influence on disposition of P-gp substrate. This study was to investigate whether continuous and multiple doses of RA extract granules had modulatory effects on human P-gp. A randomised, placebo-controlled, two-period crossover pharmacokinetic drug interaction study was conducted in healthy Chinese volunteers. Fexofenadine was used as a P-gp phenotyping probe. Fourteen volunteers received RA extract granules or placebo (4g bid) for 7 days and then received a single oral dose of 120mg fexofenadine. Fexofenadine plasma concentrations were determined by HPLC. Pharmacokinetic parameters were calculated by non-compartmental method and bioequivalence evaluation was performed. Pharamcokinetic parameters in the placebo phase were as follows: T1/2 (3.75±1.47h), Cmax (745.11±137.41μg/L), Tmax (2.25±0.47h), AUC(0-t) (3894.27±923.45μgh/L), AUC(0-∞) (3993.84±912.97μgh/L). Pharamcokinetic parameters in the RA extract phase were as follows: T1/2 (4.00±1.24h), Cmax (709.44±170.03μg/L), Tmax (2.21±0.51h), AUC(0-t) (3832.72±1077.60μgh/L), AUC(0-∞) (3983.53±1019.83μgh/L). The influence of RA extract on fexofenadine Cmax and AUC lacks statistical significance. Fexofenadine in the two phases were bioequivalent. In the placebo phase, T1/2 of fexofenadine in ABCB1 3435T mutation allele carriers was longer compared to ABCB1 3435CC carriers (4.43±1.44h vs. 2.54±0.21h, p<0.05). However, RA extract pretreatment abolished such genotype-related difference due to the lengthened T1/2 in ABCB1 3435CC carriers. There was no

  11. Temozolomide induces the production of epidermal growth factor to regulate MDR1 expression in glioblastoma cells.

    PubMed

    Munoz, Jessian L; Rodriguez-Cruz, Vivian; Greco, Steven J; Nagula, Vipul; Scotto, Kathleen W; Rameshwar, Pranela

    2014-10-01

    Glioblastoma multiforme (GBM) commonly resists the frontline chemotherapy treatment temozolomide. The multidrug resistance gene (MDR1) and its protein, P-glycoprotein (P-gp), are associated with chemoresistance. This study investigated the mechanisms underlying MDR1-mediated resistance by GBM to temozolomide. P-gp trafficking was studied by flow cytometry and Western blot analysis. MDR1 expression was analyzed by real-time PCR and reporter gene assays. AP-1 interaction with MDR1 was studied by chromatin immunoprecipitation assay. EGF production was analyzed by ELISA, EGFR signaling was determined by Western blot analysis, and in vivo response to erlotinib and/or temozolomide was studied in nude mice. During the early phase of temozolomide treatment, intracellular P-gp was trafficked to the cell membrane, followed by conformational change into active P-gp. At the later phase, gene transcription of MDR1 was induced by temozolomide-mediated production of EGF. EGF activated ERK1/2-JNK-AP-1 cofactors (c-jun and c-fos). An inhibitor of EGFR kinase (erlotinib) given to nude mice with GBM prevented temozolomide-induced resistance. The results identified an essential role for activated EGFR in the resistance of GBM to temozolomide. Temozolomide resistance occurred through a biphasic response; first, by a conformational change in P-gp into the active form and, second, by releasing EGF, which caused autocrine stimulation of GBM cells to induce MDR1. Pharmacologic inhibition of EGFR kinase blunted the ability of GBM cells to resist temozolomide. These findings may explain reports on the common occurrence of mutant EGFR (EGFRvIII) and EGFR expansion in the resistance of GBM cells.

  12. Resveratrol Increases Anti-Proliferative Activity of Bestatin Through Downregulating P-Glycoprotein Expression Via Inhibiting PI3K/Akt/mTOR Pathway in K562/ADR Cells.

    PubMed

    Wang, Li; Wang, Changyuan; Jia, Yongming; Liu, Zhihao; Shu, Xiaohong; Liu, Kexin

    2016-05-01

    Multidrug resistance (MDR) is a major obstacle in the clinical therapy of hematological malignancies. P-glycoprotein (P-gp) overexpression results in reduction of intracellular drug concentration with a consequence that the cytotoxicity of anti-tumor drugs is decreased, which leads to MDR in K562/ADR cells. In this study, we found that resveratrol enhanced the anti-proliferative activity of bestatin in K562/ADR cells. Co-treatment with resveratrol, IC50 values of bestatin in K562/ADR cells significantly decreased and activation of caspase-3 and caspase-8 increased, which indicated that resveratrol potentiated bestatin-induced apoptosis. Resveratrol increased the intracellular concentration of bestatin through inhibiting P-gp function and downregulating P-gp expression at mRNA and protein levels, which increased anti-proliferative activity of bestatin in K562/ADR cells. Resveratrol decreased the phosphorylation of Akt and mTOR but did not affect the phosphorylations of JNK or ERK1/2. These results demonstrated that resveratrol could increase the anti-proliferative activity of bestatin through downregulating P-gp expression via suppressing the PI3K/Akt/mTOR signaling pathway.

  13. Heterologous and cell free protein expression systems.

    PubMed

    Farrokhi, Naser; Hrmova, Maria; Burton, Rachel A; Fincher, Geoffrey B

    2009-01-01

    In recognition of the fact that a relatively small percentage of 'named' genes in databases have any experimental proof for their annotation, attention is shifting towards the more accurate assignment of functions to individual genes in a genome. The central objective will be to reduce our reliance on nucleotide or amino acid sequence similarities as a means to define the functions of genes and to annotate genome sequences. There are many unsolved technical difficulties associated with the purification of specific proteins from extracts of biological material, especially where the protein is present in low abundance, has multiple isoforms or is found in multiple post-translationally modified forms. The relative ease with which cDNAs can be cloned has led to the development of methods through which cDNAs from essentially any source can be expressed in a limited range of suitable host organisms, so that sufficient levels of the encoded proteins can be generated for functional analysis. Recently, these heterologous expression systems have been supplemented by more robust prokaryotic and eukaryotic cell-free protein synthesis systems. In this chapter, common host systems for heterologous expression are reviewed and the current status of cell-free expression systems will be presented. New approaches to overcoming the special problems encountered during the expression of membrane-associated proteins will also be addressed. Methodological considerations, including the characteristics of codon usage in the expressed DNA, peptide tags that facilitate subsequent purification of the expressed proteins and the role of post-translational modifications, are examined.

  14. Effect of Tacrolimus on the pharmacokinetics of bioactive lignans of Wuzhi tablet (Schisandra sphenanthera extract) and the potential roles of CYP3A and P-gp.

    PubMed

    Qin, Xiao-ling; Chen, Xiao; Zhong, Guo-ping; Fan, Xiao-mei; Wang, Ying; Xue, Xin-ping; Wang, Ying; Huang, Min; Bi, Hui-chang

    2014-04-15

    We recently reported that Wuzhi tablet (WZ), a preparation of the ethanol extract of Wuweizi (Schisandra sphenanthera), had significant effects on blood concentrations of Tacrolimus (FK506) in renal transplant recipients and rats. The active lignans in WZ are schisandrin A, schisandrin B, schisandrin C, schisandrol A, schisandrol B, schisantherin A, and schisantherin B. Until now, whether the pharmacokinetics of these lignans in WZ would be affected by FK506 remained unknown. Therefore, this study aimed to investigate whether and how FK506 affected pharmacokinetics of lignans in WZ in rats and the potential roles of CYP3A and P-gp. After a single oral co-administration of FK506 and WZ, the blood concentration of lignans in WZ was decreased by FK506; furthermore, the AUC of schisantherin A, schisandrin A, schisandrol A and schisandrol B was only 64.5%, 47.2%, 55.1% and 57.4% of that of WZ alone group, respectively. Transport study in Caco-2 cells showed that these lignans were not substrates of P-gp, suggesting decreased blood concentration of lignans by FK506 was not via P-gp pathway. Metabolism study in the human recombinant CYP 3A showed that these lignans had higher affinity to CYP3A than that of FK506, and thus had a stronger CYP3A-mediated metabolism. It was concluded that the blood concentrations of these lignans were decreased and their CYP3A-mediated metabolisms were increased in the presence of FK506 since these lignans had higher affinity to CYP3A. Copyright © 2014 Elsevier GmbH. All rights reserved.

  15. Inhibition of ALDH1A1 activity decreases expression of drug transporters and reduces chemotherapy resistance in ovarian cancer cell lines.

    PubMed

    Januchowski, Radosſaw; Wojtowicz, Karolina; Sterzyſska, Karolina; Sosiſska, Patrycja; Andrzejewska, Maſgorzata; Zawierucha, Piotr; Nowicki, Michaſ; Zabel, Maciej

    2016-09-01

    The high mortality of ovarian cancer patients results from the failure of treatment caused by the inherent or acquired chemotherapy drug resistance. It was reported that overexpression of aldehyde dehydrogenase A1 (ALDH1A1) in cancer cells can be responsible for the development of drug resistance. To add the high expression of the drug transporter proteins the ALDHA1 is considered as a molecular target in cancer therapy. Therefore, we analysed drug-resistant ovarian cancer cell lines according to ALDHA1 expression and the association with drug resistance. The expression of ALDH1A1, P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) was determined using a microarray and confirmed by Q-PCR, western blot and fluorescence analysis. ALDH1A1 activity was determined using an Aldefluor assay. The impact of all-trans retinoic acid (ATRA) and diethylaminobenzaldehyde (DEAB) on chemotherapy resistance was assessed by the MTT chemosensitivity assay. The most abundant expression of ALDH1A1 was noted in paclitaxel- and topotecan-resistant cell lines where two populations of ALDH-positive and ALDH-negative cells could be observed. Those cell lines also revealed the overexpression of P-gp and BCRP respectively, and were able to form spheres in non-adherent conditions. Pre-treatment with ATRA and DEAB reduced chemotherapy resistance in both cell lines. ATRA treatment led to downregulation of the ALDH1A1, P-gp and BCRP proteins. DEAB treatment led to downregulation of the P-gp protein and BCRP transcript and protein. Our results indicate that ALDH1A1-positive cancer cells can be responsible for drug resistance development in ovarian cancer. Developing more specific ALDH1A1 inhibitors can increase chemotherapy effectiveness in ovarian cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Human-Mouse Chimeras With Normal Expression and Function Reveal That Major Domain Swapping is Tolerated by P-glycoprotein (ABCB1)

    PubMed Central

    Pluchino, Kristen M.; Hall, Matthew D.; Moen, Janna K.; Chufan, Eduardo E.; Fetsch, Patricia A.; Shukla, Suneet; Gill, Deborah R.; Hyde, Stephen C.; Xia, Di; Ambudkar, Suresh V.; Gottesman, Michael M.

    2017-01-01

    The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-crosslinking with [125I]-Iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug-binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes. PMID:26820614

  17. Hyperammonemia enhances the function and expression of P-glycoprotein and Mrp2 at the blood-brain barrier through NF-κB.

    PubMed

    Zhang, Ji; Zhang, Mian; Sun, Binbin; Li, Ying; Xu, Ping; Liu, Can; Liu, Li; Liu, Xiaodong

    2014-12-01

    Ammonia is considered to be the main neurotoxin responsible for hepatic encephalopathy resulting from liver failure. Liver failure has been reported to alter expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2) at the blood-brain barrier (BBB). The aim of this study was to investigate whether ammonia is involved in abnormalities of expression and activity of P-gp and Mrp2 at the BBB. Hyperammonemic rats were developed by an intraperitoneal injection of ammonium acetate (NH4 Ac, 4.5 mmol/kg). Results showed that Mrp2 function markedly increased in cortex and hippocampus of rats at 6 h following NH4 Ac administration. Significant increase in function of P-gp was observed in hippocampus of rats. Meanwhile, such alterations were in line with the increase in mRNA and protein levels of P-gp and Mrp2. Significant increase in levels of nuclear amount of nuclear factor-κB (NF-κB) p65 was also observed. Primarily cultured rat brain microvessel endothelial cells (rBMECs) were used for in vitro study. Data indicated that 24 h exposure to ammonia significantly increased function and expression of P-gp and Mrp2 in rBMECs, accompanied with activation of NF-κB. Furthermore, such alterations induced by ammonia were reversed by NF-κB inhibitor. In conclusion, this study demonstrates that hyperammonemia increases the function and expression of P-gp and Mrp2 at the BBB via activating NF-κB pathway. Hyperammonemia, a proverbial main factor responsible for neurocognitive disorder and blood-brain barrier (BBB) dysfunction resulting from liver failure, could increase the expression and activity of P-glycoprotein and multidrug resistance-associated protein 2 (Mrp2) at the BBB both in vivo and in vitro. Furthermore, the NF-κB activation stimulated by hyperammonemia may be the potential mechanism underlying such abnormalities induced by hyperammonemia.

  18. S9788 modulation of P-glycoprotein- and Multidrug-related protein-mediated multidrug resistance by Servier 9788 in doxorubicin-resistant MCF7 cells.

    PubMed

    Bichat, F; Solis-Recendez, G; Poullain, M G; Poupon, M F; Khayat, D; Bastian, G

    1998-08-15

    Inherent or acquired resistance to multiple natural drugs, termed multidrug resistance (MDR), represents a major obstacle to chemotherapy. Expression of P-glycoprotein (P-gp) in MCF7mdr and MCF7R resistant cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. MCF7R, but not the MDR1 gene-transfected MCF7mdr cells, expressed multidrug-related protein (MRP) concomitantly. Efficacy of an MDR modulator, designated as Servier 9788 (S9788), was estimated by doxorubicin (Dox) sensitization, Dox incorporation, and functional rhodamine 123 assay on MCF7 cell lines. Results showed that S9788 modulates the P-gp-associated MDR of MCF7mdr cells as well as the Multidrug-related protein-associated MDR of MCF7R cells.

  19. Expression of clock proteins in developing tooth.

    PubMed

    Zheng, Li; Papagerakis, Silvana; Schnell, Santiago D; Hoogerwerf, Willemijntje A; Papagerakis, Petros

    2011-01-01

    Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. Transient Protein Expression by Agroinfiltration in Lettuce.

    PubMed

    Chen, Qiang; Dent, Matthew; Hurtado, Jonathan; Stahnke, Jake; McNulty, Alyssa; Leuzinger, Kahlin; Lai, Huafang

    2016-01-01

    Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level.

  1. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  2. Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance

    PubMed Central

    Xu, Yan; Ohms, Stephen J.; Li, Zhen; Wang, Qiao; Gong, Guangming; Hu, Yiqiao; Mao, Zhiyong; Shannon, M. Frances; Fan, Jun Y.

    2013-01-01

    Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3’-UTR of the MDR1 gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR. PMID:24303078

  3. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  4. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  5. β-Asarone promotes Temozolomide's entry into glioma cells and decreases the expression of P-glycoprotein and MDR1.

    PubMed

    Wang, Nanbu; Zhang, Qinxin; Ning, Baile; Luo, Laiyu; Fang, Yongqi

    2017-06-01

    Glioma is the most common primary brain tumor and has an undesirable prognosis due to the blood-brain barrier (BBB) and drug resistance. A thorough investigation of the changes in intracellular drug concentrations is important to observe therapeutic effects and cell resistance. P-glycoprotein (P-gp) is an essential protein of Multi-drug resistance 1 (MDR1). The over-expression of P-gp and MDR1 is associated with poor prognosis and drug-resistance in glioma. However, β-asarone can pass through the BBB easily and increase the drug concentration in the rat brain. Our aim is to study the effect of β-asarone on promoting the entry of temozolomide (TMZ) into human glioma U251 cells. The cells were divided into three groups: model group, TMZ group (300μM) and co-administration group (360μM β-asarone; 300μM TMZ). We further detected P-gp and MDR1 expression in U251 and rat glioma C6 cells in four groups: model group (U251/C6), TMZ group (U251 300μM, C6 420μM), β-asarone group (U251 360μM, C6 450μM) and co-administration group (β-asarone 360μM, TMZ 300μM for U251; β-asarone 450μM, TMZ 420μM for C6). Then, high performance liquid chromatography was used to determine the intracellular and extracellular levels of TMZ. Morphological changes in both cells were observed by the microscope. The Counting Kit-8 assay was used to measure the cell proliferation and toxicity. Cell immunohistochemistry/immunofluorescence, flowcytometry and western blot were synchronously used to examine the expression of P-gp. We also determined the levels of MDR1 mRNA by RT-PCR. The results showed that β-asarone could promote the entry of TMZ into U251 cells through the membrane. The co-administration of β-asarone and TMZ also decreased cell proliferation and the expression of P-gp and MDR1 better than single medication in U251 and C6 cells. All of the data suggest that β-asarone might contribute to treatment by promoting TMZ's entry into glioma cells, thereby contributing to anti

  6. Irradiation of rat brain reduces P-glycoprotein expression and function.

    PubMed

    Bart, J; Nagengast, W B; Coppes, R P; Wegman, T D; van der Graaf, W T A; Groen, H J M; Vaalburg, W; de Vries, E G E; Hendrikse, N H

    2007-08-06

    The blood-brain barrier (BBB) hampers delivery of several drugs including chemotherapeutics to the brain. The drug efflux pump P-glycoprotein (P-gp), expressed on brain capillary endothelial cells, is part of the BBB. P-gp expression on capillary endothelium decreases 5 days after brain irradiation, which may reduce P-gp function and increase brain levels of P-gp substrates. To elucidate whether radiation therapy reduces P-gp expression and function in the brain, right hemispheres of rats were irradiated with single doses of 2-25 Gy followed by 10 mg kg(-1) of the P-gp substrate cyclosporine A (CsA) intravenously (i.v.), with once 15 Gy followed by CsA (10, 15 or 20 mg kg(-1)), or with fractionated irradiation (4 x 5 Gy) followed by CsA (10 mg kg(-1)) 5 days later. Additionally, four groups of three rats received 25 Gy once and were killed 10, 15, 20 or 25 days later. The brains were removed and P-gp detected immunohistochemically. P-gp function was assessed by [(11)C]carvedilol uptake using quantitative autoradiography. Irradiation increased [(11)C]carvedilol uptake dose-dependently, to a maximum of 20% above non irradiated hemisphere. CsA increased [(11)C]carvedilol uptake dose-dependently in both hemispheres, but more (P<0.001) in the irradiated hemisphere. Fractionated irradiation resulted in a lost P-gp expression 10 days after start irradiation, which coincided with increased [(11)C]carvedilol uptake. P-gp expression decreased between day 15 and 20 after single dose irradiation, and increased again thereafter. Rat brain irradiation results in a temporary decreased P-gp function.

  7. Inhibition of P-glycoprotein expression and function by anti-diabetic drugs gliclazide, metformin, and pioglitazone in vitro and in situ

    PubMed Central

    Abbasi, Mehran Mesgari; Valizadeh, Hadi; Hamishehkar, Hamed; Zakeri-Milani, Parvin

    2016-01-01

    P-glycoprotein (P-gp) is a trans-membrane drug efflux pump. Several drugs are P-gp substrates. Some drugs may affect the activity of P-gp by inhibiting its function, resulting in significant drug-drug interactions (DDIs). It is critical to understand which drugs are inhibitors of P-gp so that adverse DDIs can be minimized or avoided. This study investigated the effects of gliclazide, metformin, and pioglitazone on the function and expression of P-gp. Rhodamine 123 (Rh 123) efflux assays in Caco-2 cells and western blot testing were used to study in vitro the effect of the drugs on P-gp function and expression. The in situ rat single-pass intestinal permeability model was developed to study the effect of the drugs on P-gp function. Digoxin and verapamil were used as a known substrate and inhibitor of P-gp, respectively. Digoxin levels in intestinal perfusion samples were analyzed by high-performance liquid chromatography. Intestinal effective permeability (Peff) of digoxin in the presence of 0.1, 10, and 500 μM gliclazide, 100 and 7000 μM metformin, and 50 and 300 μM pioglitazone was significantly increased relative to the digoxin treated cells (P < 0.01). P-gp expression was decreased by gliclazide, metformin and pioglitazone. Intracellular accumulation of Rh 123 by the drugs increased, but the differences were not significant relative to the control cells (P > 0.05). It was found that gliclazide, metformin, and pioglitazone inhibited P-gp efflux activity in situ and down-regulated P-gp expression in vitro. Further investigations are necessary to confirm the obtained results and to define the mechanism underlying P-gp inhibition by the drugs. PMID:27499787

  8. ESPRESSO: a system for estimating protein expression and solubility in protein expression systems.

    PubMed

    Hirose, Shuichi; Noguchi, Tamotsu

    2013-05-01

    Recombinant protein technology is essential for conducting protein science and using proteins as materials in pharmaceutical or industrial applications. Although obtaining soluble proteins is still a major experimental obstacle, knowledge about protein expression/solubility under standard conditions may increase the efficiency and reduce the cost of proteomics studies. In this study, we present a computational approach to estimate the probability of protein expression and solubility for two different protein expression systems: in vivo Escherichia coli and wheat germ cell-free, from only the sequence information. It implements two kinds of methods: a sequence/predicted structural property-based method that uses both the sequence and predicted structural features, and a sequence pattern-based method that utilizes the occurrence frequencies of sequence patterns. In the benchmark test, the proposed methods obtained F-scores of around 70%, and outperformed publicly available servers. Applying the proposed methods to genomic data revealed that proteins associated with translation or transcription have a strong tendency to be expressed as soluble proteins by the in vivo E. coli expression system. The sequence pattern-based method also has the potential to indicate a candidate region for modification, to increase protein solubility. All methods are available for free at the ESPRESSO server (http://mbs.cbrc.jp/ESPRESSO). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Mitochondrial expression and activity of P-glycoprotein under oxidative stress in outer blood-retinal barrier.

    PubMed

    Zhang, Yue-Hong; Li, Juan; Yang, Wei-Zhong; Xian, Zhuan-Hua; Feng, Qi-Ting; Ruan, Xiang-Cai

    2017-01-01

    To investigate the role of oxidative stress in regulating the functional expression of P-glycoprotein (P-gp) in mitochondria of D407 cells. D407 cells were exposed to different ranges of concentrations of H2O2. The mitochondrial location of P-gp in the cells subjected to oxidative stress was detected by confocal analysis. Expression of P-gp in isolated mitochondria was assessed by Western blot. The pump activity of P-gp was evaluated by performing the efflux study on isolated mitochondria with Rhodamine 123 (Rho-123) alone and in the presence of P-gp inhibitor (Tariquidar) using flow cytometry analysis. The cells were pretreated with 10 mmol/L N-acetylcysteine (NAC) for 30min before exposing to H2O2, and analyzed the mitochondrial extracts by Western blot and flow cytometry. P-gp was co-localized in the mitochondria by confocal laser scanning microscopy, and it was also detected in the mitochondria of D407 cells using Western blot. Exposure to increasing concentrations of H2O2 led to gradually increased expression and location of P-gp in the mitochondria of cells. Rho-123 efflux assay showed higher uptake of Rho-123 on isolated mitochondria in the presence of Tariquidar both in normal and oxidative stress state. H2O2 up-regulated P-gp in D407 cells, which could be reversed by NAC treatment. H2O2 could up-regulate the functional expression of P-gp in mitochondria of D407 cells, while antioxidants might suppress oxidative-stress-induced over-expression of functional P-gp. It is indicative that limiting the mitochondrial P-gp transport in retinal pigment epithelium cells would be to improve the effect of mitochondria-targeted antioxidant therapy in age-related macular degeneration-like retinopathy.

  10. Quantitative Assessment of the Impact of Fluorine Substitution on P-Glycoprotein (P-gp) Mediated Efflux, Permeability, Lipophilicity, and Metabolic Stability.

    PubMed

    Pettersson, Martin; Hou, Xinjun; Kuhn, Max; Wager, Travis T; Kauffman, Gregory W; Verhoest, Patrick R

    2016-06-09

    Strategic replacement of one or more hydrogen atoms with fluorine atom(s) is a common tactic to improve potency at a given target and/or to modulate parameters such as metabolic stability and pKa. Molecular weight (MW) is a key parameter in design, and incorporation of fluorine is associated with a disproportionate increase in MW considering the van der Waals radius of fluorine versus hydrogen. Herein we examine a large compound data set to understand the effect of introducing fluorine on the risk of encountering P-glycoprotein mediated efflux (as measured by MDR efflux ratio), passive permeability, lipophilicity, and metabolic stability. Statistical modeling of the MDR ER data demonstrated that an increase in MW as a result of introducing fluorine atoms does not lead to higher risk of P-gp mediated efflux. Fluorine-corrected molecular weight (MWFC), where the molecular weight of fluorine has been subtracted, was found to be a more relevant descriptor.

  11. Novel tablet formulation of amorphous candesartan cilexetil solid dispersions involving P-gp inhibition for optimal drug delivery: in vitro and in vivo evaluation.

    PubMed

    Surampalli, Gurunath; Nanjwade, Basavaraj K; Patil, P A; Chilla, Rakesh

    2016-09-01

    The aim of this study was to develop a novel tablet formulation of amorphous candesartan cilexetil (CAN) solid dispersion involving effective P-gp inhibition for optimal drug delivery by direct compression (DC) method. To accomplish DC, formulation blends were evaluated for micromeritic properties. The Carr index, Hausner ratio, flow rate and cotangent of the angle α were determined. The tablets with and without naringin prepared by DC technique were evaluated for average weight, hardness, disintegration time and friability assessments. The drug release profiles were determined to study the dissolution kinetics. In vivo pharmacokinetic studies were conducted in rabbits. Accelerated stability studies were performed for tablets at 40 ± 2 °C/75% RH ± 5% for 6 months. FTIR studies confirmed no discoloration, liquefaction and physical interaction between naringin and drug. The results indicated that tablets prepared from naringin presented a dramatic release (82%) in 30 min with a similarity factor (76.18), which is most likely due to the amorphous nature of drug and the higher micromeritic properties of blends. Our findings noticed 1.7-fold increase in oral bioavailability of tablet prepared from naringin with mean Cmax and AUC0-12 h values as 35.81 ± 0.13 μg/mL and 0.14 ± 0.09 μg h/mL, respectively. The tablets with and without naringin prepared by DC technique were physically and chemically stable under accelerated stability conditions upon storage for 6 months. These results are attractive for further development of an oral tablet formulation of CAN through P-gp inhibition using naringin, a natural flavonoid as a pharmaceutical excipient.

  12. Streamlined expressed protein ligation using split inteins.

    PubMed

    Vila-Perelló, Miquel; Liu, Zhihua; Shah, Neel H; Willis, John A; Idoyaga, Juliana; Muir, Tom W

    2013-01-09

    Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

  13. The potential predictive role of nuclear NHERF1 expression in advanced gastric cancer patients treated with epirubicin/oxaliplatin/capecitabine first line chemotherapy

    PubMed Central

    Mangia, Anita; Caldarola, Lucia; Dell'Endice, Stefania; Scarpi, Emanuela; Saragoni, Luca; Monti, Manlio; Santini, Daniele; Brunetti, Oronzo; Simone, Giovanni; Silvestris, Nicola

    2015-01-01

    Cellular resistance in advanced gastric cancer (GC) might be related to function of multidrug resistance (MDR) proteins. The adaptor protein NHERF1 (Na+/H+ exchanger regulatory factor) is an important player in cancer progression for a number of solid malignancies, even if its role to develop drug resistance remains uncertain. Herein, we aimed to analyze the potential association between NHERF1 expression and P-gp, sorcin and HIF-1α MDR-related proteins in advanced GC patients treated with epirubicin/oxaliplatin/capecitabine (EOX) chemotherapy regimen, and its relation to response. Total number of 28 untreated patients were included into the study. Expression and subcellular localization of all proteins were assessed by immunohistochemistry on formalin-fixed paraffin embedded tumor samples. We did not found significant association between NHERF1 expression and the MDR-related proteins. A trend was observed between positive cytoplasmic NHERF1 (cNHERF1) expression and negative nuclear HIF-1α (nHIF-1α) expression (68.8% versus 31.3% respectively, P = 0.054). However, cytoplasmic P-gp (cP-gp) expression was positively correlated with both cHIF-1α and sorcin expression (P = 0.011; P = 0.002, respectively). Interestingly, nuclear NHERF1 (nNHERF1) staining was statistically associated with clinical response. In detail, 66.7% of patients with high nNHERF1 expression had a disease control rate, while 84.6% of subjects with negative nuclear expression of the protein showed progressive disease (P = 0.009). Multivariate analysis confirmed a significant correlation between nNHERF1 and clinical response (OR 0.06, P = 0.019). These results suggest that nuclear NHERF1 could be related to resistance to the EOX regimen in advanced GC patients, identifying this marker as a possible independent predictive factor. PMID:26126066

  14. Cerebral uptake of mefloquine enantiomers with and without the P-gp inhibitor elacridar (GF1210918) in mice

    PubMed Central

    de Lagerie, Sylvie Barraud; Comets, Emmanuelle; Gautrand, Céline; Fernandez, Christine; Auchere, Daniel; Singlas, Eric; Mentre, France; Gimenez, François

    2004-01-01

    Mefloquine is a chiral neurotoxic antimalarial agent showing stereoselective brain uptake in humans and rats. It is a substrate and an inhibitor of the efflux protein P-glycoprotein. We investigated the stereoselective uptake and efflux of mefloquine in mice, and the consequences of the combination with an efflux protein inhibitor, elacridar (GF120918) on its brain transport. Racemic mefloquine (25 mg kg−1) was administered intraperitoneally with or without elacridar (10 mg kg−1). Six to seven mice were killed at each of 11 time-points between 30 min and 168 h after administration. Blood and brain concentrations of mefloquine enantiomers were determined using liquid chromatography. A three-compartment model with zero-order absorption from the injection site was found to best represent the pharmacokinetics of both enantiomers in blood and brain. (−)Mefloquine had a lower blood and brain apparent volume of distribution and a lower efflux clearance from the brain, resulting in a larger brain/blood ratio compared to (+)mefloquine. Elacridar did not modify blood concentrations or the elimination rate from blood for either enantiomers. However, cerebral AUCinf of both enantiomers were increased, with a stronger effect on (+)mefloquine. The efflux clearance from the brain decreased for both enantiomers, with a larger decrease for (+)mefloquine. After administration of racemic mefloquine in mice, blood and brain pharmacokinetics are stereoselective, (+)mefloquine being excreted from brain more rapidly than its antipode, showing that mefloquine is a substrate of efflux proteins and that mefloquine enantiomers undergo efflux in a stereoselective manner. Moreover, pretreatment with elacridar reduced the brain efflux clearances with a more pronounced effect on (+)mefloquine. PMID:15023856

  15. Expression and purification of membrane proteins.

    PubMed

    Kubicek, Jan; Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Labahn, Jörg

    2014-01-01

    Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended.

  16. Engineering Genes for Predictable Protein Expression

    PubMed Central

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2013-01-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering. PMID:22425659

  17. Engineering genes for predictable protein expression.

    PubMed

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  18. PARP-1 protein expression in glioblastoma multiforme

    PubMed Central

    Galia, A.; Calogero, A.E.; Condorelli, R.A.; Fraggetta, F.; La Corte, C.; Ridolfo, F.; Bosco, P.; Castiglione, R.; Salemi, M.

    2012-01-01

    One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker. PMID:22472897

  19. Tumor endothelial expression of P-glycoprotein upon microvesicular transfer of TrpC5 derived from adriamycin-resistant breast cancer cells

    SciTech Connect

    Dong, YePing; Pan, QiongXi; Jiang, Li; Chen, Zhen; Zhang, FangFang; Liu, YanJun; Xing, Hui; Shi, Mei; Li, Jiao; Li, XiYuan; Zhu, YaoDan; Chen, Yun; Bruce, Iain C.; Jin, Jian Ma, Xin

    2014-03-28

    Highlights: • TrpC5 was mainly accumulated in microvesicles of drug-resistant MCF-7/ADM cells. • Microvesicles from MCF-7/ADM transferred TrpC5 to endothelial cells. • TrpC5 inhibition reduced P-glycoprotein accumulation on tumor blood vessels in vivo. - Abstract: Treatment of carcinoma commonly fails due to chemoresistance. Studies have shown that endothelial cells acquire resistance via the tumor microenvironment. Microvesicle (MV) shedding from the cell membrane to the microenvironment plays an important role in communication between cells. The aim of the present study was to determine whether MCF-7 adriamycin-resistant cells (MCF-7/ADM) shed MVs that alter the characteristics of human microvessel endothelial cells (HMECs). MVs from tumor cells transferred a Ca{sup 2+}-permeable channel TrpC5 to HMECs, inducing the expression of P-glycoprotein (P-gp) by activation of the transcription factor NFATc3 (nuclear factor of activated T cells isoform c3). Expression of the mdr1 gene was blocked by the TrpC5-blocking antibody T5E3, and the production of P-gp in HMECs was reduced by blockade of TrpC5. Thus, we postulate that endothelial cells acquire the resistant protein upon exposure to TrpC5-containg MVs in the microenvironment, and express P-gp in the TrpC5–NFATc3 signal pathway.

  20. Regulation of Mutant p53 Protein Expression.

    PubMed

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

  1. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  2. Pien Tze Huang induces apoptosis in multidrug‑resistant U2OS/ADM cells via downregulation of Bcl‑2, survivin and P-gp and upregulation of Bax.

    PubMed

    Zhang, Yan; Wang, Qihong; Niu, Susheng; Liu, Junning; Zhang, Li

    2014-02-01

    Pien Tze Huang (PZH) is a well-known traditional Chinese formula that was first prescribed by a royal physician in the Ming Dynasty. PZH has been used to treat various types of cancers including osteosarcoma. Previous studies have shown that PZH may effectively inhibit osteosarcoma cell growth in vivo and in vitro via induction of apoptosis and inhibition of migratory and invasive abilities. However, little is known regarding the effects of PZH on osteosarcomas that are resistant to chemotherapy, which has emerged as a major clinical problem. In the present study, the cellular effects of PZH on multidrug-resistant U2OS/ADM human osteosarcoma cells were investigated. Our results showed that PZH reduced cell viability in a dose- and time-dependent manner and arrested cells in the G2/M phase of the cell cycle, suggesting that PZH inhibits the proliferation of U2OS/ADM cells. Hoechst 33258 staining and Annexin V/propidium iodide double staining revealed typical nuclear features of apoptosis, and treatment with PZH increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner. Further experiments demonstrated that apoptosis induction by PZH was accompanied by downregulation of Bcl-2 and survivin and upregulation of Bax. In addition, following treatment with PZH, intracellular Rhodamine 123 accumulation was increased and the expression of P-gp was significantly suppressed. Taken together, these results provide a possible molecular mechanism for the anticancer effect of PZH on U2OS/ADM cells and suggest that PZH may be a potent therapeutic agent for drug-resistant osteosarcoma.

  3. Trypanosoma cruzi expresses diverse repetitive protein antigens.

    PubMed Central

    Hoft, D F; Kim, K S; Otsu, K; Moser, D R; Yost, W J; Blumin, J H; Donelson, J E; Kirchhoff, L V

    1989-01-01

    We screened a Trypanosoma cruzi cDNA expression library with human and rabbit anti-T. cruzi sera and identified cDNA clones that encode polypeptides containing tandemly arranged repeats which are 6 to 34 amino acids in length. The peptide repeats encoded by these cDNAs varied markedly in sequence, copy number, and location relative to the polyadenylation site of the mRNAs from which they were derived. The repeats were specific for T. cruzi, but in each case the sizes of the corresponding mRNAs and the total number of repeat copies encoded varied considerably among different isolates of the parasite. Expression of the peptide repeats was not stage specific. One of the peptide repeats occurred in a protein with an Mr of greater than 200,000 and one was in a protein of Mr 75,000 to 105,000. The frequent occurrence and diversity of these peptide repeats suggested that they may play a role in the ability of the parasite to evade immune destruction in its invertebrate and mammalian hosts, but the primary roles of these macromolecules may be unrelated to the host-parasite relationship. Images PMID:2659529

  4. Wheat germ systems for cell-free protein expression.

    PubMed

    Harbers, Matthias

    2014-08-25

    Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.

  5. Involvement of multidrug resistance-associated protein 2 in intestinal secretion of grepafloxacin in rats.

    PubMed

    Naruhashi, Kazumasa; Tamai, Ikumi; Inoue, Natsuko; Muraoka, Hiromi; Sai, Yoshimichi; Suzuki, Nagao; Tsuji, Akira

    2002-02-01

    We investigated the contribution of multidrug resistance-associated protein 2 (MRP2) to the secretory transport of grepafloxacin and compared its functional role with that of P-glycoprotein (P-gp) by using Sprague-Dawley rats (SDRs) and Eisai hyperbilirubinemic rats (EHBRs), in which MRP2 is hereditarily defective. In intestinal tissue from SDRs mounted in Ussing chambers, the level of transport in the direction from the serosal layer to the mucosal layer was twofold greater than that in the direction from the mucosal layer to the serosal layer. This secretory transport of grepafloxacin was diminished by both probenecid, an MRP2 inhibitor, and cyclosporine, a P-gp inhibitor. In intestinal tissue from EHBRs, the secretory transport of grepafloxacin was lower than that in intestinal tissue from SDRs and was inhibited by cyclosporine but not by probenecid. The absorption of grepafloxacin from intestinal loops in SDRs was in the order of duodenum > jejunum > ileum and was increased by cyclosporine but not by probenecid. The absorption in EHBRs was not higher than that in SDRs. The intestinal secretory clearance in SDRs after intravenous administration of grepafloxacin was shown to be greater for the ileum than for the duodenum, which is in good agreement with the previously reported regional expression profile of MRP2 mRNA. The intestinal secretory clearance was lower in EHBRs than in SDRs. Accordingly, in addition to P-gp, MRP2 might play a role in the secretory transport of grepafloxacin. The function of MRP2 in facilitating grepafloxacin transport in the secretory direction is more pronounced both in vitro and in vivo, while the restriction of entry from the lumen into the cell by MRP2 seems to be negligible, compared with that by P-gp, in the case of grepafloxacin.

  6. Expression, regulation, and function of drug transporters in cervicovaginal tissues of a mouse model used for microbicide testing

    PubMed Central

    Zhou, Tian; Hu, Minlu; Pearlman, Andrew; Rohan, Lisa C.

    2016-01-01

    P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance protein 4 (MRP4) are three efflux transporters that play key roles in the pharmacokinetics of antiretroviral drugs used in the pre-exposure prophylaxis of HIV sexual transmission. In this study, we investigated the expression, regulation, and function of these transporters in cervicovaginal tissues of a mouse model. Expression and regulation were examined using real-time RT-PCR and immunohistochemical staining, in the mouse tissues harvested at estrus and diestrus stages under natural cycling or after hormone synchronization. The three transporters were expressed at moderate to high levels compared to the liver. Transporter proteins were localized in various cell types in different tissue segments. Estrous cycle and exogenous hormone treatment affected transporter mRNA and protein expression, in a tissue- and transporter-dependent manner. Depo-Provera-synchronized mice were dosed vaginally or intraperitoneally with 3H-TFV, with or without MK571 co-administration, to delineate the function of cervicovaginal Mrp4. Co-administration of MK571 significantly increased the concentration of vaginally-administered TFV in endocervix and vagina. MK571 increased the concentration of intraperitoneally-administered TFV in the cervicovaginal lavage and vagina by several fold. Overall, P-gp, Bcrp, and Mrp4 were positively expressed in mouse cervicovaginal tissues, and their expression can be regulated by the estrous cycle or by exogenous hormones. In this model, the Mrp4 transporter impacted TFV distribution in cervicovaginal tissues. PMID:27453435

  7. The Effects of Cetirizine on P-glycoprotein Expression and Function In vitro and In situ

    PubMed Central

    Mesgari Abbasi, Mehran; Valizadeh, Hadi; Hamishekar, Hamed; Mohammadnejad, Leila; Zakeri-Milani, Parvin

    2016-01-01

    Purpose: P-glycoprotein (P-gp) plays a major role in oral absorption of drugs. Induction or inhibition of P-gp by drugs contributes to variability of its transport activity and often results in clinically relevant drug-drug interactions. The purpose of this study was to investigate the effect of cetirizine, a second generation H1 antihistamine, on P-gp function and expression in vitro and in situ. Methods: The in-vitro rhodamin-123 (Rho123) efflux assay in Caco-2 cells was used to study the effect of cetirizine on P-gp function. Western blot analysis was used for surveying the effect of cetirizine on expression of P-gp in Caco-2 cells. Rat in situ single-pass intestinal permeability technique was used to calculate the intestinal permeability of a known P-gp substrate (digoxin) in the presence of cetirizine. The amounts of digoxin and cetirizine in intestinal perfusion samples were analyzed using a HPLC method. Results: The results showed significant increase in Rho123 uptake (P < 0.05) and also P-gp band intensity decrease in cetirizine-treated cells in vitro. Furthermore the intestinal permeability of digoxin was also increased significantly in the presence of cetirizine (P < 0.01). Conclusion: Therefore it is concluded that cetirizine is a P-gp inhibitor and this should be considered in co administration of cetrizine with other P-gp substrate drugs. Further investigations are required to confirm our results and to determine the mechanism underlying P-gp inhibition by cetirizine. PMID:27123426

  8. Biochemical interaction of anti-HCV telaprevir with the ABC transporters P-glycoprotein and breast cancer resistance protein.

    PubMed

    Fujita, Yuria; Noguchi, Kohji; Suzuki, Tomonori; Katayama, Kazuhiro; Sugimoto, Yoshikazu

    2013-11-06

    The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp)/ABCB1 and breast cancer resistance protein (BCRP)/ABCG2 are involved in the intestinal absorption and renal excretion of various substrate drugs. Their activities affect sub-therapeutic drug concentrations and excretion of natural transporter substrates. The new oral anti-HCV drug telaprevir has dramatically improved the efficacy of hepatitis-C virus (HCV) treatment, and recent studies have suggested a possible pharmacological interaction between telaprevir and P-gp. We studied the kinetics of in vitro interactions between telaprevir and P-gp and BCRP to understand the molecular basis of that interaction. The effect of telaprevir on P-gp- and BCRP-mediated transport was evaluated by an in vitro vesicle transporter assay using different transport substrates, and the kinetics of transporter inhibition was determined. The results showed that telaprevir could inhibit P-gp- and BCRP-mediated transport in the in vitro vesicle transport assay, with each IC50 values of ≈ 7 μmol/L and ≈ 30 μmol/L, respectively. Analyses of Lineweaver-Burk plots showed that telaprevir was likely to be a competitive inhibitor against P-gp and BCRP. Photoaffinity labeling experiments were employed to observe competitive inhibition by telaprevir using iodoarylazidoprazosin (IAAP) as a binding substrate for P-gp and BCRP. These experiments revealed that telaprevir inhibited [125I]-IAAP-binding with P-gp and BCRP. Telaprevir competitively inhibited P-gp and BCRP, and P-gp-mediated transport was more sensitive to telaprevir compared with BCRP-mediated transport. These data suggest that telaprevir represses the transporter functions of P-gp and BCRP via direct inhibition.

  9. pH dependent but not P-gp dependent bidirectional transport study of S-propranolol: the importance of passive diffusion

    PubMed Central

    Zheng, Yi; Benet, Leslie Z.; Okochi, Hideaki; Chen, Xijing

    2016-01-01

    Purpose Recent controversial publications, citing studies purporting to show that P-gp mediates the transport of propranolol, proposed that passive biological membrane transport is negligible. Based on the BDDCS, the extensively metabolized-highly permeable-highly soluble BDDCS class 1 drug, propranolol, shows a high passive permeability at concentrations unrestricted by solubility that can overwhelm any potential transporter effects. Here we reinvestigate the effects of passive diffusion and carrier-mediated transport on S-propranolol. Methods Bidirectional permeability and inhibition of efflux transport studies were carried out in MDCK, MDCK-MDR1 and Caco-2 cell lines at different concentrations. Transcellular permeability studies were conducted at different apical pHs in the rat jejunum Ussing chamber model and PAMPA system. Results S-propranolol exhibited efflux ratios lower than 1 in MDCK, MDCK-MDR1 and Caco-2 cells. No significant differences of Papp, B->A in the presence and absence of the efflux inhibitor GG918 were observed. However, an efflux ratio of 3.63 was found at apical pH 6.5 with significant decrease in Papp, A->B and increase in Papp, B->A compared to apical pH 7.4 in Caco-2 cell lines. The pH dependent permeability was confirmed in the Ussing chamber model. S-propranolol flux was unchanged during inhibition by verapamil and rifampin. Furthermore, pH dependent permeability was also observed in the PAMPA system. Conclusions S-propranolol does not exhibit active transport as proposed previously. The "false" positive efflux ratio can be explained by the pH partition theory. As expected, passive diffusion, but not active transport, plays the primary role in the permeability of the BDDCS class 1 drug propranolol. PMID:25690341

  10. pH Dependent but not P-gp Dependent Bidirectional Transport Study of S-propranolol: The Importance of Passive Diffusion.

    PubMed

    Zheng, Yi; Benet, Leslie Z; Okochi, Hideaki; Chen, Xijing

    2015-08-01

    Recent controversial publications, citing studies purporting to show that P-gp mediates the transport of propranolol, proposed that passive biological membrane transport is negligible. Based on the BDDCS, the extensively metabolized-highly permeable-highly soluble BDDCS class 1 drug, propranolol, shows a high passive permeability at concentrations unrestricted by solubility that can overwhelm any potential transporter effects. Here we reinvestigate the effects of passive diffusion and carrier-mediated transport on S-propranolol. Bidirectional permeability and inhibition of efflux transport studies were carried out in MDCK, MDCK-MDR1 and Caco-2 cell lines at different concentrations. Transcellular permeability studies were conducted at different apical pHs in the rat jejunum Ussing chamber model and PAMPA system. S-propranolol exhibited efflux ratios lower than 1 in MDCK, MDCK-MDR1 and Caco-2 cells. No significant differences of Papp, B->A in the presence and absence of the efflux inhibitor GG918 were observed. However, an efflux ratio of 3.63 was found at apical pH 6.5 with significant decrease in Papp, A->B and increase in Papp, B->A compared to apical pH 7.4 in Caco-2 cell lines. The pH dependent permeability was confirmed in the Ussing chamber model. S-propranolol flux was unchanged during inhibition by verapamil and rifampin. Furthermore, pH dependent permeability was also observed in the PAMPA system. S-propranolol does not exhibit active transport as proposed previously. The "false" positive efflux ratio can be explained by the pH partition theory. As expected, passive diffusion, but not active transport, plays the primary role in the permeability of the BDDCS class 1 drug propranolol.

  11. Co-treatment with the anti-malarial drugs mefloquine and primaquine highly sensitizes drug-resistant cancer cells by increasing P-gp inhibition.

    PubMed

    Kim, Ju-Hwa; Choi, Ae-Ran; Kim, Yong Kee; Yoon, Sungpil

    2013-11-22

    The purpose of this study was to identify conditions that will increase the sensitivity of resistant cancer cells to anti-mitotic drugs. Currently, atovaquine (ATO), chloroquine (CHL), primaquine (PRI), mefloquine (MEF), artesunate (ART), and doxycycline (DOY) are the most commonly used anti-malarial drugs. Herein, we tested whether anti-malarial drugs can sensitize drug-resistant KBV20C cancer cells. None of the six tested anti-malarial drugs was found to better sensitize the drug-resistant cells compared to the sensitive KB cells. With an exception of DOY, all other anti-malarial drugs tested could sensitize both KB and KBV20C cells to a similar extent, suggesting that anti-malarial drugs could be used for sensitive as well as resistant cancer cells. Furthermore, we examined the effects of anti-malarial drugs in combination with an antimitotic drug, vinblastine (VIN) on the sensitisation of resistant KBV20C cells. Using viability assay, microscopic observation, assessment of cleaved PARP, and Hoechst staining, we identified that two anti-malarial drugs, PRI and MEF, highly sensitized KBV20C-resistant cells to VIN treatment. Moreover, PRI- or MEF-induced sensitisation was not observed in VIN-treated sensitive KB parent cells, suggesting that the observed effect is specific to resistant cancer cells. We demonstrated that the PRI and MEF sensitisation mechanism mainly depends on the inhibition of p-glycoprotein (P-gp). Our findings may contribute to the development of anti-malarial drug-based combination therapies for patients resistant to anti-mitotic drugs.

  12. Development and Validation of an In-Cell Western for Quantifying P-Glycoprotein Expression in Human Brain Microvascular Endothelial (hCMEC/D3) Cells.

    PubMed

    McInerney, Mitchell P; Pan, Yijun; Short, Jennifer L; Nicolazzo, Joseph A

    2017-01-05

    An in-cell western (ICW) protocol detecting the relative expression of P-glycoprotein (P-gp) in human cerebro-microvascular endothelial cells (hCMEC/D3) was developed and optimized, with the intention of improving throughput relative to western blotting (WB). For validation of the ICW protocol, hCMEC/D3 cells were incubated with known P-gp upregulators (10 μM rifampicin and 5 μM SR12813) and treated with siRNA targeted against MDR1, before measuring changes in P-gp expression, using both ICW and WB in parallel. To confirm a relationship between the detected P-gp expression and function, the uptake of the P-gp substrate rhodamine-123 was assessed following SR12813 treatment. Rifampicin and SR12813 significantly upregulated P-gp expression (1.5-fold and 1.9-fold, respectively) compared to control, as assessed by the ICW protocol. WB analysis of the same treatments revealed 1.4-fold and 1.5-fold upregulations. MDR1 siRNA reduced P-gp abundance by 20% and 35% when assessed by ICW and WB, respectively. SR12813 treatment reduced rhodamine-123 uptake by 18%, indicating that the observed changes in P-gp expression by ICW were associated with comparable functional changes. The correlation of P-gp upregulation by WB, rhodamine-123 uptake, and the ICW protocol provide validation of a new ICW method as an alternative method for quantification of P-gp in hCMEC/D3 cells.

  13. Green Fluorescent Protein as a Marker for Gene Expression

    NASA Astrophysics Data System (ADS)

    Chalfie, Martin; Tu, Yuan; Euskirchen, Ghia; Ward, William W.; Prasher, Douglas C.

    1994-02-01

    A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

  14. Effects of immunosuppressive treatment on protein expression in rat kidney

    PubMed Central

    Kędzierska, Karolina; Sporniak-Tutak, Katarzyna; Sindrewicz, Krzysztof; Bober, Joanna; Domański, Leszek; Parafiniuk, Mirosław; Urasińska, Elżbieta; Ciechanowicz, Andrzej; Domański, Maciej; Smektała, Tomasz; Masiuk, Marek; Skrzypczak, Wiesław; Ożgo, Małgorzata; Kabat-Koperska, Joanna; Ciechanowski, Kazimierz

    2014-01-01

    The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents’ toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins’ synthesis. Very slight differences were observed between the group receiving cyclosporine, mycophenolate mofetil, and glucocorticoids (CMG) and the control group. In contrast, compared to the control group, animals receiving tacrolimus, mycophenolate mofetil, and glucocorticoids (TMG) exhibited higher expression of proteins responsible for renal drug metabolism and lower expression levels of cytoplasmic actin and the major urinary protein. In the TMG group, we observed higher expression of proteins responsible for drug metabolism and a decrease in the expression of respiratory chain enzymes (thioredoxin-2) and markers of distal renal tubular damage (heart fatty acid-binding protein) compared to expression in the CMG

  15. Using the BacMam Baculovirus System to Study Expression and Function of Recombinant Efflux Drug Transporters in Polarized Epithelial Cell Monolayers

    PubMed Central

    Fung, King Leung; Kapoor, Khyati; Pixley, Jessica N.; Talbert, Darrell J.; Kwit, Alexandra D.T.; Ambudkar, Suresh V.

    2016-01-01

    The ATP-binding cassette (ABC) transporter superfamily includes several membrane-bound proteins that are critical to drug pharmacokinetics and disposition. Pharmacologic evaluation of these proteins in vitro remains a challenge. In this study, human ABC transporters were expressed in polarized epithelial cell monolayers transduced using the BacMam baculovirus gene transfer system. The purpose of the study was to evaluate the efficacy of BacMam baculovirus to transduce cells grown in monolayers. In a porcine kidney cell line, LLC-PK1 cells, baculoviral transduction is successful only via the apical side of a polarized monolayer. We observed that recombinant ABC transporters were expressed on the cell surface with post-translational modification. Furthermore, sodium butyrate played a critical role in recombinant protein expression, and preincubation in the presence of tunicamycin or thapsigargin enhanced protein expression. Cells overexpressing human P-glycoprotein (P-gp) showed vectorial basolateral-to-apical transport of [3H]-paclitaxel, which could be reversed by the inhibitor tariquidar. Similarly, coexpression of human P-gp and ABCG2 in LLC-PK1 cells resulted in higher transport of mitoxantrone, which is a substrate for both transporters, than in either P-gp– or ABCG2-expressing cells alone. Taken together, our results indicate that a high level of expression of efflux transporters in a polarized cell monolayer is technically feasible with the BacMam baculovirus system PMID:26622052

  16. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  17. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P

    2015-01-13

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

  18. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  19. Strain engineering for improved expression of recombinant proteins in bacteria

    PubMed Central

    2011-01-01

    Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins. PMID:21569582

  20. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

    In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins. PMID:25230046

  1. Effects of licochalcone A on the bioavailability and pharmacokinetics of nifedipine in rats: possible role of intestinal CYP3A4 and P-gp inhibition by licochalcone A.

    PubMed

    Choi, Jin-Seok; Choi, Jun-Shik; Choi, Dong-Hyun

    2014-10-01

    The purpose of this study was to investigate the possible effects of licochalcone A (a herbal medicine) on the pharmacokinetics of nifedipine and its main metabolite, dehydronifedipine, in rats. The pharmacokinetic parameters of nifedipine and/or dehydronifedipine were determined after oral and intravenous administration of nifedipine to rats in the absence (control) and presence of licochalcone A (0.4, 2.0 and 10 mg/kg). The effect of licochalcone A on P-glycoprotein (P-gp) and cytochrome P450 (CYP) 3A4 activity was also evaluated. Nifedipine was mainly metabolized by CYP3A4. Licochalcone A inhibited CYP3A4 enzyme activity in a concentration-dependent manner with a 50% inhibition concentration (IC50 ) of 5.9 μm. In addition, licochalcone A significantly enhanced the cellular accumulation of rhodamine-123 in MCF-7/ADR cells overexpressing P-gp. The area under the plasma concentration-time curve from time 0 to infinity (AUC) and the peak plasma concentration (Cmax ) of oral nifedipine were significantly greater and higher, respectively, with licochalcone A. The metabolite (dehydronifedipine)-parent AUC ratio (MR) in the presence of licochalcone A was significantly smaller compared with the control group. The above data could be due to an inhibition of intestinal CYP3A4 and P-gp by licochalcone A. The AUCs of intravenous nifedipine were comparable without and with licochalcone A, suggesting that inhibition of hepatic CYP3A4 and P-gp was almost negligible. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Development of small-molecule P-gp inhibitors of the N-benzyl 1,4-dihydropyridine type: novel aspects in SAR and bioanalytical evaluation of multidrug resistance (MDR) reversal properties.

    PubMed

    Baumert, Christiane; Günthel, Marianne; Krawczyk, Sören; Hemmer, Marc; Wersig, Tom; Langner, Andreas; Molnár, Joséf; Lage, Hermann; Hilgeroth, Andreas

    2013-01-01

    Novel series of N-benzyl 1,4-dihydropyridines have been prepared by facile syntheses. All relevant substituents of the molecular scaffold have been varied. The resulting compounds were biologically evaluated as P-glycoprotein (P-gp) inhibitors. Substitutions of the N-benzyl residue favour biological activity beside respective 3-ester functions. Most active compounds were further evaluated as multidrug resistance (MDR) modulators to restore the cytotoxic properties of varying daunorubicin applications.

  3. BPR1K653, a Novel Aurora Kinase Inhibitor, Exhibits Potent Anti-Proliferative Activity in MDR1 (P-gp170)-Mediated Multidrug-Resistant Cancer Cells

    PubMed Central

    Cheung, Chun Hei Antonio; Lin, Wen-Hsing; Hsu, John Tsu-An; Hour, Tzyh-Chyuan; Yeh, Teng-Kuang; Ko, Shengkai; Lien, Tzu-Wen; Coumar, Mohane Selvaraj; Liu, Jin-Fen; Lai, Wen-Yang; Shiao, Hui-Yi; Lee, Tian-Ren; Hsieh, Hsing-Pang; Chang, Jang-Yang

    2011-01-01

    Background Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells. Principal Findings BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats. Conclusions and Significance BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments. PMID:21887256

  4. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.

    PubMed

    Hayashi, Kokoro; Kojima, Chojiro

    2010-11-01

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  5. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    PubMed

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  6. Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

    USDA-ARS?s Scientific Manuscript database

    The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

  7. Cell-free protein synthesis as a promising expression system for recombinant proteins.

    PubMed

    Ge, Xumeng; Xu, Jianfeng

    2012-01-01

    Cell-free protein synthesis (CFPS) has major advantages over traditional cell-based methods in the capability of high-throughput protein synthesis and special protein production. During recent decades, CFPS has become an alternative protein production platform for both fundamental and applied purposes. Using Renilla luciferase as model protein, we describe a typical process of CFPS in wheat germ extract system, including wheat germ extract preparation, expression vector construction, in vitro protein synthesis (transcription/translation), and target protein assay.

  8. Analysis of MDR genes expression and cross-resistance in eight drug resistant ovarian cancer cell lines.

    PubMed

    Januchowski, Radosław; Sterzyńska, Karolina; Zaorska, Katarzyna; Sosińska, Patrycja; Klejewski, Andrzej; Brązert, Maciej; Nowicki, Michał; Zabel, Maciej

    2016-10-18

    Multiple drug resistance (MDR) of cancer cells is the main reason of intrinsic or acquired insensitivity to chemotherapy in many cancers. In this study we used ovarian cancer model of acquired drug resistance to study development of MDR. We have developed eight drug resistant cell lines from A2780 ovarian cancer cell line: two cell lines resistant to each drug commonly used in ovarian cancer chemotherapy: cisplatin (CIS), paclitaxel (PAC), doxorubicin (DOX) and topotecan (TOP). A chemosensitivity assay - MTT was performed to assess drug cross-resistance. Quantitative real-time polymerase chain reaction and immunofluorescence were also performed to determine mRNA and protein expression of genes/proteins involved in drug resistance (P-gp, BCRP, MRP1, MRP2, MVP). Flow cytometry was used to determine the activity of drug transporters. We could observe cross-resistance between PAC- and DOX-resistant cell lines. Additionally, both PAC-resistant cell lines were cross-resistant to TOP and both TOP-resistant cell lines were cross-resistant to DOX. We observed two different mechanisms of resistance to TOP related to P-gp and BCRP expression and activity. P-gp and BCRP were also involved in DOX resistance. Expression of MRP2 was increased in CIS-resistant cell lines and increased MVP expression was observed in CIS-, PAC- and TOP-, but not in DOX-resistant cell lines. Effectiveness of TOP and DOX in second line of chemotherapy in ovarian cancer can be limited because of their cross-resistance to PAC. Moreover, cross-resistance of PAC-resistant cell line to CIS suggests that such interaction between those drugs might also be probable in clinic.

  9. Regulation of hepatic drug transporter activity and expression by organochlorine pesticides.

    PubMed

    Bucher, Simon; Le Vee, Marc; Jouan, Elodie; Fardel, Olivier

    2014-03-01

    Organochlorine (OC) pesticides constitute a major class of persistent and toxic organic pollutants, known to modulate drug-detoxifying enzymes. In the present study, OCs were demonstrated to also alter the activity and expression of human hepatic drug transporters. Activity of the sinusoidal influx transporter OCT1 (organic cation transporter 1) was thus inhibited by endosulfan, chlordane, heptachlor, lindane, and dieldrine, but not by dichlorodiphenyltrichloroethane isomers, whereas those of the canalicular efflux pumps MRP2 (multidrug resistance-associated protein 2) and BCRP (breast cancer resistance protein) were blocked by endosulfan, chlordane, heptachlor, and chlordecone; this latter OC additionally inhibited the multidrug resistance gene 1 (MDR1)/P-glycoprotein (P-gp) activity. OCs, except endosulfan, were next found to induce MDR1/P-gp and MRP2 mRNA expressions in hepatoma HepaRG cells; some of them also upregulated BCRP. By contrast, expression of sinusoidal transporters was not impaired (organic anion-transporting polypeptide (OATP) 1B1 and OATP2B1) or was downregulated (sodium taurocholate co-transporting polypeptide (NTCP) and OCT1). Such regulations of drug transporter activity and expression, depending on the respective nature of OCs and transporters, may contribute to the toxicity of OC pesticides.

  10. Cell-free expression of G-protein-coupled receptors.

    PubMed

    Orbán, Erika; Proverbio, Davide; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.

  11. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

  12. Induction of Expression and Functional Activity of P-glycoprotein Efflux Transporter by Bioactive Plant Natural Products

    PubMed Central

    Abuznait, Alaa H.; Qosa, Hisham; O’Connell, Nicholas D.; Akbarian-Tefaghi, Jessica; Sylvester, Paul W.; El Sayed, Khalid A.; Kaddoumi, Amal

    2011-01-01

    The effect of bioactive plant natural products on the expression and functional activity of P-glycoprotein (P-gp) is poorly understood. Interactions of bioactive plant-based food and dietary supplements with P-gp can cause significant alteration of pharmacokinetic properties of P-gp substrate drugs when used in combination. This can augment toxicity and/or interfere with the drug’s therapeutic outcomes. This study investigated the effects of diverse commonly used plant natural products on the expression and activity of P-gp in human adenocarcinoma cells (LS-180). These natural products included the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (cembratriene), the palm oil-derived γ-tocotrienol, the extra-virgin olive oil-derived secoiridoid oleocanthal, and the triterpene acid asiatic acid derived from Melaleuca ericifolia and abundant in several other common plant dietary supplements. Treatment with 25 μM of cembratriene, oleocanthal, γ-tocotrienol, or asiatic acid showed 2.3-3.0-fold increase in P-gp expression as demonstrated by Western blotting. These results were consistent with those obtained by quantitative analysis of fluorescent micrographs for P-gp. Accumulation studies demonstrated 31-38% decrease in rhodamine 123 intracellular levels when LS-180 cells were treated with the investigated compounds as a result of P-gp induction. Bioactive natural products can up-regulate the P-gp expression and functionality, which may induce herb/food-drug interactions when concomitantly used with P-gp substrate drugs. PMID:21851848

  13. A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

    PubMed

    Nalaskowski, Marcus M; Ehm, Patrick; Giehler, Susanne; Mayr, Georg W

    2012-09-01

    Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

  14. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  15. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    PubMed

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally.

  16. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  17. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  18. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  19. Engineering Cells to Improve Protein Expression

    PubMed Central

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J.

    2014-01-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. PMID:24704806

  20. Engineering cells to improve protein expression.

    PubMed

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J

    2014-06-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Expression of heat shock protein genes in insect stress responses

    USDA-ARS?s Scientific Manuscript database

    The heat shock proteins (HSPs) that are abundantly expressed in insects are important modulators of insect survival. Expression of HSP genes in insects is not only developmentally regulated, but also induced by various stressors in order to confer protection against such stressors. The expression o...

  2. Emerging technology of in situ cell free expression protein microarrays.

    PubMed

    Nand, Amita; Gautam, Anju; Pérez, Javier Batista; Merino, Alejandro; Zhu, Jinsong

    2012-02-01

    Recently, in situ protein microarrays have been developed for large scale analysis and high throughput studies of proteins. In situ protein microarrays produce proteins directly on the solid surface from pre-arrayed DNA or RNA. The advances in in situ protein microarrays are exemplified by the ease of cDNA cloning and cell free protein expression. These technologies can evaluate, validate and monitor protein in a cost effective manner and address the issue of a high quality protein supply to use in the array. Here we review the importance of recently employed methods: PISA (protein in situ array), DAPA (DNA array to protein array), NAPPA (nucleic acid programmable protein array) and TUSTER microarrays and the role of these methods in proteomics.

  3. Evolution, diversification, and expression of KNOX proteins in plants

    PubMed Central

    Gao, Jie; Yang, Xue; Zhao, Wei; Lang, Tiange; Samuelsson, Tore

    2015-01-01

    The KNOX (KNOTTED1-like homeobox) transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK, and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II) as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification. PMID:26557129

  4. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  5. Timosaponin A-III reverses multi-drug resistance in human chronic myelogenous leukemia K562/ADM cells via downregulation of MDR1 and MRP1 expression by inhibiting PI3K/Akt signaling pathway.

    PubMed

    Chen, Jie-Ru; Jia, Xiu-Hong; Wang, Hong; Yi, Ying-Jie; Wang, Jian-Yong; Li, You-Jie

    2016-05-01

    One of the major causes of failure in chemotherapy for patients with human chronic myelogenous leukemia (CML) is the acquisition of multidrug resistance (MDR). MDR is often associated with the overexpression of drug efflux transporters of the ATP-binding cassette (ABC) protein family. Timosaponin A-III (TAIII), a saponin isolated from the rhizome of Anemarrhena asphodeloides, has previously demonstrated the ability to suppress certain human tumor processes and the potential to be developed as an anticancer agent. Nevertheless, the ability of TAIII to reverse MDR has not yet been explored. In this study, the adriamycin (ADM) resistance reversal effect of TAIII in human CML K562/ADM cells and the underlying mechanism was investigated. The Cell Counting Kit-8 (CCK-8) assay showed that TAIII had a reversal effect on the drug resistance of K562/ADM cells. Flow cytometry assay showed increased intracellular accumulation of ADM after cells were pretreated with TAIII, and the changes in the accumulation of rhodamine-123 (Rho-123) and 5(6)-carboxyfluorescein diacetate (CFDA) dye in K562/ADM cells were determined to be similar to the changes of intracellular accumulation of ADM. After pretreatment of cells with TAIII, the decreasing expression of P-gp and MRP1 mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). Western blotting showed TAIII inhibiting P-gp and MRP1 expression depended on the PI3K/Akt signaling pathway by decreasing the activity of p-Akt. Moreover, wortmannin an inhibitor of PI3K/Akt signaling pathway has a strong inhibitory effect on the expression of p-Akt, P-gp and MRP1. Besides, the combined treatment with TAIII did not have an affect on wortmannin downregulation of p-Akt, P-gp and MRP1. Taken together, our findings demonstrate, for the first time, that TAIII induced MDR reversal through inhibition of P-gp and MRP1 expression and function with regained adriamycin sensitivity which might mainly correlate to the regulation of PI3K

  6. Tools for Co-expressing Multiple Proteins in Mammalian Cells

    PubMed Central

    Assur, Zahra; Hendrickson, Wayne A.; Mancia, Filippo

    2013-01-01

    Summary Structural and functional studies of many mammalian systems are critically dependent on abundant supplies of recombinant multi-protein complexes. Mammalian cells are often the most ideal, if not the only suitable host for such experiments. This is due to their intrinsic capability to generate functional mammalian proteins. This advantage is frequently countered by problems with yields in expression, time required to generate over-expressing lines, and elevated costs. Co-expression of multiple proteins adds another level of complexity to these experiments, as cells need to be screened and selected for expression of suitable levels of each component. Here we present an efficient fluorescence marking procedure for establishing stable cell lines that over-express two proteins in co-ordination, and we validate the method in the production of recombinant monoclonal antibody Fab fragments. This procedure may readily be expanded to systems of greater complexity, comprising more then two components. PMID:21987254

  7. Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

    PubMed

    Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

    2016-01-01

    BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

  8. Transient protein expression in three Pisum sativum (green pea) varieties.

    PubMed

    Green, Brian J; Fujiki, Masaaki; Mett, Valentina; Kaczmarczyk, Jon; Shamloul, Moneim; Musiychuk, Konstantin; Underkoffler, Susan; Yusibov, Vidadi; Mett, Vadim

    2009-02-01

    The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.

  9. [The relationship between multi-drug resistance and proportion of leukemia stem cells and expression of drug transporters in drug-resistant leukemia K562/ADM cells].

    PubMed

    Yi, Juan; Chen, Jing; Sun, Jing; Wei, Hu-Lai

    2009-07-07

    To investigate the drug resistance, proportion of leukemia stem cells (LSC) and expression of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in drug-sensitive and multidrug-resistant leukemia cell population. The multidrug-resistant leukemia K562/ADM cell and its parental K562 cell were used as the model cells. The drug sensitivity was tested with a MTT assay. Flow cytometry was employed to detect the immunophenotype of stem cells and the expression of P-gp and BCRP. The self-renewal and proliferating potential were examined with methylcellulose colony-forming unit assay. K562/ADM cells were highly resistant to adriamycin, daunorubicin and etoposide. The amount of CD34+, CD123+ and CD34+ CD38- cells in K562/ADM cells was much higher than that in K562 cells, and the proportion of CD34+ CD38- CD123+ cells (LSC) in K562/ADM cells was (5.23 +/- 0.21)% versus (1.27 + 0.17)% in K562 cells, which was 4.12-fold higher than that in K562 cells. Both P-gp and BCRP were overexpressed in K562/ADM cells relative to K562 cells, and the K562/ADM cells coexpressing P-gp and BCRP were 11.25-fold higher over K562 cells. The proportion of CD34+ CD38- CD123+ BCRP+ and CD34+ CD38- P-gp+ BCRP+ cells in K562/ADM cells were (4.13 +/- 0.40)% and (5.80 +/- 1.19)% respectively, which were 3.66- and 11.37-fold higher than the same cells in K562 cells [(1.13 +/- 0.15)% and (0.51 +/- 0.01)%]. Furthermore, drug-resistant K562/ADM cells displayed 4.17-time greater colony-forming ability over the parent K562 cells, corresponding to the proportion of LSCs in K562/ADM cells. The ABC transporters-overexpressing LSC population exists in drug-resistant leukemic K562/ADM cells relative to drug-sensitive K562 cells, and the drug-resistant LSCs may be the source of chemotherapeutic resistance of leukemia.

  10. Mobile phone radiation might alter protein expression in human skin

    PubMed Central

    Karinen, Anu; Heinävaara, Sirpa; Nylund, Reetta; Leszczynski, Dariusz

    2008-01-01

    Background Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin to RF-EMF will cause changes in protein expression in living people. Results Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were present in all 10 volunteers. This suggests that protein expression in human skin might be affected by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected proteins observed in our earlier in vitro studies. Conclusion This is the first study showing that molecular level changes might take place in human volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening approach can identify protein targets of RF-EMF in human volunteers. PMID:18267023

  11. Impact of Chronic Alcohol Ingestion on Cardiac Muscle Protein Expression

    PubMed Central

    Fogle, Rachel L.; Lynch, Christopher J.; Palopoli, Mary; Deiter, Gina; Stanley, Bruce A.; Vary, Thomas C.

    2014-01-01

    Background Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved. Methods The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT™). Following the reaction with the ICAT™ reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches. Results Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system. Conclusions Based on the changes in proteins, we speculate modulation of

  12. Exocyst Complex Protein Expression in the Human Placenta

    PubMed Central

    Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

    2014-01-01

    Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

  13. Exocyst complex protein expression in the human placenta.

    PubMed

    Gonzalez, I M; Ackerman, W E; Vandre, D D; Robinson, J M

    2014-07-01

    Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion. Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst's regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins. Copyright 2010 Elsevier Inc. All rights reserved.

  15. Improving membrane protein expression by optimizing integration efficiency.

    PubMed

    Niesen, Michiel J M; Marshall, Stephen S; Miller, Thomas F; Clemons, William M

    2017-09-16

    The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were four-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effect of double mutations, on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  16. Advantageous Solubility-Permeability Interplay When Using Amorphous Solid Dispersion (ASD) Formulation for the BCS Class IV P-gp Substrate Rifaximin: Simultaneous Increase of Both the Solubility and the Permeability.

    PubMed

    Beig, Avital; Fine-Shamir, Noa; Lindley, David; Miller, Jonathan M; Dahan, Arik

    2017-02-15

    Rifaximin is a BCS class IV (low-solubility, low-permeability) drug and also a P-gp substrate. The aims of this work were to assess the efficiency of different rifaximin amorphous solid dispersion (ASDs) formulations in achieving and maintaining supersaturation and to investigate the consequent solubility-permeability interplay. Spray-dried rifaximin ASDs were prepared with different hydrophilic polymers and their ability to achieve and maintain supersaturation was assessed. Then, rifaximin's apparent intestinal permeability was investigated as a function of increasing supersaturation both in vitro using the parallel artificial membrane permeability assay (PAMPA) and in vivo using the single-pass rat intestinal perfusion (SPIP) model. The efficiency of the different ASDs to achieve and maintain supersaturation of rifaximin was found to be highly polymer dependent, and the copovidone/HPC-SL formulation was found to be superior to the other two, allowing supersaturation of 200× that of the crystalline solubility for 20 h. In vitro, rifaximin flux was increased and the apparent permeability was constant as a function of increasing supersaturation level. In vivo, on the other hand, absorption rate coefficient (k a) was first constant as a function of increasing supersaturation, but at 250×, the crystalline solubility k a was doubled, similar to the k a in the presence of the strong P-gp inhibitor GF120918. In conclusion, a new and favorable nature of solubility-permeability interplay was revealed in this work: delivering high supersaturation level of the BCS class IV drug rifaximin via ASD, thereby saturating the drugs' P-gp-mediated efflux transport, led to the favorable unique win-win situation, where both the solubility and the permeability increased simultaneously.

  17. Interaction of Oligomeric Breast Cancer Resistant Protein (BCRP) with Adjudin: A Male Contraceptive with Anti-Cancer Activity

    PubMed Central

    Cheng, Yan Ho; Jenardhanan, Pranitha; Mathur, Premendu P.; Qian, Xiaojing; Xia, Weiliang; Silvestrini, Bruno; Cheng, Chuen Yan

    2016-01-01

    Breast cancer resistant protein (BCRP, ABCG2) is an ATP-binding cassette (ABC) transporter, which together with two other ABC efflux drug pumps, namely P-glycoprotein (P-gp, ABCB1) and multidrug resistance-related protein 1 (MRP1, ABCC1) is the most important multidrug resistance protein found in eukaryotic cells including cells in the testis. However, unlike P-gp and MRP1, which are components of the Sertoli cell blood-testis barrier (BTB), BCRP is not expressed at the BTB in rodents and human testes. Instead, BCRP is expressed by peritubular myoid cells and endothelial cells of the lymphatic vessel in the tunica propria, residing outside the BTB. As such, the testis is equipped with two levels of defense against xenobiotics or drugs, preventing these harmful substances from entering the adluminal compartment to perturb meiosis and post-meiotic spermatid development: one at the level of the BTB conferred by P-gp and MRP1 and one at the tunica propria conferred by BCRP. The presence of drug transporters at the tunica propria as well as at the Sertoli cell BTB thus poses significant obstacles in developing non-hormonal contraceptives if these drugs (e.g., adjudin) exert their effects in germ cells behind the BTB, such as in the adluminal (apical) compartment of the seminiferous epithelium. Herein, we summarize recent findings pertinent to adjudin, a non-hormonal male contraceptive, and molecular interactions of adjudin with BCRP so that this information can be helpful to devise delivery strategies to evade BCRP in the tunica propria to improve its bioavailability in the testis. PMID:25620224

  18. Protein Production for Structural Genomics Using E. coli Expression

    PubMed Central

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Li, Hui; Zhou, Min; Joachimiak, Grazyna; Babnigg, Gyorgy; Joachimiak, Andrzej

    2014-01-01

    The goal of structural biology is to reveal details of the molecular structure of proteins in order to understand their function and mechanism. X-ray crystallography and NMR are the two best methods for atomic level structure determination. However, these methods require milligram quantities of proteins. In this chapter a reproducible methodology for large-scale protein production applicable to a diverse set of proteins is described. The approach is based on protein expression in E. coli as a fusion with a cleavable affinity tag that was tested on over 20,000 proteins. Specifically, a protocol for fermentation of large quantities of native proteins in disposable culture vessels is presented. A modified protocol that allows for the production of selenium-labeled proteins in defined media is also offered. Finally, a method for the purification of His6-tagged proteins on immobilized metal affinity chromatography columns that generates high-purity material is described in detail. PMID:24590711

  19. Protein Expression Dynamics During Postnatal Mouse Brain Development

    PubMed Central

    Laeremans, Annelies; Van de Plas, Babs; Clerens, Stefan; Van den Bergh, Gert; Arckens, Lutgarde; Hu, Tjing-Tjing

    2013-01-01

    We explored differential protein expression profiles in the mouse forebrain at different stages of postnatal development, including 10-day (P10), 30-day (P30), and adult (Ad) mice, by large-scale screening of proteome maps using two-dimensional difference gel electrophoresis. Mass spectrometry analysis resulted in the identification of 251 differentially expressed proteins. Most molecular changes were observed between P10 compared to both P30 and Ad. Computational ingenuity pathway analysis (IPA) confirmed these proteins as crucial molecules in the biological function of nervous system development. Moreover, IPA revealed Semaphorin signaling in neurons and the protein ubiquitination pathway as essential canonical pathways in the mouse forebrain during postnatal development. For these main biological pathways, the transcriptional regulation of the age-dependent expression of selected proteins was validated by means of in situ hybridization. In conclusion, we suggest that proteolysis and neurite outgrowth guidance are key biological processes, particularly during early brain maturation. PMID:25157209

  20. In vitro potential modulation of baicalin and baicalein on P-glycoprotein activity and expression in Caco-2 cells and rat gut sacs.

    PubMed

    Miao, Qing; Wang, Zhiyong; Zhang, Yuanyuan; Miao, Peipei; Zhao, Yuanyuan; Zhang, Yujie; Ma, Shuangcheng

    2016-09-01

    Context Previous studies have shown that Scutellariae Radix, the dried root of Scutellaria baicalensis Georgi (Labiatae), has a certain inhibitory effect on P-glycoprotein (P-gp), but the effects of its main active constituents on P-gp are still ambiguous. Objectives In vitro studies were performed to investigate the effects of its main active constituents (baicalin and its aglycone, baicalein) on the activity and expression of P-gp in intestine using Caco-2 cells and rat gut sacs. Materials and methods In Caco-2 cell experiments, the effects of baicalin and baicalein on P-gp activity were investigated using a P-gp substrate, rhodamine 123 and non-substrate fluorescein Na, by determining their intracellular fluorescence accumulation, and their effects on P-gp expression were determined using flow cytometry. In addition, rat gut sac model was selected to investigate the effects of baicalin and baicalein on the transport of verapamil, a classical P-gp substrate. The gut sacs of male Sprague-Dawley rats were filled with 0.4 mL the test solution contained verapamil (0.2575 mg/mL) and the drugs [baicalin and baicalein, at concentrations of 1/8 IC50 (59.875, 41.5 μg/mL), 1/4 IC50 (119.75, 83 μg/mL) and 1/2 IC50 (239.5, 166 μg/mL)], and then incubated in Tyrode's solution for a period of time. After termination of the incubation, the incubated solution was processed for the subsequent detection. Results According to the results of MTT assay, the IC50 values of verapamil, baicalin and baicalein were 104, 479, 332 μg/mL, respectively. The obtained results from the two models were confirmed mutually. As a result, baicalin exhibited no obvious effect on intracellular accumulation of Rh-123, and almost had no effect on P-gp expression and verapamil transportation, while baicalein significantly increased intracellular accumulation of Rh-123 (p < 0.01), down-regulated P-gp expression (p < 0.01) and increased the transport of verapamil (p < 0

  1. Analysis of incomplete gene expression dataset through protein-protein interaction information.

    PubMed

    Massanet-Vila, Raimon; Padró, Teresa; Cardús, Anna; Badimon, Lina; Caminal, Pere; Perera, Alexandre

    2011-01-01

    This paper shows a graph based method to analyze proteomic expression data. The method allows the prediction of the expression of genes not measured by the gene expression technology based on the local connectivity properties of the measured differentially expressed gene set. The prediction of the expression jointly with the stability of this prediction as a function of the variation of the initial expressed set is computed. The method is able to correctly predict one third of the proteins with independence of variations on the selection of the initial set. The algorithm is validated through a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer (MALDI-TOF) protein expression experiment aiming the study of the protein expression patterns and post-translational modifications in human endothelial vascular cells exposed to atherosclerotic levels of Low Density Lipoproteins (LDL).

  2. Proteins and an Inflammatory Network Expressed in Colon Tumors

    PubMed Central

    Zhu, Wenhong; Fang, Changming; Gramatikoff, Kosi; Niemeyer, Christina C.; Smith, Jeffrey W.

    2011-01-01

    The adenomatous polyposis coli (APC) protein is crucial to homeostasis of normal intestinal epithelia because it suppresses the β-catenin/TCF pathway. Consequently, loss or mutation of the APC gene causes colorectal tumors in humans and mice. Here, we describe our use of Multidimensional Protein Identification Technology (MudPIT) to compare protein expression in colon tumors to that of adjacent healthy colon tissue from ApcMin/+ mice. Twenty-seven proteins were found to be up-regulated in colon tumors and twenty-five down-regulated. As an extension of the proteomic analysis, the differentially expressed proteins were used as “seeds” to search for co-expressed genes. This approach revealed a co-expression network of 45 genes that is up-regulated in colon tumors. Members of the network include the antibacterial peptide cathelicidin (CAMP), Toll-like receptors (TLRs), IL-8, and triggering receptor expressed on myeloid cells 1 (TREM1). The co-expression network is associated with innate immunity and inflammation, and there is significant concordance between its connectivity in humans versus mice (Friedman: p value = 0.0056). This study provides new insights into the proteins and networks that are likely to drive the onset and progression of colon cancer. PMID:21366352

  3. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    PubMed

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Performance benchmarking of four cell-free protein expression systems.

    PubMed

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  5. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

  6. Recombinant Brucella abortus gene expressing immunogenic protein

    SciTech Connect

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  7. Patterns of fluorescent protein expression in Scleractinian corals.

    PubMed

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  8. A Statistical Study on Oscillatory Protein Expression

    NASA Astrophysics Data System (ADS)

    Yan, Shiwei

    Motivated by the experiments on the dynamics of a common network motif, p53 and Mdm2 feedback loop, by Lahav et al. [Nat. Genet 36, 147(2004)] in individual cells and Lev Bar-or et al. [Proc. Natl. Acad. Sci. USA 97, 11250(2000)] at the population of cells, we propose a statistical signal-response model with aiming to describe the different oscillatory behaviors for the activities of p53 and Mdm2 proteins both in individual and in population of cells in a unified way. At the cellular level, the activities of p53 and Mdm2 proteins are described by a group of nonlinear dynamical equations where the damage-derived signal is assumed to have the form with abrupt transition (”on” leftrightarrow ”off”) as soon as signal strength passes forth and back across a threshold. Each cell responses to the damage with different time duration within which the oscillations persist. For the case of population of cells, the activities of p53 and Mdm2 proteins will be the population average of the individual cells, which results damped oscillations, due to the averaging over the cell population with the different response time.

  9. Expression and Purification of Mini G Proteins from Escherichia coli.

    PubMed

    Carpenter, Byron; Tate, Christopher G

    2017-04-20

    Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR-G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.

  10. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  11. Climbazole increases expression of cornified envelope proteins in primary keratinocytes.

    PubMed

    Pople, J E; Moore, A E; Talbot, D C S; Barrett, K E; Jones, D A; Lim, F L

    2014-10-01

    Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays. Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry. Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers. Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker

  12. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    PubMed Central

    2012-01-01

    Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase

  13. Improvement of Transmembrane Transport Mechanism Study of Imperatorin on P-Glycoprotein-Mediated Drug Transport.

    PubMed

    Liao, Zheng-Gen; Tang, Tao; Guan, Xue-Jing; Dong, Wei; Zhang, Jing; Zhao, Guo-Wei; Yang, Ming; Liang, Xin-Li

    2016-11-24

    P-glycoprotein (P-gp) affects the transport of many drugs; including puerarin and vincristine. Our previous study demonstrated that imperatorin increased the intestinal absorption of puerarin and vincristine by inhibiting P-gp-mediated drug efflux. However; the underlying mechanism was not known. The present study investigated the mechanism by which imperatorin promotes P-gp-mediated drug transport. We used molecular docking to predict the binding force between imperatorin and P-gp and the effect of imperatorin on P-gp activity. P-gp efflux activity and P-gp ATPase activity were measured using a rhodamine 123 (Rh-123) accumulation assay and a Pgp-Glo™ assay; respectively. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess cellular membrane fluidity in MDCK-MDR1 cells. Western blotting was used to analyze the effect of imperatorin on P-gp expression; and P-gp mRNA levels were assessed by qRT-PCR. Molecular docking results demonstrated that the binding force between imperatorin and P-gp was much weaker than the force between P-gp and verapamil (a P-gp substrate). Imperatorin activated P-gp ATPase activity; which had a role in the inhibition of P-gp activity. Imperatorin promoted Rh-123 accumulation in MDCK-MDR1 cells and decreased cellular membrane fluidity. Western blotting demonstrated that imperatorin inhibited P-gp expression; and qRT-PCR revealed that imperatorin down-regulated P-gp (MDR1) gene expression. Imperatorin decreased P-gp-mediated drug efflux by inhibiting P-gp activity and the expression of P-gp mRNA and protein. Our results suggest that imperatorin could down-regulate P-gp expression to overcome multidrug resistance in tumors.

  14. Recombinant protein expression in Escherichia coli: advances and challenges

    PubMed Central

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555

  15. Expression of rabies virus G protein in carrots (Daucus carota).

    PubMed

    Rojas-Anaya, Edith; Loza-Rubio, Elizabeth; Olivera-Flores, Maria Teresa; Gomez-Lim, Miguel

    2009-12-01

    Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

  16. Vectors for the expression of tagged proteins in Drosophila.

    PubMed

    Parker, L; Gross, S; Alphey, L

    2001-12-01

    Regulated expression systems have been extremely useful in developmental studies, allowing the expression of specific proteins in defined spatial and temporal patterns. If these proteins are fused to an appropriate molecular tag, then they can be purified or visualized without the need to raise specific antibodies. If the tag is inherently fluorescent, then the proteins can even be visualized directly, in living tissue. We have constructed a series of P element-based transformation vectors for the most widely used expression system in Drosophila, GAL4/UAS. These vectors provide a series of useful tags for antibody detection, protein purification, and/or direct visualization, together with a convenient multiple cloning site into which the cDNA of interest can be inserted.

  17. Microfluidic chips for protein differential expression profiling.

    PubMed

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  18. Variation in Protein Intake Induces Variation in Spider Silk Expression

    PubMed Central

    Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

    2012-01-01

    Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

  19. Differential Protein Expression in Congenital and Acquired Cholesteatomas

    PubMed Central

    Kim, Sung Huhn; Choi, Jae Young

    2015-01-01

    Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D) electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5–3), plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5–3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5–3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins may differ

  20. Differential protein expression in Phalaenopsis under low temperature.

    PubMed

    Yuan, Xiu-Yun; Liang, Fang; Jiang, Su-Hua; Wan, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2015-01-01

    A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

  1. Global Analysis of Protein Expression of Inner Ear Hair Cells.

    PubMed

    Hickox, Ann E; Wong, Ann C Y; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel; Yates, John R; Ryan, Allen F; Savas, Jeffrey N

    2017-02-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.

  2. Prolonged morphine administration alters protein expression in the rat myocardium

    PubMed Central

    2011-01-01

    Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. Results Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. Conclusions The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart. PMID:22129148

  3. Effects of Astragalus polysaccharides on P-glycoprotein efflux pump function and protein expression in H22 hepatoma cells in vitro

    PubMed Central

    2012-01-01

    Background Astragalus polysaccharides (APS) are active constituents of Astragalus membranaceus. They have been widely studied, especially with respect to their immunopotentiating properties, their ability to counteract the side effects of chemotherapeutic drugs, and their anticancer properties. However, the mechanism by which APS inhibit cancer and the issue of whether that mechanism involves the reversal of multidrug resistance (MDR) is not completely clear. The present paper describes an investigation of the effects of APS on P-glycoprotein function and expression in H22 hepatoma cell lines resistant to Adriamycin (H22/ADM). Methods H22/ADM cell lines were treated with different concentrations of APS and/or the most common chemotherapy drugs, such as Cyclophosphamid, Adriamycin, 5-Fluorouracil, Cisplatin, Etoposide, and Vincristine. Chemotherapeutic drug sensitivity, P-glycoprotein function and expression, and MDR1 mRNA expression were detected using MTT assay, flow cytometry, Western blotting, and quantitative RT-PCR. Results When used alone, APS had no anti-tumor activity in H22/ADM cells in vitro. However, it can increase the cytotoxicity of certain chemotherapy drugs, such as Cyclophosphamid, Adriamycin, 5-Fluorouracil, Cisplatin, Etoposide, and Vincristine, in H22/ADM cells. It acts in a dose-dependent manner. Compared to a blank control group, APS increased intracellular Rhodamine-123 retention and decreased P-glycoprotein efflux function in a dose-dependent manner. These factors were assessed 24 h, 48 h, and 72 h after administration. APS down regulated P-glycoprotein and MDR1 mRNA expression in a concentration-dependent manner within a final range of 0.8–500 mg/L and in a time-dependent manner from 24–72 h. Conclusion APS can enhance the chemosensitivity of H22/ADM cells. This may involve the downregulation of MDR1 mRNA expression, inhibition of P-GP efflux pump function, or both, which would decrease the expression of the MDR1 protein. PMID

  4. Detailed characterization of cysteine-less P-glycoprotein reveals subtle pharmacological differences in function from wild-type protein

    PubMed Central

    Taylor, Andrew M; Storm, Janet; Soceneantu, Loredana; Linton, Kenneth J; Gabriel, Mark; Martin, Catherine; Woodhouse, James; Blott, Emma; Higgins, Christopher F; Callaghan, Richard

    2001-01-01

    Subtle alterations in the coupling of drug binding to nucleotide hydrolysis were observed following mutation of all seven endogenous cysteine residues to serines in the human multidrug resistance transporter, P-glycoprotein. Wild-type (wt) and the mutant (cys-less) forms of P-gp were expressed in Trichoplusia ni (High Five) cells and purified by metal affinity chromatography in order to undertake functional studies. No significant differences were observed in substrate ([3H]-azidopine) binding to wt or cys-less P-gp. Furthermore, neither the transported substrate vinblastine, nor the modulator nicardipine, differed in their respective potencies to displace [3H]-azidopine from the wt or cys-less P-gp. These results suggest that respective binding sites for these drugs were unaffected by the introduced cysteine to serine substitutions. The Michaelis-Menten characteristics of basal ATP hydrolysis of the two isoforms of P-gp were identical. The maximal ATPase activity in the presence of vinblastine was marginally reduced whilst the Km was unchanged in cys-less P-gp compared to control. However, cys-less P-gp displayed lower overall maximal ATPase activity (62%), a decreased Km and a lower degree of stimulation (76%) in the presence of the modulator nicardipine. Therefore, the serine to cysteine mutations in P-gp may suggest that vinblastine and nicardipine transduce their effects on ATP hydrolysis through distinct conformational pathways. The wt and cys-less P-gp isoforms display similarity in their fundamental kinetic properties thereby validating the use of cys-less P-gp as a template for future cysteine-directed structure/function analysis. PMID:11739236

  5. Analysis of protein composition and protein expression in the tear fluid of patients with congenital aniridia.

    PubMed

    Ihnatko, Robert; Edén, Ulla; Lagali, Neil; Dellby, Anette; Fagerholm, Per

    2013-12-06

    Aniridia is a rare congenital genetic disorder caused by haploinsuffiency of the PAX6 gene, the master gene for development of the eye. The expression of tear proteins in aniridia is unknown. To screen for proteins involved in the aniridia pathophysiology, the tear fluid of patients with diagnosed congenital aniridia was examined using two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two-dimensional map of tear proteins in aniridia has been established and 7 proteins were differentially expressed with P<0.01 between aniridia patients and control subjects. Five of them were more abundant in healthy subjects, particularly α-enolase, peroxiredoxin 6, cystatin S, gelsolin, apolipoprotein A-1 and two other proteins, zinc-α2-glycoprotein and lactoferrin were more expressed in the tears of aniridia patients. Moreover, immunoblot analysis revealed elevated levels of vascular endothelial growth factor (VEGF) in aniridia tears which is in concordance with clinical finding of pathological blood and lymph vessels in the central and peripheral cornea of aniridia patients. The proteins with different expression in patients' tears may be new candidate molecules involved in the pathophysiology of aniridia and thus may be helpful for development of novel treatment strategies for the symptomatic therapy of this vision threatening condition. This study is first to demonstrate protein composition and protein expression in aniridic tears and identifies proteins with different abundance in tear fluid from patients with congenital aniridia vs. healthy tears. © 2013 Elsevier B.V. All rights reserved.

  6. Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

    PubMed

    Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

    2015-08-01

    Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

  7. Targeting P-glycoprotein expression and cancer cell energy metabolism: combination of metformin and 2-deoxyglucose reverses the multidrug resistance of K562/Dox cells to doxorubicin.

    PubMed

    Xue, Chaojun; Wang, Changyuan; Liu, Qi; Meng, Qiang; Sun, Huijun; Huo, Xiaokui; Ma, Xiaodong; Liu, Zhihao; Ma, Xiaochi; Peng, Jinyong; Liu, Kexin

    2016-07-01

    P-glycoprotein (P-gp) is one of the major obstacles to efficiency of cancer chemotherapy. Here, we investigated whether combination of metformin and 2-deoxyglucose reverses the multidrug resistance (MDR) of K562/Dox cells and tried to elucidate the possible mechanisms. The combination of metformin and 2-deoxyglucose selectively enhanced the cytotoxicity of doxorubicin against K562/Dox cells. Metformin was not a substrate of P-gp but suppressed the elevated level of P-gp in K562/Dox cells. The downregulation of P-gp may be partly attributed to the inhibition of extracellular signal-regulated kinase pathway. The addition of 2-deoxyglucose to metformin initiated a strong metabolic stress in both K562 and K562/Dox cells. Combination of metformin and 2-deoxyglucose inhibited glucose uptake and lactate production in K562 and K562/Dox cells leading to a severe depletion in ATP and a enhanced autophagy. Above all, P-gp substrate selectively aggravated this ATP depletion effect and increased cell apoptosis in K562/Dox cells. In conclusion, metformin decreases P-gp expression in K562/Dox cells via blocking phosphorylation of extracellular signal-regulated kinase. P-gp substrate increases K562/Dox cell apoptosis via aggravating ATP depletion induced by combination of metformin and 2-deoxyglucose. Our observations highlight the importance of combination of metformin and 2-deoxyglucose in reversing multidrug resistance.

  8. Recent patents on alphavirus protein expression and vector production.

    PubMed

    Aranda, Alejandro; Ruiz-Guillen, Marta; Quetglas, Jose I; Bezunartea, Jaione; Casales, Erkuden; Smerdou, Cristian

    2011-12-01

    Alphaviruses contain a single-strand RNA genome that can be modified to express heterologous genes at high levels. Alphavirus vectors can be packaged within viral particles (VPs) or used as DNA/RNA layered systems. The broad tropism and high expression levels of alphavirus vectors have made them very attractive for applications like recombinant protein expression, vaccination or gene therapy. Expression mediated by alphavirus vectors is generally transient due to induction of apoptosis. However, during the last years several non-cytopathic mutations have been identified within the replicase sequence of different alphaviruses, allowing prolonged protein expression in culture cells. Some of these mutants, which have been patented, have allowed the generation of stable cell lines able to express recombinant proteins for extended periods of time in a constitutive or inducible manner. Production of alphavirus VPs usually requires cotransfection of cells with vector and helper RNAs providing viral structural proteins in trans. During this process full-length wild type (wt) genomes can be generated through recombination between different RNAs. Several new strategies to reduce wt virus generation during packaging, optimize VP production, increase packaging capacity, and provide VPs with specific targeting have been recently patented. Finally, hybrid vectors between alphavirus and other types of viruses have led to a number of patents with applications in vaccination, cancer therapy or retrovirus production.

  9. Expression and purification of GST-FHL2 fusion protein.

    PubMed

    Yu, H; Ma, Q; Lin, J; Sun, Y F; Zheng, F

    2013-12-06

    Escherichia coli is the most widely used host for the production of recombinant proteins. However, most eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. The interactions between human FHL2 protein and many types of proteins, including structural proteins, kinases, and several classes of transcription factor, have been found to have important roles in a variety of fundamental processes, including arrhythmia, hypertrophy, atherosclerosis, and angiogenesis. To achieve high-level expression of soluble recombinant human FHL2 protein in E. coli, we have constructed a recombinant expression plasmid, pGEX-4T-1-FHL2, in which we merged FHL2 cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac inducible promoter. Using this plasmid, we have achieved high expression of soluble FHL2 as a GST fusion protein in E. coli BL21. We have used the engineered plasmid (pGEX-4T-1-FHL2) and the modified E. coli strain to overcome the problem of removing the GST moiety while expressing soluble FHL2. Our results show that: 1) the recombinant plasmid was successfully constructed. Sequencing results showed that FHL2 and GST are in the same reading frame; 2) at 23°C, soluble GST-FHL2 fusion protein was highly expressed after induction with 0.1 mM IPTG; and 3) GST-FHL2 can be detected by Western blotting using mouse monoclonal anti-GST antibody. Our data are the first to show that high yields of soluble FHL2 tagged with GST can be achieved in E.coli.

  10. Enhanced Expression of Hedgehog Pathway Proteins in Oral Epithelial Dysplasia.

    PubMed

    Dias, Rosane Borges; Valverde, Ludmila de Faro; Sales, Caroline Brandi Schlaepfer; Guimarães, Vanessa Sousa Nazaré; Cabral, Márcia Grillo; de Aquino Xavier, Flávia Caló; Dos Santos, Jean Nunes; Ramos, Eduardo Antônio Gonçalves; Gurgel Rocha, Clarissa Araújo

    2016-09-01

    The aim of this study was to characterize the profile of the proteins involved in the Hedgehog signaling pathway to aid in the understanding of the pathogenesis of oral epithelial dysplasia (OED). The proteins SHH, PTCH1, HHIP, SUFU, GLI1, and cyclin D1 were evaluated by immunohistochemistry in 25 cases of OED, 4 of non-neoplasic oral mucosa, 8 of inflammatory fibrous hyperplasia and 5 of hyperkeratosis. SHH proteins were predominant in OED cases. Although PTCH1 protein was observed in all cases, this molecule was more highly expressed in OED. The inhibitor protein SUFU was present in OED and HHIP protein was overexpressed in OED. GLI1 proteins were predominantly found in the nuclei of epithelial cells in OED. Basal and suprabasal cells in the epithelial lining were positive for cyclin D1 only in OED. In conclusion, comparative analysis of the proteins involved in the Hedgehog pathway suggests that enhanced expression of these proteins can play an important role in the biological behavior of OED.

  11. Cell Cycle Programs of Gene Expression Control Morphogenetic Protein Localization

    PubMed Central

    Lord, Matthew; Yang, Melody C.; Mischke, Michelle; Chant, John

    2000-01-01

    Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms. PMID:11134078

  12. Nuclear Factor-Kappa B Activity Regulates Brain Expression of P-Glycoprotein in the Kainic Acid-Induced Seizure Rats

    PubMed Central

    Yu, Nian; Di, Qing; Liu, Hao; Hu, Yong; Jiang, Ying; Yan, Yu-kui; Zhang, Yan-fang; Zhang, Ying-dong

    2011-01-01

    This study was aimed to investigate the effect of NF-κB activity on the seizure susceptibility, brain damage, and P-gp expression in kainic acid- (KA-) induced seizure rats. Male SD rats were divided into saline control group (NS group), KA induced epilepsy group (EP group), and epilepsy group intervened with NF-κB inhibitor-pyrrolidine dithiocarbamate salt (PDTC group) or with dexamethasone (DEX group). No seizures were observed in the rats of NS group. Compared with NS group, increased P-gp expression and NF-κB activation in the rat brain of the EP group were observed after KA micro-injection. Both PDTC and DEX pre-treatment significantly increased the latency to grade III or V seizure onset compared to EP group but failed to show neuron-protective effect as the number of survival neurons didn't significantly differ from that in EP group. Furthermore, PDTC pre-treatment significantly decreased P-gp expression along with NF-κB activation in the hippocampus CA3 area and amygdala complex of rats compared with the EP group, implying that NF-κB activation involved in the seizure susceptibility and seizure induced brain P-gp over-expression. Additionally, DEX pre-treatment only decreased P-gp expression level without inhibition of NF-κB activation, suggesting NF-κB independent pathway may also participate in regulating seizure induced P-gp over-expression. PMID:21403895

  13. Nuclear factor-kappa B activity regulates brain expression of P-glycoprotein in the kainic acid-induced seizure rats.

    PubMed

    Yu, Nian; Di, Qing; Liu, Hao; Hu, Yong; Jiang, Ying; Yan, Yu-kui; Zhang, Yan-fang; Zhang, Ying-dong

    2011-01-01

    This study was aimed to investigate the effect of NF-κB activity on the seizure susceptibility, brain damage, and P-gp expression in kainic acid- (KA-) induced seizure rats. Male SD rats were divided into saline control group (NS group), KA induced epilepsy group (EP group), and epilepsy group intervened with NF-κB inhibitor-pyrrolidine dithiocarbamate salt (PDTC group) or with dexamethasone (DEX group). No seizures were observed in the rats of NS group. Compared with NS group, increased P-gp expression and NF-κB activation in the rat brain of the EP group were observed after KA micro-injection. Both PDTC and DEX pre-treatment significantly increased the latency to grade III or V seizure onset compared to EP group but failed to show neuron-protective effect as the number of survival neurons didn't significantly differ from that in EP group. Furthermore, PDTC pre-treatment significantly decreased P-gp expression along with NF-κB activation in the hippocampus CA3 area and amygdala complex of rats compared with the EP group, implying that NF-κB activation involved in the seizure susceptibility and seizure induced brain P-gp over-expression. Additionally, DEX pre-treatment only decreased P-gp expression level without inhibition of NF-κB activation, suggesting NF-κB independent pathway may also participate in regulating seizure induced P-gp over-expression.

  14. Protein Co-Expression Network Analysis (ProCoNA)

    SciTech Connect

    Gibbs, David L.; Baratt, Arie; Baric, Ralph; Kawaoka, Yoshihiro; Smith, Richard D.; Orwoll, Eric S.; Katze, Michael G.; Mcweeney, Shannon K.

    2013-06-01

    Biological networks are important for elucidating disease etiology due to their ability to model complex high dimensional data and biological systems. Proteomics provides a critical data source for such models, but currently lacks robust de novo methods for network construction, which could bring important insights in systems biology. We have evaluated the construction of network models using methods derived from weighted gene co-expression network analysis (WGCNA). We show that approximately scale-free peptide networks, composed of statistically significant modules, are feasible and biologically meaningful using two mouse lung experiments and one human plasma experiment. Within each network, peptides derived from the same protein are shown to have a statistically higher topological overlap and concordance in abundance, which is potentially important for inferring protein abundance. The module representatives, called eigenpeptides, correlate significantly with biological phenotypes. Furthermore, within modules, we find significant enrichment for biological function and known interactions (gene ontology and protein-protein interactions). Biological networks are important tools in the analysis of complex systems. In this paper we evaluate the application of weighted co-expression network analysis to quantitative proteomics data. Protein co-expression networks allow novel approaches for biological interpretation, quality control, inference of protein abundance, a framework for potentially resolving degenerate peptide-protein mappings, and a biomarker signature discovery.

  15. Thyroid-Related Protein Expression in the Human Thymus

    PubMed Central

    Park, Do Joon; Jung, Kyeong Cheon

    2017-01-01

    Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall's corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS. PMID:28386277

  16. Protein-directed ribosomal frameshifting temporally regulates gene expression

    PubMed Central

    Napthine, Sawsan; Ling, Roger; Finch, Leanne K.; Jones, Joshua D.; Bell, Susanne; Brierley, Ian; Firth, Andrew E.

    2017-01-01

    Programmed −1 ribosomal frameshifting is a mechanism of gene expression, whereby specific signals within messenger RNAs direct a proportion of translating ribosomes to shift −1 nt and continue translating in the new reading frame. Such frameshifting normally occurs at a set ratio and is utilized in the expression of many viral genes and a number of cellular genes. An open question is whether proteins might function as trans-acting switches to turn frameshifting on or off in response to cellular conditions. Here we show that frameshifting in a model RNA virus, encephalomyocarditis virus, is trans-activated by viral protein 2A. As a result, the frameshifting efficiency increases from 0 to 70% (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins. PMID:28593994

  17. Using ion exchange chromatography to purify a recombinantly expressed protein.

    PubMed

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment.

  18. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    PubMed Central

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2013-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We sought to determine whether GILT’s reductase activity regulates CatS expression and function. Confocal microscopy confirmed that GILT and CatS colocalized within lysosomes of B cells. GILT expression posttranscriptionally decreased the steady-state protein expression of CatS in primary B cells and B-cell lines. GILT did not substantially alter the expression of other lysosomal proteins, including H2-M, H2-O, or CatL. GILT’s reductase active site was necessary for diminished CatS protein levels, and GILT expression decreased the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation. GILT expression decreased the proteolysis of a CatS selective substrate. This study illustrates a physiologic mechanism that regulates CatS and has implications for fine tuning MHC class II-restricted Ag processing and for the development of CatS inhibitors, which are under investigation for the treatment of autoimmune disease. PMID:23012103

  19. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

    PubMed

    Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J

    2015-01-06

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

  20. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

    PubMed

    Horstick, Eric J; Jordan, Diana C; Bergeron, Sadie A; Tabor, Kathryn M; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A

    2015-04-20

    Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

  1. Global Analysis of Protein Expression of Inner Ear Hair Cells

    PubMed Central

    Wong, Ann C.Y.; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel

    2017-01-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes. SIGNIFICANCE STATEMENT Hearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that

  2. SPINK 1 Protein Expression and Prostate Cancer Progression

    PubMed Central

    Flavin, Richard; Pettersson, Andreas; Hendrickson, Whitney K.; Fiorentino, Michelangelo; Finn, Stephen; Kunz, Lauren; Judson, Gregory L.; Lis, Rosina; Bailey, Dyane; Fiore, Christopher; Nuttall, Elizabeth; Martin, Neil E.; Stack, Edward; Penney, Kathryn L.; Rider, Jennifer R.; Sinnott, Jennifer; Sweeney, Christopher; Sesso, Howard D.; Fall, Katja; Giovannucci, Edward; Kantoff, Philip; Stampfer, Meir; Loda, Massimo; Mucci, Lorelei A.

    2014-01-01

    Purpose SPINK1 over-expression has been described in prostate cancer and is linked with poor prognosis in many cancers. The objective of this study was to characterize the association between SPINK1 over-expression and prostate cancer specific survival. Experimental Design The study included 879 participants in the US Physicians’ Health Study and Health Professionals Follow–Up Study, diagnosed with prostate cancer (1983 – 2004) and treated by radical prostatectomy. Protein tumor expression of SPINK1 was evaluated by immunohistochemistry on tumor tissue microarrays. Results 74/879 (8%) prostate cancer tumors were SPINK1 positive. Immunohistochemical data was available for PTEN, p-Akt, pS6, stathmin, androgen receptor (AR) and ERG (as a measure of the TMPRSS2:ERG translocation). Compared to SPINK1 negative tumors, SPINK1 positive tumors showed higher PTEN and stathmin expression, and lower expression of AR (p<0.01). SPINK1 over-expression was seen in 47 of 427 (11%) ERG negative samples and in 19 of 427 (4%) ERG positive cases (p=0.0003). We found no significant associations between SPINK1 status and Gleason grade or tumor stage. There was no association between SPINK1 expression and biochemical recurrence (p=0.56). Moreover, there was no association between SPINK1 expression and prostate cancer mortality (there were 75 lethal cases of prostate cancer during a mean of 13.5 years follow-up [HR 0.71 (95% confidence interval 0.29–1.76)]). Conclusions Our results suggest that SPINK1 protein expression may not be a predictor of recurrence or lethal prostate cancer amongst men treated by radical prostatectomy. SPINK1 and ERG protein expression do not appear to be entirely mutually exclusive, as some previous studies have suggested. PMID:24687926

  3. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  4. Enhancement of G Protein-Coupled Receptor Surface Expression

    PubMed Central

    Dunham, Jill H.; Hall, Randy A.

    2009-01-01

    G protein-coupled receptors (GPCRs) mediate physiological responses to a diverse array of stimuli and are the molecular targets for numerous therapeutic drugs. GPCRs primarily signal from the plasma membrane, but when expressed in heterologous cells many GPCRs exhibit poor trafficking to the cell surface. Multiple approaches have been taken to enhance GPCR surface expression in heterologous cells, including addition/deletion of receptor sequences, co-expression with interacting proteins, and treatment with pharmacological chaperones. In addition to allowing for enhanced surface expression of certain GPCRs in heterologous cells, these approaches have also shed light on the control of GPCR trafficking in vivo and in some cases have led to new therapeutic approaches for treating human diseases that result from defects in GPCR trafficking. PMID:19679364

  5. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    USDA-ARS?s Scientific Manuscript database

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal po...

  6. Hedyotis diffusa Willd overcomes 5-fluorouracil resistance in human colorectal cancer HCT-8/5-FU cells by downregulating the expression of P-glycoprotein and ATP-binding casette subfamily G member 2

    PubMed Central

    LI, QIONGYU; WANG, XIANGFENG; SHEN, ALING; ZHANG, YUCHEN; CHEN, YOUQIN; SFERRA, THOMAS J.; LIN, JIUMAO; PENG, JUN

    2015-01-01

    Previous studies have demonstrated that Hedyotis diffusa Willd (HDW), a traditional Chinese herbal medicine, exhibits potent anticancer activity in models of colorectal cancer (CRC). Aggressive forms of CRC exhibit resistance to widely used chemotherapeutic drugs, including the antimetabolite, 5-fluorouracil (5-FU); however, less is known with regard to the activity of HDW against 5-FU-resistant cancer. In the present study, the mechanism of action and the potency of ethanol extracts of HDW (EEHDW) were investigated on a multidrug-resistant CRC HCT-8/5-FU cell line. Using an MTT cell proliferation assay, EEHDW treatment was shown to significantly reduce the cell viability of HCT-8/5-FU cells in a dose- and time-dependent manner. Furthermore, EEHDW significantly increased the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine-123, as compared with the untreated controls. To further investigate the molecular mechanisms targeted by EEHDW in the resistant cells, the expression levels of the ABC drug transporter protein, P-glycoprotein (P-gp), and ABC subfamily G member 2 (ABCG2), were analyzed using reverse-transcription polymerase chain reaction and western blot analysis. The mRNA and protein expression levels of P-gp and ABCG2 were reduced in the HCT-8/5-FU cells following EEHDW treatment, indicating that EEHDW inhibits ABCG2-mediated drug resistance by downregulating the expression of ABCG2 and P-gp. Therefore, the potential application of EEHDW as a chemotherapeutic adjuvant represents a promising alternative approach to the treatment of drug-resistant CRC. PMID:26640560

  7. Hedyotis diffusa Willd overcomes 5-fluorouracil resistance in human colorectal cancer HCT-8/5-FU cells by downregulating the expression of P-glycoprotein and ATP-binding casette subfamily G member 2.

    PubMed

    Li, Qiongyu; Wang, Xiangfeng; Shen, Aling; Zhang, Yuchen; Chen, Youqin; Sferra, Thomas J; Lin, Jiumao; Peng, Jun

    2015-11-01

    Previous studies have demonstrated that Hedyotis diffusa Willd (HDW), a traditional Chinese herbal medicine, exhibits potent anticancer activity in models of colorectal cancer (CRC). Aggressive forms of CRC exhibit resistance to widely used chemotherapeutic drugs, including the antimetabolite, 5-fluorouracil (5-FU); however, less is known with regard to the activity of HDW against 5-FU-resistant cancer. In the present study, the mechanism of action and the potency of ethanol extracts of HDW (EEHDW) were investigated on a multidrug-resistant CRC HCT-8/5-FU cell line. Using an MTT cell proliferation assay, EEHDW treatment was shown to significantly reduce the cell viability of HCT-8/5-FU cells in a dose- and time-dependent manner. Furthermore, EEHDW significantly increased the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine-123, as compared with the untreated controls. To further investigate the molecular mechanisms targeted by EEHDW in the resistant cells, the expression levels of the ABC drug transporter protein, P-glycoprotein (P-gp), and ABC subfamily G member 2 (ABCG2), were analyzed using reverse-transcription polymerase chain reaction and western blot analysis. The mRNA and protein expression levels of P-gp and ABCG2 were reduced in the HCT-8/5-FU cells following EEHDW treatment, indicating that EEHDW inhibits ABCG2-mediated drug resistance by downregulating the expression of ABCG2 and P-gp. Therefore, the potential application of EEHDW as a chemotherapeutic adjuvant represents a promising alternative approach to the treatment of drug-resistant CRC.

  8. p53 and MDM2 protein expression in actinic cheilitis.

    PubMed

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  9. Argonaute Family Protein Expression in Normal Tissue and Cancer Entities

    PubMed Central

    Bruckmann, Astrid; Hauptmann, Judith; Deutzmann, Rainer; Meister, Gunter; Bosserhoff, Anja Katrin

    2016-01-01

    The members of the Argonaute (AGO) protein family are key players in miRNA-guided gene silencing. They enable the interaction between small RNAs and their respective target mRNA(s) and support the catalytic destruction of the gene transcript or recruit additional proteins for downstream gene silencing. The human AGO family consists of four AGO proteins (AGO1-AGO4), but only AGO2 harbors nuclease activity. In this study, we characterized the expression of the four AGO proteins in cancer cell lines and normal tissues with a new mass spectrometry approach called AGO-APP (AGO Affinity Purification by Peptides). In all analyzed normal tissues, AGO1 and AGO2 were most prominent, but marked tissue-specific differences were identified. Furthermore, considerable changes during development were observed by comparing fetal and adult tissues. We also identified decreased overall AGO expression in melanoma derived cell lines compared to other tumor cell lines and normal tissues, with the largest differences in AGO2 expression. The experiments described in this study suggest that reduced amounts of AGO proteins, as key players in miRNA processing, have impact on several cellular processes. Deregulated miRNA expression has been attributed to chromosomal aberrations, promoter regulation and it is known to have a major impact on tumor development and progression. Our findings will further increase our basic understanding of the molecular basis of miRNA processing and its relevance for disease. PMID:27518285

  10. Expression analysis of BRUCE protein in esophageal squamous cell carcinoma.

    PubMed

    Salehi, Somayeh; Jafarian, Amir Hossein; Forghanifard, Mohammad Mahdi

    2016-10-01

    Apoptosis is a form of cell death in response to diverse stressful physiological or pathological stimuli. One of the most important gene families involved in apoptosis is inhibitors of apoptosis. As a member of inhibitors of apoptosis, BRUCE can suppress apoptosis and promote cell division. Because esophageal squamous cell carcinoma (ESCC) cells, as well as other cancer cells, are immortal, our aim in this study was to analyze BRUCE protein expression in ESCC and evaluate its correlation with tumoral clinicopathologic features. Fifty ESCC specimens were examined for BRUCE protein expression using immunohistochemistry. A defined scoring method was applied. BRUCE protein was detected in 82% of tumors. Tumor progression stage and invasion depth correlated significantly with BRUCE protein expression (P=.019 and .005, respectively). Furthermore, association of BRUCE expression with tumor location was near significant (P=.058). The correlation of BRUCE overexpression in ESCC and disease aggressiveness may confirm the importance of BRUCE in ESCC progression and invasiveness. Therefore, BRUCE protein may be a molecular marker for aggressive ESCC and, thus, a potential therapeutic target to inhibit tumor cell progression and invasion. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

    PubMed Central

    Lilly, Jacob L.; Sheldon, Phillip R.; Hoversten, Liv J.; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J.

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium. PMID:25536421

  12. Patterns of soybean proline-rich protein gene expression.

    PubMed Central

    Wyatt, R E; Nagao, R T; Key, J L

    1992-01-01

    The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes. PMID:1525563

  13. AB223. Expression of tight junction proteins in rat vagina

    PubMed Central

    Oh, Kyung Jin; Lee, Hyun-Suk; Chung, Ho Suck; Ahn, Kyu Youn; Park, Kwangsung

    2014-01-01

    Aim Tight junction plays a role in apical cell-to-cell adhesion and epithelial polarity. In this study, we investigated the expression of tight junction proteins, such as Claudin-1, zonula occludens (ZO)-1, junction adhesion molecule (JAM)-A, and occludin in rat vagina. Methods Female Sprague-dawley rats (230-240 g, n=20) were divided into two groups: control (n=10) and bilateral ovariectomy (n=10). The expression and cellular localization of claudin-1, ZO-1, JAM-A, and occludin were determined in each group by immunohistochemistry and Western blot. Results Immunolabeling of ZO-1 was mainly expressed in the capillaries and venules of the vagina. Claudin-1, JAM-A, and occludin were expressed in the epithelium of the vagina. The immunoreactivity and protein expression of claudin-1 was significantly decreased in the ovariectomy group compared with the control group. Conclusions Our results suggest that tight junction proteins may have an important role in the vagina. Further studies are needed to clarify the role of each tight junction protein on vaginal lubrication.

  14. Functional interaction between co-expressed MAGE-A proteins

    PubMed Central

    Laiseca, Julieta E.; Ladelfa, María F.; Cotignola, Javier; Peche, Leticia Y.; Pascucci, Franco A.; Castaño, Bryan A.; Galigniana, Mario D.; Schneider, Claudio

    2017-01-01

    MAGE-A (Melanoma Antigen Genes-A) are tumor-associated proteins with expression in a broad spectrum of human tumors and normal germ cells. MAGE-A gene expression and function are being increasingly investigated to better understand the mechanisms by which MAGE proteins collaborate in tumorigenesis and whether their detection could be useful for disease prognosis purposes. Alterations in epigenetic mechanisms involved in MAGE gene silencing cause their frequent co-expression in tumor cells. Here, we have analyzed the effect of MAGE-A gene co-expression and our results suggest that MageA6 can potentiate the androgen receptor (AR) co-activation function of MageA11. Database search confirmed that MageA11 and MageA6 are co-expressed in human prostate cancer samples. We demonstrate that MageA6 and MageA11 form a protein complex resulting in the stabilization of MageA11 and consequently the enhancement of AR activity. The mechanism involves association of the Mage A6-MHD domain to MageA11, prevention of MageA11 ubiquitinylation on lysines 240 and 245 and decreased proteasome-dependent degradation. We experimentally demonstrate here for the first time that two MAGE-A proteins can act together in a non-redundant way to potentiate a specific oncogenic function. Overall, our results highlight the complexity of the MAGE gene networking in regulating cancer cell behavior. PMID:28542476

  15. Functional interaction between co-expressed MAGE-A proteins.

    PubMed

    Laiseca, Julieta E; Ladelfa, María F; Cotignola, Javier; Peche, Leticia Y; Pascucci, Franco A; Castaño, Bryan A; Galigniana, Mario D; Schneider, Claudio; Monte, Martin

    2017-01-01

    MAGE-A (Melanoma Antigen Genes-A) are tumor-associated proteins with expression in a broad spectrum of human tumors and normal germ cells. MAGE-A gene expression and function are being increasingly investigated to better understand the mechanisms by which MAGE proteins collaborate in tumorigenesis and whether their detection could be useful for disease prognosis purposes. Alterations in epigenetic mechanisms involved in MAGE gene silencing cause their frequent co-expression in tumor cells. Here, we have analyzed the effect of MAGE-A gene co-expression and our results suggest that MageA6 can potentiate the androgen receptor (AR) co-activation function of MageA11. Database search confirmed that MageA11 and MageA6 are co-expressed in human prostate cancer samples. We demonstrate that MageA6 and MageA11 form a protein complex resulting in the stabilization of MageA11 and consequently the enhancement of AR activity. The mechanism involves association of the Mage A6-MHD domain to MageA11, prevention of MageA11 ubiquitinylation on lysines 240 and 245 and decreased proteasome-dependent degradation. We experimentally demonstrate here for the first time that two MAGE-A proteins can act together in a non-redundant way to potentiate a specific oncogenic function. Overall, our results highlight the complexity of the MAGE gene networking in regulating cancer cell behavior.

  16. Protein synthesis rate is the predominant regulator of protein expression during differentiation

    PubMed Central

    Kristensen, Anders R; Gsponer, Joerg; Foster, Leonard J

    2013-01-01

    External perturbations, by forcing cells to adapt to a new environment, often elicit large-scale changes in gene expression resulting in an altered proteome that improves the cell's fitness in the new conditions. Steady-state levels of a proteome depend on transcription, the levels of transcripts, translation and protein degradation but system-level contribution that each of these processes make to the final protein expression change has yet to be explored. We therefore applied a systems biology approach to characterize the regulation of protein expression during cellular differentiation using quantitative proteomics. As a general rule, it seems that protein expression during cellular differentiation is largely controlled by changes in the relative synthesis rate, whereas the relative degradation rate of the majority of proteins stays constant. In these data, we also observe that the proteins in defined sub-structures of larger protein complexes tend to have highly correlated synthesis and degradation rates but that this does not necessarily extend to the holo-complex. Finally, we provide strong evidence that the generally poor correlation observed between transcript and protein levels can fully be explained once the protein synthesis and degradation rates are taken into account. PMID:24045637

  17. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins

    PubMed Central

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-01-01

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions. PMID:28266541

  18. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins.

    PubMed

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-03-07

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions.

  19. Spatiotemporal expression profiling of proteins in rat sciatic nerve regeneration using reverse phase protein arrays

    PubMed Central

    2012-01-01

    Background Protein expression profiles throughout 28 days of peripheral nerve regeneration were characterized using an established rat sciatic nerve transection injury model. Reverse phase protein microarrays were used to identify the spatial and temporal expression profile of multiple proteins implicated in peripheral nerve regeneration including growth factors, extracellular matrix proteins, and proteins involved in adhesion and migration. This high-throughput approach enabled the simultaneous analysis of 3,360 samples on a nitrocellulose-coated slide. Results The extracellular matrix proteins collagen I and III, laminin gamma-1, fibronectin, nidogen and versican displayed an early increase in protein levels in the guide and proximal sections of the regenerating nerve with levels at or above the baseline expression of intact nerve by the end of the 28 day experimental course. The 28 day protein levels were also at or above baseline in the distal segment however an early increase was only noted for laminin, nidogen, and fibronectin. While the level of epidermal growth factor, ciliary neurotrophic factor and fibroblast growth factor-1 and -2 increased throughout the experimental course in the proximal and distal segments, nerve growth factor only increased in the distal segment and fibroblast growth factor-1 and -2 and nerve growth factor were the only proteins in that group to show an early increase in the guide contents. As expected, several proteins involved in cell adhesion and motility; namely focal adhesion kinase, N-cadherin and β-catenin increased earlier in the proximal and distal segments than in the guide contents reflecting the relatively acellular matrix of the early regenerate. Conclusions In this study we identified changes in expression of multiple proteins over time linked to regeneration of the rat sciatic nerve both demonstrating the utility of reverse phase protein arrays in nerve regeneration research and revealing a detailed, composite

  20. Acute liver failure impairs function and expression of breast cancer-resistant protein (BCRP) at rat blood-brain barrier partly via ammonia-ROS-ERK1/2 activation.

    PubMed

    Li, Ying; Zhang, Ji; Xu, Ping; Sun, Binbin; Zhong, Zeyu; Liu, Can; Ling, Zhaoli; Chen, Yang; Shu, Nan; Zhao, Kaijing; Liu, Li; Liu, Xiaodong

    2016-07-01

    We once reported that P-glycoprotein (P-GP) and multidrug resistance-associated protein 2 (MRP2) were oppositely regulated at the blood-brain barrier (BBB) of thioacetamide-induced acute liver failure (ALF) rats. This study aimed to investigate whether ALF affected function and expression of breast cancer-resistant protein (BCRP) at the BBB of rats and the role of ammonia in the regulation. ALF rats were developed by intraperitoneal (i.p.) injection of thioacetamide (300 mg/kg) for 2 days. Hyperammonemic rats were developed by NH4 Ac (i.p. 4.5 mmol/kg). BCRP function and expression were measured by brain distribution of specific substrates (prazosin and methotrexate) and western blot, respectively. MDCK-BCRP cells and primarily cultured rat brain microvessel endothelial cells (rBMECs) were employed to investigate possible mechanisms through which ammonia regulated BCRP function and expression. The results showed that both ALF and hyperammonemia significantly weakened function and expression of BCRP in the brain of rats. The function and expression of BCRP in MDCK-BCRP cells and rBMECs were strikingly decreased after exposure to NH4 Cl and H2 O2 , accompanied by remarkable increases in the levels of phosphorylated ERK1/2 and reactive oxygen species (ROS). The altered BCRP expression and function by ammonia and H2 O2 were restored by ROS scavenger N-acetylcysteine and ERK1/2 inhibitor U0126. Markedly increased levels of ERK1/2 phosphorylation and ROS were found in the brains of ALF rats and hyperammonemic rats. All above results indicated ALF down-regulated expression and function of BCRP at BBB of rats partly via hyperammonemia. Activation of ROS-mediated ERK1/2 phosphorylation may be one of the reasons that ammonia impaired BCRP expression and function at the BBB. The present study showed that the expression and function of breast cancer resistant protein (BCRP) at blood-brain barrier (BBB) of thioacetamide-induced ALF rats were down-regulated which partly

  1. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts.

    PubMed

    Carmona-Rodríguez, Bruno; Alvarez-Pérez, Marco Antonio; Narayanan, A Sampath; Zeichner-David, Margarita; Reyes-Gasga, José; Molina-Guarneros, Juan; García-Hernández, Ana Lilia; Suárez-Franco, José Luis; Chavarría, Ivet Gil; Villarreal-Ramírez, Eduardo; Arzate, Higinio

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  2. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  3. Fe-S Proteins that Regulate Gene Expression

    PubMed Central

    Mettert, Erin L.; Kiley, Patricia J.

    2014-01-01

    Iron-sulfur (Fe-S) cluster containing proteins that regulate gene expression are present in most organisms. The innate chemistry of their Fe-S cofactors makes these regulatory proteins ideal for sensing environmental signals, such as gases (e.g. O2 and NO), levels of Fe and Fe-S clusters, reactive oxygen species, and redox cycling compounds, to subsequently mediate an adaptive response. Here we review the recent findings that have provided invaluable insight into the mechanism and function of these highly significant Fe-S regulatory proteins. PMID:25450978

  4. Expression of Aequorea green fluorescent protein in plant cells.

    PubMed

    Hu, W; Cheng, C L

    1995-08-07

    The coding region of the green fluorescent protein (GFP) from Aequorea victoria has been fused to the cauliflower mosaic virus 35S promoter and introduced into maize leaf protoplasts. Transient expression of GFP was observed. In addition, the coding region of GFP was fused to an Arabidopsis heat shock promoter and co-transformed with another construct in which GFP has been replaced with chloramphenicol acetyltransferase (CAT). The heat-induced expression of GFP in maize protoplasts parallels that of CAT. While GFP was expressed in both dark-grown and green maize leaf protoplasts, no green fluorescence was observed in similarly transformed Arabidopsis protoplasts.

  5. Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure

    EPA Science Inventory

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-bindin...

  6. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  7. Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

    PubMed

    Bonaccorso, C M; Spatuzza, M; Di Marco, B; Gloria, A; Barrancotto, G; Cupo, A; Musumeci, S A; D'Antoni, S; Bardoni, B; Catania, M V

    2015-05-01

    Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in

  8. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  9. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  10. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  11. Cementum attachment protein/protein-tyrosine phosphotase-like member A is not expressed in teeth.

    PubMed

    Schild, Christof; Beyeler, Michael; Lang, Niklaus P; Trueb, Beat

    2009-02-01

    Cementum is a highly specialized connective tissue that covers tooth roots. The only cementum-specific protein described to date is the cementum attachment protein (CAP). A putative sequence for CAP was established from a cDNA clone isolated from a human cementifying fibroma cDNA library. This sequence overlaps with a phosphatase-like protein in muscle termed the protein-tyrosine phosphatase-like member A (PTPLA). To clarify the nature of CAP/PTPLA, we cloned the homologous rat protein and determined its sequence. The rat protein shared 94% sequence identity with the human protein. On Northern blots containing RNA from various rat tissues of different developmental stages, the cDNA hybridized to an mRNA expressed in heart and skeletal muscle but not in teeth. These results were confirmed by real-time PCR. Thus, the sequence deposited in public databanks under the name 'cementum attachment protein' does not represent genuine CAP.

  12. Controlling for Gene Expression Changes in Transcription Factor Protein Networks*

    PubMed Central

    Banks, Charles A. S.; Lee, Zachary T.; Boanca, Gina; Lakshminarasimhan, Mahadevan; Groppe, Brad D.; Wen, Zhihui; Hattem, Gaye L.; Seidel, Chris W.; Florens, Laurence; Washburn, Michael P.

    2014-01-01

    The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein–protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions. PMID:24722732

  13. Secreted Proteins Defy the Expression Level-Evolutionary Rate Anticorrelation.

    PubMed

    Feyertag, Felix; Berninsone, Patricia M; Alvarez-Ponce, David

    2017-03-01

    The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E-R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein-protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E-R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E-R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E-R anticorrelation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Selection of soluble protein expression constructs: the experimental determination of protein domain boundaries.

    PubMed

    Dyson, Michael R

    2010-08-01

    Proteins can contain multiple domains each of which is capable of possessing a separate independent function and three-dimensional structure. It is often useful to clone and express individual protein domains to study their biochemical properties and for structure determination. However, the annotated domain boundaries in databases such as Pfam or SMART are not always accurate. The present review summarizes various strategies for the experimental determination of protein domain boundaries.

  15. Evaluation of TPGS-modified thermo-sensitive Pluronic PF127 hydrogel as a potential carrier to reverse the resistance of P-gp-overexpressing SMMC-7721 cell lines.

    PubMed

    Gao, Lei; Wang, Xiaoqing; Ma, Jianli; Hao, Daifeng; Wei, Pei; Zhou, Liang; Liu, Guiyang

    2016-04-01

    In the present studies locally injectable docetaxel nanocrystals loaded d-alpha tocopheryl polyethylene glycol 1000 succinate-modified Pluronic F127 (DOC-NCs-TPGS-PF127) thermo-sensitive hydrogels were prepared to reverse drug resistance of P-glycoprotein (P-gp)-overexpressing human liver cancer SMMC-7721 tumors. Firstly, DOC nanosuspensions with mean particle size of 196nm were prepared and dispersed into series of mixed solutions containing PF127 and TPGS of different ratios to obtain DOC-NCs-TPGS-PF127 hydrogels. DOC NCs, exhibiting a uniform distribution and very good physical stability during three sol-gel cycles in the hydrogel network, did not influence the gelation temperature. Swelling-dependent release pattern was found for DOC NCs from hydrogels and release profiles could be well fitted by the Peppas equation. MTT test showed that hydrogels containing 0% or 0.1% TPGS had no cytotoxicity against L929 fibroblasts. Both DOC solution and DOC-NCs-TPGS-PF127 hydrogels exhibited obvious cytotoxicity against sensitive SMMC-7721 cells. When resistant SMMC7721 cells were treated, DOC-NCs-TPGS-PF127 hydrogels showed significantly higher cytotoxicity compared with DOC solution and hydrogels containing no TPGS (DOC-NCs-PF127), with markedly lower IC50 and resistant index (RI). After intratumoral injection in SMMC-7721/RT tumor xenograft Balb/c mice model, DOC-NCs-TPGS-PF127 hydrogels exhibited about 5-fold increase and 1.8-fold increase in the inhibition rate of tumor growth compared with intravenous and intratumoral injection of DOC solution, respectively. It could be concluded that TPGS-modified PF127 thermo-sensitive hydrogel was an excellent locally injectable carrier to reverse P-gp overexpression associated multi-drug resistance.

  16. Effects of HMG-CoA reductase inhibitors on the pharmacokinetics of nifedipine in rats: Possible role of P-gp and CYP3A4 inhibition by HMG-CoA reductase inhibitors.

    PubMed

    Lee, Chong-Ki; Choi, Jun-Shik; Choi, Dong-Hyun

    2015-02-01

    This study aimed to investigate the effects of HMG-CoA reductase inhibitors on the pharmacokinetics of nifedipine in rats. We determined the pharmacokinetic parameters of nifedipine and dehydronifedipine in rats after oral and intravenous administration of nifedipine without and with HMG-CoA reductase inhibitors. We evaluated the effect of HMG-CoA reductase inhibitors on the activity of P-glycoprotein (P-gp) and cytochrome P450 (CYP)3A4. Atorvastatin, fluvastatin, pravastatin and simvastatin inhibited CYP3A4 activities; inhibitory concentration (IC50) values were 47.0, 5.2, 15.0 and 3.3 μM, respectively. Simvastatin and fluvastatin increased the cellular uptake of rhodamine-123. The area under the plasma concentration-time curve (AUC0-∞) and the peak plasma concentration (Cmax) of oral nifedipine were significantly increased by fluvastatin and simvastatin, respectively, compared to control group. The total body clearance (CL/F) of nifedipine after oral administration with fluvastatin and simvastatin were significantly decreased compared to those of control. The metabolite-parent AUC ratio (MR) of nifedipine with fluvastatin and simvastatin were significantly decreased, which suggested that fluvastatin and simvastatin inhibited metabolism of nifedipine, respectively. The AUC0-∞ of intravenouse nifedipine with fluvastatin and simvastatin was significantly higher than that of the control group. The increased bioavailability of nifedipine may be mainly due to inhibition of both P-gp in the small intestine and CYP3A subfamily-mediated metabolism of nifedipine in the small intestine and/or in the liver and to the reduction of the CL/F of nifedipine by fluvastatin and simvastatin. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  17. Effects of HMG-CoA reductase inhibitors on the pharmacokinetics of losartan and its main metabolite EXP-3174 in rats: possible role of CYP3A4 and P-gp inhibition by HMG-CoA reductase inhibitors.

    PubMed

    Yang, Si-Hyung; Choi, Jun-Shik; Choi, Dong-Hyun

    2011-01-01

    The present study was designed to investigate the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (atorvastatin, pravastatin, simvastatin) on the pharmacokinetics of losartan and its active metabolite EXP-3174 in rats. Pharmacokinetic parameters of losartan and EXP-3174 in rats were determined after oral and intravenous administration of losartan (9 mg/kg) without and with HMG-CoA reductase inhibitors (1 mg/kg). The effect of HMG-CoA reductase inhibitors on P-gp and cytochrome (CYP) 3A4 activity were also evaluated. Atorvastatin, pravastatin and simvastatin inhibited CYP3A4 activities with IC₅₀ values of 48.0, 14.1 and 3.10 μmol/l, respectively. Simvastatin (1-10 μmol/l) enhanced the cellular uptake of rhodamine-123 in a concentration-dependent manner. The area under the plasma concentration-time curve (AUC₀₋∞) and the peak plasma concentration of losartan were significantly (p < 0.05) increased by 59.6 and 45.8%, respectively, by simvastatin compared to those of control. The total body clearance (CL/F) of losartan after oral administration with simvastatin was significantly decreased (by 34.8%) compared to that of controls. Consequently, the absolute bioavailability (F) of losartan after oral administration with simvastatin was significantly increased by 59.4% compared to that of control. The metabolite-parent AUC ratio was significantly decreased by 25.7%, suggesting that metabolism of losartan was inhibited by simvastatin. In conclusion, the enhanced bioavailability of losartan might be mainly due to inhibition of P-gp in the small intestine and CYP3A subfamily-mediated metabolism of losartan in the small intestine and/or liver and to reduction of the CL/F of losartan by simvastatin.

  18. Expression Trend of Selected Ribosomal Protein Genes in Nasopharyngeal Carcinoma

    PubMed Central

    Ma, Xiang-Ru; Sim, Edmund Ui-Hang; Ling, Teck-Yee; Tiong, Thung-Sing; Subramaniam, Selva Kumar; Khoo, Alan Soo-Beng

    2012-01-01

    Background: Ribosomal proteins are traditionally associated with protein biosynthesis until recent studies that implicated their extraribosomal functions in human diseases and cancers. Our previous studies using GeneFishing™ DEG method and microarray revealed underexpression of three ribosomal protein genes, RPS26, RPS27, and RPL32 in cancer of the nasopharynx. Herein, we investigated the expression pattern and nucleotide sequence integrity of these genes in nasopharyngeal carcinoma to further delineate their involvement in tumourigenesis. The relationship of expression level with clinicopathologic factors was also statistically studied. Methods: Quantitative Polymerase Chain Reaction was performed on nasopharyngeal carcinoma and their paired normal tissues. Expression and sequence of these three genes were analysed. Results: All three ribosomal protein genes showed no significant difference in transcript expressions and no association could be established with clinicopathologic factors studied. No nucleotide aberrancy was detected in the coding regions of these genes. Conclusion: There is no early evidence to substantiate possible involvement of RPS26, RPS27, and RPL32 genes in NPC tumourigenesis. PMID:23613646

  19. Expression of Prokaryotic Integral Membrane Proteins in E. coli.

    PubMed

    Love, James D

    2017-01-01

    Production of prokaryotic membrane proteins for structural and functional studies in E. coli can be parallelized and miniaturized. All stages from cloning, expression, purification to detergent selection can be investigated using high-throughput techniques to rapidly and economically find tractable targets.

  20. Computational codon optimization of synthetic gene for protein expression

    PubMed Central

    2012-01-01

    Background The construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression. Results In this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU. Conclusions The proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology. PMID:23083100

  1. Expression and detection of LINE-1 ORF-encoded proteins.

    PubMed

    Dai, Lixin; LaCava, John; Taylor, Martin S; Boeke, Jef D

    2014-01-01

    LINE-1 (L1) elements are endogenous retrotransposons active in mammalian genomes. The L1 RNA is bicistronic, encoding two non-overlapping open reading frames, ORF1 and ORF2, whose protein products (ORF1p and ORF2p) bind the L1 RNA to form a ribonucleoprotein (RNP) complex that is presumed to be a critical retrotransposition intermediate. However, ORF2p is expressed at a significantly lower level than ORF1p; these differences are thought to be controlled at the level of translation, due to a low frequency ribosome reinitiation mechanism controlling ORF2 expression. As a result, while ORF1p is readily detectable, ORF2p has previously been very challenging to detect in vitro and in vivo. To address this, we recently tested several epitope tags fused to the N- or C-termini of the ORF proteins in an effort to enable robust detection and affinity purification from native (L1RP) and synthetic (ORFeus-Hs) L1 constructs. An analysis of tagged RNPs from both L1RP and ORFeus-Hs showed similar host-cell-derived protein interactors. Our observations also revealed that the tag sequences affected the retrotransposition competency of native and synthetic L1s differently although they encode identical ORF proteins. Unexpectedly, we observed apparently stochastic expression of ORF2p within seemingly homogenous L1-expressing cell populations.

  2. Binary and ternary combinations of anti-HIV protease inhibitors: effect on gene expression and functional activity of CYP3A4 and efflux transporters

    PubMed Central

    Kwatra, Deep; Vadlapudi, Aswani Dutt; Vadlapatla, Ramya Krishna; Khurana, Varun; Pal, Dhananjay; Mitra, Ashim K.

    2015-01-01

    Background The purpose of this study is to identify the effect of binary and ternary combinations of anti-HIV protease inhibitors (PIs) on the expression of metabolizing enzyme (CYP3A4) and efflux transporters [multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP)] in a model intestinal cell line (LS-180). Methods LS-180 cells were treated with various combinations of PIs (amprenavir, indinavir, saquinavir and lopinavir), and the mRNA expression levels of metabolizing enzyme and efflux transporters were measured using quantitative reverse transcription polymerase chain reaction. The alteration of gene expression was further correlated to the expression of nuclear hormone receptor PXR. Uptake of fluorescent and radioactive substrates was carried out to study the functional activity of these proteins. Cytotoxicity and adenosine triphosphate (ATP) assays were carried out to measure stress responses. Results Binary and ternary combinations of PIs appeared to modulate the expression of CYP3A4, MRP2, P-gp and BCRP in a considerable manner. Unlike the individual PIs, their binary combinations showed much greater induction of metabolizing enzyme and efflux proteins. However, such pronounced induction was not observed in the presence of ternary combinations. The observed trend of altered mRNA expression was found to correlate well with the change in expression levels of PXR. The gene expression was found to correlate with activity assays. Lack of cytotoxicity and ATP activity was observed in the treatment samples, suggesting that these alterations in expression levels were probably not stress responses. Conclusions In the present study, we demonstrated that combinations of drugs can have serious consequences toward the treatment of HIV infection by altering their bioavailability and disposition. PMID:24399676

  3. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  4. The expression of metabolism-related proteins in phyllodes tumors.

    PubMed

    Kwon, Ji Eun; Jung, Woo-Hee; Koo, Ja Seung

    2013-02-01

    The purpose of this study was to investigate the association between the expression of hypoxia-inducible factor (HIF)-1α, insulin-like growth factor (IGF)-1, glucose transporter 1 (Glut-1), carbonic anhydrase IX (CAIX), and monocarboxylate transporter (MCT)4, which are metabolism-related proteins in phyllodes tumors (PTs), and clinicopathologic factors and its implication. We used tissue microarrays to analyze 207 PTs and performed immunohistochemical staining against the glycolysis-related molecules HIF-1α, IGF-1, Glut-1, CAIX, and MCT4. We then compared the immunohistochemical results and clinicopathologic parameters. The expressions of HIF-1α, Glut-1, CAIX, and MCT4 in the stromal component of PTs increased (P = 0.019, P < 0.001, P = 0.045, and P < 0.001, respectively) with increasing tumor grade. According to univariate analysis, factors associated with shorter disease-free survival were Glut-1 expression (P = 0.001) and MCT4 expression (P < 0.001) in the stromal component, and the factors associated with shorter overall survival were IGF-1 expression (P = 0.012), Glut-1 expression (P < 0.001), CAIX expression (P = 0.039), and MCT4 expression (P < 0.001) in the stromal component. Our investigation of stromal expression of the metabolism-related proteins HIF-1α, IGF-1, Glut-1, CAIX, and MCT4 revealed that, as the PT grade increased, the stromal expression of HIF-1α, Glut-1, CAIX, and MCT4 significantly increased. This result suggested that increasing PT grade is associated with increased glycolysis in the stromal component.

  5. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

    Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

  6. Heterologous expression of membrane proteins: choosing the appropriate host.

    PubMed

    Bernaudat, Florent; Frelet-Barrand, Annie; Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. © 2011 Bernaudat et al.

  7. Spica prunellae and its marker compound rosmarinic acid induced the expression of efflux transporters through activation of Nrf2-mediated signaling pathway in HepG2 cells.

    PubMed

    Wu, Jinjun; Zhu, Yuanfeng; Li, Fangyuan; Zhang, Guiyu; Shi, Jian; Ou, Rilan; Tong, Yunli; Liu, Yuting; Liu, Liang; Lu, Linlin; Liu, Zhongqiu

    2016-12-04

    Spica prunellae (SP) is a well-known traditional Chinese medicinal herb with properties of antihypertensive, antihyperglycemic, antiviral, anti-inflammatory, and antitumor activities. This herb is also popularly consumed as a food additive in some drinks or other food forms for treating pyreticosis. Rosmarinic acid (RA) is the marker compound from SP, which possesses anti-oxidative and anti-inflammatory functions. This study aims to investigate the regulatory effect of the water extract of SP (WESP) and RA on efflux transports (ETs), including P-glycoprotein (p-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) in HepG2 cell line. Results would provide beneficial information for the proper application of SP in clinics. HepG2 cells were treated with different doses of the tested drugs for 24 or 96h. MTT assay was used to examine cell viability. The protein and mRNA levels of the ETs were measured by using Western blot and real-time PCR, respectively. Reporter assay was used to study the antioxidant response element (ARE)-luciferin activity by using HepG2-C8 cells, which were generated by transfecting plasmid containing ARE-luciferin gene into HepG2 cells. The transport activities of ETs were tested by using substrate probes. WESP significantly (p<0.05) increased the expression of ETs in a dose-dependent manner. The increase caused by WESP was stronger than RA alone. Both WESP and RA promoted the translocation of nuclear factor E2-related factor-2 (Nrf2) from cytoplasm to the nucleus as well as significantly (p<0.05) enhanced the ARE-luciferin activity. WESP and RA also enhanced the efflux activity of P-gp and MRP2, accompanied by marked increase (p<0.05) in the intracellular ATP levels. WESP could significantly induce the expression of ETs through the activation of Nrf2-mediated signaling pathway in HepG2 cells. RA could be one of the active compounds responsible for the induction. WESP and RA also enhanced the efflux

  8. Optimization of translation profiles enhances protein expression and solubility.

    PubMed

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  9. A characterization of structural proteins expressed by Bombyx mori bidensovirus.

    PubMed

    Lü, Peng; Xing, Yali; Hu, Zhaoyang; Yang, Yanhua; Pan, Ye; Chen, Kangmin; Zhu, Feifei; Zhou, Yajing; Chen, Keping; Yao, Qin

    2017-03-01

    Bombyx mori bidensiovirus (BmBDV) is a species of Bidensovirus that has been was placed into a new genus within the new family Bidnaviridae by the International Committee on Taxonomy of Viruses. BmBDV causes fatal flacherie disease in silkworms, which causes large losses to the sericulture industry. BmBDV contains two sets of complementary linear single-stranded DNAs of approximately 6.5kb (viral DNA 1, VD1) and 6.0kb (viral DNA 2, VD2). VD1 and VD2 are encapsidated in separate icosahedral non-enveloped capsids, which are similar in size and shape. However, the strategies used to express BmBDV structural proteins remains unclear. In this work, a total of six structural proteins were separated by two-dimensional electrophoresis and shown to be encoded by the BmBDV VP gene via mass spectrometry. The transmission electron microscopy results showed that co-expression of the BmBDV VP and SP structural proteins in Spodoptera frugiperda sf9 cells resulted in the formation of 22-24nm virus-like particles. Furthermore, a mutation of the major structural protein-encoding VP gene, in which the second in-frame ATG codon was mutated to GCG, abrogated the production of several structural proteins, indicating that this strategy of expressing BmBDV VP is dependent on a leaky scanning translation mechanism.

  10. Optimization of Translation Profiles Enhances Protein Expression and Solubility

    PubMed Central

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5’-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein. PMID:25965266

  11. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  12. Comparison of 3 assay systems using a common probe substrate, calcein AM, for studying P-gp using a selected set of compounds.

    PubMed

    Szerémy, Péter; Pál, Akos; Méhn, Dóra; Tóth, Beáta; Fülöp, Ferenc; Krajcsi, Péter; Herédi-Szabó, Krisztina

    2011-01-01

    The multidrug resistance protein 1 (MDR1) transporter is the most abundantly investigated adenosine triphosphate (ATP)-Binding Cassette (ABC) transporter protein. Multiple assay systems were developed to study MDR1-mediated transport and possible drug-drug interactions. Yet, as different probe substrates are used in these assays, it is difficult to directly compare the results. In this study, a common probe substrate was applied in 3 assay systems developed to study MDR1: the cellular dye efflux assay, the ATPase assay, and the vesicular transport assay. This probe substrate is calcein acetoxymethyl ester (calcein AM), the acetoxymethyl ester derivative of the fluorescent dye, calcein. Using a common probe allows the investigation of the effect of passive permeability on the result obtained by testing various compounds. In this study, 22 compounds with different logP values were tested in the above-mentioned 3 assay systems. The vesicular transport assay proved most sensitive, detecting 18 of 22 interactions with the protein. The ATPase assay detected 15 interactions, whereas the cellular dye efflux assay was the least sensitive with only 10 hits. A correlation was found between the hydrophobicity of the compound and the ratio of cellular and vesicular transport IC(50) values, indicating the effect of passive permeability on the result. Based on hydrophobicity, the current study provides guidelines on applying the most correct tool for studying MDR1 interactions.

  13. Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

    PubMed Central

    Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

    2012-01-01

    Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

  14. Expression of glutamine metabolism-related proteins in thyroid cancer

    PubMed Central

    Kim, Hye Min; Lee, Yu Kyung; Koo, Ja Seung

    2016-01-01

    Purpose This study aimed to investigate the expression of glutamine metabolism-related protein in tumor and stromal compartments among the histologic subtypes of thyroid cancer. Results GLS1 and GDH expression in tumor and stromal compartments were the highest in AC than in other subtypes. Tumoral ASCT2 expression was higher in MC but lower in FC (p < 0.001). In PTC, tumoral GLS1 and tumoral GDH expression was higher in the conventional type than in the follicular variant (p = 0.043 and 0.001, respectively), and in PTC with BRAF V600E mutation than in PTC without BRAF V600E mutation (p<0.001). Stromal GDH positivity was the independent factor associated with short overall survival (hazard ratio: 21.48, 95% confidence interval: 2.178-211.8, p = 0.009). Methods We performed tissue microarrays with 557 thyroid cancer cases (papillary thyroid carcinoma [PTC]: 344, follicular carcinoma [FC]: 112, medullary carcinoma [MC]: 70, poorly differentiated carcinoma [PDC]: 23, and anaplastic carcinoma [AC]: 8) and 152 follicular adenoma (FA) cases. We performed immunohistochemical staining of glutaminolysis-related proteins (glutaminase 1 [GLS1], glutamate dehydrogenase [GDH], and amino acid transporter-2 [ASCT-2]). Conclusion Glutamine metabolism-related protein expression differed among the histologic subtypes of thyroid cancer. PMID:27447554

  15. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    SciTech Connect

    Raymond, Amy; Lovell, Scott; Lorimer, Don; Walchli, John; Mixon, Mark; Wallace, Ellen; Thompkins, Kaitlin; Archer, Kimberly; Burgin, Alex; Stewart, Lance

    2009-12-01

    With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  16. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

  17. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

    PubMed Central

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-01-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  18. Dark proteins: effect of inclusion body formation on quantification of protein expression.

    PubMed

    Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R

    2008-09-01

    Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used

  19. Methods and constructs for expression of foreign proteins in photosynthetic organisms

    DOEpatents

    Laible, Philip D.; Hanson, Deborah K.

    2002-01-01

    A method for expressing and purifying foreign proteins in photosynthetic organisms comprising the simultaneous expression of both the heterologous protein and a means for compartmentalizing or sequestering of the protein.

  20. Transient expression and cellular localization of recombinant proteins in cultured insect cells

    USDA-ARS?s Scientific Manuscript database

    Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

  1. Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

    PubMed

    Sawatsky, Bevan; Bente, Dennis A; Czub, Markus; von Messling, Veronika

    2016-05-01

    The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

  2. Grape seed extract inhibits VEGF expression via reducing HIF-1alpha protein expression.

    PubMed

    Lu, Jianming; Zhang, Keqiang; Chen, Shiuan; Wen, Wei

    2009-04-01

    Grape seed extract (GSE) is a widely consumed dietary supplement that has antitumor activity. Here, we have investigated the inhibitory effect of GSE on the expression of vascular endothelial growth factor (VEGF) and the mechanism underlying this action. We found that GSE inhibited VEGF messenger RNA (mRNA) and protein expression in U251 human glioma cells and MDA-MB-231 human breast cancer cells. GSE inhibited transcriptional activation of the VEGF gene through reducing protein but not mRNA expression of hypoxia-inducible factor (HIF) 1alpha. The inhibitory effect of GSE on HIF-1alpha expression was mainly through inhibiting HIF-1alpha protein synthesis rather than promoting protein degradation. Consistent with this result, GSE-suppressed phosphorylation of several important components involved in HIF-1alpha protein synthesis, such as Akt, S6 kinase and S6 protein. Furthermore, in the MDA-MB-231 tumor, we found that GSE treatment inhibited the expression of VEGF and HIF-1alpha and the phosphorylation of S6 kinase without altering the subcellular localization of HIF-1alpha, correlating with reduced vessel density and tumor size. Depletion of polyphenol with polyvinylpyrrolidone abolished the inhibitory activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the inhibitory activity. Taken together, our results indicate that GSE inhibits VEGF expression by reducing HIF-1alpha protein synthesis through blocking Akt activation. This finding provides new insight into the mechanisms of anticancer activity of GSE and reveals a novel molecular mechanism underlying the antiangiogenic action of GSE.

  3. Grape seed extract inhibits VEGF expression via reducing HIF-1α protein expression

    PubMed Central

    Lu, Jianming; Zhang, Keqiang; Chen, Shiuan; Wen, Wei

    2009-01-01

    Grape seed extract (GSE) is a widely consumed dietary supplement that has antitumor activity. Here, we have investigated the inhibitory effect of GSE on the expression of vascular endothelial growth factor (VEGF) and the mechanism underlying this action. We found that GSE inhibited VEGF messenger RNA (mRNA) and protein expression in U251 human glioma cells and MDA-MB-231 human breast cancer cells. GSE inhibited transcriptional activation of the VEGF gene through reducing protein but not mRNA expression of hypoxia-inducible factor (HIF) 1α. The inhibitory effect of GSE on HIF-1α expression was mainly through inhibiting HIF-1α protein synthesis rather than promoting protein degradation. Consistent with this result, GSE-suppressed phosphorylation of several important components involved in HIF-1α protein synthesis, such as Akt, S6 kinase and S6 protein. Furthermore, in the MDA-MB-231 tumor, we found that GSE treatment inhibited the expression of VEGF and HIF-1α and the phosphorylation of S6 kinase without altering the subcellular localization of HIF-1α, correlating with reduced vessel density and tumor size. Depletion of polyphenol with polyvinylpyrrolidone abolished the inhibitory activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the inhibitory activity. Taken together, our results indicate that GSE inhibits VEGF expression by reducing HIF-1α protein synthesis through blocking Akt activation. This finding provides new insight into the mechanisms of anticancer activity of GSE and reveals a novel molecular mechanism underlying the antiangiogenic action of GSE. PMID:19131542

  4. [Cloning, prokaryotic expression of cattle Ghrelin gene and biological activity detection of the expressed protein].

    PubMed

    Zhang, Ailing; Zhang, Li; Chen, Hong; Zhang, Liangzhi; Lan, Xianyong; Zhang, Chunlei; Zhang, Cunfang; Zhu, Zeyi

    2009-01-01

    The cDNA of cattle Ghrelin gene was amplified from abomasum fundic gland mRNA of Qinchuan Cattle by RT-PCR. PCR product was cloned into the T vector pGM-T to construct pGh-T1 for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET32a (+) and transformed into host Escherichia coli strain BL21 (DE3) for expression. The expression of pGh-32 mature Ghrelin protein was induced by IPTG and was identified by SDS-PAGE. The expression product was observed with soluble protein and inclusion body. Western blotting showed that the recombinant protein was recognized by his-antibody specifically. The protein was purified by Ni-NTA column and was used to inject rabbits to obtain polyclona antibody. ELISA result showed that the antibody titer was 1:12 800. The immunohistochemistry test between the hypothalamus arcuate nucleus and the antibody showed that fusion protein had biological activity. This will provide a basis for further study on the biological function of Ghrelin protein to growth and development and fat deposition of cattle.

  5. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  6. Morphine Withdrawal Modifies Prion Protein Expression in Rat Hippocampus

    PubMed Central

    Mattei, Vincenzo; Martellucci, Stefano; Santilli, Francesca; Manganelli, Valeria; Garofalo, Tina; Candelise, Niccolò; Caruso, Alessandra; Sorice, Maurizio; Scaccianoce, Sergio

    2017-01-01

    The hippocampus is a vulnerable brain structure susceptible to damage during aging and chronic stress. Repeated exposure to opioids may alter the brain so that it functions normally when the drugs are present, thus, a prolonged withdrawal might lead to homeostatic changes headed for the restoration of the physiological state. Abuse of morphine may lead to Reacting Oxygen Species-induced neurodegeneration and apoptosis. It has been proposed that during morphine withdrawal, stress responses might be responsible, at least in part, for long-term changes of hippocampal plasticity. Since prion protein is involved in both, Reacting Oxygen Species mediated stress responses and synaptic plasticity, in this work we investigate the effect of opiate withdrawal in rats after morphine treatment. We hypothesize that stressful stimuli induced by opiate withdrawal, and the subsequent long-term homeostatic changes in hippocampal plasticity, might modulate the Prion protein expression. Our results indicate that abstinence from the opiate induced a time-dependent and region-specific modification in Prion protein content, indeed during morphine withdrawal a selective unbalance of hippocampal Prion Protein is observable. Moreover, Prion protein overexpression in hippocampal tissue seems to generate a dimeric structure of Prion protein and α-cleavage at the hydrophobic domain. Stress factors or toxic insults can induce cytosolic dimerization of Prion Protein through the hydrophobic domain, which in turn, it stimulates the α-cleavage and the production of neuroprotective Prion protein fragments. We speculate that this might be the mechanism by which stressful stimuli induced by opiate withdrawal and the subsequent long-term homeostatic changes in hippocampal plasticity, modulate the expression and the dynamics of Prion protein. PMID:28081197

  7. Expression and Localization of Plant Protein Disulfide Isomerase.

    PubMed Central

    Shorrosh, B. S.; Subramaniam, J.; Schubert, K. R.; Dixon, R. A.

    1993-01-01

    A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules. PMID:12231974

  8. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    PubMed Central

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

  9. The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo

    PubMed Central

    Hostettler, Lola; Grundy, Laura; Käser-Pébernard, Stéphanie; Wicky, Chantal; Schafer, William R.; Glauser, Dominique A.

    2017-01-01

    The Green Fluorescent Protein (GFP) has been tremendously useful in investigating cell architecture, protein localization, and protein function. Recent developments in transgenesis and genome editing methods now enable working with fewer transgene copies and, consequently, with physiological expression levels. However, lower signal intensity might become a limiting factor. The recently developed mNeonGreen protein is a brighter alternative to GFP in vitro. The goal of the present study was to determine how mNeonGreen performs in vivo in Caenorhabditis elegans—a model used extensively for fluorescence imaging in intact animals. We started with a side-by-side comparison between cytoplasmic forms of mNeonGreen and GFP expressed in the intestine, and in different neurons, of adult animals. While both proteins had similar photostability, mNeonGreen was systematically 3–5 times brighter than GFP. mNeonGreen was also used successfully to trace endogenous proteins, and label specific subcellular compartments such as the nucleus or the plasma membrane. To further demonstrate the utility of mNeonGreen, we tested transcriptional reporters for nine genes with unknown expression patterns. While mNeonGreen and GFP reporters gave overall similar expression patterns, low expression tissues were detected only with mNeonGreen. As a whole, our work establishes mNeonGreen as a brighter alternative to GFP for in vivo imaging in a multicellular organism. Furthermore, the present research illustrates the utility of mNeonGreen to tag proteins, mark subcellular regions, and describe new expression patterns, particularly in tissues with low expression. PMID:28108553

  10. Modular Protein Expression Toolbox (MoPET), a standardized assembly system for defined expression constructs and expression optimization libraries

    PubMed Central

    Birkenfeld, Jörg; Franz, Jürgen; Gritzan, Uwe; Linden, Lars; Trautwein, Mark

    2017-01-01

    The design and generation of an optimal expression construct is the first and essential step in in the characterization of a protein of interest. Besides evaluation and optimization of process parameters (e.g. selection of the best expression host or cell line and optimal induction conditions and time points), the design of the expression construct itself has a major impact. However, the path to this final expression construct is often not straight forward and includes multiple learning cycles accompanied by design variations and retesting of construct variants, since multiple, functional DNA sequences of the expression vector backbone, either coding or non-coding, can have a major impact on expression yields. To streamline the generation of defined expression constructs of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed. This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules with a minimal cloning burden. Combining these features with a standardized cloning strategy facilitates the identification of optimized DNA expression constructs in shorter time. The MoPET system currently consists of 53 defined DNA modules divided into eight functional classes and can be flexibly expanded. However, already with the initial set of modules, 792,000 different constructs can be rationally designed and assembled. Furthermore, this starting set was used to generate small and mid-sized combinatorial expression optimization libraries. Applying this screening approach, variants with up to 60-fold expression improvement have been identified by MoPET variant library screening. PMID:28520717

  11. Tools to cope with difficult-to-express proteins.

    PubMed

    Saccardo, Paolo; Corchero, José Luís; Ferrer-Miralles, Neus

    2016-05-01

    The identification of DNA coding sequences contained in the genome of many organisms coupled to the use of high throughput approaches has fueled the field of recombinant protein production. Apart from basic research interests, the growing relevance of this field is highlighted by the global sales of the top ten biopharmaceuticals on the market, which exceeds the trillion USD in a steady increasing tendency. Therefore, the demand of biological compounds seems to have a long run on the market. One of the most popular expression systems is based on Escherichia coli cells which apart from being cost-effective counts with a large selection of resources. However, a significant percentage of the genes of interest are not efficiently expressed in this system, or the expressed proteins are accumulated within aggregates, degraded or lacking the desired biological activity, being finally discarded. In some instances, expressing the gene in a homologous expression system might alleviate those drawbacks but then the process usually increases in complexity and is not as cost-effective as the prokaryotic systems. An increasing toolbox is available to approach the production and purification of those difficult-to-express proteins, including different expression systems, promoters with different strengths, cultivation media and conditions, solubilization tags and chaperone coexpression, among others. However, in most cases, the process follows a non-integrative trial and error strategy with discrete success. This review is focused on the design of the whole process by using an integrative approach, taken into account the accumulated knowledge of the pivotal factors that affect any of the key processes, in an attempt to rationalize the efforts made in this appealing field.

  12. Competitive method-based electrochemiluminescent assay with protein-nucleotide conversion for ratio detection to efficiently monitor the drug resistance of cancer cells.

    PubMed

    Liang, Wen-Bin; Yang, Ming-Zhen; Zhuo, Ying; Zheng, Ying-Ning; Xiong, Cheng-Yi; Chai, Ya-Qin; Yuan, Ruo

    2016-12-01

    A simple and highly-efficient approach to monitor the expression of P-glycoprotein (P-gp) in cells was urgently needed to demonstrate the drug resistance of cancer cells. Herein, a competitive method-based electrochemiluminescent (ECL) assay with a single ECL indicator was proposed for the first time to efficiently estimate the concentration ratio of two proteins. By converting the different proteins to partially coincident nucleotide sequences via a sandwich type immunoassay on magnetic beads, the concentration ratio related ECL signals could be obtained via competitive nucleotide hybridization on an electrode surface. This method could thoroughly overcome the limitations of simultaneous ECL assays via multiple ECL indicators with inevitable cross reactions. At the same time, rolling circle amplification was employed to improve the detection performances, especially the detection limit and sensitivity. With P-gp and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a model, the proposed ECL assay was successfully employed to monitor the drug resistance of cancer cells. Compared with conventional technologies, improved sensitivity and accuracy were achieved with a correlation coefficient of 0.9928 and a detection limit of 0.52%. Success in the establishment of the competitive method-based ECL assay offered an efficient strategy to demonstrate the concentration ratio of two proteins and a potential approach for detecting other proteins and nucleotide sequences, revealing a new avenue for ultrasensitive biomolecule diagnostics, especially in cell function research.

  13. Expression of low molecular weight proteins in patients with leukaemia.

    PubMed

    Sheikh, N; Abid, R; Qureshi, A W; Basheer, T

    2012-06-01

    The current study is conducted to observe the differences in the level of low molecular weight proteins in the sera of patients with leukaemia in comparison to healthy subjects (control group). The sera of patients with leukaemia showed 15 peaks in the densitometric curve in comparison to the seven peaks of the controls. The peaks in the experimental samples that coincide with those in the control were of 134.14, 113.15, 76.06, 63.25, 48.07, 22.85 and 16.47 kDa molecular weights, respectively. Most of the new peaks appeared between the proteins of molecular weight 36-29 kDa in the experimental groups. Mean density of the 134.14 kDa protein band showed an increase in the protein in experimental groups I and II only whereas 113.15 and 22.85 kDa protein were increased in all experimental groups of patients with leukaemia. The expression of 76.06 and 63.25 kDa protein fraction was downregulated in the patients with leukaemia. A decline in the level of the protein of 48.07 kDa was observed in patients with leukaemia except in group I. Unlike the other protein fractions, the level of the protein of 16.47 kDa was significantly (p < 0.05) increased with a maximum density in group II. Intergroup experimental) comparison revealed an increasing pattern of 95.44 and 89.21 kDa with maximum level in group III sera. However the protein fractions of 38.07 and 34.94 kDa varied in the serum with maximum density in Group IV Protein fractions of 32.92 and 31.24 kDa were expressed in all age groups of patients with leukaemia with a maximum density in group III whereas the percentage densities of 14.42 and 13.56 kDa protein were quite different. This preliminary study will provide a basis to study the role of different proteins in patients with leukaemia.

  14. Expression Screening of Fusion Partners from an E. coli Genome for Soluble Expression of Recombinant Proteins in a Cell-Free Protein Synthesis System

    PubMed Central

    Kim, Dong-Myung

    2011-01-01

    While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with aggregation-prone heterologous proteins and then directly expressed for quantitative estimation of the expression efficiency of soluble translation products. Through two-step screening procedures involving the expression of 552 fusion constructs targeted against a series of cytokine proteins, we were able to discover a number of endogenous E. coli proteins that dramatically enhanced the soluble expression of the target proteins. This strategy of cell-free expression screening can be extended to quantitative, global analysis of genomic resources for various purposes. PMID:22073212

  15. Expression of β-amyloid precursor protein in refractory epilepsy.

    PubMed

    Sima, Xiutian; Xu, Jianguo; Li, Jinmei; Zhong, Weiying; You, Chao

    2014-04-01

    β-amyloid precursor protein (β-APP), also known as Aβ peptide, has a key role in the pathogenesis of Alzheimer's disease, and is also likely to be involved in the development of refractory epilepsy. The mechanism behind the association between β-APP and refractory epilepsy remains to be elucidated. The aim of the present study was to examine the levels of APP mRNA and β-APP protein in patients with refractory epilepsy. Tissue samples were obtained from patients with chronic pharmacoresistant epilepsy who underwent surgery. Levels of APP mRNA and β-APP protein in epileptic temporal lobe and hippocampal tissue were assessed using quantitative polymerase chain reaction, immunohistochemistry and immunofluorescence. The expression levels of protein significantly increased in the temporal cortex and the hippocampus of the patients with epilepsy. β-APP may thus contribute to the pathogenesis of refractory epilepsy.

  16. Expression and serological reactivity of hemorrhagic enteritis virus hexon protein.

    PubMed

    Lobová, Dana; Celer, Vladimír

    2016-05-01

    The aim of this work was to express the recombinant hexon protein of the hemorrhagic enteritis virus, to establish the diagnostic value of this protein for serological detection of antibodies in turkey serum samples and to assess seroprevalence of the infection in the Czech Republic. The N' terminal part of the hexon protein was expressed in a bacterial expression system and used as an antigen in an ELISA test for the detection of hemorrhagic enteritis virus specific antibodies in turkey sera. Validation of the test was performed by comparison with a commercially available ELISA test. Serological reactivity was assessed on a panel of 126 turkey sera by a newly developed ELISA test. Serum samples were taken from turkey farms with the history of hemorrhagic enteritis virus infection, from farms with animals free of infection, and from turkey farms following vaccination. Both ELISA kits gave identical results (100 %) with the tested sera. ELISA based on the recombinant hexon protein thus proved useful and cheaper for detection of antibodies in turkey flocks infected with the hemorrhagic enteritis virus.

  17. Prion protein expression in bovine podocytes and extraglomerular mesangial cells.

    PubMed

    Amselgruber, W M; Steffl, M; Didier, A; Märtlbauer, E; Pfaff, E; Büttner, M

    2006-06-01

    The cellular form of the prion protein (PrP(c)) is thought to be a substrate for an abnormal isoform of the prion protein (PrP(sc)). One emerging hypothesis is that the proposed conversion phenomenon takes place at the site at which the infectious agent meets PrP(c). PrP(c) is abundant in the central nervous system, but little is known about the cell-type-specific distribution of PrP(c) in non-neuronal tissues of cattle. We have studied whether PrP(c), a protein found predominantly in neurons, also exists in bovine podocytes, since neurons and podocytes share a large number of similarities. We have therefore examined the expression of PrP(c) by immunohistochemistry, reverse transcription/polymerase chain reaction and enzyme-linked immunosorbent analysis. Immunostained serial sections and specific antibodies against PrP(c) have revealed that PrP(c) is selectively localized in podocytes and is particularly strongly expressed in extraglomerular mesangial cells but not in endothelial or intraglomerular mesangial cells. The selective expression of PrP(c) in podocytes is of special importance, as it suggests that these cells represent possible targets for peripheral infection with prions and demonstrates that PrP(c) can be added to the list of neuronal factors expressed in mammalian podocytes.

  18. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  19. Protein Phosphatase-1 Regulates Expression of Neuregulin-1

    PubMed Central

    Ammosova, Tatiana; Washington, Kareem; Rotimi, Jamie; Kumari, Namita; Smith, Kahli A.; Niu, Xiaomei; Jerebtsova, Marina; Nekhai, Sergei

    2016-01-01

    Protein phosphatase 1 (PP1), a cellular serine/threonine phosphatase, is targeted to cellular promoters by its major regulatory subunits, PP1 nuclear targeting subunit, nuclear inhibitor of PP1 (NIPP1) and RepoMan. PP1 is also targeted to RNA polymerase II (RNAPII) by NIPP1 where it can dephosphorylate RNAPII and cycle-dependent kinase 9 (CDK9). Here, we show that treatment of cells with a small molecule activator of PP1 increases the abundance of a neuregulin-1 (NRG-1)-derived peptide. NRG-1 mRNA and protein levels were increased in the cells stably or transiently expressing mutant NIPP1 (mNIPP1) that does not bind PP1, but not in the cells expressing NIPP1. Expression of mNIPP1 also activated the NRG-1 promoter in an NF-κB-dependent manner. Analysis of extracts from mNIPP1 expressing cells by glycerol gradient centrifugation showed a redistribution of PP1 and CDK9 between large and small molecular weight complexes, and increased CDK9 Thr-186 phosphorylation. This correlated with the increased CDK9 activity. Further, RNAPII co-precipitated with mNIPP1, and phosphorylation of RNAPII C-terminal domain (CTD) Ser-2 residues was greater in cells expressing mNIPP1. In mNIPP1 expressing cells, okadaic acid, a cell-permeable inhibitor of PP1, did not increase Ser-2 CTD phosphorylation inhibited by flavopiridol, in contrast to the NIPP1 expressing cells, suggesting that PP1 was no longer involved in RNAPII dephosphorylation. Finally, media conditioned with mNIPP1 cells induced the proliferation of wild type 84-31 cells, consistent with a role of neuregulin-1 as a growth promoting factor. Our study indicates that deregulation of PP1/NIPP1 holoenzyme activates NRG-1 expression through RNAPII and CDK9 phosphorylation in a NF-κB dependent manner. PMID:27918433

  20. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    SciTech Connect

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  1. Tissue-Specific Protein Expression in Plant Mitochondria.

    PubMed Central

    Conley, C. A.; Hanson, M. R.

    1994-01-01

    Although the physiological role of plant mitochondria is thought to vary in different tissues at progressive stages of development, there has been little documentation that the complement of mitochondrial proteins is altered in different plant organs. Because the phenomenon of cytoplasmic male sterility suggests an unusual function for mitochondria in floral buds, we examined the tissue-specific expression of mitochondrial proteins in petunia buds at several stages of development, using both fertile and cytoplasmic male sterile plants. On tissue prints of cryostat-sectioned buds, antibodies recognizing subunit A of the mitochondrial ATPase (ATPA) localized very differently from antibodies recognizing subunit II of the cytochrome oxidase (COXII), which indicated that mitochondria in the same tissue could differentially express mitochondrially encoded proteins. The petunia cytoplasmic male sterility-associated fused (pcf) gene encodes a protein that colocalized with ATPA and the nuclear-encoded mitochondrial alternative oxidase (AOA) in sporogenous tissues, where little COXII protein was found. These overlapping and differential localization patterns may provide clues to the molecular mechanism of cytoplasmic male sterility. PMID:12244222

  2. Eosinophil granule proteins expressed in ocular cicatricial pemphigoid

    PubMed Central

    Heiligenhaus, A.; Schaller, J.; Mauss, S.; Engelbrecht, S.; Dutt, J.; Foster, C; Steuhl, K.

    1998-01-01

    BACKGROUND—Blister formation and tissue damage in bullous pemphigoid have been attributed to the release of eosinophil granule proteins—namely, to eosinophil derived cationic protein (ECP) and major basic protein (MBP). In the present investigation these eosinophil granule proteins were studied in the conjunctiva of patients with ocular cicatricial pemphigoid (OCP).
METHODS—Conjunctival biopsy specimens obtained from patients with subacute (n=8) or chronic conjunctival disease (n=13) were analysed histologically and immunohistochemically using antibodies directed against EG1 (stored and secreted ECP), EG2 (secreted ECP), MBP, CD45 (common leucocyte antigen), CD3 (pan T cell marker), and HLA-DR (class II antigen).
RESULTS—Subepithelial mononuclear cells, mast cells, and neutrophils were detected in all specimens. The number of mononuclear cells, neutrophils, CD45+ cells, CD3+ cells, and the HLA-DR expression were significantly higher in the subacute than in the chronic disease group. Some eosinophils were found in specimens from five of eight patients with subacute OCP, but in none of the patients with chronic disease. The eosinophil granule proteins (ECP and MBP) were found in the epithelium and substantia propria in patients with subacute conjunctivitis.
CONCLUSIONS—Subepithelial cell infiltration in the conjunctiva greatly differs between subacute and chronic ocular cicatricial pemphigoid specimens. The findings suggest that eosinophil granule proteins may participate in tissue damage in acute phase of inflammation in OCP.

 Keywords: ocular cicatricial pemphigoid; conjunctivitis; eosinophil derived cationic protein; major basic protein PMID:9602632

  3. Somatostatin regulates tight junction proteins expression in colitis mice.

    PubMed

    Li, Xiao; Wang, Qian; Xu, Hua; Tao, Liping; Lu, Jing; Cai, Lin; Wang, Chunhui

    2014-01-01

    Tight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P<0.05); after octreotide treatment, claudin-1 and claudin-3 expression significantly increased compared with untreated CR infected mice (P<0.05). In DSS colitis model, occludin and claudin-3 expression significantly decreased compared with the control mice (P<0.05); and octreotide treatment could only significantly upregulate claudin-3 expression compared with untreated DSS colitis mice (P<0.05). To testify our results in vivo, we repeated the models in caco-2 cells by exposed with enteropathogenic Escherichia coli (E. Coli) and Tumor necrosis factor α (TNF-α). The results in vitro were consistent with in vivo study. The results suggested that somatostatin play a role in intestinal barrier protection by modulating tight junction proteins expression.

  4. Somatostatin regulates tight junction proteins expression in colitis mice

    PubMed Central

    Li, Xiao; Wang, Qian; Xu, Hua; Tao, Liping; Lu, Jing; Cai, Lin; Wang, Chunhui

    2014-01-01

    Tight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); after octreotide treatment, claudin-1 and claudin-3 expression significantly increased compared with untreated CR infected mice (P < 0.05). In DSS colitis model, occludin and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); and octreotide treatment could only significantly upregulate claudin-3 expression compared with untreated DSS colitis mice (P < 0.05). To testify our results in vivo, we repeated the models in caco-2 cells by exposed with enteropathogenic Escherichia coli (E. Coli) and Tumor necrosis factor α (TNF-α). The results in vitro were consistent with in vivo study. The results suggested that somatostatin play a role in intestinal barrier protection by modulating tight junction proteins expression. PMID:24966923

  5. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  6. Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns.

    PubMed

    Jiang, Guang-Jian; Wang, Ke; Miao, De-Qiang; Guo, Lei; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2011-01-01

    It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.

  7. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins.

  8. Epithelial membrane protein 1 expression in ovarian serous tumors.

    PubMed

    Demirag, Guzin Gonullu; Kefeli, Mehmet; Kemal, Yasemin; Yucel, Idris

    2016-03-01

    The present study aimed to analyze the clinical significance of epithelial membrane protein 1 (EMP1) expression in ovarian serous tumors. A total of 84 cases of ovarian serous tumor (50 patients with malignant ovarian serous tumors and 34 patients with borderline and benign serous tumors) were retrospectively analyzed. Differences in the expression levels of EMP1 between the malignant and non-malignant tumor groups were evaluated by immunohistochemical staining. In addition, the association between EMP1 expression and prognostic factors in malignant ovarian serous tumors was investigated. The expression levels of EMP1 were significantly reduced in all the 50 malignant ovarian serous tumors, compared with the 34 non-malignant ovarian serous tumors (P<0.000). Reduced expression of EMP1 was correlated with high grade (P=0.009) and stage (P<0.000) of malignant tumors. EMP1 expression was not observed to be correlated with any other investigated parameters, including surgery, type of operation and chemotherapy response (P>0.005). These results indicated that EMP1 may have a significant role as a negative regulator in ovarian serous tumors, and reduced EMP1 expression in serous tumors may be associated with increased disease severity.

  9. BMP-7 PROTEIN EXPRESSION IS DOWNREGULATED IN HUMAN DIABETIC NEPHROPATHY.

    PubMed

    Ivanac-Janković, Renata; Ćorić, Marijana; Furić-Čunko, Vesna; Lovičić, Vesna; Bašić-Jukić, Nikolina; Kes, Petar

    2015-06-01

    Bone morphogenetic protein-7 (BMP-7) is expressed in all parts of the normal kidney parenchyma, being highest in the epithelium of proximal tubules. It protects kidney against acute and chronic injury, inflammation and fibrosis. Diabetic nephropathy is the leading cause of chronic kidney disease, and is characterized by decreased expression of BMP-7. The aim of our study was to analyze whether the expression of BMP-7 is significantly changed in advanced stages of human diabetic nephropathy. Immunohistochemical analysis of the expression of BMP-7 was performed on archival material of 30 patients that underwent renal biopsy and had confirmed diagnosis of diabetic nephropathy. Results showed that BMP-7 was differently expressed in the cytoplasm of epithelial cells of proximal tubules and podocytes among all stages of diabetic nephropathy. At early stages of diabetic nephropathy, BMP-7 was strongly positive in proximal tubules and podocytes, while low expression was recorded in the majority of samples at advanced stages. In conclusion, increased expression of BMP-7 at initial stages of diabetic nephropathy with subsequent decrease at advanced stage highlights the role of BMP-7 in the protection of kidney structure and function. Further investigations should be focused on disturbances of BMP-7 receptors and signaling pathways in patients with diabetic nephropathy.

  10. Generation of transgenic dogs that conditionally express green fluorescent protein.

    PubMed

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

  11. Autophagy and lysosomal related protein expression patterns in human glioblastoma.

    PubMed

    Giatromanolaki, Alexandra; Sivridis, Efthimios; Mitrakas, Achileas; Kalamida, Dimitra; Zois, Christos E; Haider, Syed; Piperidou, Charitomeni; Pappa, Aglaia; Gatter, Kevin C; Harris, Adrian L; Koukourakis, Michael I

    2014-01-01

    Glioblastoma cells are resistant to apoptotic stimuli with autophagic death prevailing under cytotoxic stress. Autophagy interfering agents may represent a new strategy to test in combination with chemo-radiation. We investigated the patterns of expression of autophagy related proteins (LC3A, LC3B, p62, Beclin 1, ULK1 and ULK2) in a series of patients treated with post-operative radiotherapy. Experiments with glioblastoma cell lines (T98 and U87) were also performed to assess autophagic response under conditions simulating the adverse intratumoral environment. Glioblastomas showed cytoplasmic overexpression of autophagic proteins in a varying extent, so that cases could be grouped into low and high expression groups. 10/23, 5/23, 13/23, 5/23, 8/23 and 9/23 cases examined showed extensive expression of LC3A, LC3B, Beclin 1, Ulk 1, Ulk 2 and p62, respectively. Lysosomal markers Cathepsin D and LAMP2a, as well as the lyososomal biogenesis transcription factor TFEB were frequently overexpressed in glioblastomas (10/23, 11/23, and 10/23 cases, respectively). TFEB was directly linked with PTEN, Cathepsin D, HIF1α, LC3B, Beclin 1 and p62 expression. PTEN was also significantly related with LC3B but not LC3A expression, in both immunohistochemistry and gene expression analysis. Confocal microscopy in T98 and U87 cell lines showed distinct identity of LC3A and LC3B autophagosomes. The previously reported stone-like structure (SLS) pattern of LC3 expression was related with prognosis. SLS were inducible in glioblastoma cell lines under exposure to acidic conditions and 2DG mediated glucose antagonism. The present study provides the basis for autophagic characterization of human glioblastoma for further translational studies and targeted therapy trials.

  12. Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

    PubMed Central

    Johnston, Jonathan D; Price, Sally A; Bristow, David R

    1998-01-01

    Acute flunitrazepam (1 μM) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity. PMID:9723942

  13. Disposable bioreactors for inoculum production and protein expression.

    PubMed

    Eibl, Regine; Löffelholz, Christian; Eibl, Dieter

    2014-01-01

    Disposable bioreactors have been increasingly implemented over the past ten years. This relates to both R & D and commercial manufacture, in particular, in animal cell-based processes. Among the numerous disposable bioreactors which are available today, wave-mixed bag bioreactors and stirred bioreactors are predominant. Whereas wave-mixed bag bioreactors represent the system of choice for inoculum production, stirred systems are often preferred for protein expression. For this reason, the authors present protocols instructing the reader how to use the wave-mixed BIOSTAT CultiBag RM 20 L for inoculum production and the stirred UniVessel SU 2 L for recombinant protein production at benchtop scale. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing the human placental secreted alkaline phosphatase (SEAP).

  14. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2015-01-01

    Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins. PMID:17846637

  15. Expression data on liver metabolic pathway genes and proteins

    PubMed Central

    Raja Gopal Reddy, Mooli; Pavan Kumar, Chodisetti; Mahesh, Malleswarapu; Sravan Kumar, Manchiryala; Jeyakumar, Shanmugam M.

    2016-01-01

    Here, we present the expression data on various metabolic pathways of liver with special emphasize on lipid and carbohydrate metabolism and long chain polyunsaturated fatty acid (PUFA) synthesis, both at gene and protein levels. The data were obtained to understand the effect of vitamin A deficiency on the expression status (both gene and protein levels) of some of the key factors involved in lipogenesis, fatty acid oxidation, triglyceride secretion, long chain PUFA, resolvin D1 synthesis, glucose transport and glycogen synthesis of liver, using modern biology tools, such as quantitative real-time PCR (RT-PCR) and immunoblotting techniques. This data article provides the supporting evidence to the article “Vitamin A deficiency suppresses high fructose-induced triglyceride synthesis and elevates resolvin D1 levels” [1] and therefore, these data may be referred back, for comprehensive understanding and interpretations and for future studies. PMID:26909377

  16. Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

    PubMed

    Jung, Yuna; Jung, Hyeim; Lim, Dongbin

    2015-12-01

    Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli.

  17. Automated recombinant protein expression screening in Escherichia coli.

    PubMed

    Busso, Didier; Stierlé, Matthieu; Thierry, Jean-Claude; Moras, Dino

    2008-01-01

    To fit the requirements of structural genomics programs, new as well as classical methods have been adapted to automation. This chapter describes the automated procedure developed within the Structural Biology and Genomics Platform, Strasbourg for performing recombinant protein expression screening in Escherichia coli. The procedure consists of parallel competent cells transformation, cell plating, and liquid culture inoculation, implemented for up to 96 samples at a time.

  18. Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein.

    PubMed

    Rocha, Carolina S; Luz, Dirce F; Oliveira, Marli L; Baracat-Pereira, Maria C; Medrano, Francisco Javier; Fontes, Elizabeth P B

    2007-03-01

    The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of beta-strand structure (42%) over the alpha-helix (9.9%). The fluorescence emission maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K(d)) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K(d)=2.79+/-0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.

  19. Hippocampal expression of the calcium sensor protein visinin-like protein-1 in schizophrenia.

    PubMed

    Bernstein, Hans-Gert; Braunewell, Karl-Heinz; Spilker, Christina; Danos, Peter; Baumann, Bruno; Funke, Sieglinde; Diekmann, Silvia; Gundelfinger, Eckart D; Bogerts, Bernhard

    2002-03-25

    Hippocampal cytoarchitectural abnormalities may be part of the cerebral substrate of schizophrenia. Amongst the chemical components being abnormal in brains of schizophrenics are altered calcium concentrations and reduced expression of the neurotrophin receptor, trkB. We studied by immunohistochemical methods the distribution of visinin-like protein-1 (VILIP-1), which is a calcium sensor protein and at the same time a trkB mRNA binding protein, in hippocampi of nine schizophrenic patients and nine matched control subjects. In normal hippocampi VILIP-1 immunoreactivity was found in multiple pyramidal cells and interneurons. A portion of VILIP-1 immunoreactive interneurons co-express calretinin (60%) and parvalbumin (<10%). In schizophrenics fewer pyramidal cells but more interneurons were immunostained. Our data point to an involvement of the protein in the altered hippocampal circuitry in schizophrenia.

  20. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix

    PubMed Central

    Allen, Robert S.; Tilbrook, Kimberley; Warden, Andrew C.; Campbell, Peter C.; Rolland, Vivien; Singh, Surinder P.; Wood, Craig C.

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia. PMID:28316608

  1. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

    PubMed

    Allen, Robert S; Tilbrook, Kimberley; Warden, Andrew C; Campbell, Peter C; Rolland, Vivien; Singh, Surinder P; Wood, Craig C

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

  2. Expression and subcellular localization of a novel nuclear acetylcholinesterase protein.

    PubMed

    Santos, Susana Constantino Rosa; Vala, Inês; Miguel, Cláudia; Barata, João T; Garção, Pedro; Agostinho, Paula; Mendes, Marta; Coelho, Ana V; Calado, Angelo; Oliveira, Catarina R; e Silva, João Martins; Saldanha, Carlota

    2007-08-31

    Acetylcholine is found in the nervous system and also in other cell types (endothelium, lymphocytes, and epithelial and blood cells), which are globally termed the non-neuronal cholinergic system. In this study we investigated the expression and subcellular localization of acetylcholinesterase (AChE) in endothelial cells. Our results show the expression of the 70-kDa AChE in both cytoplasmic and nuclear compartments. We also describe, for the first time, a nuclear and cytoskeleton-bound AChE isoform with approximately 55 kDa detected in endothelial cells. This novel isoform is decreased in response to vascular endothelial growth factor via the proteosomes pathway, and it is down-regulated in human leukemic T-cells as compared with normal T-cells, suggesting that the decreased expression of the 55-kDa AChE protein may contribute to an angiogenic response and associate with tumorigenesis. Importantly, we show that its nuclear expression is not endothelial cell-specific but also evidenced in non-neuronal and neuronal cells. Concerning neuronal cells, we can distinguish an exclusively nuclear expression in postnatal neurons in contrast to a cytoplasmic and nuclear expression in embryonic neurons, suggesting that the cell compartmentalization of this new AChE isoform is changed during the development of nervous system. Overall, our studies suggest that the 55-kDa AChE may be involved in different biological processes such as neural development, tumor progression, and angiogenesis.

  3. The E4 protein; structure, function and patterns of expression.

    PubMed

    Doorbar, John

    2013-10-01

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1^E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein's flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1^E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1^E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1^E4, these kinases regulate one of

  4. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    PubMed

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

  5. The expression and induction of heat shock proteins in molluscs.

    PubMed

    Liu, Dongwu; Chen, Zhiwei

    2013-05-01

    Living cells respond to stress stimuli by triggering rapid changes in the protein profiles, and the induction of heat shock proteins (HSPs) plays an important part in this process. HSPs, mainly acting as molecular chaperones, are constitutively expressed in cells and involved in protein folding, assembly, degradation, and intracellular localization. The overexpression of HSPs represents a ubiquitous molecular mechanism to cope with stress. Compared to vertebrates, molluscs have a biphasic life cycle where pelagic larvae go through settlement and metamorphosis. HSPs may play an important role in the survival strategy of molluscs during the biphasic life stages. Since aquatic environments are highly dynamic, molluscs may be subject to a variety of sources of stress and HSPs might play a more important role in the adaptation of these animals. Moreover, the mechanisms of stress tolerance in molluscs can offer fundamental insights into the adaptation of organisms for a wide range of environmental challenges. The cDNA of HSPs has been cloned from some molluscs, and HSPs can be induced by heat stress, hypoxia, heavy metal contamination, and aestivation, etc. The expression of HSPs was detected in the neuroendocrine system, mollusc development, and reproductive process. Furthermore, the induction of HSPs is related with the phosphorylation of stress-activated p38 mitogen-activated protein kinase (p38 MAPK) and cJun-N-terminal kinases (JNKs) in molluscs.

  6. Phylogeny and expression of carbonic anhydrase-related proteins

    PubMed Central

    2010-01-01

    Background Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate α-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR. Results We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain

  7. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression

    PubMed Central

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-01-01

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm’-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. PMID:27231931

  8. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression.

    PubMed

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-05-24

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm'-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment.

  9. A novel Escherichia coli solubility enhancer protein for fusion expression of aggregation-prone heterologous proteins.

    PubMed

    Song, Jong-Am; Lee, Dae-Sung; Park, Jin-Seung; Han, Kyung-Yeon; Lee, Jeewon

    2011-07-10

    Through the proteome analysis of Escherichia coli BL21(DE3), we previously identified the stress-responsive protein, arsenate reductase (ArsC), that showed a high cytoplasmic solubility and a folding capacity even in the presence of stress-inducing reagents. In this study, we used ArsC as an N-terminal fusion partner to synthesize nine aggregation-prone proteins as water-soluble forms. As a result, solubility of the aggregation-prone proteins increased dramatically by the fusion of ArsC, due presumably to its tendency to facilitate the folding of target proteins. Also, we evaluated and confirmed the efficacy of ArsC-fusion expression in making the fusion-expressed target proteins have their own native function or structure. That is, the self-assembly function of human ferritin light chain, l-arginine-degrading function of arginine deiminase, and the correct secondary structure of human granulocyte colony stimulating factor were clearly observed through transmission electron microscope analysis, colorimetric enzyme activity assay, and circular dichroism, respectively. It is strongly suggested that ArsC can be in general an efficient fusion expression partner for the production of soluble and active heterologous proteins in E. coli. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Sweeping away protein aggregation with entropic bristles: Intrinsically disordered protein fusions enhance soluble expression

    PubMed Central

    Santner, Aaron A.; Croy, Carrie H.; Vasanwala, Farha H.; Uversky, Vladimir N.; Van, Ya-Yue J.; Dunker, A. Keith

    2014-01-01

    Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can enable the target protein to fold free from interference. Using both naturally-occurring and artificial polypeptides we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have low sequence complexity and high net charge, but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently-used fusions such as maltose-binding-protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physio-chemical properties they did not perturb the structure, conformational stability nor function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically-disordered fusions, and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins. PMID:22924672

  11. Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.

    PubMed

    Santner, Aaron A; Croy, Carrie H; Vasanwala, Farha H; Uversky, Vladimir N; Van, Ya-Yue J; Dunker, A Keith

    2012-09-18

    Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both a large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can allow the target protein to fold free from interference. Using both naturally occurring and artificial polypeptides, we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have a low level of sequence complexity and a high net charge but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently used fusions such as maltose-binding protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physiochemical properties, they did not perturb the structure, conformational stability, or function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically disordered fusions and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.

  12. Amyloid Precursor Protein Expression Modulates Intestine Immune Phenotype

    PubMed Central

    Puig, Kendra L.; Swigost, Adam J.; Zhou, Xudong; Sens, MaryAnn; Combs, Colin K.

    2014-01-01

    Amyloid precursor protein (APP) is widely expressed across many tissue and cell types. Proteolytic processing of the protein gives rise to a plethora of protein fragments with varied biological activities. Although a large amount of data has been generated describing the metabolism of the protein in neurons, its role in regulating the phenotype of other cells remains unclear. Based upon prior work demonstrating that APP regulates the activation phenotype of monocytic lineage cells, we hypothesized that APP can regulate macrophage activation phenotype in tissues other than brain. Ileums of the small intestines from C57BL6/J wild type and APP−/− mice were compared as a representative tissue normally associated with abundant macrophage infiltration. APP−/− intestines demonstrated diminished CD68 immunoreactivity compared to wild type mice. This correlated with significantly less cycloxygenase-2 (cox-2), CD68, CD40, CD11c, and βIII-tubulin protein levels. Peritoneal macrophage from APP−/− mice demonstrated decreased in vitro migratory ability compared to wild type cells and diminished basal KC cytokine secretion. Whereas, APP−/− intestinal macrophage had an increase in basal KC cytokine secretion compared to wild type cells. Conversely, there was a significant decrease in multiple cytokine levels in APP−/− compared to wild type ileums. Finally, APP−/− mice demonstrated impaired absorption and increased motility compared to wild type mice. These data demonstrate the APP expression regulates immune cell secretions and phenotype and intestinal function. This data set describes a novel function for this protein or its metabolites that may be relevant not only for Alzheimer’s disease but a range of immune-related disorders. PMID:22124967

  13. Matrix Gla Protein expression pattern in the early avian embryo.

    PubMed

    Correia, Elizabeth; Conceição, Natércia; Cancela, M Leonor; Belo, José A

    2016-01-01

    MGP (Matrix Gla Protein) is an extracellular matrix vitamin K dependent protein previously identified as a physiological inhibitor of calcification and shown to be well conserved among vertebrates during evolution. MGP is involved in other mechanisms such as TGF-β and BMP activity, and a proposed modulator of cell-matrix interactions. MGP is expressed early in vertebrate development although its role has not been clarified. Previous work in the chicken embryo found MGP localization predominantly in the aorta and aortic valve base, but no data is available earlier in development. Here we examined MGP expression pattern using whole-mount in situ hybridization and histological sectioning during the initial stages of chick development. MGP was first detected at HH10 in the head and in the forming dorsal aorta. At the moment of the onset of blood circulation, MGP was expressed additionally in the venous plexus which will remodel into the vitelline arteries. By E2.25, it is clear that the vitelline arteries are MGP positive. MGP expression progresses centrifugally throughout the area vasculosa of the yolk sac. Between stages HH17 and HH19 MGP is seen in the dorsal aorta, heart, notochord, nephric duct, roof plate, vitelline arteries and in the yolk sac, beneath main arterial branches and in the vicinity of several vessels and venules. MGP expression persists in these areas at least until E4.5. These data suggest that MGP expression could be associated with cell migration and differentiation and to the onset of angiogenesis in the developing chick embryo. This data has biomedical relevance by pointing to the potential use of chick embryo explants to study molecules involved in artery calcification.

  14. Differential expression of proteins in renal cortex and medulla: a proteomic approach.

    PubMed

    Arthur, John M; Thongboonkerd, Visith; Scherzer, Janice A; Cai, Jian; Pierce, William M; Klein, Jon B

    2002-10-01

    Western blotting has previously been used to identify changes in protein expression in renal tissue. However, only a few proteins can be studied in each experiment by Western blot. We have used proteomic tools to construct protein maps of rat kidney cortex and medulla. Expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were excised and digested with trypsin. Peptide masses were identified by MALDI-TOF mass spectrometry. The Mascot search engine was used to analyze the peptide masses and identify the proteins. Seventy-two proteins were identified (54 unique proteins) out of approximately 1000 spots visualized on each gel. Most of the spots were expressed both in cortex and medulla. Of the identified proteins, three were expressed only in medulla and one only in cortex. Nine proteins were expressed in both regions but to a greater extent in cortex and three proteins were expressed more in medulla. Differential expression was confirmed for three proteins by Western blot. A large group of proteins and their relative expression levels from cortical and medullary portions of rat kidneys were found. Sixteen proteins are differentially expressed. Proteomics can be used to identify differential expression of proteins in the kidney on a large scale. Proteomics should be useful to detect changes in renal protein expression in response to a large range of physiological and pathophysiological stimuli.

  15. Heat-shock protein expression in canine corneal wound healing.

    PubMed

    Peterson, Cornelia W M; Carter, Renee T; Bentley, Ellison; Murphy, Christopher J; Chandler, Heather L

    2016-05-01

    Heat-shock proteins, particularly the 70-kDa member (Hsp70), have been implicated in facilitating wound healing in multiple tissues. Expression and localization of three HSPs were assessed in normal and wounded canine corneas to elucidate a role in epithelial healing. Paraffin-embedded normal corneas, acute and repeatedly abraded corneas, and keratectomies of spontaneous chronic corneal epithelial defects (SCCEDs) were subjected to routine immunohistochemistry for Hsp27, 47, and 70 expression. Ex vivo corneal defects were created and treated with anti-HSPs or IgG controls, and wound healing was monitored. Primary cultures of canine corneal stromal fibroblasts and corneal epithelial cells were treated with exogenous Hsp70, and an artificial wound was created in vitro to monitor restoration of the monolayer. Normal canine corneas exhibited constitutive expression of all HSPs evaluated. Inducible expression was demonstrated in acutely wounded tissues, and expression in the chronically abraded corneas was relocalized. All HSP expression was below the limits of detection in the epithelium of SCCED samples. Inhibition of HSPs in culture resulted in delayed wound healing when compared to controls. Hsp70-treated fibroblasts demonstrated significantly (P < 0.001) increased migration and proliferation compared to the vehicle control; however, there was no significant effect of exogenous Hsp70 on corneal epithelial cells. These findings suggest that HSPs are induced in the normal canine cornea during re-epithelialization. Hsp70 expression is likely important for inducing the cytoarchitectural remodeling, migration, and proliferation necessary early in the canine corneal healing response, and suppressed expression may contribute to the pathophysiology of nonhealing defects. © 2015 American College of Veterinary Ophthalmologists.

  16. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant f