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1

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

PubMed

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. PMID:24058883

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

2013-01-01

2

P-gp expression in brown trout erythrocytes: evidence of a detoxification mechanism in fish erythrocytes  

PubMed Central

Blood is a site of physiological transport for a great variety of molecules, including xenobiotics. Blood cells in aquatic vertebrates, such as fish, are directly exposed to aquatic pollution. P-gp are ubiquitous “membrane detoxification proteins” implicated in the cellular efflux of various xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), which may be pollutants. The existence of this P-gp detoxification system inducible by benzo [a] pyrene (BaP), a highly cytotoxic PAH, was investigated in the nucleated erythrocytes of brown trout. Western blot analysis showed the expression of a 140-kDa P-gp in trout erythrocytes. Primary cultures of erythrocytes exposed to increasing concentrations of BaP showed no evidence of cell toxicity. Yet, in the same BaP-treated erythrocytes, P-gp expression increased significantly in a dose-dependent manner. Brown trout P-gp erythrocytes act as membrane defence mechanism against the pollutant, a property that can be exploited for future biomarker development to monitor water quality. PMID:24305632

Valton, Emeline; Amblard, Christian; Wawrzyniak, Ivan; Penault-Llorca, Frederique; Bamdad, Mahchid

2013-01-01

3

Curcuma drugs and curcumin regulate the expression and function of P-gp in Caco-2 cells in completely opposite ways.  

PubMed

Curcumin is a phenolic compound isolated from rhizomes of C. longa, C. aromatica and other Curcumas except C. zedoaria. Recently, both curcumin and Curcumas have become prevalent as supplement. P-gp has been reported as an important determinant for drug absorption in small intestine. In this study, Caco-2 cell monolayers were treated with methanol extracts of Curcumas (0.1 mg/ml) or curcumin (30 microM) for 72h to investigate the relationship between the potential affects of Curcumas and curcumin on P-gp. [(3)H]-digoxin and rhodamine 123 were used to evaluate P-gp activity. All Curcumas significantly increased the activity of P-gp by up-regulating the expressions of P-gp protein and MDR1 mRNA levels. Interestingly, contrary to Curcumas, curcumin treatment inhibited the activity of P-gp with a decrease in P-gp protein and MDR1 mRNA expression levels. Curcumas might alter the pharmacokinetics of co-administrated drugs by up-regulating the function and expression levels of intestinal P-gp. However, curcumin has no relationship with the inductive effect of Curcumas since curcumin showed an opposite effects. Caution should be exercised when Curcumas or curcumin are to be consumed with drugs that are P-gp substrates because Curcumas and curcumin might regulate the function of P-gp in completely opposite ways. PMID:18439772

Hou, Xiao-Long; Takahashi, Kyoko; Tanaka, Ken; Tougou, Katsuhiko; Qiu, Feng; Komatsu, Katsuko; Takahashi, Koichi; Azuma, Junichi

2008-06-24

4

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

SciTech Connect

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.

Han Yi [Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543 (Singapore); Chin Tan, Theresa May [Department of Biochemistry, National University of Singapore, 18 Science Drive 4, 117543 (Singapore); Lim, Lee-Yong [Pharmacy, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, WA 6009 (Australia)], E-mail: limly@cyllene.uwa.edu.au

2008-08-01

5

Expression of P-gp in acute myeloid leukemia and the reversal function of As2O3 on drug resistance  

PubMed Central

To study the expression of P-glycoprotein (P-gp) and the reversal function of As2O3, the active ingredient of arsenic, on drug resistance in acute myeloid leukemia (AML) patients, P-gp and cluster of differentiation 34 (CD34) were examined in primary mononuclear and resistant cells, with or without As2O3. In addition, multidrug resistance gene 1 (MDR1) mRNA expression was investigated in K562/D cells and AML patients. In total, 28.6% of newly-treated (NT) patients and 59.1% of relapsed/refractory (RR) patients were P-gpfunction+, and 31.7% of NT patients and 59.1% of RR patients were CD34+. The positivity rate of P-gpfunction and CD34+ expression in the RR group were significantly higher compared with that in the NT group (P<0.05). In addition, higher CD34+, P-gpexpression+ and P-gpfunction+ values were observed in older patients compared with younger patients. MDR1 expression was downregulated in certain patients following treatment with AS2O3. In the present study, the overexpression of P-gp was the primary cause of drug resistance in the AML patients, and MDR1 expression was downregulated by As2O3 in primary leukemia and drug-resistant cells. PMID:25435954

GAO, FENG; DONG, WANWEI; YANG, WEI; LIU, JIA; ZHENG, ZHIHONG; SUN, KAILAI

2015-01-01

6

Expression of P-gp in acute myeloid leukemia and the reversal function of As2O3 on drug resistance.  

PubMed

To study the expression of P-glycoprotein (P-gp) and the reversal function of As2O3, the active ingredient of arsenic, on drug resistance in acute myeloid leukemia (AML) patients, P-gp and cluster of differentiation 34 (CD34) were examined in primary mononuclear and resistant cells, with or without As2O3. In addition, multidrug resistance gene 1 (MDR1) mRNA expression was investigated in K562/D cells and AML patients. In total, 28.6% of newly-treated (NT) patients and 59.1% of relapsed/refractory (RR) patients were P-gpfunction (+), and 31.7% of NT patients and 59.1% of RR patients were CD34(+). The positivity rate of P-gpfunction and CD34(+) expression in the RR group were significantly higher compared with that in the NT group (P<0.05). In addition, higher CD34(+), P-gpexpression (+) and P-gpfunction (+) values were observed in older patients compared with younger patients. MDR1 expression was downregulated in certain patients following treatment with AS2O3. In the present study, the overexpression of P-gp was the primary cause of drug resistance in the AML patients, and MDR1 expression was downregulated by As2O3 in primary leukemia and drug-resistant cells. PMID:25435954

Gao, Feng; Dong, Wanwei; Yang, Wei; Liu, Jia; Zheng, Zhihong; Sun, Kailai

2015-01-01

7

Reversal Effect of ST6GAL 1 on Multidrug Resistance in Human Leukemia by Regulating the PI3K/Akt Pathway and the Expression of P-gp and MRP1  

PubMed Central

?-galactoside ?2, 6-sialyltransferse gene (ST6GAL) family has two members, which encode corresponding enzymes ST6Gal I and ST6Gal II. The present atudy was to investigate whether and how ST6GAL family involved in multidrug resistance (MDR) in human leukemia cell lines and bone marrow mononuclear cells (BMMC) of leukemia patients. Real-time PCR showed a high expression level of ST6GAL1 gene in both MDR cells and BMMCs (*P<0.05). Alternation of ST6GAL1 levels had a significant impact on drug-resistant phenotype changing of K562 and K562/ADR cells both in vitro and in vivo. However, no significant changes were observed of ST6GAL2 gene. Further data revealed that manipulation of ST6GAL1 modulated the activity of phosphoinositide 3 kinase (PI3K)/Akt signaling and consequently regulated the expression of P-glycoprotein (P-gp, *P<0.05) and multidrug resistance related protein 1 (MRP1, *P<0.05), which are both known to be associated with MDR. Therefore we postulate that ST6GAL1 is responsible for the development of MDR in human leukemia cells probably through medicating the activity of PI3K/Akt signaling and the expression of P-gp and MRP1. PMID:24454800

Hao, Keji; Li, Yanping; Song, Xiaobo; Zhou, Huimin; Jia, Li

2014-01-01

8

Alternation of adriamycin penetration kinetics in MCF-7 cells from 2D to 3D culture based on P-gp expression through the Chk2/p53/NF-?B pathway.  

PubMed

Monolayer cells are largely different from tumor masses, and might misguide drug screenings. 3D in vitro cell culture models simulate the characteristics of tumor masses in vivo and have recently been used in many studies of anti-cancer drugs. Among various 3D cell culture models, multi-cellular layer (MCL) models allow for the direct quantitative assessment of the penetration of chemotherapeutic agents through solid tissue environments without requiring the use of fluorescently labeled drugs or imaging molecules. Therefore, in our present study, a 3D-no base and embedded MCF-7 MCL model was successfully developed for a 14-day culture. Over time, its thickness and cell layers increased and exhibited highly proliferative properties and drug resistance to adriamycin (ADR) with markedly elevated IC50 values. Meanwhile, G2/M stage cycle arrest was also observed, which likely up-regulated P-gp expression through the Chk2/p53/NF-?B pathway. The elevated P-gp expression altered the ADR penetration kinetics in MCF-7 MCLs in vitro by accelerating the apparent penetration of ADR through the intercellular spaces of the MCLs. Additionally, a decreased ADR retention within tumor cells was observed, but could be significantly reversed by a P-gp inhibitor. The attenuated ADR retention in the deeper cells of tumor masses was confirmed in xenografted mice in vivo. This phenomenon could be elucidated by the mathematical modeling of penetration kinetics parameters. Our study provided a new model that evaluated and improved the quantification of the drug penetration kinetics, revealed the relationship between P-gp and drug penetration through tumor masses, and suggested the potential molecular mechanisms. PMID:25478729

Lu, Meng; Zhou, Fang; Hao, Kun; Liu, Jiali; Chen, Qianying; Ni, Ping; Zhou, Honghao; Wang, Guangji; Zhang, Jingwei

2015-01-15

9

Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein.  

PubMed

Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae. Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0. 4-0.7 micromol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated. By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone. Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp. Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K(MgATP)(M), V(max), and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies. PMID:10729188

Figler, R A; Omote, H; Nakamoto, R K; Al-Shawi, M K

2000-04-01

10

Protoberberine alkaloids and their reversal activity of P-gp expressed multidrug resistance (MDR) from the rhizome of Coptis japonica Makino.  

PubMed

Six protoberberine alkaloids were isolated from the chloroform layer of the rhizome of Coptis japonica Makino (Ranunculaceae). The structures of the isolated compounds were determined to be 6-([1,3]dioxolo[4,5-g]isoquinoline-5-carbonyl)-2,3-dimethoxy-benzoic acid methyl ester (1), oxyberberine (2), 8-oxo-epiberberine (3), 8-oxocoptisine (4), berberine (5) and palmatine (6) by physicochemical and spectroscopic methods. The compound 3 (8-oxo-epiberberine) was first isolated from natural sources. The compounds were tested for cytotoxicity against five tumor cell lines in vitro by SRB method, and also tested for the MDR reversal activities. Compound 4 was of significant P-gp MDR inhibition activity with ED50 value 0.018 microg/mL in MES-SA/DX5 cell and 0.0005 microg/mL in HCT15 cell, respectively. PMID:17024849

Min, Yong Deuk; Yang, Min Cheol; Lee, Kyu Ha; Kim, Kyung Ran; Choi, Sang Un; Lee, Kang Ro

2006-09-01

11

Involvement of CUL4A in regulation of multidrug resistance to P-gp substrate drugs in breast cancer cells.  

PubMed

CUL4A encodes a core component of a cullin-based E3 ubiquitin ligase complex that regulates many critical processes such as cell cycle progression, DNA replication, DNA repair and chromatin remodeling by targeting a variety of proteins for ubiquitination and degradation. In the research described in this report we aimed to clarify whether CUL4A participates in multiple drug resistance (MDR) in breast cancer cells. We first transfected vectors carrying CUL4A and specific shCUL4A into breast cancer cells and corresponding Adr cells respectively. Using reverse transcription polymerase chain reactions and western blots, we found that overexpression of CUL4A in MCF7 and MDA-MB-468 cells up-regulated MDR1/P-gp expression on both the transcription and protein levels, which conferred multidrug resistance to P-gp substrate drugs, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. On the other hand, silencing CUL4A in MCF7/Adr and MDA-MB-468/Adr cells led to the opposite effect. Moreover, ERK1/2 in CUL4A-overexpressing cells was highly activated and after treatment with PD98059, an ERK1/2-specific inhibitor, CUL4A-induced expression of MDR1/P-gp was decreased significantly. Lastly, immunohistochemistry in breast cancer tissues showed that P-gp expression had a positive correlation with the expression of CUL4A and ERK1/2. Thus, these results implied that CUL4A and ERK1/2 participated in multi-drug resistance in breast cancer through regulation of MDR1/P-gp expression. PMID:24368600

Wang, Yunshan; Ma, Guangxin; Wang, Qin; Wen, Mingxin; Xu, Yangyang; He, Xiuquan; Zhang, Pengju; Wang, Yuli; Yang, Taomei; Zhan, Panpan; Wei, Guangwei

2013-01-01

12

Restoration of chemosensitivity by multifunctional micelles mediated by P-gp siRNA to reverse MDR.  

PubMed

One of the main obstacles in tumor therapy is multiple drug resistance (MDR) and an underlying mechanism of MDR is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins, especially P-glycoprotein (P-gp). In the synergistic treatment of siRNA and anti-cancer drug doxorubicin, it is crucial that both the siRNA and doxorubicin are simultaneously delivered to the tumor cells and the siRNA can fleetly down-regulate P-g before doxorubicin inactivates the P-gp and is pumped out. Herein, a type of micelles comprising a polycationic PEI-CyD shell to condense the siRNA and hydrophobic core to package doxorubicin is reported. The structure of the polymer is determined by (1)H NMR, FT-IR, DSC, and XRD and the micelles are characterized by DLS, 2D-NOESY NMR, and TEM to study the self-assembly of the micelles with siRNA and drugs. In vitro studies demonstrate controlled release and temporal enhancement of the therapeutic efficacy of P-gp siRNA and doxorubicin. Release of siRNA down-regulates the mRNA and protein levels of P-gp in the MCF-7/ADR cell lines effectively and the accumulated doxorubicin facilitates apoptosis of the cells to reverse MDR. Moreover, in vivo research reveals that the siRNA and doxorubicin loaded micelles induce tumor cell apoptosis and inhibit the growth of MDR tumor. The western blotting and RT-PCR results illustrate that the synergistic treatment of siRNA and doxorubicin leads to efficient reduction of the P-gp expression as well as cell apoptotic induction in MDR tumors at a small dosage of 0.5 mg/kg. The micelles have large clinical potential in drug/RNAi synergistic treatment via restoration of the chemosensitivity in MDR cancer therapy. PMID:25002258

Shen, Jie; Wang, Qiwen; Hu, Qida; Li, Yongbing; Tang, Guping; Chu, Paul K

2014-10-01

13

P-gp activity is a critical resistance factor against AVE9633 and DM4 cytotoxicity in leukaemia cell lines, but not a major mechanism of chemoresistance in cells from acute myeloid leukaemia patients  

PubMed Central

Background AVE9633 is a new immunoconjugate comprising a humanized monoclonal antibody, anti-CD33 antigen, linked through a disulfide bond to the maytansine derivative DM4, a cytotoxic agent and potent tubulin inhibitor. It is undergoing a phase I clinical trial. Chemoresistance to anti-mitotic agents has been shown to be related, in part, to overexpression of ABC proteins. The aim of the present study was to investigate the potential roles of P-gp, MRP1 and BCRP in cytotoxicity in AVE9633-induced acute myeloid leukaemia (AML). Methods This study used AML cell lines expressing different levels of P-gp, MRP1 or BCRP proteins and twenty-five samples from AML patients. Expression and functionality of the transporter protein were analyzed by flow cytometry. The cytotoxicity of the drug was evaluated by MTT and apoptosis assays. Results P-gp activity, but not MRP1 and BCRP, attenuated AVE9633 and DM4 cytotoxicity in myeloid cell lines. Zosuquidar, a potent specific P-gp inhibitor, restored the sensitivity of cells expressing P-gp to both AVE9633 and DM4. However, the data from AML patients show that 10/25 samples of AML cells (40%) were resistant to AVE9633 or DM4 (IC50 > 500 nM), and this was not related to P-gp activity (p-Value: 0.7). Zosuquidar also failed to re-establish drug sensitivity. Furthermore, this resistance was not correlated with CD33 expression (p-Value: 0.6) in those cells. Conclusion P-gp activity is not a crucial mechanism of chemoresistance to AVE9633. For patients whose resistance to conventional anthracycline AML regimens is related to ABC protein expression, a combination with AVE9633 could be beneficial. Other mechanisms such as microtubule alteration could play an important role in chemoresistance to AVE9633. PMID:19549303

2009-01-01

14

P-gp efflux pump inhibition potential of common environmental contaminants determined in vitro.  

PubMed

Across different species, cellular efflux pumps such as P-glycoprotein (P-gp; also termed multidrug resistance protein 1 [MDR1]) serve as a first line of defense by transporting toxic xenobiotics out of the cell. This mechanism is also active in aquatic organisms such as mussels, fish, and their larvae. Modulation of this resistance mechanism by chemical agents occurring in the environment could result in either higher or lower internal concentrations of toxic or endogenous compounds in cells. The aim of the present study was to explore and quantify the inhibition of the P-gp efflux pumps by several ubiquitous aquatic contaminants. The calcein-acetoxymethyl ester (calcein-AM) assay commonly used in pharmacological research was established with P-gp-overexpressing Madin-Darby canine kidney cells (MDCKII-MDR1) in a 96-well plate, avoiding extra washing, centrifugation, and lysis steps. This calcein-AM-based P-gp cellular efflux pump inhibition assay (CEPIA) was used to study the inhibition by commonly occurring environmental contaminants. Among others, the compounds pentachlorophenol, perfluorooctane sulfonate, and perfluorooctanoate strongly inhibited the P-gp-mediated efflux of calcein-AM while the chloninated alkanes did not seem to interact with the transporter. The fact that common pollutants can be potent modulators of the efflux transporters is a motive to further study whether this increases the toxicity of other contaminants present in the same matrices. PMID:24375866

Georgantzopoulou, Anastasia; Skoczy?ska, Ewa; Van den Berg, Johannes H J; Brand, Walter; Legay, Sylvain; Klein, Sebastian G; Rietjens, Ivonne M C M; Murk, Albertinka J

2014-04-01

15

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines  

Microsoft Academic Search

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs.This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1

Michela Solazzo; Ornella Fantappiè; Nadia Lasagna; Chiara Sassoli; Daniele Nosi; Roberto Mazzanti

2006-01-01

16

Clin Pharmacol Ther . Author manuscript Donor P-gp polymorphisms strongly influence renal function and graft  

E-print Network

3A and the efflux transporter P-glycoprotein (P-gp; ), bothABCB1 abundantly expressed in the kidney. We retrospectively investigated the role of polymorphisms in , andCYP3A4 CYP3A5 ABCB1 kidney graft-Meiers Estimate ; Kidney ; physiology ; Kidney Function Tests ; Kidney Transplantation ; physiology ; Male

Boyer, Edmond

17

Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF  

PubMed Central

Background The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. Results Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFN? and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines - but not IFN? - stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFN? produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF -which is internalised with its receptor - produced an overexpression of P-gp in CNTF-deficient astrocytes. Conclusions These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF. PMID:12150717

Monville, Christelle; Fages, Christiane; Feyens, Anne-Marie; d'Hondt, Véronique; Guillet, Catherine; Vernallis, Ann; Gascan, Hugues; Peschanski, Marc

2002-01-01

18

Expression and significance of glucose transporter-1, P-glycoprotein, multidrug resistance-associated protein and glutathione S-transferase-? in laryngeal carcinoma  

PubMed Central

Increasing glucose transporter-1 (GLUT-1) activity is one of the most important ways to increase the cellular influx of glucose. We previously demonstrated that increased GLUT-1 expression was an independent predictor of survival in patients with laryngeal carcinoma. Thus, GLUT-1 may present a novel therapeutic target in laryngeal carcinoma. In this study, the expression of GLUT-1, P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and glutathione S-transferase-? (GST-?) in laryngeal carcinomas was investigated by immunohistochemistry. Additionally, possible correlations between GLUT-1 and P-gp, MRP1 and GST-? and various clinicopathological parameters were analyzed. In this study, 52.9% (18/34), 58.8% (20/34), 20.6% (7/34) and 58.8% (20/34) of the laryngeal carcinomas were positive for GLUT-1, P-gp, MRP1 and GST-?, respectively. The expression of GLUT-1, P-gp, MRP1 and GST-? was higher in laryngeal carcinoma specimens when compared with laryngeal precancerous lesions (P<0.05). Pearson’s correlation analysis showed correlations between GLUT-1 and P-gp (r=0.364; P=0.034), GLUT-1 and MRP1 (r=0.359; P=0.037) and P-gp and GST-? (r=0.426; P=0.012). GLUT-1 expression was found to significantly correlate with tumor-node-metastasis classification (P=0.02) and clinical stage (P=0.037). Furthermore, P-gp was found to significantly correlate with clinical stage (P=0.026). Univariate analysis showed that MRP1 expression was significantly associated with poor survival (c2=5.16; P=0.023). Multivariate analysis revealed that lymph node metastasis (P=0.009) and MRP1 overexpression (P=0.023) were significant predictors of poor survival. In the present study, the expression of GLUT-1, P-gp, MRP1 and GST-? in laryngeal carcinomas was investigated, as well as the correlations between these proteins. P-gp was found to significantly correlate with clinical stage, while MRP1 overexpression was significantly associated with poor survival.

MAO, ZHONG-PING; ZHAO, LI-JUN; ZHOU, SHUI-HONG; LIU, MENG-QIN; TAN, WEI-FENG; YAO, HONG-TIAN

2015-01-01

19

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

PubMed

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 ?M) 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 ?M) 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. PMID:23742857

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

2013-09-01

20

Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact with calmodulin.  

PubMed Central

Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide during transport into the lumen of the endoplasmic reticulum. Using site-specific photo-crosslinking we have followed the fate of the signal sequence of preprolactin in a cell-free system. This signal sequence has an unusually long hydrophilic n-region containing several positively charged amino acid residues. We found that after cleavage by signal peptidase the signal sequence is in contact with lipids and subunits of the signal peptidase complex. The cleaved signal sequence is processed further and an N-terminal fragment is released into the cytosol. This signal peptide fragment was found to interact efficiently with calmodulin. Similar to preprolactin, the signal sequence of the HIV-1 envelope protein p-gp160 has the characteristic feature for calmodulin binding in its n-region. We found that a signal peptide fragment of p-gp160 was released into the cytosol and interacts with calmodulin. Our results suggest that signal peptide fragments of some cellular and viral proteins can interact with cytosolic target molecules. The functional consequences of such interactions remain to be established. However, our data suggest that signal sequences may be functionally more versatile than anticipated up to now. PMID:9362478

Martoglio, B; Graf, R; Dobberstein, B

1997-01-01

21

Activation of ?-catenin signalling by GSK-3 inhibition increases p-glycoprotein expression in brain endothelial cells  

PubMed Central

This study investigates involvement of ?-catenin signalling in regulation of p-glycoprotein (p-gp) expression in endothelial cells derived from brain vasculature. Pharmacological interventions that enhance or that block ?-catenin signalling were applied to primary rat brain endothelial cells and to immortalized human brain endothelial cells, hCMEC/D3, nuclear translocation of ?-catenin being determined by immunocytochemistry and by western blot analysis to confirm effectiveness of the manipulations. Using the specific glycogen synthase kinase-3 (GSK-3) inhibitor 6-bromoindirubin-3?-oxime enhanced ?-catenin and increased p-gp expression including activating the MDR1 promoter. These increases were accompanied by increases in p-gp-mediated efflux capability as observed from alterations in intracellular fluorescent calcein accumulation detected by flow cytometry. Similar increases in p-gp expression were noted with other GSK-3 inhibitors, i.e. 1-azakenpaullone or LiCl. Application of Wnt agonist [2-amino-4-(3,4-(methylenedioxy) benzylamino)-6-(3-methoxyphenyl)pyrimidine] also enhanced ?-catenin and increased transcript and protein levels of p-gp. By contrast, down-regulating the pathway using Dickkopf-1 or quercetin decreased p-gp expression. Similar changes were observed with multidrug resistance protein 4 and breast cancer resistance protein, both known to be present at the blood–brain barrier. These results suggest that regulation of p-gp and other multidrug efflux transporters in brain vasculature can be influenced by ?-catenin signalling. PMID:18624906

Lim, Joseph C.; Kania, Katarzyna D.; Wijesuriya, Hasini; Chawla, Sangeeta; Sethi, Jaswinder K.; Pulaski, Lukasz; Romero, Ignacio A.; Couraud, Pierre O.; Weksler, Babette B.; Hladky, Stephen B.; Barrand, Margery A.

2015-01-01

22

Critical role for P-glycoprotein expression in hematopoietic cells in the FVB.mdr1a?/? model of colitis  

PubMed Central

Objective P-glycoprotein (P-gp), the functional product of the multi-drug resistance gene (mdr), is a transmembrane protein that extrudes substrates from the intracellular environment. P-gp is expressed on the apical surface of epithelial cells and on cells from the hematopoietic lineage. Human MDR polymorphisms have been associated with increased risk inflammatory bowel disease, and FVB/N animals deficient in mdr1a expression develop spontaneous colitis. Previous studies utilizing adult bone marrow chimeras indicated colitis development in this animal model was contingent on P-gp deficiency in radiation resistant epithelial cells. However, the use of adult animals may mask the role of hematopoietic immune cells in colitis initiation, due to pre-existing epithelial abnormalities. Methods To assess the importance of P-gp expression in intestinal epithelial and hematopoietic derived cells on colitis induction in FVB.mdr1a?/?animal we developed a neonatal model of bone marrow reconstitution. FVB/N and FVB.mdr1a?/?adult and neonatal animals were lethally irradiated and reconstituted with bone marrow from FVB/N or FVB.mdr1a?/?donors. Animals were observed for 20 weeks. Results Adult FVB/N animals deficient in P-gp expression in hematopoietically derived immune cells developed colitis similar to adult animals deficient in P-gp expression in radiation resistant epithelial/stromal cells. However, neonatal animals deficient in P-gp expression in hematopoietically derived immune cells developed a more histologically significant colitis than those deficient in P-gp expression in epithelial tissue. Conclusions The use of a neonatal model of bone marrow reconstitution has revealed a critical role for P-gp expression in hematopoietically derived immune cells in colitis development in the FVB.mdr1a?/? model. PMID:21681110

Staley, Elizabeth M.; Dimmitt, Reed A.; Schoeb, Trenton R.; Tanner, Scott M.; Lorenz, Robin G.

2013-01-01

23

Vincristine-induced expression of P-glycoprotein in MOLM-13 and SKM-1 acute myeloid leukemia cell lines is associated with coexpression of nestin transcript.  

PubMed

Nestin is a class 6 filament protein typically expressed in neural stem/progenitor cells. However, nestin expression has been observed in other tissues during mammalian embryogenesis. In human neural stem/progenitor cells, coexpression of nestin and P-glycoprotein (P-gp, ABCB1 member of the ABC transporter family) was detected. P-gp-mediated drug efflux is the most common molecular cause of multidrug resistance in neoplastic cells. Nestin expression has also been detected in various human solid tumours as well as in the corresponding established cell lines. Interestingly, expression of nestin in different leukemia cells has been recently reported. Here, we show that expression of P-gp is associated with the simultaneous expression of nestin in acute myeloid leukemia cell lines (MOLM-13 and SKM-1) under the selective pressure of vincristine, a substance that may induce P-gp expression in neoplastic cells. PMID:24968412

Imrichova, Denisa; Coculova, Martina; Messingerova, Lucia; Sulova, Zdena; Breier, Albert

2014-10-01

24

The Inhibitory Effect of Pseudolaric Acid B on Gastric Cancer and Multidrug Resistance via Cox-2/PKC-?/P-gp Pathway  

PubMed Central

Aim To investigate the inhibitory effect of pseudolaric acid B on subcutaneous xenografts of human gastric adenocarcinoma and the underlying molecular mechanisms involved in its multidrug resistance. Methods Human gastric adenocarcinoma SGC7901 cells and drug-resistant SGC7901/ADR cells were injected into nude mice to establish a subcutaneous xenograft model. The effects of pseudolaric acid B with or without adriamycin treatment were compared by determining the tumor size and weight. Cyclo-oxygenase-2, protein kinaseC-? and P-glycoprotein expression levels were determined by immunohistochemistry and western blot. Results Pseudolaric acid B significantly suppressed the tumor growth induced by SGC7901 cells and SGC7901/ADR cells. The combination of pseudolaric acid B and the traditional chemotherapy drug adriamycin exhibited more potent inhibitory effects on the growth of gastric cancer in vivo than treatment with either pseudolaric acid B or adriamycin alone. Protein expression levels of cyclo-oxygenase-2, protein kinaseC-? and P-glycoprotein were inhibited by pseudolaric acid B alone or in combination with adriamycin in SGC7901/ADR cell xenografts. Conclusion Pseudolaric acid B has a significant inhibitory effect and an additive inhibitory effect in combination with adriamycin on the growth of gastric cancer in vivo, which reverses the multidrug resistance of gastric neoplasm to chemotherapy drugs by downregulating the Cox-2/PKC-?/P-gp/mdr1 signaling pathway. PMID:25250794

Sun, Qian; Li, Yan

2014-01-01

25

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

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-1?, 1-? (IL-1?), -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 significant increase of the activity of both transporters was evidenced at the BBB in the adult brain. Moreover, juvenile and adult brain showed differences in their expression profiles of cytokines and chemokines mediated by ET-1. Conclusions BBB transporter activity during neuroinflammation differs between the juvenile and adult brains. These findings emphasize the importance of considering differential P-gp and BCRP transport regulation mechanisms between adult and juvenile BBB in the context of neuroinflammation. PMID:23253775

2012-01-01

26

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

PubMed

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 ?s each. Cells were also treated with low doxorubicin concentration ([DOX]=1.7 ?M). 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. PMID:24767854

Kulbacka, Julita; Daczewska, Ma?gorzata; Dubi?ska-Magiera, Magda; Choroma?ska, Anna; Rembia?kowska, Nina; Surowiak, Pawe?; Kulbacki, Marek; Kotulska, Ma?gorzata; Saczko, Jolanta

2014-12-01

27

P-gp modulating drugs greatly potentiate the in vitro effect of ivermectin against resistant larvae of Haemonchus placei.  

PubMed

Since its production in the 1980s, ivermectin (IVM) has been used indiscriminately and the selection pressure to which bovine gastrointestinal nematodes have been exposed has been intense, resulting in considerable economic losses due to parasitic resistance. One possibility for the control of resistant parasites is the use of P-glycoprotein (P-gp) modulators, because one of the main biochemical changes in ivermectin-resistant parasites is the increased activity of membrane proteins responsible for the efflux of drugs and xenobiotics. This study aimed to evaluate the in vitro effect of eight P-gp modulating drugs to potentiate IVM efficacy against an IVM-resistant field isolate of Haemonchus placei (Nematoda: Trichostrongylidae). The association of IVM with cyclosporin-A, ceftriaxone, dexamethasone, diminazene aceturate, quercetin, trifluoperazine, verapamil, or vinblastine resulted in increased IVM (10(-4)M) efficacy of 5.1%, 49.06%, 76.42%, 3.31%, 28.85%, 13.74%, 45.64% and 43.61%, respectively, and reduced the IVM half maximal effective concentration (EC50) from 4.381×10(-6)M to 9.877×10(-8), 2.739×10(-7), 1.240×10(-6), 1.651×10(-6), 2.710×10(-7), 1.159×10(-7), 1.026×10(-6) and 7.136×10(-7)M, respectively. Only diminazene aceturate did not significantly reduce the number of migrating larvae when associated with IVM (P>0.05). The effect of P-gp modulating drugs depended on IVM concentration, with greater potentiating effect at lower IVM concentrations. The in vitro application of trifluoperazine, dexamethasone, quercetin, verapamil, cyclosporin A, vinblastine, and ceftriaxone potentiated IVM efficacy against an IVM-resistant field isolate of H. placei, resulting in higher efficacy and lower IVM EC50. PMID:25178553

Heckler, R P; Almeida, G D; Santos, L B; Borges, D G L; Neves, J P L; Onizuka, M K V; Borges, F A

2014-10-15

28

A novel approach to overcome multidrug resistance: utilization of P-gp mediated efflux of paclitaxel to attack neighboring vascular endothelial cells in tumors.  

PubMed

We tried to overcome the paclitaxel (PTX) resistance of cancer cells due to P-glycoprotein (P-gp) overexpression in the in vivo anti-tumor chemotherapy by utilizing polyethylene glycol-modified liposomal paclitaxel (PL-PTX). First of all, established were PTX-resistant Colon-26 cancer cells (C26/PTX) overexpressing P-gp, which provided IC50 value of PTX solution about 30 times larger than that obtained for control C26 (C26/control) in the in vitro MTT assay. Western blot analysis confirmed P-gp expression in C26/PTX 10 times higher than that in C26/control, indicating that the resistance acquisition of C26/PTX to PTX would be ascribed to the enhanced efflux of PTX by P-gp overexpressed in C26/PTX. However, the in vivo anti-tumor effect of PL-PTX in C26/PTX-bearing mice was similar to that in C26/control-bearing mice. Double immunohistochemical staining of vascular endothelial cells and apoptotic cells within tumor tissues demonstrated that the apoptotic cell death was preferentially observed in vascular endothelial cells in C26/PTX tumors after intravenous administration of PL-PTX, while that was in tumor cells in C26/control tumors. These results suggest that the in vivo anti-tumor effect of PL-PTX in C26/PTX-bearing mice would be ascribed to the cytotoxic action of PTX pumped out of tumor cells by overexpressed P-gp to vascular endothelial cells in tumor tissues. PMID:24956463

Yoshizawa, Yuta; Ogawara, Ken-ichi; Kimura, Toshikiro; Higaki, Kazutaka

2014-10-01

29

In silico modeling for the nonlinear absorption kinetics of UK-343,664: a P-gp and CYP3A4 substrate.  

PubMed

The aim of this work was to extrapolate in vitro and preclinical animal data to simulate the pharmacokinetic parameters of UK-343,664, a P-glycoprotein (P-gp) and CYP3A4 substrate, in human. In addition, we aimed to develop a simulation model to demonstrate the involvement and the controversial complex interaction of intestinal P-gp and CYP3A4 in its nonlinear absorption, first-pass extraction, and pharmacokinetics using the advanced compartmental absorption and transit (ACAT) model. Finally, we aimed to compare the results predicted from the model to the reported findings in human clinical studies. In situ perfusion, allometric scaling, PBPK Rodger mechanistic approach, in vitro metabolism, and fitting to in vivo data were used to mechanistically explain the absorption, distribution and metabolism, respectively. GastroPlus was used to build the integrated simulation model in human for UK-343,664 to mechanistically explain the observed clinical data at 30, 100, 200, 400, and 800 mg oral doses. The measured in vitro value for CYP3A4 K(m) (465 ?M) in rCYPs was converted to units of ?g/mL, corrected for assumed microsomal binding (17.8%) and applied to all metabolic processes. The measured in vitro values of V(max) for CYP3A4 (38.9 pmol/min/pmol), 2C8, 2C9, 2C19, and 2D6 were used along with the in vitro CYP3A4 K(m) to simulate liver first pass extraction and systemic clearance. The measured in vitro values of V(max) for CYP3A4 and 2D6 were used along with the in vitro CYP3A4 K(m) to simulate gut first pass extraction. V(max) and K(m) values for P-gp were obtained by fitting to in vivo data and used to simulate gut efflux transport activity. Investigation of the interaction mechanism of P-gp and CYP3A4 in the intestine was achieved by comparing the influence of a virtual knockout of P-gp or gut metabolism on the fraction absorbed, fraction reaching the portal vein, and fraction metabolized in the gut. Comparison between simulation and in vivo results showed that the in silico simulation provided a mechanistic explanation of the observed nonlinear absorption kinetics of UK-343,664 in human following its administration in the range of 30-800 mg as oral solutions. The simulation results of the pharmacokinetic parameters, AUC and C(max), by GastroPlus were comparable with those observed in vivo. This simulation model is one possible mechanistic explanation of the observed in vivo data and suggests that the nonlinear dose dependence could be attributed to saturation of both the efflux transport by P-gp and the intestinal metabolism. However, the concentration ranges for either protein saturation did not overlap and resulted in much greater than dose proportional increases in AUC. At low doses, producing intraenterocyte concentrations below the fitted value of K(m) for P-gp, the influence of P-gp appears to be protective and results in a lower fraction of gut 3A4 metabolism. At higher doses, as P-gp becomes saturated the fraction of gut 3A4 extraction increases, and eventually at the highest doses, where 3A4 becomes saturated, the fraction of gut 3A4 extraction again decreases. Such a complex interpretation of this in vitro-in vivo extrapolation (IVIVE) is another example of the value and insight obtained by physiologically based absorption simulation. PMID:22264132

Abuasal, Bilal S; Bolger, Michael B; Walker, Don K; Kaddoumi, Amal

2012-03-01

30

Effect of Emergence of Fluoroquinolone Resistance on Intrinsic Expression of P-Glycoprotein Phenotype in Corneal Epithelial Cells  

PubMed Central

Abstract Purpose Multidrug resistance (MDR) represents a major obstacle to the success of antimicrobial fluoroquinolone (FQ) therapy. MDR-associated efflux protein pumps antimicrobial agents out of the corneal cells, leading to suboptimal eradication of microbes. This article examines whether long-term FQ (levofloxacin, ofloxacin, and gatifloxacin) therapy can modify the MDR phenotype (P-glycoprotein [P-gp]) on corneal epithelial cells (Statens Seruminstitut Rabbit Cornea [SIRC]). Methods To study the effect of FQ, SIRC cells without any exposure to FQ (control) were compared with the cells exposed to ofloxacin, levofloxacin, and gatifloxacin at a concentration of 10??g/mL for 3 weeks. Efflux activity of P-gp was assessed by in vitro uptake studies (fluorescent and radioactive), flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR). Results In the presence of FQ, elevated P-gp expression was noted with uptake, flow cytometry, and qRT-PCR analyses. This study confirms that long-term exposure to antibiotics, particularly FQ, can induce overexpression of P-gp efflux transporter present on the corneal cells. P-gp overexpression is commonly noticed in anticancer drug resistance cell lines; however, for the first time, this report describes overexpression of P-gp due to FQ exposure. Conclusions Based on this result, it is suggested that strategies should be developed and implemented not only to overcome resistance to ocular pathogen but also to FQ-induced cellular resistance. PMID:21830912

Barot, Megha; Gokulgandhi, Mitan R.; Haghnegahdar, Megan; Dalvi, Pranjali

2011-01-01

31

Deconstruction of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety to separate P-glycoprotein (P-gp) activity from ?2 receptor affinity in mixed P-gp/?2 receptor agents.  

PubMed

6,7-Dimethoxytetrahydroisoquinoline is widely used as basic moiety in ?2 receptor ligands, in order to provide ?2versus ?1 selectivity. This same moiety is also widely exploited in modulators of P-glycoprotein (P-gp) efflux pump, so that mixed ?2/P-gp agents are often obtained. Deconstruction of 6,7-dimethoxytetrahydroisoquinoline moiety present in the potent mixed ?2/P-gp agent 6,7-dimethoxy-2-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]butyl]-1,2,3,4-tetrahydroisoquinoline (1) could lead to the separation of ?2 affinity from P-gp activity. Therefore, phenethylamino-, benzylamino- and indanamine series were obtained. The NH group was also methylated in the N-phenethylamino series, and ethylated in the benzylamino series, to better match 6,7-dimethoxytetrahydroisoquinoline. The ?2 affinity drastically decreased with the increase of conformational freedom, whereas alkylation of the NH-group was beneficial for ?2 receptor interaction. By contrast, deconstruction of 6,7-dimethoxytetrahydroisoquinoline slightly reduced P-gp activity, with dimethoxy-substituted derivatives displaying potent P-gp interaction. Therefore, 'ring-opened' 6,7-dimethoxytetrahydroisoquinoline derivatives represent a promising strategy to obtain P-gp selective agents devoid of ?2 receptor affinity. PMID:25462276

Pati, Maria Laura; Abate, Carmen; Contino, Marialessandra; Ferorelli, Savina; Luisi, Renzo; Carroccia, Laura; Niso, Mauro; Berardi, Francesco

2015-01-01

32

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

PubMed

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. PMID:24311454

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

2014-02-01

33

Leptospira Protein Expression During Infection  

Technology Transfer Automated Retrieval System (TEKTRAN)

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...

34

Upregulation of Stat1-HDAC4 confers resistance to etoposide through enhanced multidrug resistance 1 expression in human A549 lung cancer cells.  

PubMed

Despite efforts to develop efficient chemotherapeutic drug strategies to treat cancer, acquired drug resistance is a commonly encountered problem. In the present study, to investigate this phenomenon, human A549 lung cancer cells resistant to the topoisomerase inhibitor etoposide (A549RT?eto) were used and compared with A549 parental cells. A549RT?eto cells demonstrated increased resistance to etoposide?induced apoptosis when compared with A549 parental cells. Notably, A549RT?eto cells were observed to exhibit greater levels of histone deacetylase 4 (HDAC4), phospho?Stat1 and P?glycoprotein [P?gp; encoded by the multidrug resistance 1 (MDR1) gene], compared with A549 cells. To address whether HDAC4 protein is involved in etoposide resistance in A549 cells, A549RT?eto cells were treated with trichostatin A (TSA; an HDAC inhibitor) during etoposide treatment. The combined treatment was demonstrated to enhance etoposide?induced apoptosis and reduce expression levels of HDAC4, P?gp and phospho?Stat1. In addition, the suppression of Stat1 with siRNA enhanced etoposide?induced apoptosis and reduced the expression levels of HDAC4 and P?gp, suggesting that Stat1 is essential in the regulation of resistance to etoposide, and in the upregulation of P?gp. Notably, TSA treatment reduced P?gp transcript levels but Stat1 siRNA treatment did not, suggesting that P?gp is regulated by HDAC at the transcriptional level and by Stat1 at the post?transcriptional level. These results suggest that the upregulation of Stat1 and HDAC4 determines etoposide resistance through P?gp expression in human A549 lung cancer cells. PMID:25395162

Kaewpiboon, Chutima; Srisuttee, Ratakorn; Malilas, Waraporn; Moon, Jeong; Oh, Sangtaek; Jeong, Hye Gwang; Johnston, Randal N; Assavalapsakul, Wanchai; Chung, Young-Hwa

2015-03-01

35

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

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 ?M, a 20-fold enhancement compared to the first generation chemosensitizer, verapamil (4 ?M). 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

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

2012-01-01

36

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

PubMed

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-?B (NF-?B) 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-?B. Furthermore, such alterations induced by ammonia were reversed by NF-?B inhibitor. In conclusion, this study demonstrates that hyperammonemia increases the function and expression of P-gp and Mrp2 at the BBB via activating NF-?B 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-?B activation stimulated by hyperammonemia may be the potential mechanism underlying such abnormalities induced by hyperammonemia. PMID:25200138

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

2014-12-01

37

The coordinated role of CYP450 enzymes and P-gp in determining cancer resistance to chemotherapy.  

PubMed

The relationship between CYP450 and P-gp occurs at different levels. It is known that certain substrates of P-gp undergo metabolic transformations by various CYP450 isoforms; in addition some of them demonstrated to be activators of both P-gp and CYP450. The majority of such compounds are well-known chemotherapeutics, therefore the purpose of this review is to clarify whether there is a relationship between the simultaneous modulation of CYP450 and P-gp and the onset of drug resistance in tumors treatment. Here, we discuss the biological aspects of the topic in relation to the various tissues distribution of CYP450 and P-gp, the recent findings regarding the ability of some chemotherapeutics in modulating both P-gp and CYP450, whether this modulation is ultimately responsible for the onset of drug resistance in cancer treatment and the promising role of gene polymorphisms in determining the interindividual variability in drug responses in clinical practice. PMID:21434858

Azzariti, Amalia; Porcelli, Letizia; Quatrale, Anna Elisa; Silvestris, Nicola; Paradiso, Angelo

2011-10-01

38

Cargoing P-gp inhibitors via nanoparticle sensitizes tumor cells against doxorubicin.  

PubMed

Inhibitors against multidrug resistance (MDR) efflux transporters have failed in most clinical settings due to unfavorable pharmacokinetic interactions with co-administered anti-cancer drug and their inherent toxicities. Nanoparticles (NPs) have shown potential to overcome drug efflux by delivering and localizing therapeutic molecules within tumor mass. In this work, we investigated effect of nanocarrier surface charge and formulation parameters for a hydrophilic and lipophilic MDR inhibitor on their ability to reverse drug resistance. Active inhibition of efflux pumps was achieved by encapsulating first and third generation P-gp inhibitors- verapamil and elacridar respectively in non-ionic, anionic and cationic surfactant-based NPs. The ability of NPs to reverse P-glycoprotein (P-gp)-mediated MDR efflux was evaluated in sensitive (A2780) and resistant (A2780Adr) ovarian cancer cell lines by various in vitro accumulation and cytotoxicity assays. Uptake mechanism for NP appears to be caveolae-dependent with 20%-higher internalization in A2780Adr than A2780 cell lines which can be co-related to the biophysical membrane composition. Cationic- CTAB NPs showed highest reversal efficacy followed by PVA and SDS-NP (P+S NP) and PVA-NPs. As compared to doxorubicin treated drug resistant cells lines, blank-, verapamil- and elacridar-CTAB-NPs showed 2.6-, 20- and 193-fold lower IC50 values. This work highlights the importance of inhibitor-loaded charged particles to overcome cancer drug resistance. PMID:25437111

Singh, Manu Smriti; Lamprecht, Alf

2015-01-30

39

Breast cancer-derived microparticles display tissue selectivity in the transfer of resistance proteins to cells.  

PubMed

Microparticles (MPs) play a vital role in cell communication by facilitating the horizontal transfer of cargo between cells. Recently, we described a novel "non-genetic" mechanism for the acquisition of multidrug resistance (MDR) in cancer cells by intercellular transfer of functional P-gp, via MPs. MDR is caused by the overexpression of the efflux transporters P-glycoprotein (P-gp) and Multidrug Resistance-Associated Protein 1 (MRP1). These transporters efflux anticancer drugs from resistant cancer cells and maintain sublethal intracellular drug concentrations. By conducting MP transfer experiments, we show that MPs derived from DX breast cancer cells selectively transfer P-gp to malignant MCF-7 breast cells only, in contrast to VLB100 leukaemic cell-derived MPs that transfer P-gp and MRP1 to both malignant and non-malignant cells. The observed transfer selectivity is not the result of membrane restrictions for intercellular exchange, limitations in MP binding to recipient cells or the differential expression of the cytoskeletal protein, Ezrin. CD44 (isoform 10) was found to be selectively present on the breast cancer-derived MPs and not on leukaemic MPs and may contribute to the observed selective transfer of P-gp to malignant breast cells observed. Using the MCF-7 murine tumour xenograft model we demonstrated the stable transfer of P-gp by MPs in vivo, which was found to localize to the tumour core as early as 24 hours post MP exposure and to remain stable for at least 2 weeks. These findings demonstrate a remarkable capacity by MPs to disseminate a stable resistant trait in the absence of any selective pressure. PMID:23593486

Jaiswal, Ritu; Luk, Frederick; Dalla, Penelope V; Grau, Georges Emile Raymond; Bebawy, Mary

2013-01-01

40

The use of microdialysis techniques in mice to study P-gp function at the blood-brain barrier.  

PubMed

An integrated assay system involving dual/triple-probe microdialysis techniques in rats was developed earlier for testing interactions with P-glycoprotein (P-gp) at the blood-brain barrier using quinidine/PSC-833 as a P-gp substrate/inhibitor combination. The aim of the present study was to expand our assay system to mice using microdialysis with simultaneous sampling of blood and brain and to compare the result with a primary mouse brain endothelial cell monolayer (pMBMEC) assay. Brain penetration of quinidine was dose dependent in both anesthetized and awake mice after intraperitoneal drug administration. PSC-833 pretreatment caused a 2.5- to 3.4-fold increase in quinidine levels of brain dialysate samples in anesthetized or awake animals, after single or repeated administration of PSC-833. In pMBMEC, a 2.0- to 2.5-fold efflux ratio was observed in the transcellular transport of quinidine. The P-gp-mediated vectorial transport of quinidine was eliminated by PSC-833. These results indicate that quinidine with PSC-833 is a good probe substrate-reference inhibitor combination for testing drug-drug interactions with P-gp in the in vivo and in vitro mouse systems. With increasing number of humanized transgenic mice, a test system with mouse microdialysis experimentation becomes more important to predict drug-drug interactions in humans. PMID:23204072

Sziráki, István; Erd?, Franciska; Trampus, Péter; Sike, Mirabella; Molnár, Petra Magdolna; Rajnai, Zsuzsanna; Molnár, Judit; Wilhelm, Imola; Fazakas, Csilla; Kis, Emese; Krizbai, István; Krajcsi, Péter

2013-04-01

41

Reduced ABCB1 Expression and Activity in the Presence of Acrylic Copolymers  

PubMed Central

Purpose: P-glycoprotein (P-gp; ABCB1), an integral membrane protein in the apical surface of human intestinal epithelial cells, plays a crucial role in the intestinal transport and efflux leading to changes in the bioavailability of oral pharmaceutical compounds. This study was set to examine the potential effects of three Eudragits RL100, S100 and L100 on the intestinal epithelial membrane transport of rhodammine-123 (Rho-123), a substrate of P-gp using a monolayer of human colon cancer cell line (Caco-2). Methods: The least non-cytotoxic concentrations of the excipients were assessed in Caco-2 cells by the MTT assay. Then the transepithelial transport of Rho-123 across Caco-2 monolayers was determined with a fluorescence spectrophotometer. Besides, the expression of the P-gp in cells exposed to the polymers was demonstrated using Western-blotting analysis. Results: Treatment of cells with Eudragit RL100 and L100 led to a very slight change while Eudragit S100 showed 61% increase in Rho-123 accumulation (P<0.001) and also reduced transporter expression. Conclusion: Our studies suggest that using proper concentrations of the Eudragit S100 in drug formulation would improve intestinal permeability and absorption of p-gp substrate drugs. PMID:24754004

Mohammadzadeh, Ramin; Baradaran, Behzad; Valizadeh, Hadi; Yousefi, Bahman; Zakeri-Milani, Parvin

2014-01-01

42

Comparison of 99mTc-Tetrofosmin and 99mTc-Sestamibi Uptake in Glioma Cell Lines: The Role of P-Glycoprotein Expression  

PubMed Central

99mTc-Tetrofosmin (99mTc-TF) and 99mTc-Sestamibi (99mTc-MIBI) are SPECT tracers that have been used for brain tumor imaging. Tumor's multidrug resistance phenotype, namely, P-glycoprotein (p-gp), and the multidrug resistance related proteins (MRPs) expression have been suggested to influence both tracers' uptake. In the present study we set out to compare 99mTc-TF and 99mTc-MIBI uptake in high-grade glioma cell lines and to investigate the influence of gliomas p-gp expression on both tracers' uptake. We used four glioma cell lines (U251MG, A172, U87MG, and T98G). The expression of p-gp protein was evaluated by flow cytometry. Twenty ?Ci (7.4·105?Bq) of 99mTc-TF and 99mTc-MIBI were used. The radioactivity in the cellular lysate was measured with a dose calibrator. P-gp was significantly expressed only in the U251MG cell line (P < 0.001). In all gliomas cell lines (U251MG, U87MG, A172, and T98G) the 99mTc-TF uptake was significantly higher than 99mTc-sestamibi. The U251MG cell line, in which significant p-gp expression was documented, exhibited the strongest uptake difference. 99mTc-TF uptake was higher than 99mTc-MIBI in all studied high-grade glioma cell lines. Thus, 99mTc-TF may be superior to 99mTc-MIBI for glioma imaging in vivo. PMID:25436147

Alexiou, George A.; Xourgia, Xanthi; Vartholomatos, Evrysthenis; Kalef-Ezra, John A.; Fotopoulos, Andreas D.; Kyritsis, Athanasios P.

2014-01-01

43

Comparison of (99m)tc-tetrofosmin and (99m)tc-sestamibi uptake in glioma cell lines: the role of p-glycoprotein expression.  

PubMed

(99m)Tc-Tetrofosmin ((99m)Tc-TF) and (99m)Tc-Sestamibi ((99m)Tc-MIBI) are SPECT tracers that have been used for brain tumor imaging. Tumor's multidrug resistance phenotype, namely, P-glycoprotein (p-gp), and the multidrug resistance related proteins (MRPs) expression have been suggested to influence both tracers' uptake. In the present study we set out to compare (99m)Tc-TF and (99m)Tc-MIBI uptake in high-grade glioma cell lines and to investigate the influence of gliomas p-gp expression on both tracers' uptake. We used four glioma cell lines (U251MG, A172, U87MG, and T98G). The expression of p-gp protein was evaluated by flow cytometry. Twenty ?Ci (7.4·10(5)?Bq) of (99m)Tc-TF and (99m)Tc-MIBI were used. The radioactivity in the cellular lysate was measured with a dose calibrator. P-gp was significantly expressed only in the U251MG cell line (P < 0.001). In all gliomas cell lines (U251MG, U87MG, A172, and T98G) the (99m)Tc-TF uptake was significantly higher than (99m)Tc-sestamibi. The U251MG cell line, in which significant p-gp expression was documented, exhibited the strongest uptake difference. (99m)Tc-TF uptake was higher than (99m)Tc-MIBI in all studied high-grade glioma cell lines. Thus, (99m)Tc-TF may be superior to (99m)Tc-MIBI for glioma imaging in vivo. PMID:25436147

Alexiou, George A; Xourgia, Xanthi; Vartholomatos, Evrysthenis; Tsiouris, Spyridon; Kalef-Ezra, John A; Fotopoulos, Andreas D; Kyritsis, Athanasios P

2014-01-01

44

Protein misinteraction avoidance causes highly expressed proteins to evolve slowly  

E-print Network

Protein misinteraction avoidance causes highly expressed proteins to evolve slowly Jian-Rong Yanga, 2012 (received for review October 21, 2011) The tempo and mode of protein evolution have been central questions in biology. Genomic data have shown a strong influence of the expression level of a protein on its

Zhang, Jianzhi

45

Functional Characterization of Glycosylation-Deficient Human P-Glycoprotein Using A Vaccinia Virus Expression System  

Microsoft Academic Search

.   P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and\\u000a 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient\\u000a (Gly?) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly? P-gp (91,

J. J. Gribar; M. Ramachandra; C. A. Hrycyna; S. Dey; S. V. Ambudkar

2000-01-01

46

The elimination of P-glycoprotein over-expressing cancer cells by antimicrobial cationic peptide NK-2: the unique way of multi-drug resistance modulation.  

PubMed

Most chemotherapeutics harm normal cells causing severe side effects and induce the development of resistance in cancer cells. Antimicrobial peptides (AMPs), recognized as anti-cancer agents, may overcome these limitations. The most studied mechanism underlying multi-drug resistance (MDR) is the over-expression of cell membrane transporter P-glycoprotein (P-gp), which extrudes a variety of hydrophobic drugs. Additionally, P-gp contributes to cell membrane composition and increases the net negative charge on cell surface. We postulated that NK-lysin derived cationic peptide NK-2 might discriminate and preferentially eliminate P-gp over-expressing cancer cells. To test this hypothesis, we employed MDR non-small cell lung carcinoma (NCI-H460/R) and colorectal carcinoma (DLD1-TxR) cell lines with high P-gp expression. MDR cancer cells that survived NK-2 treatment had decreased P-gp expression and were more susceptible to doxorubicin. We found that NK-2 more readily eliminated P-gp high-expressing cells. Acting in 'carpet-like' manner NK-2 co-localized with P-gp on the MDR cancer cell membrane. The inhibition of P-gp reduced the NK-2 effect in MDR cancer cells and, vice versa, NK-2 decreased P-gp transport activity. In conclusion, NK-2 could modulate MDR in unique way, eliminating the P-gp high-expressing cells from heterogeneous cancers and making them more vulnerable to classical drug treatment. PMID:23298945

Bankovi?, Jasna; Andrä, Jörg; Todorovi?, Nataša; Podolski-Reni?, Ana; Miloševi?, Zorica; Miljkovi?, Dor?e; Krause, Jannike; Ruždiji?, Sabera; Tani?, Nikola; Peši?, Milica

2013-04-15

47

Expression of multiple proteins in transgenic plants  

DOEpatents

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.

Vierstra, Richard D. (Madison, WI); Walker, Joseph M. (Madison, WI)

2002-01-01

48

Relationship between the expression of cyclooxygenase 2 and MDR1\\/P-glycoprotein in invasive breast cancers and their prognostic significance  

Microsoft Academic Search

INTRODUCTION: Recent reports suggest that expression of the cyclooxygenase 2 (COX-2) enzyme may up-regulate expression of MDR1\\/P-glycoprotein (MDR1\\/P-gp), an exponent of resistance to cytostatic drugs. The present study aimed at examining the relationship between the expression of COX-2 and of MDR1\\/P-gp in a group of breast cancer cases. METHODS: Immunohistochemical reactions were performed using monoclonal antibodies against COX-2 and MDR1\\/P-gp

Pawel Surowiak; Verena Materna; Rafal Matkowski; Katarzyna Szczuraszek; Jan Kornafel; Andrzej Wojnar; Marek Pudelko; Manfred Dietel; Carsten Denkert; Maciej Zabel; Hermann Lage

2005-01-01

49

Coevolution of gene expression among interacting proteins  

SciTech Connect

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.

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

2004-03-01

50

Drug-efflux and target-site gene expression patterns in Haemonchus contortus larvae able to survive increasing concentrations of levamisole in vitro  

PubMed Central

While there is some evidence that changes in nicotinic acetylcholine receptor (nAChR) subunits confer resistance to levamisole in gastrointestinal helminth parasites, the exact nature of the resistance mechanism(s) is unclear. We utilised the presence of a resistant fraction within the Wallangra 2003 isolate of Haemonchus contortus larvae in order to subdivide the population into three subpopulations of larvae able to survive increasing concentrations of the drug. We then measured gene expression levels in the subpopulations and the larval population as a whole, focusing on genes encoding the subunit components of levamisole-sensitive receptors, genes encoding ancillary proteins involved in receptor assembly, and P-glycoprotein (P-gp) genes. The subpopulation surviving the lowest levamisole concentration showed increases of 1.5- to 3-fold in a number of P-gp genes (Hco-pgp-3, -4, -10, and -14) alongside unchanged receptor genes, compared to the whole Wallangra larval population. On the other hand, the subpopulation surviving the intermediate levamisole concentration showed an increase in only a single P-gp (Hco-pgp-14), alongside decreases in some receptor subunit (Hco-unc-63a) and ancillary protein genes (Hco-unc-50, Hco-ric-3.1 and 3.1). The subpopulation surviving the highest levamisole concentration showed further decreases in receptor subunit genes (Hco-unc-63a and Hco-unc-29 paralogs) as well as genes involved in receptor assembly (Hco-unc-74, Hco-unc-50, Hco-ric-3.1 and 3.1), alongside no increased P-gp gene levels. This suggests a biphasic pattern of drug resistance in the larvae of this worm isolate, in which a non-specific P-gp-mediated mechanism confers low levels of resistance, while higher level resistance is due to altered receptor subunit composition as a result of changes in both subunit composition and in the levels of proteins involved in receptor assembly. PMID:25057457

Sarai, Ranbir S.; Kopp, Steven R.; Coleman, Glen T.; Kotze, Andrew C.

2014-01-01

51

Alterations in function and expression of ABC transporters at blood-brain barrier under diabetes and the clinical significances  

PubMed Central

Diabetes is a systematic metabolic disease, which often develops a number of well-recognized vascular complications including brain complications which may partly result from the dysfunction of blood-brain barrier (BBB). BBB is generally considered as a mechanism for protecting the brain from unwanted actions resulting from substances in the blood and maintaining brain homeostasis via monitoring the entry or efflux of compounds. ATP-binding cassette (ABC) family of transporters including P-glycoprotein (P-GP) and breast cancer-related protein (BCRP), widely expressed in the luminal membrane of the microvessel endothelium and in the apical membrane of the choroids plexus epithelium, play important roles in the function of BBB. However, these transporters are easily altered by some diseases. The present article was focused on the alteration in expression and function of both P-GP and BCRP at BBB by diabetes and the clinical significances. PMID:25540622

Liu, Li; Liu, Xiao-Dong

2014-01-01

52

Cell signaling and heat shock protein expression  

Microsoft Academic Search

Exposure of cells and organs to heat shock is associated with numerous changes in various cellular metabolic parameters and overexpression of proteins collectively known as heat shock proteins (HSP). In this communication we review the cell-signaling events that are altered in response to heat shock as they relate to the subsequent induction of HSP 70 kd (HSP-70) expression. We also

Juliann Gong Kiang; George C. Tsokos

1996-01-01

53

Expression systems for production of heterologous proteins  

Microsoft Academic Search

With the advent of our ability to clone and express a foreign gene in the heterologous host, came a remar ka- ble capability to make almost any protein in abundant quantity to be used as therapeutic or diagnostic agents. It quickly led to the realization that proteins made in different hosts are different in many ways, particularly in their post-translation

Meena Rai; Harish Padh

54

SUMO Technology Fusion Tag Protein Expression System  

E-print Network

/active protein #12;SUMO Vectors SUMOpro · Based on the yeast SUMO · For expression in E.Coli · Cleaved by SUMO Protease 1 SUMOpro-3 · Based on the human SUMO · For expression in E.Coli or Pichia Pastoris · Cleaved in E.Coli, S.cerevisiae, insect, or mammalian cells · Only cleaved by SUMOstar Protease #12;SUMOpro

Lebendiker, Mario

55

Interindividual Variability in Hepatic Organic Anion-Transporting Polypeptides and P-Glycoprotein (ABCB1) Protein Expression: Quantification by Liquid Chromatography Tandem Mass Spectroscopy and Influence of Genotype, Age, and Sex  

PubMed Central

Interindividual variability in protein expression of organic anion-transporting polypeptides (OATPs) OATP1B1, OATP1B3, OATP2B1, and multidrug resistance-linked P-glycoprotein (P-gp) or ABCB1 was quantified in frozen human livers (n = 64) and cryopreserved human hepatocytes (n = 12) by a validated liquid chromatography tandem mass spectroscopy (LC-MS/MS) method. Membrane isolation, sample workup, and LC-MS/MS analyses were as described before by our laboratory. Briefly, total native membrane proteins, isolated from the liver tissue and cryopreserved hepatocytes, were trypsin digested and quantified by LC-MS/MS using signature peptide(s) unique to each transporter. The mean ± S.D. (maximum/minimum range in parentheses) protein expression (fmol/µg of membrane protein) in human liver tissue was OATP1B1- 2.0 ± 0.9 (7), OATP1B3- 1.1 ± 0.5 (8), OATP2B1- 1 1.7 ± 0.6 (5), and P-gp- 0.4 ± 0.2 (8). Transporter expression in the liver tissue was comparable to that in the cryopreserved hepatocytes. Most important is that livers with SLCO1B1 (encoding OATP1B1) haplotypes *14/*14 and *14/*1a [i.e., representing single nucleotide polymorphisms (SNPs), c.388A > G, and c.463C > A] had significantly higher (P < 0.0001) protein expression than the reference haplotype (*1a/*1a). Based on these genotype-dependent protein expression data, we predicted (using Simcyp) an up to ?40% decrease in the mean area under the curve of rosuvastatin or repaglinide in subjects harboring these variant alleles compared with those harboring the reference alleles. SLCO1B3 (encoding OATP1B3) SNPs did not significantly affect protein expression. Age and sex were not associated with transporter protein expression. These data will facilitate the prediction of population-based human transporter-mediated drug disposition, drug-drug interactions, and interindividual variability through physiologically based pharmacokinetic modeling. PMID:24122874

Prasad, Bhagwat; Evers, Raymond; Gupta, Anshul; Hop, Cornelis E. C. A.; Salphati, Laurent; Shukla, Suneet; Ambudkar, Suresh V.

2014-01-01

56

Interindividual variability in hepatic organic anion-transporting polypeptides and P-glycoprotein (ABCB1) protein expression: quantification by liquid chromatography tandem mass spectroscopy and influence of genotype, age, and sex.  

PubMed

Interindividual variability in protein expression of organic anion-transporting polypeptides (OATPs) OATP1B1, OATP1B3, OATP2B1, and multidrug resistance-linked P-glycoprotein (P-gp) or ABCB1 was quantified in frozen human livers (n = 64) and cryopreserved human hepatocytes (n = 12) by a validated liquid chromatography tandem mass spectroscopy (LC-MS/MS) method. Membrane isolation, sample workup, and LC-MS/MS analyses were as described before by our laboratory. Briefly, total native membrane proteins, isolated from the liver tissue and cryopreserved hepatocytes, were trypsin digested and quantified by LC-MS/MS using signature peptide(s) unique to each transporter. The mean ± S.D. (maximum/minimum range in parentheses) protein expression (fmol/µg of membrane protein) in human liver tissue was OATP1B1- 2.0 ± 0.9 (7), OATP1B3- 1.1 ± 0.5 (8), OATP2B1- 1 1.7 ± 0.6 (5), and P-gp- 0.4 ± 0.2 (8). Transporter expression in the liver tissue was comparable to that in the cryopreserved hepatocytes. Most important is that livers with SLCO1B1 (encoding OATP1B1) haplotypes *14/*14 and *14/*1a [i.e., representing single nucleotide polymorphisms (SNPs), c.388A > G, and c.463C > A] had significantly higher (P < 0.0001) protein expression than the reference haplotype (*1a/*1a). Based on these genotype-dependent protein expression data, we predicted (using Simcyp) an up to ?40% decrease in the mean area under the curve of rosuvastatin or repaglinide in subjects harboring these variant alleles compared with those harboring the reference alleles. SLCO1B3 (encoding OATP1B3) SNPs did not significantly affect protein expression. Age and sex were not associated with transporter protein expression. These data will facilitate the prediction of population-based human transporter-mediated drug disposition, drug-drug interactions, and interindividual variability through physiologically based pharmacokinetic modeling. PMID:24122874

Prasad, Bhagwat; Evers, Raymond; Gupta, Anshul; Hop, Cornelis E C A; Salphati, Laurent; Shukla, Suneet; Ambudkar, Suresh V; Unadkat, Jashvant D

2014-01-01

57

Proteomics for Protein Expression Profiling in Neuroscience*  

PubMed Central

As the technology of proteomics moves from a theoretical approach to a practical reality, neuroscientists will have to determine the most appropriate applications for this technology. Neuroscientists will have to surmount difficulties particular to their research, such as limited sample amounts, heterogeneous cellular compositions in samples, and the fact that many proteins of interest are rare, hydrophobic proteins. This review examines protein isolation and protein fractionation and separation using two-dimensional electrophoresis (2-DE) and mass spectrometry proteomic methods. Methods for quantifying relative protein expression between samples (e.g., 2-DIGE, and ICAT) are also described. The coverage of the proteome, ability to detect membrane proteins, resource requirements, and quantitative reliability of different approaches is also discussed. Although there are many challenges in proteomic neuroscience, this field promises many rewards in the future. PMID:15176464

Freeman, Willard M.; Hemby, Scott E.

2013-01-01

58

Expressing Mammalian Signaling Proteins in Bacteria  

Microsoft Academic Search

Rhodopsin is the only G-protein coupled receptor whose X-ray crystal structure has been solved. Due to its inexpensiveness and the availability of simple methods to purify it in large quantities, bovine rhodopsin has been successfully used in obtaining diffraction-quality 3-D crystals. In our experiments, the bovine rhodopsin protein was cloned into the commercially available E. coli expression plasmid pBAD-TOPO using

Soyika Richardson; Mark S. Braiman

2006-01-01

59

Protein structure protection commits gene expression patterns  

Microsoft Academic Search

BACKGROUND: 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. RESULTS: 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

Jianping Chen; Han Liang; Ariel Fernández

2008-01-01

60

Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.  

PubMed

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes. PMID:21382497

Busso, Didier; Peleg, Yoav; Heidebrecht, Tatjana; Romier, Christophe; Jacobovitch, Yossi; Dantes, Ada; Salim, Loubna; Troesch, Edouard; Schuetz, Anja; Heinemann, Udo; Folkers, Gert E; Geerlof, Arie; Wilmanns, Matthias; Polewacz, Andrea; Quedenau, Claudia; Büssow, Konrad; Adamson, Rachel; Blagova, Elena; Walton, Julia; Cartwright, Jared L; Bird, Louise E; Owens, Raymond J; Berrow, Nick S; Wilson, Keith S; Sussman, Joel L; Perrakis, Anastassis; Celie, Patrick H N

2011-08-01

61

Determination of P-glycoprotein surface expression and functional ability after in vitro treatment with darunavir or raltegravir in lymphocytes of healthy donors.  

PubMed

It has been shown that P-glycoprotein (P-gp) can greatly affect the cell uptake of antiretroviral drugs, thus hampering their access to HIV-1 replication sites. Lymphocytes are important sites of replication of HIV and target of other drugs, modification on these cells of P-gp could have an effect on pharmacokinetic of antiretrovirals and drug substrates. Blood samples from 16 healthy volunteers were used to determine the expression of P-gp on total, T and T helper lymphocytes after exposure to darunavir, a second generation protease inhibitor, and raltegravir, the first approved integrase inhibitor. Moreover, the effect of the drugs on P-gp functional activity was also studied by the rhodamine-123 efflux test. Darunavir, but not raltegravir, exposure caused a moderate, dose-dependent increment in P-gp expression in total, T and T helper lymphocytes, as demonstrated by the relative frequency of P-gp+ cells and by the amount of P-gp molecules present on cell surface. Functionally, incubation with darunavir led to a marked inhibition of P-gp activity measured by the efflux of rhodamine-123 similar to that observed by verapamil, a specific P-gp inhibitor. Raltegravir was not able to modify the efflux of rhodamine-123 level. Data show that darunavir, unlike raltegravir, may modify the expression and functionality of P-gp on human lymphocytes, thus leading to potential changes in intracellular concentrations of darunavir in patients treated with other drugs substrate of P-gp and vice versa. Our study highlights the need for studies on drug interactions via the P-gp modulation mechanism, especially with the current multi-drug regimens. PMID:23707228

Tempestilli, Massimo; Gentilotti, Elisa; Tommasi, Chiara; Nicastri, Emanuele; Martini, Federico; De Nardo, Pasquale; Narciso, Pasquale; Pucillo, Leopoldo P

2013-08-01

62

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

PubMed

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 ?g/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. PMID:24927684

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

2014-08-01

63

Expression and purification of membrane proteins.  

PubMed

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. PMID:24674067

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

2014-01-01

64

The effects of prion protein expression on metal metabolism.  

PubMed

The prion protein is a glycoprotein that binds metals such as copper and manganese. When converted to a proteinase resistant isoform it is associated with prion diseases such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Although, the co-ordination and metal affinity of the prion protein has been well studied, the association of the protein with cellular metal metabolism has been less well investigated. We used transgenic manipulation of prion protein expression and other recombinant techniques to alter expression of known copper binding proteins to investigate the role of the prion protein in copper metabolism. We found that changing the expression of the prion protein alters proteins associated with copper uptake, storage and export from the cell. In addition, alteration in the expression of superoxide dismutases increased prion protein expression dramatically. Reducing copper in the diet decreased expression of the prion protein in the brain while increased dietary manganese dramatically increased the protein's expression. Cellular prion infection also increased the expression of metal transporting proteins and increased cellular manganese concentrations. Overall our results show a close link between cellular resistance to oxidative stress and also copper metabolism. These findings are in line with previous data suggesting that the prion protein is an antioxidant and associated with copper uptake into cells. The disturbance to copper metabolism, as a result of altered prion protein expression clearly demonstrates the important role of the prion protein in copper metabolism. The implication is that prion protein expression has a homeostatic role in copper metabolism. PMID:19233277

Kralovicova, Silvia; Fontaine, Sarah N; Alderton, Alexandra; Alderman, Julia; Ragnarsdottir, K Vala; Collins, Steven J; Brown, David R

2009-06-01

65

Telethonin protein expression in neuromuscular disorders.  

PubMed

Telethonin is a 19-kDa sarcomeric protein, localized to the Z-disc of skeletal and cardiac muscles. Mutations in the telethonin gene cause limb-girdle muscular dystrophy type 2G (LGMD2G). We investigated the sarcomeric integrity of muscle fibers in LGMD2G patients, through double immunofluorescence analysis for telethonin with three sarcomeric proteins: titin, alpha-actinin-2, and myotilin and observed the typical cross striation pattern, suggesting that the Z-line of the sarcomere is apparently preserved, despite the absence of telethonin. Ultrastructural analysis confirmed the integrity of the sarcomeric architecture. The possible interaction of telethonin with other proteins responsible for several forms of neuromuscular disorders was also analyzed. Telethonin was clearly present in the rods in nemaline myopathy (NM) muscle fibers, confirming its localization to the Z-line of the sarcomere. Muscle from patients with absent telethonin showed normal expression for the proteins dystrophin, sarcoglycans, dysferlin, and calpain-3. Additionally, telethonin showed normal localization in muscle biopsies from patients with LGMD2A, LGMD2B, sarcoglycanopathies, and Duchenne muscular dystrophy (DMD). Therefore, the primary deficiency of calpain-3, dysferlin, sarcoglycans, and dystrophin do not seem to alter telethonin expression. PMID:12379311

Vainzof, Mariz; Moreira, Eloisa S; Suzuki, Oscar T; Faulkner, Georgine; Valle, Georgio; Beggs, Alan H; Carpen, Olli; Ribeiro, Alberto F; Zanoteli, Edmar; Gurgel-Gianneti, Juliana; Tsanaclis, Ana Maria; Silva, Helga C A; Passos-Bueno, Maria Rita; Zatz, Mayana

2002-10-01

66

Microgravity alters the expression of salivary proteins.  

PubMed

Spaceflight provides a unique opportunity to study how physiologic responses are influenced by the external environment. Microgravity has been shown to alter the function of a number of tissues and organ systems. Very little, however, is known about how microgravity affects the oral cavity. The rodent model is useful for study in that their salivary gland morphology and physiology is similar to that of humans. Useful also is the fact that saliva, a product of the salivary glands with a major role in maintaining oral health, can be easily collected in humans whereas the glands can be studied in experimental animals. Our working hypothesis is that expression of secretory proteins in saliva will respond to microgravity and will be indicative of the nature of physiologic reactions to travel in space. This study was designed to determine which components of the salivary proteome are altered in mice flown on the US space shuttle missions and to determine if a subset with predictive value can be identified using microscopy and biochemistry methods. The results showed that the expression of secretory proteins associated with beta-adrenergic hormone regulated responses and mediated via the cyclic AMP pathway was significantly altered, whereas that of a number of unrelated proteins was not. The findings are potentially applicable to designing a biochemical test system whereby specific salivary proteins can be biomarkers for stress associated with travel in space and eventually for monitoring responses to conditions on earth. PMID:24984624

Mednieks, Maija; Khatri, Aditi; Rubenstein, Renee; Burleson, Joseph A; Hand, Arthur R

2014-06-01

67

Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein  

PubMed Central

Sixteen of 22 low molecular weight integral membrane proteins from Mycobacterium tuberculosis with previously poor or undetectable levels of expression were expressed in Escherichia coli as fusions with both the maltose-binding protein (MBP) and a His8-tag. 68% of targeted proteins were expressed in high yield (?30 mg/L) in soluble and/or inclusion body form. Thrombin cleavage of the MBP fusion protein was successful for 10 of 13 proteins expressed as soluble proteins and for three proteins expressed only as inclusion bodies. The use of autoinduction growth media increased yields over Luria-Bertani (LB) growth media in 75% of the expressed proteins. Expressing integral membrane proteins with yields suitable for structural studies from a set of previously low and non-expressing proteins proved highly successful upon attachment of the maltose-binding protein as a fusion tag. PMID:17275326

KOREPANOVA, A.; MOORE, J.D.; NGUYEN, H.; HUA, Y.; CROSS, T. A.; GAO, F.

2009-01-01

68

Expression of Contractile Protein Isoforms in Microgravity  

NASA Technical Reports Server (NTRS)

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.

Anderson, Page A. W.

1996-01-01

69

Expression of MDR1/P glycoprotein in human sarcomas.  

PubMed

Conflicting reports of MDR1 gene expression in human tumours are observed according to whether studies are performed at the mRNA or P-glycoprotein level. We have investigated this expression in 22 clinically drug-resistant sarcomas at the mRNA level by Northern blot (NB), Dot blot (DB), in situ hybridisation (ISH), and at the protein level by immunohistochemistry (IHC) using three monoclonal antibodies (MoAbs): C219, JSB1, MRK16. Increased MDR1 mRNA expression was detected by NB, DB, and ISH in 1/22 sarcoma (an Ewing's sarcoma). ISH was perfectly correlated with DB hybridisation and confirmed the expression of tumoral cells alone. Specific staining of 100% of tumoral cells was obtained with the three MoAbs in the same sarcoma. Expression in tumoral cells of 12 other sarcomas was detected with MRK16, and positive staining of stromal cells with both C219 (1/22) and MRK16 (8/22) was observed. This study confirms that MDR1 overexpression occurs in human sarcomas but is not the principal mechanism of drug-resistance. Furthermore, positivity with one antibody does not necessarily imply the presence of P glycoprotein (P-gp) and a disparity may exist between the levels of P-gp and its mRNA in the same sample. So care must be taken in interpreting results and more sensitive techniques such as the polymerase chain reaction (PCR) could prove useful. PMID:7903154

Vergier, B; Cany, L; Bonnet, F; Robert, J; de Mascarel, A; Coindre, J M

1993-12-01

70

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

PubMed Central

Background 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. Findings 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 ?mol/L and???30 ?mol/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. Conclusion 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. PMID:24196382

2013-01-01

71

Original Research Communication Prion Protein Expression and Functional Importance in  

E-print Network

1 1 Original Research Communication Prion Protein Expression and Functional Importance illustrations: 7 Color illustrations: 2 (online 2) Page 1 of 48 Antioxidants&RedoxSignaling PrionProtein prion protein (PrPC ), a GPI-anchored glycoprotein, which we reported to be highly expressed in human

Paris-Sud XI, Université de

72

Green Fluorescent Protein as a Marker for Gene Expression  

Microsoft Academic Search

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.

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

1994-01-01

73

Effects of immunosuppressive treatment on protein expression in rat kidney  

PubMed Central

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 group. The consequences of the reported changes in protein expression require further study. PMID:25328384

K?dzierska, Karolina; Sporniak-Tutak, Katarzyna; Sindrewicz, Krzysztof; Bober, Joanna; Doma?ski, Leszek; Parafiniuk, Miros?aw; Urasi?ska, El?bieta; Ciechanowicz, Andrzej; Doma?ski, Maciej; Smekta?a, Tomasz; Masiuk, Marek; Skrzypczak, Wies?aw; O?go, Ma?gorzata; Kabat-Koperska, Joanna; Ciechanowski, Kazimierz

2014-01-01

74

Altered expression of mRNAs for apoptosis-modulating proteins in a low level multidrug resistant variant of a human lung carcinoma cell line that also expresses mdr1 mRNA.  

PubMed

An in vitro model that might be relevant to cancer cell chemoresistance in vivo was generated by exposing the human lung carcinoma clonal cell line DLKP-SQ to 10 sequential pulses of pharmacologically attainable doses of doxorubicin. The resistant variant, DLKP-SQ/10p, was found to be cross-resistant to doxorubicin (10x), vincristine (43x), etoposide (3x), sodium arsenate (3x), paclitaxel (38x) [which could imply overexpression of P-glycoprotein (P-gp) and possibly increased multidrug resistance-associated protein activity] and 5-fluorouracil (4x), but slightly sensitized to carboplatin. Analysis of mRNA levels in the resistant variant revealed overexpression of mdr1 mRNA without significant alteration in mrp, Topo. IIalpha, GSTpi, dhfr or thymidylate synthase mRNA levels. Overexpression of the anti-apoptotic bcl-xL transcript and the pro-apoptotic bax mRNA was also detected but no alterations in bcl-2 or bag-1 mRNA levels were observed. Resistance to a P-gp-associated drug, doxorubicin, could be reversed with P-gp circumventing agents such as cyclosporin A and verapamil, but these substances had no effect on resistance to 5-fluorouracil. Overexpression of the pro-apoptotic bcl-xS gene in the DLKP-SQ/10p line partially reversed resistance not only to P-gp-associated drugs but also to 5-fluorouracil, indicating that the ratio of bcl family members may be important in determining sensitivity to chemotherapeutic drug-induced apoptosis. PMID:10399954

NicAmhlaoibh, R; Heenan, M; Cleary, I; Touhey, S; O'Loughlin, C; Daly, C; Nuñez, G; Scanlon, K J; Clynes, M

1999-07-30

75

Expression of hepatitis C virus proteins in epithelial intestinal cells in vivo Short title: HCV protein expression in intestine  

E-print Network

Expression of hepatitis C virus proteins in epithelial intestinal cells in vivo Short title: HCV protein expression in intestine Séverine Deforges1* , Alexey Evlashev1* , Magali Perret1 , Mireille RNA and core protein. These findings suggest that LVP synthesis could occur in liver and intestine

Paris-Sud XI, Université de

76

Over-expression of secreted proteins from mammalian cell lines  

PubMed Central

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

Dalton, Annamarie C; Barton, William A

2014-01-01

77

Strain engineering for improved expression of recombinant proteins in bacteria  

PubMed Central

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

2011-01-01

78

Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast  

DOEpatents

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.

Mayfield, Stephen P. (Cardiff, CA)

2010-03-16

79

Relationship between the expression of cyclooxygenase 2 and MDR1/P-glycoprotein in invasive breast cancers and their prognostic significance  

PubMed Central

Introduction Recent reports suggest that expression of the cyclooxygenase 2 (COX-2) enzyme may up-regulate expression of MDR1/P-glycoprotein (MDR1/P-gp), an exponent of resistance to cytostatic drugs. The present study aimed at examining the relationship between the expression of COX-2 and of MDR1/P-gp in a group of breast cancer cases. Methods Immunohistochemical reactions were performed using monoclonal antibodies against COX-2 and MDR1/P-gp on samples originating from 104 cases of primary invasive breast cancer. Results COX-2-positive cases were shown to demonstrate higher expression of MDR1/P-gp (P < 0.0001). The studies also demonstrate that COX-2 expression was typical for cases of a higher grade (P = 0.01), a shorter overall survival time (P < 0.0001) and a shorter progression-free time (P < 0.0001). In the case of MDR1/P-gp, its higher expression characterised cases of a higher grade (P < 0001), with lymph node involvement (P < 0001), and shorter overall survival (P < 0.0001) and progression-free time (P < 0.0001). Conclusion Our studies confirmed the unfavourable prognostic significance of COX-2 and MDR1/P-gp. We also document a relationship between COX-2 and MDR1/P-gp, which suggests that COX-2 inhibitors should be investigated in trials as a treatment supplementary to chemotherapy of breast cancers. PMID:16168133

Surowiak, Pawel; Materna, Verena; Matkowski, Rafal; Szczuraszek, Katarzyna; Kornafel, Jan; Wojnar, Andrzej; Pudelko, Marek; Dietel, Manfred; Denkert, Carsten; Zabel, Maciej; Lage, Hermann

2005-01-01

80

Nicotine Regulates Streptococcus mutans Extracellular Polysaccharide and Related Protein Expression.  

E-print Network

Nicotine Regulates Streptococcus mutans Extracellular Polysaccharide and Related Protein Expression of Dentistry; 2 Department of Pathology and Laboratory Medicine, IU School of Medicine Streptococcus mutans

Zhou, Yaoqi

81

Strain engineering for improved expression of recombinant proteins in bacteria  

Microsoft Academic Search

Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes.\\u000a So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying\\u000a parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several\\u000a new approaches for the

Tomohiro Makino; Georgios Skretas; George Georgiou

2011-01-01

82

Calreticulin: Roles in Cell-Surface Protein Expression  

PubMed Central

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

Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

2014-01-01

83

P-glycoprotein expression and distribution in the rat placenta during pregnancy.  

PubMed

P-glycoprotein (P-gp) is a drug efflux transporter that limits the entry of various potentially toxic drugs and xenobiotics into the fetus and is thus considered a placental protective mechanism. In this study, P-gp expression was investigated in the rat chorioallantoic placenta over the course of pregnancy. Three methods have been employed: real-time RT-PCR, western blotting and immunohistochemistry. The expression of mdr1a and mdr1b genes was demonstrated as early as on the 11th gestation day (gd) and increased with advancing gestation. Western blotting analysis revealed the presence of P-gp in the rat placenta starting from gd 13 onwards. P-gp was localized in the developing labyrinth zone of the placenta on gd 13; from gd 15 up to the term P-gp was seen as a dot like continuous line in the syncytiotrophoblast layers. Our data confirm the presence of P-gp in the rat chorioallantoic placenta starting soon after its development, which may signify the involvement of P-gp in transplacental pharmacokinetics during the whole period of placental maturing. PMID:15279876

Novotna, Martina; Libra, Antonin; Kopecky, Martin; Pavek, Petr; Fendrich, Zdenek; Semecky, Vladimir; Staud, Frantisek

2004-01-01

84

Immunogenicity of foreign peptide epitopes expressed in bacterial envelope proteins.  

PubMed

We have used two bacterial proteins from Escherichia coli to express heterologous peptides. Both proteins are situated in the E. coli cell envelope but have different properties: LamB is an integral outer membrane protein, and MalE a soluble periplasmic protein. The peptides were expressed as genetic inserts within "permissive sites" of these recipient proteins, i.e. sites which allow the insertion of foreign peptides without affecting the biological properties of the host protein. In this paper, we summarize preliminary rules governing the immunogenicity of resulting LamB and MalE hybrid proteins when expressed in E. coli. We focus on two model epitopes: either peptide 132-145 from the preS(2) region of hepatitis B virus or peptide 93-103 from poliovirus VP1 capsid protein. We also present first results obtained when the same hybrid proteins were expressed in attenuated Salmonella typhimurium. Plasmids encoding the hybrid proteins were transferred to aroA S.typhimurium by electroporation. In vitro, the hybrid proteins could be expressed at high levels by S. typhimurium. Mice were immunized by parenteral and oral routes. The effect of the carrier protein and the level of its expression on the in vivo behaviour of the immunizing bacteria and on the immune response induced will be discussed. PMID:1714094

O'Callaghan, D; Charbit, A; Martineau, P; Leclerc, C; van der Werf, S; Nauciel, C; Hofnung, M

1990-01-01

85

GST-Tagged Protein Expression and Purification 1. Protein Expression (BL21)  

E-print Network

minutes Pour off waste and either begin lysis or store pellet at ­80°C #12;2. GST-Tagged ProteinM IPTG Shake for 2.5 hrs at 37°C Spin 7K 4C for 10 minutes Pour off waste and either begin lysis or store pellet at ­80°C For Low Temperature Expression: Inoculate a starter culture of 10ml in a 250ml flask

Gozani, Or

86

Expression of multidrug resistance P-glycoprotein in kidney allografts from cyclosporine A-treated patients  

Microsoft Academic Search

Expression of multidrug resistance P-glycoprotein in kidney allografts from cyclosporine A-treated patients.BackgroundThe multidrug resistance (MDR) gene product P-glycoprotein (P-gp) is a transmembrane efflux pump for hydrophobic, potentially toxic compounds, including the immunosuppressant cyclosporine A (CsA). We have previously shown that CsA increases P-gp expression in proximal tubule and endothelial cells in vitro. The aim of the present study was to

Michael J Koziolek; Regine Riess; Helmut Geiger; Frank Thévenod; Ingeborg A Hauser

2001-01-01

87

Assessing the expression of chicken anemia virus proteins in plants  

Microsoft Academic Search

Chicken anemia virus (CAV) is an important pathogen of chicken worldwide, causing severe anemia and immunodeficiency. Its small single-stranded DNA genome (2.3kb) encodes three proteins: VP1, the only structural protein, VP2, a protein phosphatase, and VP3, also known as apoptin, which induces apoptosis. In this study, CAV proteins were expressed in plants as an alternative for recombinant protein production in

Cristiano Lacorte; Hendrikus Lohuis; Rob Goldbach; Marcel Prins

2007-01-01

88

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

PubMed

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. PMID:25502200

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

2015-01-01

89

Creation of genome-wide protein expression libraries using random activation of gene expression  

Microsoft Academic Search

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 × 106 individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE

Bruce Sherf; Stephen Rundlett; P. David Jackson; Rob Perry; Scott Cain; Christina Leventhal; Mark Thornton; Rakesh Ramachandran; Jessica Whittington; Laura Lerner; Dana Costanzo; Karen McElligott; Sherry Boozer; Robert Mays; Emery Smith; Neil Veloso; Alison Klika; Jennifer Hess; Kevin Cothren; Kalok Lo; Jason Offenbacher; Joel Danzig; Matt Ducar; John J. Harrington

2001-01-01

90

mir-29 Regulates Mcl-1 Protein Expression and Apoptosis  

PubMed Central

Cellular expression of Mcl-1, an anti-apoptotic Bcl-2 family member, is tightly regulated. Recently, Bcl-2 expression was shown to be regulated by microRNAs, small endogenous RNA molecules that regulate protein expression through sequence-specific interaction with messenger RNA. By analogy, we reasoned that Mcl-1 expression may also be regulated by microRNAs. We chose human immortalized, but non-malignant, H69 cholangiocyte and malignant KMCH cholangiocarcinoma cell lines for these studies because Mcl-1 is dysregulated in cells with the malignant phenotype. In silico analysis identified a putative target site in the Mcl-1 mRNA for the mir-29 family, and we found that mir-29b was highly expressed in cholangiocytes. Interestingly, mir-29b was downregulated in malignant cells, consistent with Mcl-1 protein upregulation. Enforced mir-29b expression reduced Mcl-1 protein expression in KMCH cells. This effect was direct, as mir-29b negatively regulated expression of an Mcl-1 3’ untranslated region (UTR)-based reporter construct. Enforced mir-29b expression reduced Mcl-1 cellular protein levels and sensitized the cancer cells to TRAIL cytotoxicity. Transfection of non-malignant cells (that express high levels of mir-29) with a locked-nucleic acid antagonist of mir-29b increased Mcl-1 levels and reduced TRAIL-mediated apoptosis. Thus mir-29 is an endogenous regulator of Mcl-1 protein expression and, thereby, apoptosis. PMID:17404574

Mott, Justin L.; Kobayashi, Shogo; Bronk, Steven F.; Gores, Gregory J.

2008-01-01

91

Effects of quercetin on the bioavailability of doxorubicin in rats: role of CYP3A4 and P-gp inhibition by quercetin.  

PubMed

Quercetin, a flavonoid, is an inhibitor of P-glycoprotein-mediated efflux transport, and its oxidative metabolism is catalyzed by CYP enzymes. Thus, it is expected that the pharmacokinetics of both intravenous and oral doxorubicin can be changed by quercetin. The purpose of this study was to investigate the effect of oral quercetin on the bioavailability and pharmacokinetics of orally and intravenously administered doxorubicin in rats. The effects of quercetin on the P-glycoprotein (P-gp) and CYP3A4 activities were also evaluated. Quercetin inhibited CYP3A4 enzyme activity in a concentration-dependent manner with a 50% inhibition concentration (IC(50)) of 1.97 ?M. In addition, quercetin significantly enhanced the intracellular accumulation of rhodamine-123 in MCF-7/ADR cells overexpressing P-gp. The pharmacokinetic parameters of doxorubicin were determined in rats after oral (50 mg/kg) or intravenous (10 mg/kg) administration of doxorubicin to rats in the presence and absence of quercetin (0.6, 3 or 15 mg/kg). Compared to control, quercetin significantly (p < 0.05 for 0.6 mg/kg, p < 0.01 for 3 and 15 mg/kg) increased the area under the plasma concentration-time curve (AUC(0-?), 31.2-136.0% greater) of oral doxorubicin. Quercetin also significantly increased the peak plasma concentration (C(max)) of doxorubicin, while there was no significant change in T(max) and T(1/2) of doxorubicin. Consequently, the absolute bioavailability of doxorubicin was increased by quercetin compared to control, and the relative bioavailability of oral doxorubicin was increased by 1.32 to 2.36 fold. In contrast, the pharmacokinetics of intravenous doxorubicin were not affected by quercetin. These results suggest that the quercetin-induced increase in bioavailability of oral doxorubicin can be attributed to enhanced doxorubicin absorption in the gastrointestinal tract via quercetin-induced inhibition of P-gp and reduced first-pass metabolism of doxorubicin due to quercetin-induced inhibition of CYP3A in the small intestine and/or in the liver rather than reduced renal and/or hepatic elimination of doxorubicin. Therefore, it appears that the development of oral doxorubicin preparations is possible, which will be more convenient than the intravenous dosage forms. Therefore, concurrent use of quercetin provides a therapeutic benefit - it increases the bioavailability of doxorubicin administered orally. PMID:21544726

Choi, Jun-Shik; Piao, Yong-Ji; Kang, Keon Wook

2011-04-01

92

Functional expression of P-glycoprotein in primary cultures of human cytotrophoblasts and BeWo cells  

E-print Network

The objective of this study was to investigate the functional expression of the efflux transporter, P-glycoprotein (P-gp), in primary cultures of human cytotrophoblasts and BeWo cell monolayers. Uptake studies with primary ...

Utoguchi, Naoki; Chandorkar, Gurudatt A.; Avery, Michael; Audus, Kenneth L.

2000-01-01

93

Protein expression in Arabidopsis thaliana after chronic clinorotation  

NASA Technical Reports Server (NTRS)

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.

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

1995-01-01

94

Protein expression in Arabidopsis thaliana after chronic clinorotation  

NASA Technical Reports Server (NTRS)

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.

Piastuch, William C.; Brown, Christopher S.

1994-01-01

95

Identification of highly expressed, soluble proteins using an improved, high-throughput pooled ORF expression technology.  

PubMed

This article describes an improved pooled open reading frame (ORF) expression technology (POET) that uses recombinational cloning and solution-based tandem mass spectrometry (MS/MS) to identify ORFs that yield high levels of soluble, purified protein when expressed in Escherichia coli. Using this method, three identical pools of 512 human ORFs were subcloned, purified, and transfected into three separate E. coli cultures. After bulk expression and purification, the proteins from the three separate pools were digested into tryptic peptides. Each of these samples was subsequently analyzed in triplicate using reversed-phase high-performance liquid chromatography (LC) coupled directly online with MS/MS. The abundance of each protein was determined by calculating the average exponentially modified protein abundance index (emPAI) of each protein across the three protein pools. Human proteins that consistently gave high emPAI values were subjected to small-scale expression and purification. These clones showed high levels of expression of soluble protein. Conversely, proteins that were not observed by LC-MS/MS did not show any detectable soluble expression in small-scale validation studies. Using this improved POET method allows the expression characteristics of hundreds of proteins to be quickly determined in a single experiment. PMID:18778254

Waybright, Timothy; Gillette, William; Esposito, Dominic; Stephens, Robert; Lucas, David; Hartley, James; Veenstra, Timothy

2008-09-01

96

Co-delivery of doxorubicin and P-gp inhibitor by a reduction-sensitive liposome to overcome multidrug resistance, enhance anti-tumor efficiency and reduce toxicity.  

PubMed

Abstract To overcome multidrug resistance (MDR) in cancer chemotherapy with high efficiency and safety, a reduction-sensitive liposome (CL-R8-LP), which was co-modified with reduction-sensitive cleavable PEG and octaarginine (R8) to increase the tumor accumulation, cellular uptake and lysosome escape, was applied to co-encapsulate doxorubicin (DOX) and a P-glycoprotein (P-gp) inhibitor of verapamil (VER) in this study. The encapsulation efficiency (EE) of DOX and VER in the binary-drug loaded CL-R8-LP (DOX?+?VER) was about 95 and 70% (w/w), respectively. The uptake efficiencies, the cytotoxicity, and the apoptosis and necrosis-inducing efficiency of CL-R8-LP (DOX?+?VER) were much higher than those of DOX and the other control liposomes in MCF-7/ADR cells or tumor spheroids. Besides, CL-R8-LP (DOX?+?VER) was proven to be uptaken into MCF-7/ADR cells by clathrin-mediated and macropinocytosis-mediated endocytosis, followed by efficient lysosomal escape. In vivo, CL-R8-LP (DOX?+?VER) effectively inhibited the growth of MCF-7/ADR tumor and reduce the toxicity of DOX and VER, which could be ascribed to increased accumulation of drugs in drug-resistant tumor cells and reduced distribution in normal tissues. In summary, the co-delivery of chemotherapeutics and P-gp inhibitors by our reduction-sensitive liposome was a promising approach to overcome MDR, improve anti-tumor effect and reduce the toxicity of chemotherapy. PMID:25491241

Tang, Jie; Zhang, Li; Gao, Huile; Liu, Yayuan; Zhang, Qianyu; Ran, Rui; Zhang, Zhirong; He, Qin

2014-12-10

97

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

PubMed

Abstract Objective: 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. Methods: To accomplish DC, formulation blends were evaluated for micromeritic properties. The Carr index, Hausner ratio, flow rate and cotangent of the angle ? 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. Results: 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??g/mL and 0.14?±?0.09??g 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. Conclusion: 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. PMID:25080228

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

2014-07-31

98

Exact protein distributions for stochastic models of gene expression  

NASA Astrophysics Data System (ADS)

Stochasticity in gene expression gives rise to variations in protein levels across a population of genetically identical cells. Such fluctuations can drive phenotypic variation in clonal populations, hence there is considerable interest in quantifying noise in gene expression using stochastic models. However, obtaining exact analytical results for protein distributions has been an intractable task for all but the simplest models. We develop a novel mapping that significantly simplifies the analysis of stochastic models of gene expression. Using this mapping, we derive exact analytical results for steady-state and time-dependent protein distributions for the basic 2-stage model of gene expression. Considering extensions of the basic model, we obtain exact protein steady-state distributions for models that include the effects of post-transcriptional and post-translational regulation. The approach developed in this work is widely applicable and can contribute to a quantitative understanding of stochasticity in gene expression and its regulation.

Kulkarni, Rahul; Pendar, Hodjat; Platini, Thierry

2013-03-01

99

The Statistics of Protein Expression Ratios for Cellular Fluorescence Studies  

PubMed Central

Fluorescence studies of cellular protein-protein interactions commonly employ transient cotransfection to express two proteins carrying distinct fluorescent labels. Because transiently transfected cells differ significantly in their expression level, the concentration ratio of the two expressed proteins varies, which in turn influences the measured fluorescence signal. Knowledge of the statistics of protein expression ratios is of considerable interest both from a fundamental point of view and for cellular fluorescence studies. Despite the perceived randomness of transient transfection, we were able to develop a quantitative model that describes the average and distribution of the protein expression ratio from a cell population. We show that the expression ratio is proportional to the molar plasmid ratio and relate the distribution to the finite number of active plasmids in the cell. The process of cationic lipid-mediated transfection is explored in more detail. Specifically, the influence of lipoplexes on the statistics of the expression ratio is examined. We further demonstrate that the transfection model provides a quantitative description of fluorescence fluctuation experiments, where only a fraction of the proteins are labeled. PMID:22307451

Smith, Elizabeth M.; Mueller, Joachim D.

2012-01-01

100

Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris  

PubMed Central

The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate-screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N-methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing “jackpot” clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in-culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins. PMID:23339074

Brooks, Cory L; Morrison, Melissa; Joanne Lemieux, M

2013-01-01

101

Protein expression analyses at the single cell level.  

PubMed

The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level. PMID:25197931

Ohno, Masae; Karagiannis, Peter; Taniguchi, Yuichi

2014-01-01

102

Gene expression pattern Cloning and expression of a novel cysteine-rich secreted protein  

E-print Network

Gene expression pattern Cloning and expression of a novel cysteine-rich secreted protein family member expressed in thyroid and pancreatic mesoderm within the chicken embryo Devyn M. Smitha , Lisa A 4 January 2001; accepted 14 January 2001 Abstract We have isolated a new chicken gene

Tabin, Cliff

103

Protein Production for Structural Genomics Using E. coli Expression  

PubMed Central

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

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

2014-01-01

104

Protein Expression Dynamics During Postnatal Mouse Brain Development  

PubMed Central

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

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

2013-01-01

105

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

PubMed Central

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

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

2004-01-01

106

Multiple folding pathways for heterologously expressed human prion protein  

E-print Network

Multiple folding pathways for heterologously expressed human prion protein Graham S. Jackson , Anthony R. Clarke a , John Collinge aY * a Prion Disease Group, Department of Neurogenetics, Imperial-conformation in free solution. The data we present here shows that the human prion protein can exist in multiple

Hosszu, Laszlo

107

A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice  

PubMed Central

Proteomics has opened a new horizon in biological sciences. Global proteomic analysis is a promising technology for the discovery of thousands of proteins, post-translational modifications, polymorphisms, and molecular interactions in a variety of biological systems. The activities and roles of the identified proteins must also be elucidated, but this is complicated by the inability of conventional proteomic methods to yield quantitative information for protein expression. Thus, a variety of biological systems remain “black boxes”. Quantitative targeted absolute proteomics (QTAP) enables the determination of absolute expression levels (mol) of any target protein, including low-abundance functional proteins, such as transporters and receptors. Therefore, QTAP will be useful for understanding the activities and roles of individual proteins and their differences, including normal/disease, human/animal, or in vitro/in vivo. Here, we describe the study protocols and precautions for QTAP experiments including in silico target peptide selection, determination of peptide concentration by amino acid analysis, setup of selected/multiple reaction monitoring (SRM/MRM) analysis in liquid chromatography–tandem mass spectrometry, preparation of protein samples (brain capillaries and plasma membrane fractions) followed by the preparation of peptide samples, simultaneous absolute quantification of target proteins by SRM/MRM analysis, data analysis, and troubleshooting. An application of QTAP in biological sciences was introduced that utilizes data from inter-strain differences in the protein expression levels of transporters, receptors, tight junction proteins and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice. Among 18 molecules, 13 (abcb1a/mdr1a/P-gp, abcc4/mrp4, abcg2/bcrp, slc2a1/glut1, slc7a5/lat1, slc16a1/mct1, slc22a8/oat3, insr, lrp1, tfr1, claudin-5, Na+/K+-ATPase, and ?-gtp) were detected in the isolated brain capillaries, and their protein expression levels were within a range of 0.637-101 fmol/?g protein. The largest difference in the levels between the three strains was 2.2-fold for 13 molecules, although bcrp and mct1 displayed statistically significant differences between C57BL/6J and the other strain(s). Highly sensitive simultaneous absolute quantification achieved by QTAP will increase the usefulness of proteomics in biological sciences and is expected to advance the new research field of pharmacoproteomics (PPx). PMID:23758935

2013-01-01

108

Expression of the Sex combs reduced protein in Drosophila larvae.  

PubMed

We have generated a monoclonal antibody that binds specifically to the protein product of the homeotic Sex combs reduced (Scr) gene of Drosophila, and have mapped the patterns of Scr expression in late third instar larvae. Virtually the entire prothoracic leg imaginal disc expresses the gene, although the levels of expression vary in different disc regions. This heterogeneity does not reflect the compartmental domains defined by engrailed gene expression. Expression is also observed in the cells of the humeral and labial discs, and there is a small patch of Scr-expressing cells in the antenna disc. The gene is expressed in adepithelial cells of the three thoracic leg discs, but not in the wing or haltere discs. In the central nervous system, Scr expression is confined to a narrow band of cells in the subesophageal region of the ventral ganglion. The results are discussed with respect to the known genetic requirements for Scr+ function. PMID:2896135

Glicksman, M A; Brower, D L

1988-05-01

109

Recent developments in therapeutic protein expression technologies in plants.  

PubMed

Infectious diseases and cancers are some of the commonest causes of deaths throughout the world. The previous two decades have witnessed a combined endeavor across various biological sciences to address this issue in novel ways. The advent of recombinant DNA technologies has provided the tools for producing recombinant proteins that can be used as therapeutic agents. A number of expression systems have been developed for the production of pharmaceutical products. Recently, advances have been made using plants as bioreactors to produce therapeutic proteins directed against infectious diseases and cancers. This review highlights the recent progress in therapeutic protein expression in plants (stable and transient), the factors affecting heterologous protein expression, vector systems and recent developments in existing technologies and steps towards the industrial production of plant-made vaccines, antibodies, and biopharmaceuticals. PMID:25326175

Fahad, Shah; Khan, Faheem Ahmed; Pandupuspitasari, Nuruliarizki Shinta; Ahmed, Muhammad Mahmood; Liao, Yu Cai; Waheed, Muhammad Tahir; Sameeullah, Muhammad; Darkhshan; Hussain, Saddam; Saud, Shah; Hassan, Shah; Jan, Amanullah; Jan, Mohammad Tariq; Wu, Chao; Chun, Ma Xiao; Huang, Jianliang

2015-02-01

110

Variation in Protein Intake Induces Variation in Spider Silk Expression  

PubMed Central

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

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

2012-01-01

111

Expression of Focal Adhesion Proteins in the Developing Rat Kidney  

PubMed Central

Focal adhesions play a critical role as centers that transduce signals by cell-matrix interactions and regulate fundamental processes such as proliferation, migration, and differentiation. Focal adhesion kinase (FAK), paxillin, integrin-linked kinase (ILK), and hydrogen peroxide–inducible clone-5 (Hic-5) are major proteins that contribute to these events. In this study, we investigated the expression of focal adhesion proteins in the developing rat kidney. Western blotting analysis revealed that the protein levels of FAK, p-FAK397, paxillin, p-paxillin118, and Hic-5 were high in embryonic kidneys, while ILK expression persisted from the embryonic to the mature stage. Immunohistochemistry revealed that FAK, p-FAK397, paxillin, and p-paxillin118 were strongly expressed in condensed mesenchymal cells and the ureteric bud. They were detected in elongating tubules and immature glomerular cells in the nephrogenic zone. Hic-5 was predominantly expressed in mesenchymal cells as well as immature glomerular endothelial and mesangial cells, suggesting that Hic-5 might be involved in mesenchymal cell development. ILK expression was similar to that of FAK in the developmental stages. Interestingly, ILK was strongly expressed in podocytes in mature glomeruli. ILK might play a role in epithelial cell differentiation as well as kidney growth and morphogenesis. In conclusion, the temporospatially regulated expression of focal adhesion proteins during kidney development might play a role in morphogenesis and cell differentiation. PMID:21705647

Matsuura, Sato; Kondo, Shuji; Suga, Kenichi; Kinoshita, Yukiko; Urushihara, Maki; Kagami, Shoji

2011-01-01

112

Identification of Differentially Expressed Serum Proteins in Infectious Purpura Fulminans  

PubMed Central

Purpura fulminans (PF) is a life-threatening hemorrhagic condition. Because of the rarity and randomness of the disease, no improvement in treatment has been made for a long time. In this study, we assessed the serum proteome response to PF by comparing serum proteins between healthy controls and PF patient. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach was used after depleting 6 abundant proteins of serum. In total, 262 proteins were confidently identified with 2 unique peptides, and 38 proteins were identified significantly up- (?2) or downregulated (?0.5) based on spectral counting ratios (SpCPF/N). In the 38 proteins with significant abundance changes, 11 proteins were previously known to be associated with burn or sepsis response, but 27 potentially novel proteins may be specifically associated with PF process. Two differentially expressed proteins, alpha-1-antitrypsin (SERPINA1) and alpha-2 antiplasmin (SERPINF2), were validated by Western blot. This is the first study where PF patient and healthy controls are compared in a proteomic study to elucidate proteins involved in the response to PF. This study provides an initial basis for future studies of PF, and the differentially expressed proteins might provide new therapeutic targets to decrease the mortality of PF. PMID:24659849

Hu, Jiong-yu; Han, Jian; Zhang, Dong-xia; Jiang, Xu-pin; Chen, Bing; Huang, Yue-sheng

2014-01-01

113

Differential protein expression in phalaenopsis under low temperature.  

PubMed

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. PMID:25349090

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

2015-01-01

114

Functional expression of plant membrane proteins in Lactococcus lactis.  

PubMed

The study of most membrane proteins remains challenging due to their hydrophobicity and their low natural abundance in cells. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations and is nowadays widely used in biotechnology for large-scale production of heterologous proteins. This system has been successfully used for the production of prokaryotic and eukaryotic membrane proteins. The purpose of this chapter is to provide detailed protocols for (1) the expression of plant peripheral or intrinsic membrane proteins and then for (2) their solubilization, from Lactococcus membranes, for further purification steps and biochemical characterization. PMID:25447863

Boutigny, Sylvain; Sautron, Emeline; Frelet-Barrand, Annie; Moyet, Lucas; Salvi, Daniel; Rolland, Norbert; Seigneurin-Berny, Daphné

2015-01-01

115

Expression of genes encoding extracellular matrix proteins: A macroarray study  

PubMed Central

Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain ?1 and type XI chain ?2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs. PMID:25231141

FUTYMA, KONRAD; MIOT?A, PAWE?; RÓ?Y?SKA, KRYSTYNA; ZDUNEK, MA?GORZATA; SEMCZUK, ANDRZEJ; RECHBERGER, TOMASZ; WOJCIEROWSKI, JACEK

2014-01-01

116

Membrane cofactor protein (MCP; CD46) expression in transgenic mice  

PubMed Central

Human membrane cofactor protein (MCP; CD46) is a widely distributed complement regulator. In the mouse, expression of MCP is largely restricted to the testis while a related, widely expressed protein (Crry) appears to perform MCP's (CD46) regulatory activity. We have developed two mouse strains transgenic for human MCP (CD46) utilizing an ? 400 kb YAC clone carrying the complete gene. A third mouse strain was generated using an overlapping YAC clone isolated from a second library. The expression of human MCP (CD46) in these mouse strains was characterized by immunohistochemistry, FACS, Western blotting and RT-PCR. No differences were detected in the isoform pattern or distribution among the three strains, although the expression level varied according to how many copies of the gene were integrated. The expression profile closely mimicked that observed in humans, including the same pattern of isoform expression as the donor. In addition, tissue-specific isoform expression in the kidney, salivary gland and brain paralleled that observed in man. The transgenic mice expressed low levels of MCP (CD46) on their E, in contrast to humans but in line with most other primates. These mice should be a useful tool to analyse tissue-specific expression, to establish animal models of infections and to characterize the role of MCP (CD46) in reproduction. PMID:11422193

Kemper, C; Leung, M; Stephensen, C B; Pinkert, C A; Liszewski, M K; Cattaneo, R; Atkinson, J P

2001-01-01

117

Protein co-expression network analysis (ProCoNA)  

PubMed Central

Background 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. Results 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). Conclusions 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. PMID:23724967

2013-01-01

118

Astragaloside ? reduces the expression level of P-glycoprotein in multidrug-resistant human hepatic cancer cell lines.  

PubMed

Astragaloside is a saponin widely used in traditional Chinese medicine and has been reported to be a potent multidrug resistance (MDR) reversal agent. The present study investigated the role of astragaloside ? (ASIV) in the regulation of P-glycoprotein (P-gp, encoded by the mdr1 gene) and its effect on the reversal of MDR. The activity of ASIV was evaluated using human hepatic cancer cells Bel-7402 and the corresponding 5-fluorouracil (5-FU) resistant cells Bel-7402/FU. ASIV (0.08 mg/ml) potentiated the cytotoxicity of 5-FU which was demonstrated using the MTT assay on Bel-7402/FU cells. ASIV reduced the expression of P-gp as was revealed by immunocytochemistry. Accumulation and efflux studies with the P-gp substrate, rhodamine 123 (Rh123), demonstrated that ASIV inhibited P-gp-mediated drug efflux. Furthermore, it was demonstrated that AS? enhanced the drug accumulation of 5-FU using a high performance liquid chromatography (HPLC) assay for drug resistant cells. Furthermore, ASIV may downregulate the expression of P-gp, which was examined using western blot analysis and polymerase chain reaction. In conclusion, the results of the present study indicated that ASIV reverses the drug resistance of Bel-7402/FU cells by downregulating the expression of mdr1. ASIV may represent a potent modulator of P-gp-mediated MDR in hepatic cancer therapy. PMID:24676670

Wang, Pei-Pei; Xu, Du-Juan; Huang, Can; Wang, Wei-Ping; Xu, Wen-Ke

2014-06-01

119

Comparison of Heavy Labeled (SIL) Peptide versus SILAC Protein Internal Standards for LC-MS/MS Quantification of Hepatic Drug Transporters.  

PubMed

We studied the precision of quantification of organic anion-transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, and P-glycoprotein (P-gp) in human livers by surrogate peptide based LC-MS/MS approach using two different internal standards: stable isotope labeled peptide (SIL) versus stable isotope labeled protein (SILAC). The SIL peptides were procured commercially and the SILAC proteins were generated in-house by labeling arginine and/or lysine residues in cells expressing these transporters. Liver tissue (n = 20) was homogenized and the membrane fraction was isolated. The membranes were trypsin digested and the peptides were analyzed using LC-MS/MS under optimized conditions. The precision in the quantification of proteins in three independently trypsin digested samples from each liver was calculated as the standard deviation of the log transformed protein concentration. The precision of the SIL internal standard method was either slightly (P < 0.05, paired t-test) better than that of the SILAC method (OATP1B1, OATP1B3, and P-gp) or not different (OATP2B1). Trypsin digestion, as measured by the response of the labeled peptide derived from the SILAC protein, was consistent across liver samples. These results indicate that when maximum trypsin digestion is ensured, the SIL internal standard method can be used with confidence for quantification of drug transporters. PMID:24719762

Prasad, Bhagwat; Unadkat, Jashvant D

2014-01-01

120

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli.  

PubMed

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. PMID:25590335

Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; L?we, Jan; Moraes, Isabel; Owens, Raymond J

2015-01-01

121

Relating Whole-Genome Expression Data with Protein-Protein Interactions  

PubMed Central

We investigate the relationship of protein-protein interactions with mRNA expression levels, by integrating a variety of data sources for yeast. We focus on known protein complexes that have clearly defined interactions between their subunits. We find that subunits of the same protein complex show significant coexpression, both in terms of similarities of absolute mRNA levels and expression profiles, e.g., we can often see subunits of a complex having correlated patterns of expression over a time course. We classify the yeast protein complexes as either permanent or transient, with permanent ones being maintained through most cellular conditions. We find that, generally, permanent complexes, such as the ribosome and proteasome, have a particularly strong relationship with expression, while transient ones do not. However, we note that several transient complexes, such as the RNA polymerase II holoenzyme and the replication complex, can be subdivided into smaller permanent ones, which do have a strong relationship to gene expression. We also investigated the interactions in aggregated, genome-wide data sets, such as the comprehensive yeast two-hybrid experiments, and found them to have only a weak relationship with gene expression, similar to that of transient complexes. (Further details on genecensus.org/expression/interactions and bioinfo.mbb.yale.edu/expression/interactions.) PMID:11779829

Jansen, Ronald; Greenbaum, Dov; Gerstein, Mark

2002-01-01

122

The protein expression landscape of the Arabidopsis root.  

PubMed

Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein-protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development. PMID:22447775

Petricka, Jalean J; Schauer, Monica A; Megraw, Molly; Breakfield, Natalie W; Thompson, J Will; Georgiev, Stoyan; Soderblom, Erik J; Ohler, Uwe; Moseley, Martin Arthur; Grossniklaus, Ueli; Benfey, Philip N

2012-05-01

123

p53 targets identified by protein expression profiling  

PubMed Central

p53 triggers cell cycle arrest and apoptosis through transcriptional regulation of specific target genes. We have investigated the effect of p53 activation on the proteome using 2D gel electrophoresis analysis of mitomycin C-treated HCT116 colon carcinoma cells carrying wild-type p53. Approximately 5,800 protein spots were separated in overlapping narrow-pH-range gel strips, and 115 protein spots showed significant expression changes upon p53 activation. The identity of 55 protein spots was obtained by mass spectrometry. The majority of the identified proteins have no previous connection to p53. The proteins fall into different functional categories, such as mRNA processing, translation, redox regulation, and apoptosis, consistent with the idea that p53 regulates multiple cellular pathways. p53-dependent regulation of five of the up-regulated proteins, eIF5A, hnRNP C1/C2, hnRNP K, lamin A/C, and Nm23-H1, and two of the down-regulated proteins, Prx II and TrpRS, was examined in further detail. Analysis of mRNA expression levels demonstrated both transcription-dependent and transcription-independent regulation among the identified targets. Thus, this study reveals protein targets of p53 and highlights the role of transcription-independent effects for the p53-induced biological response. PMID:17372198

Rahman-Roblick, Rubaiyat; Johannes Roblick, Uwe; Hellman, Ulf; Conrotto, Paolo; Liu, Tao; Becker, Susanne; Hirschberg, Daniel; Jörnvall, Hans; Auer, Gert; Wiman, Klas G.

2007-01-01

124

Flavonoids inhibit the expression of heat shock proteins.  

PubMed

Cells exposed to several forms of stress, such as heat shock, transiently synthesize a group of proteins called heat shock proteins (hsps). Although many stressors other than heat shock are known to induce hsps, inhibitors of hsp expression have never been reported. Here we show that quercetin and several other flavonoids inhibit the synthesis of hsps induced by heat shock in two human cell lines, Hela cells and COLO320 DM cells. Quercetin inhibited the induction of hsp70 at the level of mRNA accumulation. This is the first report to describe the inhibition of hsp expression by reagents. PMID:2085852

Hosokawa, N; Hirayoshi, K; Nakai, A; Hosokawa, Y; Marui, N; Yoshida, M; Sakai, T; Nishino, H; Aoike, A; Kawai, K

1990-12-01

125

Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells  

PubMed Central

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

Lilly, Jacob L.; Sheldon, Phillip R.; Hoversten, Liv J.; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J.

2014-01-01

126

Interfacial polymerization for colorimetric labeling of protein expression in cells.  

PubMed

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

Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

2014-01-01

127

Beyond protein expression, MOPED goes multi-omics.  

PubMed

MOPED (Multi-Omics Profiling Expression Database; http://moped.proteinspire.org) has transitioned from solely a protein expression database to a multi-omics resource for human and model organisms. Through a web-based interface, MOPED presents consistently processed data for gene, protein and pathway expression. To improve data quality, consistency and use, MOPED includes metadata detailing experimental design and analysis methods. The multi-omics data are integrated through direct links between genes and proteins and further connected to pathways and experiments. MOPED now contains over 5 million records, information for approximately 75 000 genes and 50 000 proteins from four organisms (human, mouse, worm, yeast). These records correspond to 670 unique combinations of experiment, condition, localization and tissue. MOPED includes the following new features: pathway expression, Pathway Details pages, experimental metadata checklists, experiment summary statistics and more advanced searching tools. Advanced searching enables querying for genes, proteins, experiments, pathways and keywords of interest. The system is enhanced with visualizations for comparing across different data types. In the future MOPED will expand the number of organisms, increase integration with pathways and provide connections to disease. PMID:25404128

Montague, Elizabeth; Janko, Imre; Stanberry, Larissa; Lee, Elaine; Choiniere, John; Anderson, Nathaniel; Stewart, Elizabeth; Broomall, William; Higdon, Roger; Kolker, Natali; Kolker, Eugene

2015-01-28

128

Development of Avian Tibial Dyschondroplasia: Gene Expression and Protein Synthesis  

Microsoft Academic Search

.   Age-dependent gene expression and protein synthesis associated with chondrocyte differentiation were evaluated in the epiphyseal\\u000a growth plates of normal and tibial dyschondroplasia (TD)-afflicted chickens. In the normal growth plate, collagen type II\\u000a gene is expressed mainly by chondrocytes at the upper zone of the growth plate and by the chondrocytes in the articular cartilage.\\u000a Collagen type X and osteopontin

M. Pines; V. Knopov; O. Genina; S. Hurwitz; A. Faerman; L. C. Gerstenfeld; R. M. Leach

1998-01-01

129

?Np63 protein expression in uterine cervical and endometrial cancers  

Microsoft Academic Search

Purpose\\u000a   To investigate the significance of p63 expression in uterine cervical and endometrial cancers.\\u000a Materials and methods\\u000a   ?Np63 protein expression was studied in a variety of 127 cases of uterine cervical lesions (20 non-neoplastic cervices, 43 cervical intraepithelial neoplasia [CIN], 54 squamous cell carcinomas (SCCs), 40 adenocarcinomas, and 13 other histologic types) and 30 endometrioid type of endometrial adenocarcinomas by

Zhenhua Lin; Mingzhu Liu; Zhuhu Li; Changheon Kim; Eungseok Lee; Insun Kim

2006-01-01

130

Methods and constructs for expression of foreign proteins in photosynthetic organisms  

DOEpatents

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.

Laible, Philip D. (Villa Park, IL); Hanson, Deborah K. (Downers Grove, IL)

2002-01-01

131

Enhanced Brain Disposition and Effects of ?9Tetrahydrocannabinol in P-Glycoprotein and Breast Cancer Resistance Protein Knockout Mice  

Microsoft Academic Search

The ABC transporters P-glycoprotein (P-gp, Abcb1) and breast cancer resistance protein (Bcrp, Abcg2) regulate the CNS disposition of many drugs. The main psychoactive constituent of cannabis ?9-tetrahydrocannabinol (THC) has affinity for P-gp and Bcrp, however it is unknown whether these transporters modulate the brain accumulation of THC and its functional effects on the CNS. Here we aim to show that

Adena S. Spiro; Alexander Wong; Aurélie A. Boucher; Jonathon C. Arnold

2012-01-01

132

High cell-density expression system: A novel method for extracellular production of “difficult-to-expressproteins  

Microsoft Academic Search

Yeast's extracellular expression provides a cost-efficient means of producing industrially useful recombinant proteins. However, depending on the protein to be expressed, the production results in a poor yield, which is occasionally accompanied with loss of the expression plasmid and hence hampered growth of the host in the inducing medium. Here we propose an alternative approach, high cell-density expression, to improve

Koichi Kimata; Masaya Yamaguchi; Yuta Saito; Hiroki Hata; Kasumi Miyake; Tsuneo Yamane; Yuko Nakagawa; Akira Yano; Keisuke Ito; Yasuaki Kawarasaki

133

Protein Dynamics: Implications for Nuclear Architecture and Gene Expression  

NSDL National Science Digital Library

Studies of nuclear architecture reveal that the dynamic properties of proteins in the nucleus are critical for their function. The high mobility of proteins ensures their availability throughout the nucleus; their dynamic interplay generates an ever-changing, but overall stable, architectural framework, within which nuclear processes take place. As a consequence, overall nuclear morphology is determined by the functional interactions of nuclear components. The observed dynamic properties of nuclear proteins are consistent with a central role for stochastic mechanisms in gene expression and nuclear architecture.

Tom Mistelli (National Cancer Institute; )

2001-02-02

134

The co-delivery of a low-dose P-glycoprotein inhibitor with doxorubicin sterically stabilized liposomes against breast cancer with low P-glycoprotein expression  

PubMed Central

Introduction P-glycoprotein (P-gp) inhibitors are usually used to treat tumors that overexpress P-gps. However, most common types of breast cancers, such as Luminal A, are low-P-gp expressing, at least during the initial phases of treatment. Therefore, it would be interesting to know if P-gp inhibitors are still useful in treating low-P-gp-expressing tumors. Methods In the study reported here, the human breast-cancer cell line MCF-7, chosen as a model of Luminal A, was found to be low-P-gp expressing. We designed a novel doxorubicin (DOX) sterically stabilized liposome system co-loaded with the low-dose P-gp inhibitor cyclosporine A (CsA) (DOX/CsA/SSL). Results The co-delivery system showed good size uniformity, high encapsulation efficiency, and a desirable release profile. The cell-uptake and cytotoxicity studies demonstrated that CsA could significantly enhance the intracellular accumulation and toxicity of free DOX and the liposomal DOX in MCF-7 cells. The confocal microscopy and in vivo imaging study confirmed the intracellular and in vivo targeting effect of DOX/CsA/SSL, respectively. Finally, the in vivo study proved that DOX/CsA/SSL could achieve significantly better antitumor effect against MCF-7 tumor than controls, without inducing obvious systemic toxicity. Conclusion This study demonstrated that the co-delivery of a low-dose P-gp inhibitor and liposomal DOX could improve the therapy of low-P-gp-expressing cancer, which is of significance in clinical tumor therapy. PMID:25092974

Gao, Wei; Lin, Zhiqiang; Chen, Meiwan; Yang, Xiucong; Cui, Zheng; Zhang, Xiaofei; Yuan, Lan; Zhang, Qiang

2014-01-01

135

Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization  

ERIC Educational Resources Information Center

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…

Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

2008-01-01

136

Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses.  

PubMed

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

Kroemer, Jeremy A; Bonning, Bryony C; Harrison, Robert L

2015-01-01

137

Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses  

PubMed Central

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

Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

2015-01-01

138

Expression of targeting protein for Xenopus kinesin-like protein 2 is associated with progression of human malignant astrocytoma  

Microsoft Academic Search

In humans, the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cell cycle-associated protein, and altered TPX2 expression has been found in various malignancies. However, the contribution of TPX2 expression to astrocytoma progression is unclear. The aim of this study was to investigate TPX2 expression in human astrocytoma samples and cell lines. TPX2 protein expression was detected in

Bin Li; Xiang-Qian Qi; Xin Chen; Xin Huang; Guo-Ying Liu; Huai-Rui Chen; Cheng-Guang Huang; Chun Luo; Yi-Cheng Lu

2010-01-01

139

Simple and Efficient Method for Heterologous Expression of Clostridial Proteins  

PubMed Central

Many clostridial proteins are poorly produced in Escherichia coli. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli and the quantities of the corresponding tRNAs in E. coli are not sufficient to ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three E. coli genes, ileX, argU, and leuW, in E. coli; these genes encode tRNAs that are rarely used in E. coli (the tRNAs for the ATA, AGA, and CTA codons, respectively). Our data demonstrate that amplification of ileX dramatically increased the level of production of most of the clostridial proteins tested, while amplification of argU had a moderate effect and amplification of leuW had no effect. Thus, amplification of certain tRNA genes for rare codons in E. coli improves the expression of clostridial genes in E. coli, while amplification of other tRNAs for rare codons might not be needed for improved expression. We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence. Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E. coli. PMID:10919765

Zdanovsky, Alexey G.; Zdanovskaia, Marina V.

2000-01-01

140

Expression and activation of STAT proteins during mouse retina development  

Microsoft Academic Search

Cytokines and growth factors play important roles in mammalian ocular development and maintenance. Recent studies have indicated that some of these ligands can activate signal transducer and activator of transcription factors (STATs) and modulate gene transcription. The purpose of this study was to investigate the expression and activation of STAT proteins in the developing mouse retina. Anti-STAT and anti-phosphorylated STAT

Samuel Shao-Min Zhang; Ji-ye Wei; Chaojun Li; Colin J Barnstable; Xin-Yuan Fu

2003-01-01

141

IDENTIFICATION OF PROTEINS EXPRESSED IN GERMINATING PHAKOPSORA PACHYRHIZI UREDINIOSPORES  

Technology Transfer Automated Retrieval System (TEKTRAN)

Phakopsora pachyrhizi is the causal agent of Asian soybean rust, a devastating disease that was recently identified in 9 U.S. states during the fall of 2004. In order to develop long-term strategies to combat this disease, we are focusing on proteins expressed in germinating spores and secreted by t...

142

Expression and detection of LINE-1 ORF-encoded proteins  

PubMed Central

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. PMID:25054082

Dai, Lixin; LaCava, John; Taylor, Martin S; Boeke, Jef D

2014-01-01

143

VIRAL HEPATITIS Expression of Paramyxovirus V Proteins Promotes  

E-print Network

VIRAL HEPATITIS Expression of Paramyxovirus V Proteins Promotes Replication and Spread of Hepatitis with laboratory strains of hepatitis C virus (HCV), although levels of virus replication vary significantly spreads in its natural host cell population. (HEPATOLOGY 2011;54:1901-1912) A cute hepatitis C virus (HCV

Bhatia, Sangeeta

144

Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins  

PubMed Central

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

Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

2012-01-01

145

Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer  

SciTech Connect

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.

Raymond, Amy; Lovell, Scott; Lorimer, Don; Walchli, John; Mixon, Mark; Wallace, Ellen; Thompkins, Kaitlin; Archer, Kimberly; Burgin, Alex; Stewart, Lance; (Accel. Tech.); (deCODE)

2009-12-01

146

LC–MS Based Detection of Differential Protein Expression  

PubMed Central

While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics. Significant technical advances at both sample preparation/separation and mass spectrometry levels have revolutionized comprehensive proteome analysis. Moreover, automation and robotics for sample handling process permit multiple sampling with high throughput. For LC-MS based quantitative proteomics, sample preparation turns out to be critical step, as it can significantly influence sensitivity of downstream analysis. Several sample preparation strategies exist, including depletion of high abundant proteins or enrichment steps that facilitate protein quantification but with a compromise of focusing on a smaller subset of a proteome. While several experimental strategies have emerged, certain limitations such as physiochemical properties of a peptide/protein, protein turnover in a sample, analytical platform used for sample analysis and data processing, still imply challenges to quantitative proteomics. Other aspects that make analysis of a proteome a challenging task include dynamic nature of a proteome, need for efficient and fast analysis of protein due to its constant modifications inside a cell, concentration range of proteins that exceed dynamic range of a single analytical method, and absence of appropriate bioinformatics tools for analysis of large volume and high dimensional data. This paper gives an overview of various LC-MS methods currently used in quantitative proteomics and their potential for detecting differential protein expression. Fundamental steps such as sample preparation, LC separation, mass spectrometry, quantitative assessment and protein identification are discussed. For quantitative assessment of protein expression, both label and label free approaches are evaluated for their set of merits and demerits. While most of these methods edge on providing “relative abundance” information, absolute quantification is achieved with limitation as it caters to fewer proteins. Isotope labeling is extensively used for quantifying differentially expressed proteins, but is severely limited by successful incorporation of its heavy label. Lengthy labeling protocols restrict the number of samples that can be labeled and processed. Alternatively, label free approach appears promising as it can process many samples with any number of comparisons possible but entails reproducible experimental data for its application. PMID:20473349

Tuli, Leepika; Ressom, Habtom W.

2010-01-01

147

Changes in protein expression during honey bee larval development  

PubMed Central

Background The honey bee (Apis mellifera), besides its role in pollination and honey production, serves as a model for studying the biochemistry of development, metabolism, and immunity in a social organism. Here we use mass spectrometry-based quantitative proteomics to quantify nearly 800 proteins during the 5- to 6-day larval developmental stage, tracking their expression profiles. Results We report that honey bee larval growth is marked by an age-correlated increase of protein transporters and receptors, as well as protein nutrient stores, while opposite trends in protein translation activity and turnover were observed. Levels of the immunity factors prophenoloxidase and apismin are positively correlated with development, while others surprisingly were not significantly age-regulated, suggesting a molecular explanation for why bees are susceptible to major age-associated bee bacterial infections such as American Foulbrood or fungal diseases such as chalkbrood. Previously unreported findings include the reduction of antioxidant and G proteins in aging larvae. Conclusion These data have allowed us to integrate disparate findings in previous studies to build a model of metabolism and maturity of the immune system during larval development. This publicly accessible resource for protein expression trends will help generate new hypotheses in the increasingly important field of honey bee research. PMID:18959778

Chan, Queenie WT; Foster, Leonard J

2008-01-01

148

Expression and Targeting of Secreted Proteins from Chlamydia trachomatis  

PubMed Central

Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a vacuole termed the inclusion. Many of the interactions of chlamydiae with the host cell are dependent upon bacterial protein synthesis and presumably exposure of these proteins to the cytosol. Because of the dearth of genetic tools for chlamydiae, previous studies examining secreted proteins required the use of heterologous bacterial systems. Recent advances in genetic manipulation of chlamydia now allow for transformation of the bacteria with plasmids. We describe here a shuttle vector system, pBOMB4, that permits expression of recombinant proteins under constitutive or conditional promoter control. We show that the inclusion membrane protein IncD is secreted in a type III-dependent manner from Yersinia pseudotuberculosis and also secreted from C. trachomatis in infected cells where it localizes appropriately to the inclusion membrane. IncD truncated of the first 30 amino acids containing the secretion signal is no longer secreted and is retained by the bacteria. Cytosolic exposure of secreted proteins can be confirmed by using CyaA, GSK, or microinjection assays. A protein predicted to be retained within the bacteria, NrdB is indeed localized to the chlamydia. In addition, we have shown that the chlamydial effector protein, CPAF, which is secreted into the host cell cytosol by a Sec-dependent pathway, also accesses the cytosol when expressed from this system. These assays should prove useful to assess the secretion of other chlamydial proteins that are potentially exposed to the cytosol of the host cell. PMID:24443531

Bauler, Laura D.

2014-01-01

149

G-protein coupled receptor expression patterns delineate medulloblastoma subgroups  

PubMed Central

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 is a secondary benefit to identifying differential GPCR expression patterns in medulloblastoma tumors. PMID:24252460

2013-01-01

150

Expression and activation of STAT proteins during mouse retina development.  

PubMed

Cytokines and growth factors play important roles in mammalian ocular development and maintenance. Recent studies have indicated that some of these ligands can activate signal transducer and activator of transcription factors (STATs) and modulate gene transcription. The purpose of this study was to investigate the expression and activation of STAT proteins in the developing mouse retina. Anti-STAT and anti-phosphorylated STAT antibodies were used to detect the expression and activation of STATs in embryonic and postnatal neuronal retina, ciliary margin, and retinal pigment epithelium (RPE). In situ hybridization and Western blot were also employed. In embryonic stages, all STAT proteins were expressed in the neuronal retina in distinct cell populations at different embryonic stages. For example, Stat3 expression and activation gradually increased in the inner neuroblast layer and ciliary margin during development. In adult retina, Stat3 was detected in the inner nuclear layer and ganglion cells layers. Stat1 was strongly expressed in both outer and inner plexiform layers. Stat5a was clearly expressed in the outer/inner nuclear layer, the ganglion cell layer, and the inner plexiform layer. Strong expression of Stat3, Stat5a, and Stat6 was observed in the RPE. Activated Stat3 and Stat5a were found in the neural retina and the RPE. Distinct STAT proteins were present in different cell populations in neuronal retina and RPE suggesting multiple functions of STATs in mammalian eye development. Studies of STAT signal pathways in the eye may contribute to the understanding of molecular mechanisms in control of ocular development and pathogenesis. PMID:12634107

Zhang, Samuel Shao-Min; Wei, Ji-ye; Li, Chaojun; Barnstable, Colin J; Fu, Xin-Yuan

2003-04-01

151

Differentially Expressed Proteins in Malignant and Benign Adrenocortical Tumors  

PubMed Central

We have compared the microsomal protein composition of eight malignant and six benign adrenocortical tumors with proteomic methods. IGF2 had increased level in the malignant tumors, confirming previous microarray studies on the same material. Aldolase A, a glycolytic enzyme, also showed increased levels in the malignant tissue compared to the benign. Additionally, several proteins belonging to complex I in the mitochondrial respiration chain showed decreased levels in the malignant tissue. Taken together, this may indicate a shift in energy metabolism where glycolysis may be favored over tight coupling of glycolysis and mitochondrial respiration, a phenomenon known as the Warburg effect. One of the complex I proteins that showed decreased levels in the malignant tissue was GRIM-19. This protein has been suggested as a tumor suppressive protein by being a negative regulator of STAT3. In summary, an analysis of the microsomal proteome in adrenocortical tumors identifies groups of proteins as well as specific proteins differentially expressed in the benign and malignant forms. These proteins shed light on the biology behind malignancy and could delineate future drug targets. PMID:24498411

Kjellin, Hanna; Johansson, Henrik; Höög, Anders; Lehtiö, Janne

2014-01-01

152

Dictyostelium discoideum--a promising expression system for the production of eukaryotic proteins  

Microsoft Academic Search

In general, four different expression systems, namely, bacterial, yeast, baculovirus, and mammalian, are widely used for the overproduction of biochemical enzymes and therapeutic proteins. Clearly, bacterial expression systems offer ease of maneuver- ability with respect to large-scale production of recom- binant proteins, while, a baculovirus expression system ensures proper protein modifications, processing, and refolding of complex proteins. Despite these advan-

Ranjana Arya; Alok Bhattacharya; Kulvinder Singh Saini

2008-01-01

153

Survivin and Related Proteins in Canine Mammary Tumors: Immunohistochemical Expression.  

PubMed

Survivin is reexpressed in most human breast cancers, where its expression has been associated with tumor aggressiveness, poor prognosis, and poor response to therapy. Survivin expression was evaluated in 41 malignant canine mammary tumors (CMTs) by immunohistochemistry, in relation to histological grade and stage, and correlated with that of some related molecules (?-catenin, caspase 3, heat shock proteins) to understand their possible role in canine mammary tumorigenesis. An increase in nuclear survivin expression, compared with healthy mammary glands, was observed in CMTs, where nuclear immunolabeling was related to the presence of necrosis. No statistically significant relation was found between the expression of the investigated molecules and the histological grade or stage. The present study may suggest an important involvement of survivin in CMT tumorigenesis. Its overexpression in most of the cases evaluated might suggest that targeting survivin in CMTs may be a valid anticancer therapy. PMID:24686389

Bongiovanni, L; Romanucci, M; Malatesta, D; D'Andrea, A; Ciccarelli, A; Della Salda, L

2014-03-31

154

Knocking Down Breast Cancer Resistance Protein (Bcrp) by Adenoviral Vector-Mediated RNA Interference (RNAi) in Sandwich-Cultured Rat Hepatocytes: A Novel Tool to Assess the Contribution of Bcrp to Drug Biliary Excretion  

PubMed Central

BCRP transports numerous drugs/derived metabolites and toxins, and exhibits overlapping substrate specificity with P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2). Assessing the contribution of BCRP to drug/metabolite biliary excretion in the intact hepatocytes remains a challenge. Current studies were designed to develop a novel in vitro tool to specifically assess the contribution of Bcrp to drug biliary excretion. Adenoviral vectors expressing short hairpin (sh) RNA targeting Bcrp (Ad-si01Bcrp) or a non-target control (Ad-siNT) were packaged and infected into sandwich-cultured rat hepatocytes (SCRH). Protein levels were detected by immunoblot. Biliary excretion index (BEI) and in vitro biliary clearance (Clbiliary) of nitrofurantoin (BCRP substrate) and digoxin (P-gp substrate) were compared among non-infected, Ad-siNT- and Ad-si01Bcrp-infected cells in SCRH. shRNA targeting Bcrp efficiently knocked down Bcrp in SCRH, while levels of other transport proteins (P-gp, Mrp2, Bsep, Mrp4 and Oatp1a1) were unaffected. In SCRH exhibiting Bcrp knockdown, cellular accumulation of nitrofurantoin was increased markedly and nitrofurantoin BEI and in vitro Clbiliary were decreased to 11% and 14% of control, respectively. Digoxin values were unaffected by knockdown of Bcrp. Results indicated that Bcrp contributed predominantly to nitrofurantoin biliary excretion, but played a negligible role in digoxin biliary excretion. In summary, Bcrp knockdown in SCRH is the first in vitro model utilizing intact hepatocytes to assess the contribution of Bcrp to the biliary excretion of drugs. This approach may be useful in predicting drug-drug interactions in biliary excretion and the consequence of impaired BCRP function on the hepatic exposure of drugs/derived metabolites. PMID:19105722

Yue, Wei; Abe, Koji; Brouwer, Kim L. R.

2011-01-01

155

Knocking down breast cancer resistance protein (Bcrp) by adenoviral vector-mediated RNA interference (RNAi) in sandwich-cultured rat hepatocytes: a novel tool to assess the contribution of Bcrp to drug biliary excretion.  

PubMed

BCRP transports numerous drugs/derived metabolites and toxins, and exhibits overlapping substrate specificity with P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2). Assessing the contribution of BCRP to drug/metabolite biliary excretion in intact hepatocytes remains a challenge. Current studies were designed to develop a novel in vitro tool to specifically assess the contribution of Bcrp to drug biliary excretion. Adenoviral vectors expressing short hairpin (sh) RNA targeting Bcrp (Ad-si01Bcrp) or a nontarget control (Ad-siNT) were packaged and infected into sandwich-cultured rat hepatocytes (SCRH). Protein levels were detected by immunoblot. Biliary excretion index (BEI) and in vitro biliary clearance (Cl(biliary)) of nitrofurantoin (BCRP substrate) and digoxin (P-gp substrate) were compared among noninfected, Ad-siNT- and Ad-si01Bcrp-infected SCRH. shRNA targeting Bcrp efficiently knocked down Bcrp in SCRH, while levels of other transport proteins (P-gp, Mrp2, Bsep, Mrp4 and Oatp1a1) were unaffected. In SCRH exhibiting Bcrp knockdown, cellular accumulation of nitrofurantoin was increased markedly and nitrofurantoin BEI and in vitro Cl(biliary) were decreased to 11% and 14% of control, respectively. Digoxin values were unaffected by knockdown of Bcrp. Results indicated that Bcrp in SCRH contributed predominantly to nitrofurantoin biliary excretion, but played a negligible role in digoxin biliary excretion. In summary, Bcrp knockdown in SCRH is the first in vitro model utilizing intact hepatocytes to assess the contribution of Bcrp to the biliary excretion of drugs. This approach may be useful in predicting drug-drug interactions in biliary excretion and the consequence of impaired BCRP function on the hepatic exposure of drugs/derived metabolites. PMID:19105722

Yue, Wei; Abe, Koji; Brouwer, Kim L R

2009-01-01

156

Pooled ORF expression technology (POET): using proteomics to screen pools of open reading frames for protein expression.  

PubMed

We have developed a pooled ORF expression technology, POET, that uses recombinational cloning and proteomic methods (two-dimensional gel electrophoresis and mass spectrometry) to identify ORFs that when expressed are likely to yield high levels of soluble, purified protein. Because the method works on pools of ORFs, the procedures needed to subclone, express, purify, and assay protein expression for hundreds of clones are greatly simplified. Small scale expression and purification of 12 positive clones identified by POET from a pool of 688 Caenorhabditis elegans ORFs expressed in Escherichia coli yielded on average 6 times as much protein as 12 negative clones. Larger scale expression and purification of six of the positive clones yielded 47-374 mg of purified protein/liter. Using POET, pools of ORFs can be constructed, and the pools of the resulting proteins can be analyzed and manipulated to rapidly acquire information about the attributes of hundreds of proteins simultaneously. PMID:16113400

Gillette, William K; Esposito, Dominic; Frank, Peter H; Zhou, Ming; Yu, Li-Rong; Jozwik, Catherine; Zhang, Xiuying; McGowan, Brighid; Jacobowitz, David M; Pollard, Harvey B; Hao, Tong; Hill, David E; Vidal, Marc; Conrads, Thomas P; Veenstra, Timothy D; Hartley, James L

2005-11-01

157

Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants1  

Microsoft Academic Search

A major goal of plant biotechnology is the production of genet- ically engineered crops that express natural or foreign proteins at high levels. To enhance protein accumulation in transgenic plants, we developed a set of vectors that express proteins and peptides as C-terminal translational fusions with ubiquitin (UBQ). Studies of several proteins in tobacco (Nicotiana tabacum) showed that: (a) proteins

David Hondred; Joseph M. Walker; Dennis E. Mathews; Richard D. Vierstra

1999-01-01

158

Protein inhibitor of activated STAT3 inhibits adipogenic gene expression  

SciTech Connect

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.

Deng Jianbei [Department of Nutrition, CB 7461, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Hua Kunjie [Department of Nutrition, CB 7461, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Caveney, Erica J. [Department of Medicine, CB 7005, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Takahashi, Nobuyuki [Department of Pathology and Laboratory Medicine, CB 7525, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Harp, Joyce B. [Department of Nutrition, CB 7461, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States)]. E-mail: jharp@unc.edu

2006-01-20

159

Somatostatin regulates tight junction proteins expression in colitis mice  

PubMed Central

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 ? (TNF-?). 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

Li, Xiao; Wang, Qian; Xu, Hua; Tao, Liping; Lu, Jing; Cai, Lin; Wang, Chunhui

2014-01-01

160

Somatostatin regulates tight junction proteins expression in colitis mice.  

PubMed

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 ? (TNF-?). 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

Li, Xiao; Wang, Qian; Xu, Hua; Tao, Liping; Lu, Jing; Cai, Lin; Wang, Chunhui

2014-01-01

161

EFFECT OF UNCOUPLING PROTEIN–1 EXPRESSION ON 3T3-L1 ADIPOCYTE GENE EXPRESSION  

PubMed Central

The mitochondrial respiratory uncoupling protein 1 (UCP1) partially uncouples substrate oxidation and oxidative phosphorylation to promote the dissipation of cellular biochemical energy as heat in brown adipose tissue. We have recently shown that expression of UCP1 in 3T3-L1 white adipocytes reduces the accumulation of triglycerides. Here, we investigated the molecular basis underlying UCP1 expression in 3T3-L1 adipocytes. Gene expression data show that forced UCP1 expression down-regulated several energy metabolism pathways; but ATP levels were constant. A metabolic flux analysis model was used to reflect the gene expression changes onto metabolic processes and concordance was observed in the down-regulation of energy consuming pathways. Our data suggest that adipocytes respond to long-term mitochondrial uncoupling by minimizing ATP utilization. PMID:18061577

Senocak, Fatih S.; Si, Yaguang; Moya, Colby; Russell, William K.; Russell, David H.; Lee, Kyongbum; Jayaraman, Arul

2008-01-01

162

Protein Profile Changes during Porcine Oocyte Aging and Effects of Caffeine on Protein Expression Patterns  

PubMed Central

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. PMID:22194971

Jiang, Guang-Jian; Wang, Ke; Miao, De-Qiang; Guo, Lei; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

2011-01-01

163

Expression and activation of signal regulatory protein alpha on astrocytomas.  

PubMed

High-grade astrocytomas and glioblastomas are usually unresectable because they extensively invade surrounding brain tissue. Here, we report the expression and function of a receptor on many astrocytomas that may alter both the proliferative and invasive potential of these tumors. Signal regulatory protein (SIRP) alpha1 is an immunoglobulin superfamily transmembrane glycoprotein that is normally expressed in subsets of myeloid and neuronal cells. Transfection of many cell types with SIRPalpha1, including glioblastomas, has been shown to inhibit their proliferation in response to a range of growth factors. Furthermore, the expression of a murine SIRPalpha1 mutant has been shown to enhance cell adhesion and initial cell spreading but to inhibit cell extension and movement. The extracellular portion of SIRPalpha1 binds CD47 (integrin-associated protein), although this interaction is not required for integrin-mediated activation of SIRPalpha1. On phosphorylation, SIRPalpha1 recruits the tyrosine phosphatases SHP-1 and SHP-2, which are important in its functions. Although SHP-1 is uniquely expressed on hematopoietic cells, SHP-2 is ubiquitously expressed, so that SIRPalpha1 has the potential to function in many cell types, including astrocytomas. Because SIRPalpha1 regulates cell functions that may contribute to the malignancy of these tumors, we examined the expression of SIRPs in astrocytoma cell lines by flow cytometry using a monoclonal antibody against all SIRPs. Screening of nine cell lines revealed clear cell surface expression of SIRPs on five cell lines, whereas Northern blotting for SIRPalpha transcripts showed mRNA present in eight of nine cell lines. All nine cell lines expressed the ligand for SIRPalpha1, CD47. To further examine the expression and function of SIRPs, we studied the SF126 and U373MG astrocytoma cell lines, both of which express SIRPs, in greater detail. SIRP transcripts in these cells are identical in sequence to SIRPalpha1. The expressed deglycosylated protein is the same size as SIRPalpha1, but in the astrocytoma cells, it is underglycosylated compared with SIRPalpha1 produced in transfected Chinese hamster ovary cells. It is nonetheless still capable of binding soluble CD47. Moreover, SIRPalpha1 in each of the two cell lines recruited SHP-2 on phosphorylation, and SIRPalpha1 phosphorylation in cultured cells is CD47 dependent. Finally, examination of frozen sections from 10 primary brain tumor biopsies by immunohistochemistry revealed expression of SIRPs on seven of the specimens, some of which expressed high levels of SIRPs. Most of the tumors also expressed CD47. This is the first demonstration that astrocytomas can express SIRPalpha. Given the known role of SIRPalpha in regulating cell adhesion and responses to mitogenic growth factors, the expression of SIRPalpha1 on astrocytomas may be of considerable importance in brain tumor biology, and it offers the potential of a new avenue for therapeutic intervention. PMID:14729615

Chen, Thomas T; Brown, Eric J; Huang, Eric J; Seaman, William E

2004-01-01

164

Autophagy and lysosomal related protein expression patterns in human glioblastoma.  

PubMed

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, HIF1?, 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. PMID:25482944

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

2014-11-01

165

Bone morphogenetic protein expression in human atherosclerotic lesions.  

PubMed Central

Artery wall calcification associated with atherosclerosis frequently contains fully formed bone tissue including marrow. The cellular origin is not known. In this study, bone morphogenetic protein-2a, a potent factor for osteoblastic differentiation, was found to be expressed in calcified human atherosclerotic plaque. In addition, cells cultured from the aortic wall formed calcified nodules similar to those found in bone cell cultures and expressed bone morphogenetic protein-2a with prolonged culture. The predominant cells in these nodules had immunocytochemical features characteristic of microvascular pericytes that are capable of osteoblastic differentiation. Pericyte-like cells were also found by immunohistochemistry in the intima of bovine and human aorta. These findings suggest that arterial calcification is a regulated process similar to bone formation, possibly mediated by pericyte-like cells. Images PMID:8473518

Boström, K; Watson, K E; Horn, S; Wortham, C; Herman, I M; Demer, L L

1993-01-01

166

Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system.  

PubMed

Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription-translation-replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription-translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-Ichiro M

2013-08-01

167

Oleosin fusion expression systems for the production of recombinant proteins  

Microsoft Academic Search

For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are\\u000a capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products\\u000a to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the

Hua Ling

2007-01-01

168

Protein expression pattern of collagen type XV in mouse cornea  

Microsoft Academic Search

PurposeTo examine the alteration of protein expression pattern of collagen type XV in cornea during embryonic development and adult tissue repair. Collagen type XV is a basement membrane collagen of a subfamily of multiplexins (multiple triple helix domains and interruptions). Its COOH-terminal peptide has an anti-angiogenic effect and its distribution in avascular tissue of cornea is of interest.MethodsEyes of mouse

Shizuya Saika; Yuka Okada; Takeshi Miyamoto; Osamu Yamanaka; Yoshitaka Ohnishi; Akio Yamanaka; Akira Ooshima

2004-01-01

169

Prion protein expression in senile plaques in Alzheimer's disease  

Microsoft Academic Search

Prion protein (PrPC) is a glycolipid-anchored cell membrane sialoglycoprotein that localises in presynaptic membranes. Since synapses are vulnerable to Alzheimer's disease (AD), the present study examines PrPC expression in senile plaques, one of the major structural abnormalities in AD, by single- and double-labelling immunohistochemistry. Punctate PrPC immunoreactivity is found in diffuse plaques, whereas isolated large coarse PrPC-positive granules reminiscent of

I. Ferrer; R. Blanco; M. Carmona; B. Puig; R. Ribera; M. J. Rey; T. Ribalta

2001-01-01

170

Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.  

PubMed Central

Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV > laminin = fibronectin = vitronectin > collagen type III > undulin > collagen type I) or protein level (collagen type IV = collagen type III > vitronectin > laminin > collagen type I = fibronectin > undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8286197

Löhr, M.; Trautmann, B.; Göttler, M.; Peters, S.; Zauner, I.; Maillet, B.; Klöppel, G.

1994-01-01

171

Expression of XPG protein in human normal and tumor tissues  

PubMed Central

XPG (Xeroderma pigmentosum group G complementing factor) is a protein associated with DNA repair and transcription. Point mutations in ERCC5, the gene coding for XPG, cause the cancer-prone disorder xeroderma pigmentosum (XP) while truncation mutations give rise to individuals with the combined clinical features of XP and Cockayne syndrome. Polymorphisms of ERCC5 or alterations in XPG mRNA expression were also associated to an increase risk of different cancers types and to prognosis of cancer patients. However, the expression of XPG protein in different normal or tumor human tissues is not well known. In the present work, we have validated an immunohistochemistry (IHC) assay for detection of expression levels of XPG protein in FFPE human tissue samples. We have also tested this IHC assay in different normal and tumor human tissues. On a microarray containing 28 normal cores, positive staining was observed in 60% of the samples. The highest staining was detected in adrenal gland, breast, colon, heart, kidney, thyroid and tongue. In tumors, positive staining was observed in 9 of 10 breast cancer samples and in all 5 ovarian cancer and 5 sarcomas samples. Subcellular localization was predominantly nuclear. The use of this validated methodology would help to interpret the role of XPG in tumorogenesis and its use as a possible prognostic or predictive factor. PMID:23330005

Aracil, Miguel; Dauffenbach, Lisa M; Diez, Marta Martínez; Richeh, Rana; Moneo, Victoria; Leal, Juan Fernando Martínez; Fernández, Luis Francisco García; Kerfoot, Christopher A; Galmarini, Carlos M

2013-01-01

172

Proteasome inhibitors suppress the protein expression of mutant p53.  

PubMed

Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53. PMID:25485499

Halasi, Marianna; Pandit, Bulbul; Gartel, Andrei L

2014-10-15

173

Construction of a plasmid for heterologous protein expression with a constitutive promoter in Trichoderma reesei.  

PubMed

Trichoderma reesei is widely used as a host for homologus and heterologus protein expression because of its well-known capability of protein secretion, especially cellulases secretion. In this study, a binary vector which could be used for protein expression was constructed with a constitutive promoter of rp2 gene. This vector contained the expression cassette Prp2-target gene-Trp2 and hygromycin B selection marker based on pCAMBIA1300 for T-DNA random insertion. The feasibility of the promoter was determined by eGFP (enhanced green fluorescence protein) expression in T. reesei. For heterologus protein expression, the expression of bgla from Aspergillus niger in the transformant was 3 folds higher than that of the wild type strain. The results demonstrate that this constitutive promoter could be applied for protein expression and thus this protein express system may contribute to the industrial protein production. PMID:24120481

He, Ronglin; Zhang, Can; Guo, Wei; Wang, Lixian; Zhang, Dongyuan; Chen, Shunlin

2013-11-01

174

Sendai viruses with altered P, V, and W protein expression.  

PubMed

Wild-type Sendai virus expresses three proteins containing the N-terminal half of the P protein open reading frame due to mRNA editing; a full-length P protein (ca. 70% of the total), a V protein with the N-terminal half fused to a Cys-rich Zn(2+)-binding domain (ca. 25% of the total), and a W protein representing the N-terminal half alone (ca. 5% of the total). To examine the role of these proteins in the virus life cycle, we have prepared recombinant viruses in which the normal V mRNA expresses a W protein (V-stop; 70% P, 30% W), one which cannot edit its P gene mRNA (delta 6A; 100% P), and one which overedits its mRNA like parainfluenza virus type 3 (swap/8;20-40% P, 30% V, 30% W). All these viruses were readily recovered and grew to similar titers in eggs, and except for the P gene products, cell lines individually infected with these viruses accumulated similar amounts of viral macromolecules. The relative competitive advantage of each virus was determined by multiple cycle coinfections of eggs and found to be rSeV-Vstop = rSeV-wt > rSeV-delta 6A > rSeV-swap/8. On the other hand, rSeV-swap/8 underwent multiple cycles of replication in C57BI/6 mouse lungs and was highly virulent for these animals, whereas rSeV-delta 6A was avirulent in mice and this infection was quickly cleared. Remarkably, rSeV-Vstop appeared to be more virulent for inbred C57BI/6 mice than rSeV-wt, but was partially attenuated in infections of outbred ICR mice. Thus, the expression of either the V or the W proteins is sufficient for multiple cycles of infection and pathogenesis in C57BI/6 mice, whereas W can only partially substitute for V for pathogenesis in ICR mice. PMID:9514977

Delenda, C; Taylor, G; Hausmann, S; Garcin, D; Kolakofsky, D

1998-03-15

175

Expression pattern of Protein Kinase C ? during mouse embryogenesis  

PubMed Central

Background Protein kinase C epsilon (PKC?) belongs to the novel PKC subfamily, which consists of diacylglycerol dependent- and calcium independent-PKCs. Previous studies have shown that PKC? is important in different contexts, such as wound healing or cancer. In this study, we contribute to expand the knowledge on PKC? by reporting its expression pattern during murine midgestation using the LacZ reporter gene and immunostaining procedures. Results Sites showing highest PKC? expression were heart at ealier stages, and ganglia in older embryos. Other stained domains included somites, bone, stomach, kidney, and blood vessels. Conclusions The seemingly strong expression of PKC? in heart and ganglia shown in this study suggests a important role of this isoform in the vascular and nervous systems during mouse development. However, functional redundancy with other PKCs during midgestation within these domains and others reported here possibly exists since PKC? deficient mice do not display obvious embryonic developmental defects. PMID:23639204

2013-01-01

176

Gi/o proteins: expression for direct activation enquiry.  

PubMed

G protein-mediated pathways are fundamental mechanisms of cell signaling. In this paper, the expression and the characterization of the alphai1, alphai3, alphao1, beta1, and gamma2 subunits of the human G protein are described. This approach was developed to evaluate the G protein activation profile of new compounds. pCR-TOPO T7 vectors, engineered to contain the target sequences, were used to transform Escherichia coli competent cells. Subunits were over-expressed in a preparative scale as fusion proteins with a six-histidine tag, and subsequently purified by metal chelate chromatography. Afterward, the His-tag was removed by enterokinase digestion, and the secondary structures of the recombinant subunits were analyzed by circular dichroism. To assess the functionality of the subunits, the rate of GTP hydrolysis and GTPgammaS binding were evaluated both in the absence and in the presence of two modulators: the peptidic activator Mastoparan and the non-peptidic activator N-dodecyl-lysinamide (ML250). Tests were conducted on isolated alpha-subunit and on heterotrimeric alphabetagamma complex, alone or reconstituted in phospholipidic vesicles. Our results show that recombinant subunits are stable, properly folded and, fully active, which makes them suitable candidates for functional studies. PMID:16364655

Di Cesare Mannelli, Lorenzo; Pacini, Alessandra; Toscano, Annarita; Fortini, Martina; Berti, Debora; Ghelardini, Carla; Galeotti, Nicoletta; Baglioni, Piero; Bartolini, Alessandro

2006-05-01

177

Abscisic acid (ABA) regulation of Arabidopsis SR protein gene expression.  

PubMed

Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

Cruz, Tiago M D; Carvalho, Raquel F; Richardson, Dale N; Duque, Paula

2014-01-01

178

Expression and characterization of a lepidopteran general odorant binding protein.  

PubMed

Olfaction in months involves the transport of volatile, hydrophobic odorant molecules through the aqueous interior of the antennal sensory hairs by soluble odorant binding proteins. Two subfamilies of the 17 kDa general odorant binding proteins, GOBP1 and GOBP2, are 47-57% identical to each other and 21-57% to the pheromone binding proteins (PBPs); identity within a GOBP subfamily exceeds 78% in all lepidopteran species examined. However, the ligands for GOBPs are unknown. In order to investigate odorant specificities of GOBPs, recombinant proteins were expressed in Escherichia coli using PCR-prepared expression cassettes based on the cDNA sequences of GOBP1 and GOBP2 from Manduca sexta. Both soluble and insoluble recombinant GOBPs (rGOBPs) were obtained, and the inclusion body GOBPs were solubilized, refolded and purified. The soluble and refolded rGOBPs were purified by preparative isoelectric focusing (IEF), gel filtration, and finally by ion-exchange fast protein liquid chromatography (FPLC). Only rGOBP2, but not rGOBP1, was crossreactive with an anti-GOBP2 (Antheraea polyphemus) antiserum. rGOBP2, but not rGOBP1, could be photoaffinity labelled by the diazoacetate pheromone analog [3H]-6E, 11Z, 16:Dza. For rGOBP2, plant odors such as (Z)-3-hexen-1-ol (3Z-6:OH), geraniol, geranyl acetate, and limonene showed significant competition for binding; binding specificity was sensitive to pH and to salt concentrations. Circular dichroism (CD) confirms that, as with the pheromone binding proteins, GOBP2 is predominantly alpha-helical. Although the characterization of rGOBP1 has resisted analysis, rGOBP2 is readily prepared and studied. We suggest that GOBP2 may be broadly tuned to a class of "green" and floral odors. PMID:9219366

Feng, L; Prestwich, G D

1997-05-01

179

Cocaine increases endoplasmic reticulum stress protein expression in striatal neurons.  

PubMed

Cocaine administration upregulates the levels of extracellular glutamate and dopamine in the striatum. Activation of the receptors alters calcium homeostasis in striatal neurons leading to the expression of the endoplasmic reticulum (ER) stress proteins. It was therefore hypothesized that cocaine upregulates the expression of the ER stress proteins, immunoglobulin heavy chain binding protein (BiP), Ire1alpha and perk via glutamate and dopamine receptor activation. A novel glutamate microbiosensor and Western immunoblot analyses were mainly performed to test the hypothesis in the rat dorsal striatum. The results showed that i.p. injection of repeated cocaine (20 mg/kg) for nine consecutive days significantly increased extracellular glutamate levels while acute cocaine injection did not. However, the immunoreactivities (IR) of the ER stress proteins in the dorsal striatum were significantly increased by either acute or repeated cocaine injections as compared with saline controls. Intrastriatal injection (i.s.) of the selective group I metabotropic glutamate receptor (mGluR) antagonist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; 25 nmol) or the mGluR5 subtype antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP; 2 and 25 nmol) significantly decreased repeated cocaine-induced increases in the IR of the ER stress proteins in the injected dorsal striatum. Similarly, the selective D1 antagonist (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390; 0.1 mg/kg, i.p.) or the N-methyl-d-aspartate antagonist dizocilpine/(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-ibenzo[a,d]cyclohepten-5,10-imine maleate (MK801; 2 nmol, i.s.) decreased acute or repeated cocaine-induced the IR of the ER stress proteins in the dorsal striatum. These data suggest that cocaine upregulates expression of the ER stress proteins in striatal neurons via a mechanism involving activation of glutamate and dopamine receptors. PMID:17303341

Shin, E H; Bian, S; Shim, Y-B; Rahman, M A; Chung, K T; Kim, J Y; Wang, J Q; Choe, E S

2007-03-16

180

The Use of Xenopus laevis Oocytes for the Functional Characterization of Heterologously Expressed Membrane Proteins  

Microsoft Academic Search

The oocytes of the South African clawed frog X. laevis are widely used for the expression of heterologous proteins. The functional characterization of membrane proteins in particular has significantly profited from the use of this expression system. Heterologous cRNA can easily be injected and protein expression and function be studied with several techniques. This review will give a short overview

Carsten A. Wagner; Björn Friedrich; Iwan Setiawan; Florian Lang; Stefan Bröer

2000-01-01

181

Differential expression of LMO4 protein in Alzheimer's disease.  

PubMed

The molecular bases of late-onset and sporadic Alzheimer's disease (AD) still have to be unraveled. Among putative candidates for molecular variations in AD, we propose LMO4 protein, a transcription regulator, involved in multiple protein complexes. We investigated changes in LMO4 immunoreactivity in vulnerable brain regions of AD cases and controls of comparable age. Immunocytochemical analysis revealed a high level of LMO4 expression in the entorhinal cortex (EC) and in the CA1 hippocampal region of the control brains and a consistent decrease in the AD brains, correlated with the amount of neurofibrillary tangles (NFT) degenerating neurones and the severity of senile plaques deposition. The decrease in LMO4 immunoreactivity resulted both from weaker immunoreactive signals and from a loss of immunoreactive neurones. LMO4 immunocytochemical staining appeared not to be colocalized with NFT in a majority of neurones. Its expression was weak in the dentate gyrus and stronger in CA3-4, two regions with no or low numbers of NFT, but there was no decrease in AD compared to control cases. In the frontal cortex, the ventro-infero-median region (area 12) showed a greater LMO4 expression than the polar one (area 9), but no decrease in AD was observed. As LMO4 has been proposed to inhibit cellular differentiation, it can be hypothesized that a reduced expression is associated in EC and CA1 with attempts of diseased neurones to differentiate (e.g. compensatory neuritogenesis). Taken together, these data indicate that LMO4 protein is involved in the complexity of the disease phenotype, at least as a secondary factor. PMID:14720177

Leuba, G; Vernay, A; Vu, D; Walzer, C; Belloir, B; Kraftsik, R; Bouras, C; Savioz, A

2004-02-01

182

Optimizing Escherichia coli as a protein expression platform to produce Mycobacterium tuberculosis immunogenic proteins  

PubMed Central

Background A number of valuable candidates as tuberculosis vaccine have been reported, some of which have already entered clinical trials. The new vaccines, especially subunit vaccines, need multiple administrations in order to maintain adequate life-long immune memory: this demands for high production levels and degree of purity. Results In this study, TB10.4, Ag85B and a TB10.4-Ag85B chimeric protein (here-after referred as full) - immunodominant antigens of Mycobacterium tuberculosis - were expressed in Escherichia coli and purified to homogeneity. The rational design of expression constructs and optimization of fermentation and purification conditions allowed a marked increase in solubility and yield of the recombinant antigens. Indeed, scaling up of the process guaranteed mass production of all these three antigens (2.5-25 mg of pure protein/L cultivation broth). Quality of produced soluble proteins was evaluated both by mass spectrometry to assess the purity of final preparations, and by circular dichroism spectroscopy to ascertain the protein conformation. Immunological tests of the different protein products demonstrated that when TB10.4 was fused to Ag85B, the chimeric protein was more immunoreactive than either of the immunogenic protein alone. Conclusions We reached the goal of purifying large quantities of soluble antigens effective in generating immunological response against M. tuberculosis by a robust, controlled, scalable and economically feasible production process. PMID:24252280

2013-01-01

183

Overexpression of heat shock proteins differentially modulates protein kinase C expression in rat neonatal cardiomyocytes  

PubMed Central

Previous studies have suggested that protein kinase C (PKC) is involved in heat shock protein (Hsp)–mediated cardioprotection. Therefore, we wanted to determine whether overexpression of Hsps modulates PKC expression, which will give us further insight into understanding the mechanism by which Hsps and PKC interact to protect cells from stress-induced injury. Specifically, we overexpressed the inducible form of Hsp70 (Hsp70i) or Hsp90 in rat neonatal cardiomyocytes and evaluated PKC? or PKC? expression by immunoblotting and immunofluorescent confocal microscopy. Western analysis showed that overexpression of Hsp70i or Hsp90 decreased PKC? expression. However, overexpression of Hsp70i or Hsp90 did not modify PKC? expression over control levels. Overexpression of constitutively active PKC? or PKC? increased Hsp70i expression over control levels. The data suggest that overexpression of Hsps differentially modulates expression of PKC isoforms in rat neonatal cardiomyocytes. Furthermore, PKC may directly play a role in Hsp-mediated cardioprotection by upregulating Hsp70i expression. PMID:15115281

Coaxum, Sonya D.; Martin, Jody L.; Mestril, Ruben

2003-01-01

184

Bacterial expression and photoaffinity labeling of a pheromone binding protein.  

PubMed

The first high-level production of a binding-active odorant binding protein is described. The expression cassette polymerase chain reaction was used to generate a DNA fragment encoding the pheromone binding protein (PBP) of the male moth Antheraea polyphemus. Transformation of Escherichia coli cells with a vector containing this construct generated clones which, when induced with isopropyl beta-D-thiogalactopyranoside, produced the 14-kDa PBP in both the soluble fraction and in inclusion bodies. Purification of the soluble recombinant PBP by preparative isoelectric focusing and gel filtration gave > 95% homogeneous protein, which was immunoreactive with an anti-PBP antiserum and exhibited specific, pheromone-displaceable covalent modification by the photoaffinity label [3H]6E,11Z-hexadecadienyl diazoacetate. Recombinant PBP was indistinguishable from the insect-derived PBP, as determined by both native and denaturing gel electrophoresis, immunoreactivity, and photoaffinity labeling properties. Moreover, the insoluble inclusion body protein could be solubilized, refolded, and purified by the same procedures to give a recombinant PBP indistinguishable from the soluble PBP. Proton NMR spectra of the soluble and refolded protein provide further evidence that they possess the same folded structure. PMID:8453379

Prestwich, G D

1993-03-01

185

Structure and expression of a novel compact myelin protein - small VCP-interacting protein (SVIP).  

PubMed

SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes. PMID:24055875

Wu, Jiawen; Peng, Dungeng; Voehler, Markus; Sanders, Charles R; Li, Jun

2013-10-11

186

Studying weak and dynamic interactions of posttranslationally modified proteins using expressed protein ligation.  

PubMed

Many cellular processes are regulated by posttranslational modifications that are recognized by specific domains in protein binding partners. These interactions are often weak, thus allowing a highly dynamic and combinatorial regulatory network of protein-protein interactions. We report an efficient strategy that overcomes challenges in structural analysis of such a weak transient interaction between the Tudor domain of the Survival of Motor Neuron (SMN) protein and symmetrically dimethylated arginine (sDMA). The posttranslational modification is chemically introduced and covalently linked to the effector module by a one-pot expressed protein ligation (EPL) procedure also enabling segmental incorporation of NMR-active isotopes for structural analysis. Covalent coupling of the two interacting moieties shifts the equilibrium to the bound state, and stoichiometric interactions are formed even for low affinity interactions. Our approach should enable the structural analysis of weak interactions by NMR or X-ray crystallography to better understand the role of posttranslational modifications in dynamic biological processes. PMID:24299430

Tripsianes, Konstantinos; Chu, Nam K; Friberg, Anders; Sattler, Michael; Becker, Christian F W

2014-02-21

187

Protein sorting and expression of a unique soybean cotyledon protein, GmSBP, destined for the protein storage vacuole.  

PubMed

The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a re-characterization of GmSBP via sequence homology, cell biology, immunolocalization, and semi-quantitative analysis. The GmSBP proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and beta-conglycinin established several storage protein-like characteristics for GmSBP. All three proteins were present in a prevacuolar compartment and protein storage vacuole. All three proteins increased in expression during seed development and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, beta-conglycinin (alpha/alpha' subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitative differences between GmSBP and glycinin may be attributed to the unconserved order and spacing of cis-acting regulatory elements present within the promoter regions. Ultimately, GmSBP is transported to the mature protein storage vacuole. The biological function of GmSBP within the protein storage vacuole remains uncertain, but its localization is a remnant of its evolutionary link to a globulin-like or vicilin-like ancestor that gave rise to the 7S family of storage proteins. PMID:14558667

Elmer, Aaron; Chao, Wun; Grimes, Howard

2003-07-01

188

Gene expression pattern for putative chloroplast localized COPII related proteins with emphasis on Rab related proteins  

PubMed Central

Vesicle transport occurs in the cytosol through a COPI, COPII and clathrin coated vesicle system for transport of lipids and proteins to different subcellular compartments. All three systems consist of several different protein components to maintain a functional transport. In chloroplasts photosynthesis takes place in thylakoids. Thylakoids contain a large amount of lipids and proteins but none of these components are produced there. Transport of lipids occurs from the envelope membrane where they are produced and through the aqueous stroma before being directed to the thylakoids. Nuclear encoded proteins use distinct pathways for entering thylakoids after import into chloroplasts. Transport of lipids through stroma requires either lipid transfer proteins, association between the envelope and the thylakoid membrane, or a vesicle transport system similar to the cytosolic one. No evidence exists for lipid transfer proteins in chloroplasts, or for a consistent association between the envelope and the thylakoid membrane. However, vesicle transport has support from e.g., biochemical and genetics data as well as transelectron microscopy data. Moreover, a recent bioinformatics study revealed COPII related proteins to be putatively chloroplast localized in Arabidopsis and thus function in vesicle transport in chloroplasts. Here we present gene expression profiles of these COPII related putatively chloroplast localized proteins using Genevestigator (https://www.genevestigator.com/gv/) with special emphasis on Rab related proteins since they represent several stages of vesicle transport e.g., uncoating, tethering and fusion. PMID:24577429

Alezzawi, Mohamed; Karim, Sazzad; Khan, Nadir Zaman; Aronsson, Henrik

2014-01-01

189

Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias.  

PubMed

The aberrant overexpression of Wilms tumor 1 (WT1) in myeloid leukemia plays an important role in blast cell survival and resistance to chemotherapy. High expression of WT1 is also associated with relapse and shortened disease-free survival in patients. However, the mechanisms by which WT1 expression is regulated in leukemia remain unclear. Here, we report that heat shock protein 90 (Hsp90), which plays a critical role in the folding and maturation of several oncogenic proteins, associates with WT1 protein and stabilizes its expression. Pharmacologic inhibition of Hsp90 resulted in ubiquitination and subsequent proteasome-dependant degradation of WT1. RNAi-mediated silencing of WT1 reduced the survival of leukemia cells and increased the sensitivity of these cells to chemotherapy and Hsp90 inhibition. Furthermore, Hsp90 inhibitors 17-AAG [17-(allylamino)-17-demethoxygeldanamycin] and STA-9090 significantly reduced the growth of myeloid leukemia xenografts in vivo and effectively down-regulated the expression of WT1 and its downstream target proteins, c-Myc and Bcl-2. Collectively, our studies identify WT1 as a novel Hsp90 client and support the crucial role for the WT1-Hsp90 interaction in maintaining leukemia cell survival. These findings have significant implications for developing effective therapies for myeloid leukemias and offer a strategy to inhibit the oncogenic functions of WT1 by clinically available Hsp90 inhibitors. PMID:20651072

Bansal, Hima; Bansal, Sanjay; Rao, Manjeet; Foley, Kevin P; Sang, Jim; Proia, David A; Blackman, Ronald K; Ying, Weiwen; Barsoum, James; Baer, Maria R; Kelly, Kevin; Swords, Ronan; Tomlinson, Gail E; Battiwalla, Minoo; Giles, Francis J; Lee, Kelvin P; Padmanabhan, Swaminathan

2010-11-25

190

Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins  

Microsoft Academic Search

BACKGROUND: Studies on Rift Valley Fever Virus (RVFV) infection process and morphogenesis have been hampered due to the biosafety conditions required to handle this virus, making alternative systems such as recombinant virus-like particles, that may facilitate understanding of these processes are highly desirable. In this report we present the expression and characterization of RVFV structural proteins N, Gn and Gc

Li Liu; Polly Roy

2008-01-01

191

Effect of Culture Time on the Basal Expression Levels of Drug Transporters in Sandwich-Cultured Primary Rat HepatocytesS?  

PubMed Central

Sandwich-cultured rat hepatocytes are used in drug discovery for pharmacological and toxicological assessment of drug candidates, yet their utility as a functional model for drug transporters has not been fully characterized. To evaluate the system as an in vitro model for drug transport, expression changes of hepatic transporters relative to whole liver and freshly isolated hepatocytes (day 0) were examined by real-time quantitative reverse transcription-polymerase chain reaction for 4 consecutive days of culture. No significant differences in transporter expression levels were observed between freshly isolated hepatocytes and whole liver. Two distinct mRNA profiles were detected over time showing 1) a more than 5-fold decline in levels of uptake transporters such as Na+-taurocholate cotransporting polypeptide (Ntcp), organic anion transporter (Oat) 2, organic anion-transporting polypeptide (Oatp) 1a1, Oatp1a4, and Oatp1b2 and 2) a greater than 5-fold increase of efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-related proteins (Mrp) 1, 2, 3, and 4. In addition, protein levels and functional activities for selected transporters were also determined. Protein levels for Mrp2, Bcrp, P-gp, Ntcp, and Oatp1a4 corresponded to changes in mRNA. Functional activities of Oatps and Oct1 exhibited a 3- and 4-fold decrease on day 2 and day 4, respectively, relative to that on day 0, whereas a more than 10-fold reduction in Oat2 activity was observed. These results indicate that the cell culture conditions used herein did not provide an optimal environment for expression of all hepatic transporters. Significant time-dependent alterations in basal gene expression patterns of transporters were detected compared with those in liver or freshly isolated hepatocytes. Further work and new strategies are required to improve the validity of this model as an in vitro tool for in vivo drug transport or biliary clearance prediction. PMID:21865320

Houghton, Jessica S.; Uyeda, Craig; Grillo, Mark P.; Jin, Lixia

2011-01-01

192

Cysteine string protein expression in mammary epithelial cells.  

PubMed

Cysteine string protein (Csp) is a secretory vesicle protein previously demonstrated to be required for Ca2+-regulated exocytosis in neurons and endocrine cells. It has been suggested to function by regulating voltage-gated Ca2+ channels or, alternatively, to have a more direct effect on the regulated exocytotic machinery. Here we demonstrate the expression of Csp in mammary epithelial cells and in the KIM-2 mammary cell line. In KIM-2 cells, Csp was found to be associated with a population of small vesicles and showed partial co-distribution with the vesicle protein cellubrevin. KIM-2 cells do not express detectable levels of voltage-gated Ca2+ channels, ruling these out as a site of action. Using the release of transfected growth hormone (GH) as an assay of secretion, we found that GH is secreted in an exclusively constitutive manner from KIM-2 cells. Overexpression of Csp1 inhibits regulated exocytosis in other cell types but has no effect on constitutive GH release by KIM-2 cells. These results suggest that Csp does not have a major function in constitutive exocytosis. PMID:11294245

Gleave, T L; Beechey, R B; Burgoyne, R D

2001-02-01

193

Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.  

PubMed

The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-? (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-?B, Wnt/?-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via different subsignaling pathways. Analyses of the expression levels of dozens of genes and the protein-protein interactions among them demonstrated that CD and UC have relatively similar gene expression signatures, whereas the gene expression signatures of T1D and JRA relatively differ from the signatures of the other autoimmune diseases. These diseases are the only ones activated via the Fc? pathway. The relevant genes and pathways reported in this study are discussed at length, and may be helpful in the diagnoses and understanding of autoimmunity and/or specific autoimmune diseases. PMID:23190644

Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A

2013-03-01

194

Regulator of G-protein signalling expression and function in ovarian cancer cell lines  

Microsoft Academic Search

Regulator of G-protein signalling (RGS)2 proteins critically regulate signalling cascades initiated by G-protein coupled receptors (GPCRs) by accelerating the deactivation\\u000a of heterotrimeric G-proteins. Lysophosphatidic acid (LPA) is the predominant growth factor that drives the progression of\\u000a ovarian cancer by activating specific GPCRs and G-proteins expressed in ovarian cancer cells. We have recently reported that\\u000a RGS proteins endogenously expressed in SKOV-3

Jillian H. Hurst; Nisha Mendpara; Shelley B. Hooks

2009-01-01

195

Structural genomics of human proteins – target selection and generation of a public catalogue of expression clones  

PubMed Central

Background The availability of suitable recombinant protein is still a major bottleneck in protein structure analysis. The Protein Structure Factory, part of the international structural genomics initiative, targets human proteins for structure determination. It has implemented high throughput procedures for all steps from cloning to structure calculation. This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia coli host cells. Results and Conclusion Protein expression and sequence data of 1414 E. coli expression clones representing 537 different proteins are presented. 139 human proteins (18%) could be expressed and purified in soluble form and with the expected size. All E. coli expression clones are publicly available to facilitate further functional characterisation of this set of human proteins. PMID:15998469

Büssow, Konrad; Scheich, Christoph; Sievert, Volker; Harttig, Ulrich; Schultz, Jörg; Simon, Bernd; Bork, Peer; Lehrach, Hans; Heinemann, Udo

2005-01-01

196

Regulation of Aryl Hydrocarbon Receptor Interacting Protein (AIP) Protein Expression by MiR-34a in Sporadic Somatotropinomas  

PubMed Central

Introduction Patients with germline AIP mutations or low AIP protein expression have large, invasive somatotroph adenomas and poor response to somatostatin analogues (SSA). Methods To study the mechanism of low AIP protein expression 31 sporadic somatotropinomas with low (n = 13) or high (n = 18) AIP protein expression were analyzed for expression of AIP messenger RNA (mRNA) and 11 microRNAs (miRNAs) predicted to bind the 3’UTR of AIP. Luciferase reporter assays of wild-type and deletion constructs of AIP-3’UTR were used to study the effect of the selected miRNAs in GH3 cells. Endogenous AIP protein and mRNA levels were measured after miRNA over- and underexpression in HEK293 and GH3 cells. Results No significant difference was observed in AIP mRNA expression between tumors with low or high AIP protein expression suggesting post-transcriptional regulation. miR-34a was highly expressed in low AIP protein samples compared high AIP protein adenomas and miR-34a levels were inversely correlated with response to SSA therapy. miR-34a inhibited the luciferase-AIP-3’UTR construct, suggesting that miR-34a binds to AIP-3’UTR. Deletion mutants of the 3 different predicted binding sites in AIP-3’UTR identified the c.*6–30 site to be involved in miR-34a’s activity. miR-34a overexpression in HEK293 and GH3 cells resulted in inhibition of endogenous AIP protein expression. Conclusion Low AIP protein expression is associated with high miR-34a expression. miR-34a can down-regulate AIP-protein but not RNA expression in vitro. miR-34a is a negative regulator of AIP-protein expression and could be responsible for the low AIP expression observed in somatotropinomas with an invasive phenotype and resistance to SSA. PMID:25658813

Dénes, Judit; Kasuki, Leandro; Trivellin, Giampaolo; Colli, Leandro M.; Takiya, Christina M.; Stiles, Craig E.; Barry, Sayka; de Castro, Margaret; Gadelha, Mônica R.; Korbonits, Márta

2015-01-01

197

Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment  

PubMed Central

Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development. PMID:24699514

Chen, Fangyu; Jiang, Liangrong; Zheng, Jingsheng; Huang, Rongyu; Wang, Houcong; Hong, Zonglie; Huang, Yumin

2014-01-01

198

Structure and expression of a novel compact myelin protein – Small VCP-interacting protein (SVIP)  

SciTech Connect

Highlights: •SVIP (small p97/VCP-interacting protein) co-localizes with myelin basic protein (MBP) in compact myelin. •We determined that SVIP is an intrinsically disordered protein (IDP). •The helical content of SVIP increases dramatically during its interaction with negatively charged lipid membrane. •This study provides structural insight into interactions between SVIP and myelin membranes. -- Abstract: SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes.

Wu, Jiawen [Department of Neurology, Vanderbilt University School of Medicine (United States)] [Department of Neurology, Vanderbilt University School of Medicine (United States); Peng, Dungeng [Department of Biochemistry, Vanderbilt University School of Medicine (United States)] [Department of Biochemistry, Vanderbilt University School of Medicine (United States); Voehler, Markus [Center for Structural Biology, Vanderbilt University (United States)] [Center for Structural Biology, Vanderbilt University (United States); Sanders, Charles R. [Department of Biochemistry, Vanderbilt University School of Medicine (United States) [Department of Biochemistry, Vanderbilt University School of Medicine (United States); Center for Structural Biology, Vanderbilt University (United States); Li, Jun, E-mail: jun.li.2@vanderbilt.edu [Department of Neurology, Vanderbilt University School of Medicine (United States) [Department of Neurology, Vanderbilt University School of Medicine (United States); Tennessee Valley Healthcare System (TVHS) – Nashville VA (United States)

2013-10-11

199

Complexity of Gene Expression Evolution after Duplication: Protein Dosage Rebalancing  

PubMed Central

Ongoing debates about functional importance of gene duplications have been recently intensified by a heated discussion of the “ortholog conjecture” (OC). Under the OC, which is central to functional annotation of genomes, orthologous genes are functionally more similar than paralogous genes at the same level of sequence divergence. However, a recent study challenged the OC by reporting a greater functional similarity, in terms of gene ontology (GO) annotations and expression profiles, among within-species paralogs compared to orthologs. These findings were taken to indicate that functional similarity of homologous genes is primarily determined by the cellular context of the genes, rather than evolutionary history. Subsequent studies suggested that the OC appears to be generally valid when applied to mammalian evolution but the complete picture of evolution of gene expression also has to incorporate lineage-specific aspects of paralogy. The observed complexity of gene expression evolution after duplication can be explained through selection for gene dosage effect combined with the duplication-degeneration-complementation model. This paper discusses expression divergence of recent duplications occurring before functional divergence of proteins encoded by duplicate genes. PMID:25197576

Rogozin, Igor B.

2014-01-01

200

Evaluation of photodynamic therapy in adhesion protein expression  

PubMed Central

Photodynamic therapy (PDT) is a treatment modality that has clinical applications in both non-neoplastic and neoplastic diseases. PDT involves a light-sensitive compound (photosensitizer), light and molecular oxygen. This procedure may lead to several different cellular responses, including cell death. Alterations in the attachment of cancer cells to the substratum and to each other are important consequences of photodynamic treatment. PDT may lead to changes in the expression of cellular adhesion structure and cytoskeleton integrity, which are key factors in decreasing tumor metastatic potential. HEp-2 cells were photosensitized with aluminum phthalocyanine tetrasulfonate and zinc phthalocyanine, and the proteins ?1-integrin and focal adhesion kinase (FAK) were assayed using fluorescence microscopy. The verification of expression changes in the genes for FAK and ?1 integrin were performed by reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that HEp-2 cells do not express ?-integrin or FAK 12 h following PDT. It was concluded that the PDT reduces the adhesive ability of HEp-2 cells, inhibiting their metastatic potential. The present study aimed to analyze the changes in the expression and organization of cellular adhesion elements and the subsequent metastatic potential of HEp-2 cells following PDT treatment. PMID:25013490

PACHECO-SOARES, CRISTINA; MAFTOU-COSTA, MAIRA; DA CUNHA MENEZES COSTA, CAROLINA GENÚNCIO; DE SIQUEIRA SILVA, ANDREZA CRISTINA; MORAES, KAREN C.M.

2014-01-01

201

Evaluation of photodynamic therapy in adhesion protein expression.  

PubMed

Photodynamic therapy (PDT) is a treatment modality that has clinical applications in both non-neoplastic and neoplastic diseases. PDT involves a light-sensitive compound (photosensitizer), light and molecular oxygen. This procedure may lead to several different cellular responses, including cell death. Alterations in the attachment of cancer cells to the substratum and to each other are important consequences of photodynamic treatment. PDT may lead to changes in the expression of cellular adhesion structure and cytoskeleton integrity, which are key factors in decreasing tumor metastatic potential. HEp-2 cells were photosensitized with aluminum phthalocyanine tetrasulfonate and zinc phthalocyanine, and the proteins ?1-integrin and focal adhesion kinase (FAK) were assayed using fluorescence microscopy. The verification of expression changes in the genes for FAK and ?1 integrin were performed by reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that HEp-2 cells do not express ?-integrin or FAK 12 h following PDT. It was concluded that the PDT reduces the adhesive ability of HEp-2 cells, inhibiting their metastatic potential. The present study aimed to analyze the changes in the expression and organization of cellular adhesion elements and the subsequent metastatic potential of HEp-2 cells following PDT treatment. PMID:25013490

Pacheco-Soares, Cristina; Maftou-Costa, Maira; DA Cunha Menezes Costa, Carolina Genúncio; DE Siqueira Silva, Andreza Cristina; Moraes, Karen C M

2014-08-01

202

Sperm protein 17 is expressed in human nervous system tumours  

PubMed Central

Background Human sperm protein 17 (Sp17) is a highly conserved protein that was originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. It has recently been included in the cancer/testis (CT) antigen family, and shown to be expressed in multiple myeloma and ovarian cancer. We investigated its immunolocalisation in specimens of nervous system (NS) malignancies, in order to establish its usefulness as a target for tumour-vaccine strategies. Methods The expression of Sp17 was assessed by means of a standardised immunohistochemical procedure [(mAb/antigen) MF1/Sp17] in formalin-fixed and paraffin embedded surgical specimens of NS malignancies, including 28 neuroectodermal primary tumours (6 astrocytomas, 16 glioblastoma multiforme, 5 oligodendrogliomas, and 1 ependymoma), 25 meningeal tumours, and five peripheral nerve sheath tumours (4 schwannomas, and 1 neurofibroma),. Results A number of neuroectodermal (21%) and meningeal tumours (4%) were found heterogeneously immunopositive for Sp17. None of the peripheral nerve sheath tumours was immunopositive for Sp17. The expression pattern was heterogeneous in all of the positive samples, and did not correlate with the degree of malignancy. Conclusion The frequency of expression and non-uniform cell distribution of Sp17 suggest that it cannot be used as a unique immunotherapeutic target in NS cancer. However, our results do show the immunolocalisation of Sp17 in a proportion of NS tumour cells, but not in their non-pathological counterparts. The emerging complex function of Sp17 makes further studies necessary to clarify the link between it and immunopositive cells. PMID:16438728

Grizzi, Fabio; Gaetani, Paolo; Franceschini, Barbara; Di Ieva, Antonio; Colombo, Piergiuseppe; Ceva-Grimaldi, Giorgia; Bollati, Angelo; Frezza, Eldo E; Cobos, E; Baena, Riccardo Rodriguez y; Dioguardi, Nicola; Chiriva-Internati, Maurizio

2006-01-01

203

Purification of the Sendai virus nonstructural C protein expressed in E. coli, and preparation of antiserum against C protein  

Microsoft Academic Search

Summary An expression plasmid, ptac-C, was constructed by inserting the cDNA of the coding region of the Sendai virus nonstructural C protein downstream of the tac promoter ofE. coli expression plasmid ptac12-Bam. A new protein produced inE. coli after induction was purified to near homogeneity. The purified protein was found to be identical with the C protein predicted from the

T. Omata-Yamada; K. Hagiwara; K. Katoh; H. Yamada; K. Iwasaki

1988-01-01

204

Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent protein is not.  

PubMed

Validated gene transfer and expression tracers are essential for elucidating functions of mammalian genes. Here, we have determined the suitability and unintended side effects of enhanced green fluorescent protein (EGFP) and DsRed-Express fluorescent protein as expression tracers in long-term hematopoietic stem cells (HSCs). Retrovirally transduced mouse bone marrow cells expressing either EGFP or DsRed-Express in single or mixed dual-color cell populations were clearly discerned by flow cytometry and fluorescence microscopy. The results from in vivo competitive repopulation assays demonstrated that EGFP-expressing HSCs were maintained nearly throughout the lifespan of the transplanted mice and retained long-term multilineage repopulating potential. All mice assessed at 15 months post-transplantation were EGFP positive, and, on average, 24% total peripheral white blood cells expressed EGFP. Most EGFP-expressing recipient mice lived at least 22 months. In contrast, Discosoma sp. red fluorescent protein (DsRed)-expressing donor cells dramatically declined in transplant-recipient mice over time, particularly in the competitive setting, in which mixed EGFP- and DsRed-expressing cells were cotransplanted. Moreover, under in vitro culture condition favoring preservation of HSCs, purified EGFP-expressing cells grew robustly, whereas DsRed-expressing cells did not. Therefore, EGFP has no detectable deteriorative effects on HSCs, and is nearly an ideal long-term expression tracer for hematopoietic cells; however, DsRed-Express fluorescent protein is not suitable for these cells. PMID:17138958

Tao, Wen; Evans, Barbara-Graham; Yao, Jing; Cooper, Scott; Cornetta, Kenneth; Ballas, Christopher B; Hangoc, Giao; Broxmeyer, Hal E

2007-03-01

205

Expression of cell cycle proteins in male breast carcinoma  

PubMed Central

Introduction Male breast cancer (MBC) is a rare, yet potentially aggressive disease. Although literature regarding female breast cancer (FBC) is extensive, little is known about the etiopathogenesis of male breast cancer. Studies from our laboratory show that MBCs have a distinct immunophenotypic profile, suggesting that the etiopathogenesis of MBC is different from FBCs. The aim of this study was to evaluate and correlate the immunohistochemical expression of cell cycle proteins in male breast carcinoma to significant clinico-biological endpoints. Methods 75 cases of MBC were identified using the records of the Saskatchewan Cancer Agency over 26 years (1970-1996). Cases were reviewed and analyzed for the immunohistochemical expression of PCNA, Ki67, p27, p16, p57, p21, cyclin-D1 and c-myc and correlated to clinico-biological endpoints of tumor size, node status, stage of the disease, and disease free survival (DFS). Results Decreased DFS was observed in the majority of tumors that overexpressed PCNA (98%, p = 0.004). The overexpression of PCNA was inversely correlated to the expression of Ki67 which was predominantly negative (78.3%). Cyclin D1 was overexpressed in 83.7% of cases. Cyclin D1 positive tumors were smaller than 2 cm (55.6%, p = 0.005), had a low incidence of lymph node metastasis (38.2%, p = 0.04) and were associated with increased DFS of >150 months (p = 0.04). Overexpression of c-myc (90%) was linked with a higher incidence of node negativity (58.3%, p = 0.006) and increased DFS (p = 0.04). p27 over expression was associated with decreased lymph node metastasis (p = 0.04). P21 and p57 positive tumors were related to decreased DFS (p = 0.04). Though p16 was overexpressed in 76.6%, this did not reach statistical significance with DFS (p = 0.06) or nodal status (p = 0.07). Conclusion Aberrant cell cycle protein expression supports our view that these are important pathways involved in the etiopathogenesis of MBC. Tumors with overexpression of Cyclin D1 and c-myc had better outcomes, in contrast to tumors with overexpression of p21, p57, and PCNA with significantly worse outcomes. P27 appears to be a predictive marker for lymph nodal status. Such observation strongly suggests that dysregulation of cell cycle proteins may play a unique role in the initiation and progression of disease in male breast cancer. Such findings open up new avenues for the treatment of MBC as a suitable candidate for novel CDK-based anticancer therapies in the future. PMID:20152033

2010-01-01

206

Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.  

PubMed

We describe an efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins. Using a combination of sequence and structure consensus analyses, we designed a 33 amino acid residue AR module with seven randomized positions having a theoretical diversity of 7.2x10(7). Different numbers of this module were cloned between N and C-terminal capping repeats, i.e. ARs designed to shield the hydrophobic core of stacked AR modules. In this manner, combinatorial libraries of designed AR proteins consisting of four to six repeats were generated, thereby potentiating the theoretical diversity. All randomly chosen library members were expressed in soluble form in the cytoplasm of Escherichia coli in amounts up to 200 mg per 1 l of shake-flask culture. Virtually pure proteins were obtained in a single purification step. The designed AR proteins are monomeric and display CD spectra identical with those of natural AR proteins. At the same time, our AR proteins are highly thermostable, with T(m) values ranging from 66 degrees C to well above 85 degrees C. Thus, our combinatorial library members possess the properties required for biotechnological applications. Moreover, the favorable biophysical properties and the modularity of the AR fold may account, partly, for the abundance of natural AR proteins. PMID:12948497

Binz, H Kaspar; Stumpp, Michael T; Forrer, Patrik; Amstutz, Patrick; Plückthun, Andreas

2003-09-12

207

Expressed Protein Ligation: A Resourceful Tool to Study Protein Structure and Function  

PubMed Central

This review outlines the use of expressed protein ligation (EPL) to study protein structure, function and stability. EPL is a chemoselective ligation method that allows the selective ligation of unprotected polypeptides from synthetic and recombinant origin for the production of semi-synthetic protein samples of well-defined and homogeneous chemical composition. This method has been extensively used for the site-specific introduction of biophysical probes, unnatural amino acids, and increasingly complex post-translational modifications. Since it was introduced 10 years ago, EPL applications have grown increasingly more sophisticated in order to address even more complex biological questions. In this review we highlight how this powerful technology combined with standard biochemical analysis techniques has been used to improve our ability to understand protein structure and function. PMID:19685006

Berrade, Luis; Camarero, Julio A.

2013-01-01

208

Synergistic enhancement of globin gene expression by activator protein-1-like proteins.  

PubMed Central

DNA sequences corresponding to the four major DNase I hypersensitive sites upstream of the beta-globin gene cluster are essential for the achievement of high levels of globin gene expression and development regulation. In this study, we focused on one of these sites, hypersensitive site 2, which behaves as a powerful enhancer in transient expression and transgenic mouse experiments. We identified a tandem repeat of the activator protein 1 (AP-1) consensus sequence that binds AP-1-like proteins from nuclear extracts of K562 and HeLa cells. These proteins have the same binding properties as HeLa AP-1 but differ in the electrophoretic mobility and in functional assays. Transient-expression experiments in K562 of various deletion and point mutation constructs derived from hypersensitive site 2 indicate that the enhancer activity and the inducibility of a linked gamma-globin promoter are dependent upon the synergistic action of proteins bound to the tandem AP-1 repeat. Images PMID:2123346

Moi, P; Kan, Y W

1990-01-01

209

Imaging bacterial protein expression using genetically encoded sensors composed of RNA  

PubMed Central

We show that the difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome using fluorescent sensors based on Spinach, an RNA that activates the fluorescence of a small-molecule fluorophore. These RNAs selectively bind target proteins, and exhibit fluorescence increases that enable protein expression to be imaged in living cells. These sensors provide a general strategy to image protein expression in single bacteria in real-time. PMID:23872791

Song, Wenjiao; Strack, Rita L.; Jaffrey, Samie R.

2013-01-01

210

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins  

NASA Technical Reports Server (NTRS)

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1994-01-01

211

Expression of Trans-Membrane Proteins in vitro Using a Cell Free System  

NASA Astrophysics Data System (ADS)

Trans-membrane proteins represent a significant portion of the proteins expressed by cells. The expression of proteins in vitro, however, remains a challenge. Numerous expression approaches have been developed with cell free expression (CFE) being one of the most promising. CFE is based on a transcription-translation system that has been extracted from E. coli bacteria. Adding the desired DNA allows expression of a selected protein, and in the presence of phospholipids the expression of trans-membrane proteins becomes possible. In order to express trans-membrane proteins in a closed native environment, the cell free system (CFS) is encapsulated with a phospholipid bilayer, creating an artificial cell. To verify protein expression, AquaporinZ (AqpZ), a well-known trans-membrane protein tagged with a green fluorescent protein (eGFP), was used so the expressed proteins could be seen under a fluorescent microscope. These artificial cells will serve as an experimental platform for testing the viability of the expressed trans-membrane proteins. Results from the manipulation of these artificial cells by attaching them to the slide surface through streptavidin-biotin bonding will be presented.

Weisse, Natalie; Noireaux, Vincent; Chalmeau, Jerome

2010-10-01

212

Expression and Function of Iron-Regulatory Proteins in Retina  

PubMed Central

Summary Iron is essential for cell survival and function; yet excess iron is toxic to cells. Therefore, the cellular and whole-body levels of iron are regulated exquisitely. At least a dozen proteins participate in the regulation of iron homeostasis. Hemochromatosis, a genetic disorder of iron overload, is caused by mutations in at least five genes, namely HFE, hemojuvelin, Transferrin receptor 2, ferroportin, and hepcidin. Retina is separated from systemic circulation by inner and outer blood-retinal barriers; therefore it is widely believed that this tissue is immune to changes in systemic circulation. Even though hemochromatosis is associated with iron overload and dysfunction of a variety of systemic organs, little is known on the effects of this disease on the retina. Recent studies have shown that all five genes that are associated with hemochromatosis are expressed in the retina in a cell type-specific manner. The retinal pigment epithelium, which forms the outer blood-retinal barrier, expresses all of these five genes. It is therefore clearly evident that iron homeostasis in the retina is maintained locally by active participation of various iron-regulatory proteins. Excess iron is detrimental to the retina as evidenced from human studies and from mouse models of iron overload. Retinal iron homeostasis is disrupted in various clinical conditions such as hemochromatosis, aceruloplasminemia, age-related macular degeneration, and bacterial and viral infections. PMID:20408179

Gnana-Prakasam, Jaya P.; Martin, Pamela M.; Smith, Sylvia B.; Ganapathy, Vadivel

2013-01-01

213

Assessment of uncoupling activity of uncoupling protein 3 using a yeast heterologous expression system  

Microsoft Academic Search

Uncoupling protein 3L, uncoupling protein 1 and the mitochondrial oxoglutarate carrier were expressed in Saccharomyces cerevisae. Effects on different parameters related to the energy expenditure were studied. Both uncoupling protein 3L and uncoupling protein 1 reduced the growth rate by 49% and 32% and increased the whole yeast O2 consumption by 31% and 19%, respectively. In isolated mitochondria, uncoupling protein

Chen-Yu Zhang; Thilo Hagen; Vamsi K Mootha; Lawrence J Slieker; Bradford B Lowell

1999-01-01

214

Enhanced green fluorescent protein-expressing human mesenchymal stem cells retain neural marker expression.  

PubMed

Mesenchymal stem cells (MSCs) have the potential to play a role in autologous treatment of central nervous system injury or disease. Here we transduced human MSCs with enhanced green fluorescent protein (EGFP). We compared the capacity of control and EGFP-positive cells to proliferate under normal culture conditions, as well as express neural markers following trans-differentiation. EGFP-positive cells proliferated comparably to controls, retained EGFP expression over the course of multiple passages, and retained neural marker expression at levels comparable to control MSCs. Further neurogenic capacity of EGFP-positive human MSCs was examined by growth as neural stem cell-like neurospheres. No significant difference was observed in the ability of control or EGFP-positive cells to generate primary neurospheres or to expand during passage. When examined by immunostaining for the presence of neuroectodermal markers, neurosphere-derived cells similarly expressed neural markers. We show that human MSCs expressing EGFP represent an attractive and practical source of stem cells for the study of repair and regeneration in neurological models. PMID:18061686

Gordon, David; Glover, Colin P; Merrison, Andria M; Uney, James B; Scolding, Neil J

2008-01-01

215

Decreased glucokinase protein expression in the aged gerbil hippocampus.  

PubMed

Glucokinase (GK) and its regulatory protein (GKRP) play roles in glucose utilization as well as glucose-sensing process in the brain. In the present study, we compared GK and GKRP protein expressions in the hippocampus of adult (postnatal month 6) and aged (postnatal month 24) gerbils using immunohistochemistry and western blot analysis. Both GK and GKRP immunoreactivities were observed primarily in the pyramidal cells of the hippocampus proper and in the granule cells of the dentate gyrus of the adult and aged hippocampus. GK, not GKRP, immunoreactivity was apparently decreased in the pyramidal and granule cells of the aged group compared with that in the adult group. In addition, western blot analysis also showed that the GK, not GKRP, protein level was significantly decreased in the aged hippocampus. These results indicate that the decrease of GK may be closely related to the reduction of glucose utilization and uptake, although the ability for regulation of GK is maintained in the aged hippocampus. PMID:23515967

Lee, Choong Hyun; Park, Joon Ha; Won, Moo-Ho

2013-05-01

216

Hordein Gene Expression in a Low Protein Barley Cultivar 1  

PubMed Central

The low protein barley (Hordeum vulgare L.) cultivar `Karl' is deficient in hordeins, the major storage protein fraction, but has normal levels of other endosperm protein fractions. We compared hordein gene expression in Karl with that in `Traill,' a related cultivar that contains normal amounts of hordein. In plants grown in controlled environmental conditions, hordein accumulation in Karl was 55% of that in Traill. The amount of hordein synthesized at all developmental stages tested was lower in Karl, as shown by pulse labeling of excised spikes with [3H]leucine. To determine whether levels of hordein mRNAs were related to amounts of hordein synthesis, total RNA preparations from endosperms at three or four developmental stages were hybridized to cDNA probes corresponding to B and C hordein genes. Both B and C hordein mRNA levels were significantly lower in Karl than in Traill at intermediate developmental stages. It was concluded that the low-hordein character of Karl was regulated by the size of the hordein mRNA pool. Transcriptional and/or post-transcriptional controls may be involved in the regulation of hordein mRNA levels. Images Fig. 1 Fig. 2 PMID:16666325

Dailey, Jessica E.; Peterson, David M.; Osborn, Thomas C.

1988-01-01

217

Statins inhibit monocyte chemotactic protein 1 expression in endometriosis.  

PubMed

Statins are potent inhibitors of the endogenous mevalonate pathway. Besides inhibiting cholesterol biosynthesis, statins may also demonstrate anti-inflammatory properties. Inflammation is implicated in the attachment and invasion of endometrial cells to the peritoneal surface and growth of ectopic endometrium by inducing proliferation and angiogenesis. In this study, the effect of statins on monocyte chemotactic protein 1 (MCP-1) expression in endometriotic implants in nude mouse model and in cultured endometriotic cells was evaluated. In mouse model, simvastatin decreased MCP-1 expression in a dose-dependent manner in endometriotic implants (P < .05). Similarly, both simvastatin and mevastatin revealed a dose-dependent inhibition of MCP-1 production in cultured endometriotic cells (P < .01). This inhibitory effect of the statins on MCP-1 production was reversed by the downstream substrates of the mevalonate pathway. Moreover, statins decreased MCP-1 messenger RNA expression in cultured endometriotic cells (P < .05). In conclusion, statins exert anti-inflammatory effect in endometriotic cells and could provide a potential treatment of endometriosis in the future. PMID:22267540

Cakmak, Hakan; Basar, Murat; Seval-Celik, Yasemin; Osteen, Kevin G; Duleba, Antoni J; Taylor, Hugh S; Lockwood, Charles J; Arici, Aydin

2012-06-01

218

Expression of wild-type and mutant p53 proteins by recombinant vaccinia viruses.  

PubMed Central

To facilitate the purification of wild type p53 protein, we established a recombinant p53 vaccinia viral expression system. Using this efficient eukaryotic expression vector, we found that the expressed p53 proteins retained their specific structural characteristics. A comparison between wild type and mutant p53 proteins showed the conservation of the typical subcellular localization and the expression of specific antigenic determinants. Furthermore, wild type p53 exhibited a typical binding with large T antigen, whereas no binding was detected with mutant p53. Both wild type and mutant p53 proteins were highly stable and constituted 5-7% of total protein expressed in the infected cells. These expression recombinant viruses offer a simple, valuable system for the purification of wild type and mutant p53 proteins that are expressed abundantly in eukaryotic cells. Images PMID:1630914

Ronen, D; Teitz, Y; Goldfinger, N; Rotter, V

1992-01-01

219

Multi-Host Expression System for Recombinant Production of Challenging Proteins  

PubMed Central

Recombinant production of complex eukaryotic proteins for structural analyses typically requires a profound screening process to identify suitable constructs for the expression of ample amounts of properly folded protein. Furthermore, the evaluation of an optimal expression host has a major impact on protein yield and quality as well as on actual cost of the production process. Here we present a novel fast expression system for multiple hosts based on a single donor vector termed pFlp-Bac-to-Mam. The range of applications of pFlp-Bac-to-Mam comprises highly efficient transient transfection of HEK293-6E in serum-free suspension culture and subsequent large-scale production of challenging proteins expressing in mg per Liter level using either the baculoviral expression vector system or stable CHO production cell lines generated by Flp-mediated cassette exchange. The success of the multi-host expression vector to identify the optimal expression strategy for efficient production of high quality protein is demonstrated in a comparative expression study of three model proteins representing different protein classes: intracellular expression using a fluorescent protein, secretion of a single-chain-Fv-hIgG1Fc fusion construct and production of a large amount of highly homogeneous protein sample of the extracellular domain of a Toll-like receptor. The evaluation of the production efficiency shows that the pFlp-Bac-to-Mam system allows a fast and individual optimization of the expression strategy for each protein class. PMID:23874717

Meyer, Steffen; Lorenz, Carmen; Baser, Bahar; Wördehoff, Mona; Jäger, Volker; van den Heuvel, Joop

2013-01-01

220

Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma  

PubMed Central

Background Complex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes have been limited. Deciphering the molecular mechanism that differentiate between normal and disease state may lead to identification of biomarkers for carcinoma. Results Many proteins displayed differential expression when nuclear membrane proteome of hepatocellular carcinoma (HCC), fibrotic liver, and HepG2 cell line were assessed using 2-DE and ESI-Q-TOF MS/MS. From the down regulated set in HCC, we have identified for the first time a 15 KDa cytochrome b5A (CYB5A), ATP synthase subunit delta (ATPD) and Hemoglobin subunit beta (HBB) with 11, 5 and 22 peptide matches respectively. Furthermore, nitrosylation studies with S-nitrosocysteine followed by immunoblotting with anti SNO-cysteine demonstrated a novel and biologically relevant post translational modification of thiols of CYB5A in HCC specimens only. Immunofluorescence images demonstrated increased protein S-nitrosylation signals in the tumor cells and fibrotic region of HCC tissues. The two other nuclear membrane proteins which were only found to be nitrosylated in case of HCC were up regulated ATP synthase subunit beta (ATPB) and down regulated HBB. The decrease in expression of CYB5A in HCC suggests their possible role in disease progression. Further insight of the functional association of the identified proteins was obtained through KEGG/ REACTOME pathway analysis databases. String 8.3 interaction network shows strong interactions with proteins at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor of liver pathology. Conclusion These findings may have broader implications for understanding the mechanism of development of carcinoma. However, large scale studies will be required for further verification of their critical role in development and progression of HCC. PMID:23724895

2013-01-01

221

A Statistical Model to Identify Differentially Expressed Proteins in 2D PAGE Gels  

PubMed Central

Two dimensional polyacrylamide gel electrophoresis (2D PAGE) is used to identify differentially expressed proteins and may be applied to biomarker discovery. A limitation of this approach is the inability to detect a protein when its concentration falls below the limit of detection. Consequently, differential expression of proteins may be missed when the level of a protein in the cases or controls is below the limit of detection for 2D PAGE. Standard statistical techniques have difficulty dealing with undetected proteins. To address this issue, we propose a mixture model that takes into account both detected and non-detected proteins. Non-detected proteins are classified either as (a) proteins that are not expressed in at least one replicate, or (b) proteins that are expressed but are below the limit of detection. We obtain maximum likelihood estimates of the parameters of the mixture model, including the group-specific probability of expression and mean expression intensities. Differentially expressed proteins can be detected by using a Likelihood Ratio Test (LRT). Our simulation results, using data generated from biological experiments, show that the likelihood model has higher statistical power than standard statistical approaches to detect differentially expressed proteins. An R package, Slider (Statistical Likelihood model for Identifying Differential Expression in R), is freely available at http://www.cebl.auckland.ac.nz/slider.php. PMID:19763172

Wu, Steven H.; Black, Michael A.; North, Robyn A.; Atkinson, Kelly R.; Rodrigo, Allen G.

2009-01-01

222

Expression of an active tobacco mitogen-activated protein kinase kinase kinase enhances freezing  

E-print Network

Expression of an active tobacco mitogen-activated protein kinase kinase kinase enhances freezing mitogen- activated protein kinase kinase kinase provided benefits in transgenic tobacco at freezing the effect of expressing a heter- ologous tobacco mitogen-activated protein kinase kinase kinase (Nicotiana

Sheen, Jen

223

Cell-free protein expression in a microchannel array with passive pumping Ruba Khnouf,a  

E-print Network

Cell-free protein expression in a microchannel array with passive pumping Ruba Khnouf,a David J expression in a microfluidic device using passive pumping. The polystyrene device contains 192 microchannels of using the microchannel array for protein expression with the following advantages: (1) simultaneous

Beebe, David J.

224

A developmentally regulated DNA-binding protein from mouse brain stimulates myelin basic protein gene expression.  

PubMed Central

Transcription of the myelin basic protein (MBP) gene is regulated in a cell-type-specific and developmental stage-specific manner during myelin formation in the murine central nervous system. The 5'-flanking region of the MBP gene contains several regulatory elements that differentially contribute to the cell-type-specific transcription of MBP in cells derived from the central nervous system. The proximal element, termed MB1, which is located between nucleotides -14 and -50 with respect to the RNA start site, has previously been shown to have characteristics of a cell-type-specific enhancer element. In this study, we used band shift and UV cross-linking assays to identify DNA-binding proteins in mouse brain nuclear extract which interact with the MB1 element. Fractionation of these extracts has allowed the identification of a 38- to 41-kDa nuclear protein, derived from mouse brain tissue at the peak of myelination, which specifically binds the MB1 DNA sequence. Fractions enriched in the MB1-binding protein have been shown to stimulate transcription of the MBP promoter in extract derived from HeLa cells. MB1 binding protein activity is expressed in a tissue-specific and development stage-specific pattern which coincides with the pattern of MBP transcription, suggesting that this protein may be a biologically relevant transcription factor for the MBP gene in vivo. Images PMID:7682655

Haas, S; Gordon, J; Khalili, K

1993-01-01

225

Identification of potential biomarkers of P-glycoprotein substrate neurotoxicity in transgenic mice expressing the mutated canine ABCB1 gene.  

PubMed

Objective-To identify biomarkers of P-glycoprotein (P-gp) substrate neurotoxicity in transgenic mice expressing the mutant canine ABCB1 gene (ABCB1-1?). Animals-8 ABCB1 knock-in and knock-out transgenic mice expressing the ABCB1-1? gene and 8 control mice expressing the wild-type canine ABCB1 gene (ABCB1-WT). Procedures-Groups including 2 ABCB1-1? mutant mice and 2 ABCB1-WT mice were administered the P-gp substrates ivermectin (10 mg/kg, SC), doramectin (10 mg/kg, SC), moxidectin (10 mg/kg, PO), or digoxin (1.53 mg/kg, SC). A toxicogenomic approach based on DNA microarrays was used to examine whole-genome expression changes in mice administered P-gp substrates. Results-Compared with control ABCB1-WT mice, ABCB1-1? mutant mice developed neurotoxic signs including ataxia, lethargy, and tremors similar to those reported for dogs with the ABCB1-1? mutation. Microarray analysis revealed that gene expression was altered in ABCB1-1? mutant mice, compared with findings for ABCB1-WT mice, following administration of the same P-gp substrates. Gene pathway analysis revealed that genes with a ? 2-fold gene expression change were associated with behavior and nervous system development and function. Moreover, 34 genes were altered in the ABCB1-1? mutant mice in all 4 drug treatment groups. These genes were also associated with behavior, which was identified as the top-ranked gene network. Conclusions and Clinical Relevance-These study data have facilitated understanding of the molecular mechanisms of neurotoxicosis in ABCB1-1? mutant mice following exposure to various P-gp substrates. Some genes appear to be potential biomarkers of P-gp substrate neurotoxicity that might be used to predict the safety of those drugs in dogs with the ABCB1-1? mutation. PMID:25419811

Zhu, Min; Ming, Yi; Swaim, Heidi; Swain, Marla D; Myers, Michael J; Deaver, Christine; Wu, Xiaolin; Jones, Yolanda L; Yancy, Haile F

2014-12-01

226

Protein 53 expression in a mixed Labrador subcutaneous lymphoma  

PubMed Central

An 11 year – old mixed female Labrador was presented with two masses in trunk and neck. The tumoral masses were excised and sent for histopathological and immunohistochemical analyses. Histopathological examination of masses revealed diffuse infiltration of small sized lymphoid cells in subcutaneous tissue which were intense around the blood vessels. More than 10% lymphoid cells were CD3 positive in the immunohistochemical staining and most of them were accumulated around vessels. Protein 53 (p53) expression was detected by brown nuclei in immunohistochemical staining. Subcutaneous lymphoma was diagnosed according to histopathological results. After 6 months the case was referred with multicentric lymphoma and based on the owner request euthanasia was performed. These findings emphasize on poor prognosis for tumors with p53 mutation.

Rezaie, Annahita; Tavassoli, Abbas

2012-01-01

227

Rapid screening of membrane protein expression in transiently transfected insect cells.  

PubMed

Membrane proteins play critical roles in many biological processes and are the focus of intense biomedical research. One bottleneck for studying membrane proteins is the difficulty in expressing correctly folded and stable proteins, which often requires extensive protein engineering and multiple rounds of optimization, a time and resource intensive process. Here, we describe a method for rapidly screening membrane protein expression in insect cells. The method uses a green fluorescent protein (GFP) covalently fused to target membrane proteins and the resulting fusion proteins are then transiently expressed in insect cells. This approach enables us to dramatically reduce the time and resources required for expression screening by eliminating the need to create recombinant baculovirus. We show that transiently expressed membrane proteins can be directly monitored for their subcellular localizations by fluorescence microscopy. Moreover, their expression levels, approximate molecular mass, and stability can be evaluated with nanogram levels of unpurified proteins by ultrasensitive fluorescence-detection size exclusion chromatography (FSEC). We present our proof of principle studies using a homotrimeric ion channel (ASIC3) and a heterodimeric transporter (SLC7A5/SLC3A2) as examples, and demonstrate the utility of transient expression coupled with FSEC in optimizing membrane protein expression. PMID:23268112

Chen, Hao; Shaffer, Paul L; Huang, Xin; Rose, Paul E

2013-03-01

228

Expression of an insect ( Dendroides canadensis ) antifreeze protein in Arabidopsis thaliana results in a decrease in plant freezing temperature  

Microsoft Academic Search

Transgenic Arabidopsis thaliana plants which express genes encoding insect, Dendroides canadensis, antifreeze proteins (AFP) were produced by Agrobacterium-mediated transformation. The antifreeze protein genes, both with and without the signal peptide sequence (for protein secretion), were expressed in transformed plants. Thermal hysteresis activity (indicating the presence of active AFPs) was present in protein extracts from plants expressing both proteins and was

Tao Huang; Jessie Nicodemus; Daniel G. Zarka; Michael F. Thomashow; Michael Wisniewski; John G. Duman

2002-01-01

229

Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity  

PubMed Central

Background Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified. Results In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910?±?0.007 mM; Vmax, 0.514?±?0.038 ?Mmin-1; and Kcat =?0.099?±?0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. Conclusion We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis. PMID:24975018

2014-01-01

230

Coat protein expression strategy of oat blue dwarf virus.  

PubMed

Oat blue dwarf virus (OBDV) is a member of the genus Marafivirus whose genome encodes a 227 kDa polyprotein (p227) ostensibly processed post-translationally into its functional components. Encoded near the 3' terminus and coterminal with the p227 ORF are ORFs specifying major and minor capsid proteins (CP). Since the CP expression strategy of marafiviruses has not been thoroughly investigated, we produced a series of point mutants in the OBDV CP encoding gene and examined expression in protoplasts. Results support a model in which the 21 kDa major CP is the product of direct translation of a sgRNA, while the 24 kDa minor CP is a cleavage product derived from both the polyprotein and a larger ~26 kDa precursor translated directly from the sgRNA. Cleavage occurs at an LXG[G/A] motif conserved in many viruses that use papain-like proteases for polyprotein processing and protection against degradation via the ubiquitin-proteasome system. PMID:24503092

Edwards, Michael C; Weiland, John J

2014-02-01

231

EXPRESSION OF AN INSECT (DENDROIDES CANADENSIS) ANTIFREEZE PROTEIN IN ARABIDOPSIS THALIANA RESULTS IN A DECREASE IN PLANT FREEZING TEMPERATURE  

Technology Transfer Automated Retrieval System (TEKTRAN)

Transgenic Arabidopsis thaliana plants which express genes encoding insect, Dendroides canadensis, antifreeze proteins (AFP) were produced by Agrobacterium-mediated transformation. The antifreeze protein genes, both with and without the signal peptide sequence (for protein secretion), were expresse...

232

Redox Regulation of Thylakoid Protein Kinases and Photosynthetic Gene Expression  

PubMed Central

Abstract Significance: Photosynthetic organisms are subjected to frequent changes in their environment that include fluctuations in light quality and quantity, temperature, CO2 concentration, and nutrient availability. They have evolved complex responses to these changes that allow them to protect themselves against photo-oxidative damage and to optimize their growth under these adverse conditions. In the case of light changes, these acclimatory processes can occur in either the short or the long term and are mainly mediated through the redox state of the plastoquinone pool and the ferredoxin/thioredoxin system. Recent Advances: Short-term responses involve a dynamic reorganization of photosynthetic complexes, and long-term responses (LTRs) modulate the chloroplast and nuclear gene expression in such a way that the levels of the photosystems and their antennae are rebalanced for an optimal photosynthetic performance. These changes are mediated through a complex signaling network with several protein kinases and phosphatases that are conserved in land plants and algae. The phosphorylation status of the light-harvesting proteins of photosystem II and its core proteins is mainly determined by two complementary kinase–phosphatase pairs corresponding to STN7/PPH1 and STN8/PBCP, respectively. Critical Issues: The activity of the Stt7 kinase is principally regulated by the redox state of the plastoquinone pool, which in turn depends on the light irradiance, ambient CO2 concentration, and cellular energy status. In addition, this kinase is also involved in the LTR. Future Directions: Other chloroplast kinases modulate the activity of the plastid transcriptional machinery, but the global signaling network that connects all of the identified kinases and phosphatases is still largely unknown. Antioxid. Redox Signal. 18, 2184–2201. PMID:23339452

2013-01-01

233

Neuroendocrine secretory protein 7B2: structure, expression and functions.  

PubMed Central

7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2-7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin ('ACTH') hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2 has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation. PMID:11439082

Mbikay, M; Seidah, N G; Chrétien, M

2001-01-01

234

Modeling Signal Transduction from Protein Phosphorylation to Gene Expression  

PubMed Central

BACKGROUND Signaling networks are of great importance for us to understand the cell’s regulatory mechanism. The rise of large-scale genomic and proteomic data, and prior biological knowledge has paved the way for the reconstruction and discovery of novel signaling pathways in a data-driven manner. In this study, we investigate computational methods that integrate proteomics and transcriptomic data to identify signaling pathways transmitting signals in response to specific stimuli. Such methods can be applied to cancer genomic data to infer perturbed signaling pathways. METHOD We proposed a novel Bayesian Network (BN) framework to integrate transcriptomic data with proteomic data reflecting protein phosphorylation states for the purpose of identifying the pathways transmitting the signal of diverse stimuli in rat and human cells. We represented the proteins and genes as nodes in a BN in which edges reflect the regulatory relationship between signaling proteins. We designed an efficient inference algorithm that incorporated the prior knowledge of pathways and searched for a network structure in a data-driven manner. RESULTS We applied our method to infer rat and human specific networks given gene expression and proteomic datasets. We were able to effectively identify sparse signaling networks that modeled the observed transcriptomic and proteomic data. Our methods were able to identify distinct signaling pathways for rat and human cells in a data-driven manner, based on the facts that rat and human cells exhibited distinct transcriptomic and proteomics responses to a common set of stimuli. Our model performed well in the SBV IMPROVER challenge in comparison to other models addressing the same task. The capability of inferring signaling pathways in a data-driven fashion may contribute to cancer research by identifying distinct aberrations in signaling pathways underlying heterogeneous cancers subtypes. PMID:25392684

Cai, Chunhui; Chen, Lujia; Jiang, Xia; Lu, Xinghua

2014-01-01

235

Expression of epithelial adhesion proteins and integrins in chronic inflammation.  

PubMed Central

Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of tooth-supporting structures (periodontal disease), increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, laminin-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and laminin-5 (also known as kalinin, epiligrin, or nicein) were poorly preserved in the basement membrane zone, but both were found in unusual streak-like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the beta 1 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for beta 1 integrins. No members of the alpha v integrin family were found in any epithelia studied. Expression of the alpha 6 beta 4 integrin was high in basal cells of healthy tissue, but weak in epithelium associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7541610

Haapasalmi, K.; Mäkelä, M.; Oksala, O.; Heino, J.; Yamada, K. M.; Uitto, V. J.; Larjava, H.

1995-01-01

236

GFP-Based Expression Screening of Membrane Proteins in Insect Cells Using the Baculovirus System.  

PubMed

A key step in the production of recombinant membrane proteins for structural studies is the optimization of protein yield and quality. One commonly used approach is to fuse the protein to green fluorescent protein (GFP), enabling expression to be tracked without the need to purify the protein. Combining fusion to green fluorescent protein with the baculovirus expression system provides a useful platform for both screening and production of eukaryotic membrane proteins. In this chapter we describe our protocol for the expression screening of membrane proteins in insect cells using fusion to GFP as a reporter. We use both SDS-PAGE with in-gel fluorescence imaging and fluorescence-detection size-exclusion chromatography (FSEC) to screen for expression. PMID:25502201

Hu, Nien-Jen; Rada, Heather; Rahman, Nahid; Nettleship, Joanne E; Bird, Louise; Iwata, So; Drew, David; Cameron, Alexander D; Owens, Raymond J

2015-01-01

237

RNA protein interactions governing expression of the most abundant protein in human body, type I collagen  

PubMed Central

Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half life of collagen mRNAs and their increased translatability. Type I collagen is composed of two ?1 and one ?2 polypeptides that fold into a triple helix. This stochiometry is strictly regulated to prevent detrimental synthesis of ?1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen ?1(I) and ?2(I) polypeptides. Collagen ?1(I) mRNA has in the 3’ UTR a C-rich sequence that binds protein ?CP, this binding stabilizes the mRNA in collagen producing cells. In the 5’UTR both collagen mRNAs have a conserved stem-loop structure (5’SL). The 5’SL is critical for high collagen expression, knock in mice with disruption of the 5’SL are resistant to liver fibrosis. the 5’SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of ?1(I) and ?2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only ?1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. PMID:23907854

Stefanovic, Branko

2013-01-01

238

A novel yeast expression system for the overproduction of quality-controlled membrane proteins  

Microsoft Academic Search

Saturation of the cell’s protein folding capacity and accumulation of inactive incompletely folded protein often accompanying the overexpression of membrane proteins (MPs) presents an obstacle to their efficient purification in a functional form for structural studies. We present a novel strategy for optimization of functional MP expression in Saccharomyces cerevisiae. This approach exploits the unfolded protein response (UPR) pathway, a

Douglas A Griffith; Christina Delipala; Jane Leadsham; Simon M Jarvis; Dieter Oesterhelt

2003-01-01

239

p53 protein expression in human breast carcinoma: relationship to expression of epidermal growth factor receptor, c-erbB-2 protein overexpression, and oestrogen receptor.  

PubMed Central

The expression of p53 protein, oestrogen receptor protein, epidermal growth factor receptor (EGFR) and overexpression of the c-erbB-2 oncoprotein was examined in a series of 149 primary symptomatic breast carcinomas. Expression of p53 was present in 62 of 146 cases (42.5%) of the invasive carcinoma and one of three cases (33.3%) of ductal carcinoma in situ (DCIS) examined. Statistical associations of tumour oestrogen receptor positivity and lack of p53 protein expression, chi 2 = 19.78 (d.f. = 1), P less than 0.001, positive tumour p53 status and poor tumour grade; chi 2 = 14.1 (d.f. = 2), P less than 0.001, EGFR expression chi 2 = 7.07, (d.f. = 1), P less than 0.01 and tumour c-erbB-2 protein overexpression; chi 2 = 4.61 (d.f. = 1), P = 0.032 were identified. Expression of p53 is rare in invasive lobular carcinoma of classical type (8.3% of cases examined) in contrast to other common types of mammary carcinoma. Non-significant trends of p53 protein expression and increased regional tumour recurrence; chi 2 = 3.20 (d.f. = 1), P = 0.074 and also poorer patient survival; chi 2 = 3.76 (d.f. = 1), P = 0.053 were identified. p53 protein expression is a common event in human breast cancer and is present in both DCIS and invasive mammary carcinoma. Abnormal expression of p53 protein is a feature of both in situ and invasive breast carcinoma, implying that the abnormal p53 protein expression may be implicated in the early stages of mammary carcinoma progression. Images Figure 1 PMID:1355662

Poller, D. N.; Hutchings, C. E.; Galea, M.; Bell, J. A.; Nicholson, R. A.; Elston, C. W.; Blamey, R. W.; Ellis, I. O.

1992-01-01

240

PROTEIN INTERACTIONS AND DISEASE PHENOTYPES IN THE ABC TRANSPORTER SUPERFAMILY  

E-print Network

the multidrug transporter P-gp, associated with drug resistance phenotypes in cancer therapy [1], and the cystic fibrosis transmembrane conductance regulator (CFTR) that transports chloride ions [2]. In Escherischia coli, labeled ABCA through ABCG. There are half transporters, such as the breast cancer resistance protein BCRP

Sali, Andrej

241

Connexin expression in human acute myeloid leukemia cells: Identification of patient subsets based on protein and global gene expression profiles.  

PubMed

Bone marrow stromal cells support both normal and malignant hematopoiesis. ?his support is mediated through the local cytokine network and by direct cell?cell interactions mediated via adhesion molecules and the formation of gap junctions by connexins. Previous studies on connexins in human acute myeloid leukemia (AML) have mainly focused on the investigation of leukemia cell lines. In the present study, we therefore investigated the expression of various connexins at the protein (i.e., cell surface expression) and mRNA level in primary human AML cells. The cell surface expression of the connexins, Cx26, Cx32, Cx37, Cx43 and Cx45, varied considerably between patients, and detectable levels were observed only for subsets of patients. On the whole, Cx43 and Cx45 showed the highest cell surface expression. Connexin expression was dependent on AML cell differentiation, but showed no association with cytogenetic abnormalities or mutations of the fms-related tyrosine kinase 3 (FLT3) or nucleophosmin (NPM)?1 genes. By contrast, only Cx45 showed a significant variation between patients at the mRNA level. A high Cx45 expression was associated with the altered regulation of the mitogen?activated protein kinase (MAPK) pathway and the release of pro-inflammatory cytokines [interleukin (IL)?17, tumor necrosis factor (TNF), interferon??], whereas a low Cx45 expression was associated with the altered regulation of protein functions (i.e., ligase activity, protein folding and catabolism). There was no significant correlation observed between the connexin mRNA and protein levels. Thus, differences in connexin expression can be used to subclassify AML patients. Differences in connexin cell surface expression profiles are not reflected at the mRNA level and have to be directly examined, whereas variations in Cx45 mRNA expression are associated with differences in cell signaling and the regulation of protein functions. PMID:25529637

Reikvam, Håkon; Ryningen, Anita; Sæterdal, Lars Rune; Nepstad, Ina; Foss, Brynjar; Bruserud, Øystein

2015-03-01

242

Connexin expression in human acute myeloid leukemia cells: Identification of patient subsets based on protein and global gene expression profiles  

PubMed Central

Bone marrow stromal cells support both normal and malignant hematopoiesis. ?his support is mediated through the local cytokine network and by direct cell-cell interactions mediated via adhesion molecules and the formation of gap junctions by connexins. Previous studies on connexins in human acute myeloid leukemia (AML) have mainly focused on the investigation of leukemia cell lines. In the present study, we therefore investigated the expression of various connexins at the protein (i.e., cell surface expression) and mRNA level in primary human AML cells. The cell surface expression of the connexins, Cx26, Cx32, Cx37, Cx43 and Cx45, varied considerably between patients, and detectable levels were observed only for subsets of patients. On the whole, Cx43 and Cx45 showed the highest cell surface expression. Connexin expression was dependent on AML cell differentiation, but showed no association with cytogenetic abnormalities or mutations of the fms-related tyrosine kinase 3 (FLT3) or nucleophosmin (NPM)?1 genes. By contrast, only Cx45 showed a significant variation between patients at the mRNA level. A high Cx45 expression was associated with the altered regulation of the mitogen-activated protein kinase (MAPK) pathway and the release of pro-inflammatory cytokines [interleukin (IL)-17, tumor necrosis factor (TNF), interferon-?], whereas a low Cx45 expression was associated with the altered regulation of protein functions (i.e., ligase activity, protein folding and catabolism). There was no significant correlation observed between the connexin mRNA and protein levels. Thus, differences in connexin expression can be used to subclassify AML patients. Differences in connexin cell surface expression profiles are not reflected at the mRNA level and have to be directly examined, whereas variations in Cx45 mRNA expression are associated with differences in cell signaling and the regulation of protein functions. PMID:25529637

REIKVAM, HÅKON; RYNINGEN, ANITA; SÆTERDAL, LARS RUNE; NEPSTAD, INA; FOSS, BRYNJAR; BRUSERUD, ØYSTEIN

2015-01-01

243

High-throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label.  

PubMed

Quantifying the concentration and purity of a target protein is essential for high-throughput protein expression test and rapid screening of highly soluble proteins. However, conventional methods such as PAGE and dot blot assay generally involve multiple time-consuming tasks requiring hours or do not allow instant quantification. Here, we demonstrate a new method based on the Photoactive yellow protein turn Off/On Label (POOL) system that can instantly quantify the concentration and purity of a target protein. The main idea of POOL is to use Photoactive Yellow Protein (PYP), or its miniaturized version, as a fusion partner of the target protein. The characteristic blue light absorption and the consequent yellow color of PYP is absent when initially expressed without its chromophore, but can be turned on by binding its chromophore, p-coumaric acid. The appearance of yellow color upon adding a precursor of chromophore to the co-expressed PYP can be used to check the expression amount of the target protein via visual inspection within a few seconds as well as to quantify its concentration and purity with the aid of a spectrometer within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show excellent agreement with those by the BCA Kit, which usually takes ?1 h. We demonstrate the applicability of POOL in E. coli, insect, and mammalian cells, and for high-throughput protein expression screening. PMID:23740751

Kim, Youngmin; Ganesan, Prabhakar; Ihee, Hyotcherl

2013-08-01

244

Analysis of the protein-protein interaction networks of differentially expressed genes in pulmonary embolism.  

PubMed

The aim of the present study was to explore the function and interaction of differentially expressed genes (DEGs) in pulmonary embolism (PE). The gene expression profile GSE13535, was downloaded from the Gene Expression Omnibus database. The DEGs 2 and 18 h post?PE initiation were identified using the affy package in R software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the DEGs were analyzed using Database for Annotation Visualization and Integrated Discovery (DAVID) online analytical tools. In addition, protein?protein interaction (PPI) networks of the DEGs were constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins. The PPI network at 18 h was modularized using Clusterone, and a functional enrichment analysis of the DEGs in the top three modules was performed with DAVID. Overall, 80 and 346 DEGs were identified 2 and 18 h after PE initiation, respectively. The KEGG pathways, including chemokine signaling and toll?like receptor signaling, were shown to be significantly enriched. The five highest degree nodes in the PPI networks at 2 or 18 h were screened. The module analysis of the PPI network at 18 h revealed 11 hub nodes. A Gene Ontology terms analysis demonstrated that the DEGs in the top three modules were associated with the inflammatory, defense and immune responses. The results of the present study suggest that the DEGs identified, including chemokine?related genes TFPI2 and TNF, may be potential target genes for the treatment of PE. The chemokine signaling pathway, inflammatory response and immune response were explored, and it may be suggested that these pathways have important roles in PE. PMID:25434468

Wang, Hao; Wang, Chen; Zhang, Lei; Lu, Yinghua; Duan, Qianglin; Gong, Zhu; Liang, Aibin; Song, Haoming; Wang, Lemin

2015-04-01

245

Differential expression of hemolymph proteins between susceptible and insecticide-resistant Blattella germanica (Blattodea: Blattellidae).  

PubMed

A proteomic approach combining two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry was used to compare hemolymph expression profiles of a beta-cypermethrin-resistant Blattella germanica L. strain and a beta-cypermethrin-susceptible strain. Twenty-eight hemolymph proteins were differentially expressed in the resistant cockroach strain; 19 proteins were upregulated and 9 proteins were downregulated compared with the susceptible strain. Protein identification indicated that expression of putative cuticular protein, nitric oxide synthase, triosephosphate isomerase, alpha-amylase, ABC transporter, and Per a 3 allergen was elevated, and expression of arginine kinase and glycosidase was reduced. The differential expression of these proteins reflects the overall change in cellular structure and metabolism related to the resistance of pyrethroid insecticides. PMID:25182623

Zhang, F; Wang, X J; Huang, Y H; Zhao, Z G; Zhang, S S; Gong, X S; Xie, L; Kang, D M; Jing, X

2014-08-01

246

Altered brain protein expression profiles are associated with molecular neurological dysfunction in the PKU mouse model  

PubMed Central

Phenylketonuria (PKU), if not detected and treated in newborns, causes severe neurological dysfunction and cognitive and behavioral deficiencies. Despite the biochemical characterization of PKU, the molecular mechanisms underlying PKU-associated brain dysfunction remain poorly understood. The aim of this study was to gain insights into the pathogenesis of this neurological damage by analyzing protein expression profiles in brain tissue of Black and Tan BRachyury-PahEnu2 mice (a mouse model of PKU). We compared the cerebral protein expression of homozygous PKU mice with that of their heterozygous counterparts using two-dimensional difference gel electrophoresis analysis, and identified 21 differentially expressed proteins, four of which were over-expressed and 17 under-expressed. An in silico bioinformatic approach indicated that protein under-expression was related to neuronal differentiation and dendritic growth, and to such neurological disorders as progressive motor neuropathy and movement disorders. Moreover, functional annotation analyses showed that some identified proteins were involved in oxidative metabolism. To further investigate the proteins involved in the neurological damage, we validated two of the proteins that were most strikingly under-expressed, namely, Syn2 and Dpysl2, which are involved in synaptic function and neurotransmission. We found that Glu2/3 and NR1 receptor subunits were over-expressed in PKU mouse brain. Our results indicate that differential expression of these proteins may be associated with the processes underlying PKU brain dysfunction, namely, decreased synaptic plasticity and impaired neurotransmission. PMID:24548049

Imperlini, Esther; Orrù, Stefania; Corbo, Claudia; Daniele, Aurora; Salvatore, Francesco

2014-01-01

247

Activation and repression of prion protein expression by key regions of intron 1.  

PubMed

Expression of the prion protein is necessary for infection with prion diseases. Altered expression levels may play an important role in susceptibility to infection. Therefore, understanding the mechanisms that regulate prion protein expression is of great importance. It was previously shown that expression of the prion protein is to some degree regulated by an alternative promoter within intron 1. Studies using GFP and luciferase reporter systems were undertaken to determine key sites for the repression and activation of expression of the prion protein driven by intron 1. We identified a region within intron 1 sufficient to drive prion protein expression. Our findings highlight two potential repressor regions. Both regions have binding sites for the known repressor Hes-1. Hes-1 overexpression caused a dramatic decrease in PrP protein expression. Additionally, we have identified Atox-1 as a transcription factor that upregulates prion protein expression. These findings clearly indicate that intron 1 plays a key role in regulation of prion protein expression levels. PMID:19756378

Wright, Josephine A; McHugh, Patrick C; Stockbridge, Mark; Lane, Samantha; Kralovicova, Silvia; Brown, David R

2009-12-01

248

[The use of phase diagrams in the crystallization of oligonucleotide duplexes. I. A model of crystallization of the (pGpT)n.(pApC)n + spermine system].  

PubMed

A set of experimental phase diagrams revealing the region of existence of microcrystals in mixture "(pGpT)n.(pApC)n+spermine", n = 2,3,4, was obtained. All diagrams are wedge-like with the slope of the upper branch and the level of the lower one depending on the oligonucleotidd length. The presence of MPD, MgCl2 and NaCl changes the form of the diagrams in a different manner. A model explaining the peculiar features of the diagrams for mixture "oligonucleotide duplex+spermine" is proposed. The analysis of the diagrams was carried out on the basis of this model and the values of the binding constants for binding of spermine and Mg2+ to duplexes were estimated. Some conclusions about the types of complexes, which may form microcrystals in different regions of diagrams were made. PMID:3600626

Malinina, L V; Tereshko, V A; Makhaldiani, V V; Zarytova, V F; Ivanova, E M

1987-01-01

249

Primate Transcript and Protein Expression Levels Evolve under Compensatory Selection Pressures  

PubMed Central

Changes in gene regulation have likely played an important role in the evolution of primates. Differences in mRNA expression levels across primates have often been documented, however, it is not yet known to what extent measurements of divergence in mRNA levels reflect divergence in protein expression levels, which are probably more important in determining phenotypic differences. We used high-resolution, quantitative mass spectrometry to collect protein expression measurements from human, chimpanzee, and rhesus macaque lymphoblastoid cell lines (LCLs) and compared them to transcript expression data from the same samples. We found dozens of genes with significant expression differences between species at the mRNA level yet little or no difference in protein expression. Overall, our data suggest that protein expression levels evolve under stronger evolutionary constraint than mRNA levels. PMID:24136357

Khan, Zia; Ford, Michael J.; Cusanovich, Darren A.; Mitrano, Amy; Pritchard, Jonathan K.; Gilad, Yoav

2014-01-01

250

Comparative effects of doxercalciferol (1?-hydroxyvitamin D?) versus calcitriol (1?,25-dihydroxyvitamin D?) on the expression of transporters and enzymes in the rat in vivo.  

PubMed

Effects of 1.28 nmol/kg doxercalciferol [1?(OH)D?], a synthetic vitamin D? analog that undergoes metabolic activation to 1?,25-dihydroxyvitamin D?, the naturally occurring, biologically active form of vitamin D?, on rat transporters and enzymes were compared with those of 1?,25-dihydroxyvitamin D? [1,25(OH)?D?, active form of vitamin D?; 4.8 and 6.4 nmol/kg] given on alternate days intraperitoneally for 8 days. Changes were mostly confined to the intestine and kidney where the vitamin D receptor (VDR) was highly expressed: increased intestinal Cyp24 and Cyp3a1 messenger RNA (mRNA) and a modest elevation of apical sodium-dependent bile salt transporter (Asbt) and P-glycoprotein (P-gp) protein; increased renal VDR, Cyp24, Cyp3a9, Mdr1a, and Asbt mRNA, as well as Asbt and P-gp protein expression; and decreased renal PepT1 and Oat1 mRNA expression. In comparison, 1?(OH)D? treatment exerted a greater effect than 1,25(OH)?D? on Cyp3a and Cyp24 mRNA. However, the farnesoid X receptor -related repressive effects on liver Cyp7a1 were absent because intestinal Asbt, FGF15 and portal bile acid concentrations were unchanged. Rats on the alternate day regimen showed milder changes and lessened signs of hypercalcemia and weight loss compared with rats receiving daily injections (similar or greater amounts of 0.64-2.56 nmol/kg daily ×4) described in previous reports, showing that the protracted pretreatment regimen was associated with milder inductive and lesser toxic effects in vivo. PMID:20967888

Chow, Edwin C Y; Sondervan, Myrte; Jin, Cheng; Groothuis, Geny M M; Pang, K Sandy

2011-04-01

251

Recombinant plasmid expressing a truncated dengue-2 virus E protein without co-expression of prM protein induces partial protection in mice  

Microsoft Academic Search

A nucleic acid vaccine candidate against dengue-2 virus was constructed to express a truncated dengue-2 E glycoprotein without concomitant expression of prM. The truncated E protein was properly expressed even in the absence of prM. Mice inoculated intramuscularly with the recombinant plasmid containing 94% of the E gene did not respond with anti-dengue antibodies, cellular proliferation, or synthesis of cytokines

Raquel Ocazionez Jimenez; Benedito Antônio Lopes da Fonseca

2000-01-01

252

Analysis of differentially expressed proteins in colorectal cancer using hydroxyapatite column and SDS-PAGE.  

PubMed

Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method. PMID:21863284

Lim, Shi-Rou; Gooi, Boon-Hui; Singh, Manjit; Gam, Lay-Harn

2011-11-01

253

Teaching Molecular Biology to Undergraduate Biology Students: An Illustration of Protein Expression and Purification  

ERIC Educational Resources Information Center

Practical classes on protein expression and purification were given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering." The heterologous expression of the green fluorescent protein (GFP)* of "Aequorea victoria" is an interesting system for didactic purposes because it can be viewed easily during…

Sommer, Cesar Adolfo; Silva, Flavio Henrique; Novo, Maria Teresa Marques

2004-01-01

254

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system  

Microsoft Academic Search

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that

Marino DiFranco; Patricia Ñeco; Joana Capote; Pratap Meera; Julio L. Vergara

2006-01-01

255

Loss of expression of glial fibrillary acidic protein in acute hyperammonemia  

Microsoft Academic Search

Glial fibrillary acid protein (GFAP) is a major component of the glial filament network and alterations in expression of this protein in cultured astrocytes have been reported in response to acute ammonia exposure in vitro. In order to determine the effects of acute hyperammonemia in vivo on GFAP expression, brain extracts from rats with acute liver failure due to hepatic

Mireille Bélanger; Paul Desjardins; Nicolas Chatauret; Roger F. Butterworth

2002-01-01

256

DNA Vaccination against Tuberculosis: Expression of a Ubiquitin-Conjugated Tuberculosis Protein Enhances Antimycobacterial Immunity  

Microsoft Academic Search

Genetic immunization is a promising new technology for developing vaccines against tuberculosis that are more effective. In the present study, we evaluated the effects of intracellular turnover of antigens expressed by DNA vaccines on the immune response induced by these vaccines in a mouse model of pulmonary tuberculosis. The mycobacterial culture filtrate protein MPT64 was expressed as a chimeric protein

GIOVANNI DELOGU; ANGELA HOWARD; FRANK M. COLLINS; SHELDON L. MORRIS

2000-01-01

257

cAMP, an Activator of Protein Kinase A, Suppresses the Expression of Sonic Hedgehog  

E-print Network

cAMP, an Activator of Protein Kinase A, Suppresses the Expression of Sonic Hedgehog Alexander Received December 8, 1995 In Drosophila, it has been shown that protein kinase A and hedgehog have antagonistic actions during the formation of imaginal disks. In vertebrate skin, sonic hedgehog is expressed

Chuong, Cheng-Ming

258

Heat shock protein expression enhances heat tolerance of reptile embryos.  

PubMed

The role of heat shock proteins (HSPs) in heat tolerance has been demonstrated in cultured cells and animal tissues, but rarely in whole organisms because of methodological difficulties associated with gene manipulation. By comparing HSP70 expression patterns among representative species of reptiles and birds, and by determining the effect of HSP70 overexpression on embryonic development and hatchling traits, we have identified the role of HSP70 in the heat tolerance of amniote embryos. Consistent with their thermal environment, and high incubation temperatures and heat tolerance, the embryos of birds have higher onset and maximum temperatures for induced HSP70 than do reptiles, and turtles have higher onset and maximum temperatures than do lizards. Interestingly, the trade-off between benefits and costs of HSP70 overexpression occurred between life-history stages: when turtle embryos developed at extreme high temperatures, HSP70 overexpression generated benefits by enhancing embryo heat tolerance and hatching success, but subsequently imposed costs by decreasing heat tolerance of surviving hatchlings. Taken together, the correlative and causal links between HSP70 and heat tolerance provide, to our knowledge, the first unequivocal evidence that HSP70 promotes thermal tolerance of embryos in oviparous amniotes. PMID:25080340

Gao, Jing; Zhang, Wen; Dang, Wei; Mou, Yi; Gao, Yuan; Sun, Bao-Jun; Du, Wei-Guo

2014-09-22

259

Expression of myelin basic protein isoforms in nonglial cells  

PubMed Central

The myelin basic proteins (MBPs) mediate the cytoplasmic apposition of the oligodendrocyte plasma membrane to form the major dense line of central nervous system myelin. Four major isoforms of murine MBP, obtained by alternative splicing of seven exons from a single primary transcript, display distinct developmental profiles. We expressed these major MBPs individually in HeLa cells and mapped their distributions by immunofluorescence and confocal microscopy. The 14- and 18.5-kD MBPs that are the predominant forms in compact myelin distributed primarily in the perinuclear regions of the cell in configurations highly suggestive of close association with membranes. We infer that these MBP isoforms possess strong, nonspecific membrane-binding properties that have been adapted by the oligodendrocyte to mediate compaction of the sheaths of plasma membrane that form myelin. In contrast, the 17- and 21.5-kD isoforms distributed diffusely in both the cytoplasm and the nucleoplasm and often accumulated within the nucleus. This distribution can be correlated with the presence of the peptide segment encoded by exon II, which is unique to these isoforms. The physiological significance of the nuclear targeting displayed by the 17- and 21.5-kD MBP isoforms in HeLa cells remains to be determined. PMID:1692328

1990-01-01

260

Assessing drug distribution in tissues expressing P-glycoprotein through physiologically based pharmacokinetic modeling: model structure and parameters determination  

PubMed Central

Background The expression and activity of P-glycoproteins due to genetic or environmental factors may have a significant impact on drug disposition, drug effectiveness or drug toxicity. Hence, characterization of drug disposition over a wide range of conditions of these membrane transporters activities is required to better characterize drug pharmacokinetics and pharmacodynamics. This work aims to improve our understanding of the impact of P-gp activity modulation on tissue distribution of P-gp substrate. Methods A PBPK model was developed in order to examine activity and expression of P-gp transporters in mouse brain and heart. Drug distribution in these tissues was first represented by a well-stirred (WS) model and then refined by a mechanistic transport-based (MTB) model that includes P-gp mediated transport of the drug. To estimate transport-related parameters, we developed an original three-step procedure that allowed extrapolation of in vitro measurements of drug permeability to the in vivo situation. The model simulations were compared to a limited set of data in order to assess the model ability to reproduce the important information of drug distributions in the considered tissues. Results This PBPK model brings insights into the mechanism of drug distribution in non eliminating tissues expressing P-gp. The MTB model accounts for the main transport mechanisms involved in drug distribution in heart and brain. It points out to the protective role of P-gp at the blood-brain barrier and represents thus a noticeable improvement over the WS model. Conclusion Being built prior to in vivo data, this approach brings an interesting alternative to fitting procedures, and could be adapted to different drugs and transporters. The physiological based model is novel and unique and brought effective information on drug transporters. PMID:19146691

Fenneteau, Frédérique; Turgeon, Jacques; Couture, Lucie; Michaud, Véronique; Li, Jun; Nekka, Fahima

2009-01-01

261

Expression of Drug Transporters and Drug Metabolizing Enzymes in the Bladder Urothelium in Man and Affinity of the Bladder Spasmolytic Trospium Chloride to Transporters Likely Involved in Its Pharmacokinetics.  

PubMed

The cationic, water-soluble quaternary trospium chloride (TC) is incompletely absorbed from the gut and undergoes wide distribution but does not pass the blood-brain barrier. It is secreted by the kidneys, liver, and intestine. To evaluate potential transport mechanisms for TC, we measured affinity of the drug to the human uptake and efflux transporters known to be of pharmacokinetic relevance. Affinity of TC to the uptake transporters OATP1A2, -1B1, -1B3, -2B1, OCT1, -2, -3, OCTN2, NTCP, and ASBT and the efflux carriers P-gp, MRP2 and MRP3 transfected in HEK293 and MDCK2 cells was measured. To identify relevant pharmacokinetic mechanisms in the bladder urothelium, mRNA expression of multidrug transporters, drug metabolizing enzymes, and nuclear receptors, and the uptake of TC into primary human bladder urothelium (HBU) cells were measured. TC was shown to be a substrate of OATP1A2 (Km = 6.9 ± 1.3 ?mol/L; Vmax = 41.6 ± 1.8 pmol/mg·min), OCT1 (Km = 106 ± 16 ?mol/L; Vmax = 269 ± 18 pmol/mg·min), and P-gp (Km = 34.9 ± 7.5 ?mol/L; Vmax = 105 ± 9.1 pmol/mg·min, lipovesicle assay). The genetic OATP1A2 variants *2 and *3 were loss-of-function transporters for TC. The mRNA expression analysis identified the following transporter proteins in the human urothelium: ABCB1 (P-gp), ABCC1-5 (MRP1-5), ABCG2 (BCRP), SLCO2B1 (OATP2B1), SLCO4A1 (OATP4A1), SLC22A1 (OCT1), SLC22A3 (OCT3), SLC22A4 (OCTN1), SLC22A5 (OCTN2), and SLC47A1 (MATE1). Immuno-reactive P-gp and OATP1A2 were localized to the apical cell layers. Drug metabolizing enzymes CYP3A5, -2B6, -2B7 -2E1, SULT1A1-4, UGT1A1-10, and UGT2B15, and nuclear receptors NR1H3 and NR1H4 were also expressed on mRNA level. TC was taken up into HBU cells (Km = 18.5 ± 4.8 ?mol/L; Vmax = 106 ± 11.3 pmol/mg·min) by mechanisms that could be synergistically inhibited by naringin (IC50 = 10.8 (8.4; 13.8) ?mol/L) and verapamil (IC50 = 4.6 (2.8; 7.5) ?mol/L), inhibitors of OATP1A2 and OCT1, respectively. Affinity of TC to OCT1 and P-glycoprotein may be the reason for incomplete oral absorption, wide distribution into liver and kidneys, and substantial intestinal and renal secretions. Absence of brain distribution may result from affinity to P-gp and a low affinity to OATP1A2. The human urothelium expresses many drug transporters and drug metabolizing enzymes that may interact with TC and other drugs eliminated into the urine. PMID:25466967

Bexten, Maria; Oswald, Stefan; Grube, Markus; Jia, Jia; Graf, Tanja; Zimmermann, Uwe; Rodewald, Kathrin; Zolk, Oliver; Schwantes, Ulrich; Siegmund, Werner; Keiser, Markus

2014-12-10

262

Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines  

SciTech Connect

High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

1997-07-01

263

Insect cells-baculovirus system for the production of difficult to express proteins.  

PubMed

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed. PMID:25447865

Osz-Papai, Judit; Radu, Laura; Abdulrahman, Wassim; Kolb-Cheynel, Isabelle; Troffer-Charlier, Nathalie; Birck, Catherine; Poterszman, Arnaud

2015-01-01

264

Verapamil and Rifampin Effect on P-Glycoprotein Expression in Hepatocellular Carcinoma  

PubMed Central

Background: High expression of p-glycoprotein (P-gp) has been associated with a poor prognosis in patients with hepatocellular carcinoma (HCC). It is likely that P-gp overexpression is responsible for multidrug resistance in HCC. Objectives: The aim of this study was to elucidate the effect of potent carcinogen nitrosamine with and without verapamil and rifampin drugs on P-gp expression at the mRNA level in HCC. Materials and Methods: Four groups of rats (n = 5) were selected with different treatments and one group as control. mRNA concentration changes were monitored using quantitative PCR (QPCR). Results: A significant difference was found between verapamil treated group and the control regarding the mRNA level. The mdr1a mRNA was significantly decreased in the verapamil group (P ? 0.001). Rifampin administrated group had a decreased level of the mdr1a mRNA compared to the control group (P ? 0.006). No significant changes were observed in HCC induced rats regarding the mdr1a mRNA level when treated with verapamil and rifampin. An enhanced expression of the mdr1a gene was found In the HCC induced animals when treated with drugs. Conclusions: Verapamil and rifampin were found specific and effective against P-gp expression in HCC. In conclusion, treatment efficacy of most anticancer drugs is increased in combination with verapamil and rifampin against most advanced HCC.

Jalali, Amir; Ghasemian, Sepideh; Najafzadeh, Hossein; Galehdari, Hamid; Seifi, Masoud Reza; Zangene, Fateme; Dehdardargahi, Shaiesteh

2014-01-01

265

Protein-protein interaction and gene co-expression maps of ARFs and Aux/IAAs in Arabidopsis  

PubMed Central

The phytohormone auxin regulates nearly all aspects of plant growth and development. Based on the current model in Arabidopsis thaliana, Auxin/indole-3-acetic acid (Aux/IAA) proteins repress auxin-inducible genes by inhibiting auxin response transcription factors (ARFs). Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins are related to their unique biological functions. The objective of this study was to generate the Aux/IAA-ARF protein-protein interaction map using full length sequences and locate the interacting protein pairs to specific gene co-expression networks in order to define tissue-specific responses of the Aux/IAA-ARF interactome. Pairwise interactions between 19 ARFs and 29 Aux/IAAs resulted in the identification of 213 specific interactions of which 79 interactions were previously unknown. The incorporation of co-expression profiles with protein-protein interaction data revealed a strong correlation of gene co-expression for 70% of the ARF-Aux/IAA interacting pairs in at least one tissue/organ, indicative of the biological significance of these interactions. Importantly, ARF4-8 and 19, which were found to interact with almost all Aux-Aux/IAA showed broad co-expression relationships with Aux/IAA genes, thus, formed the central hubs of the co-expression network. Our analyses provide new insights into the biological significance of ARF-Aux/IAA associations in the morphogenesis and development of various plant tissues and organs. PMID:25566309

Piya, Sarbottam; Shrestha, Sandesh K.; Binder, Brad; Stewart, C. Neal; Hewezi, Tarek

2014-01-01

266

A Bayesian model for classifying all differentially expressed proteins simultaneously in 2D PAGE gels  

PubMed Central

Background Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is commonly used to identify differentially expressed proteins under two or more experimental or observational conditions. Wu et al (2009) developed a univariate probabilistic model which was used to identify differential expression between Case and Control groups, by applying a Likelihood Ratio Test (LRT) to each protein on a 2D PAGE. In contrast to commonly used statistical approaches, this model takes into account the two possible causes of missing values in 2D PAGE: either (1) the non-expression of a protein; or (2) a level of expression that falls below the limit of detection. Results We develop a global Bayesian model which extends the previously described model. Unlike the univariate approach, the model reported here is able treat all differentially expressed proteins simultaneously. Whereas each protein is modelled by the univariate likelihood function previously described, several global distributions are used to model the underlying relationship between the parameters associated with individual proteins. These global distributions are able to combine information from each protein to give more accurate estimates of the true parameters. In our implementation of the procedure, all parameters are recovered by Markov chain Monte Carlo (MCMC) integration. The 95% highest posterior density (HPD) intervals for the marginal posterior distributions are used to determine whether differences in protein expression are due to differences in mean expression intensities, and/or differences in the probabilities of expression. Conclusions Simulation analyses showed that the global model is able to accurately recover the underlying global distributions, and identify more differentially expressed proteins than the simple application of a LRT. Additionally, simulations also indicate that the probability of incorrectly identifying a protein as differentially expressed (i.e., the False Discovery Rate) is very low. The source code is available at https://github.com/stevenhwu/BIDE-2D. PMID:22712439

2012-01-01

267

Development of Novel Rifampicin-Derived P-Glycoprotein Activators/Inducers. Synthesis, In Silico Analysis and Application in the RBE4 Cell Model, Using Paraquat as Substrate  

PubMed Central

P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modulate P-gp expression and activity in a cellular model of the rat’s blood–brain barrier, the RBE4 cell line P-gp expression was assessed by western blot using C219 anti-P-gp antibody. P-gp function was evaluated by flow cytometry measuring the accumulation of rhodamine123. Whenever P-gp activation/induction ability was detected in a tested compound, its antidotal effect was further tested using paraquat as cytotoxicity model. Interactions between Rif or its derivatives and P-gp were also investigated by computational analysis. Rif led to a significant increase in P-gp expression at 72 h and RedRif significantly increased both P-gp expression and activity. No significant differences were observed for the other derivatives. Pre- or simultaneous treatment with RedRif protected cells against paraquat-induced cytotoxicity, an effect reverted by GF120918, a P-gp inhibitor, corroborating the observed P-gp activation ability. Interaction of RedRif with P-gp drug-binding pocket was consistent with an activation mechanism of action, which was confirmed with docking studies. Therefore, RedRif protection against paraquat-induced cytotoxicity in RBE4 cells, through P-gp activation/induction, suggests that it may be useful as an antidote for cytotoxic substrates of P-gp. PMID:23991219

Vilas-Boas, Vânia; Silva, Renata; Palmeira, Andreia; Sousa, Emília; Ferreira, Luísa Maria; Branco, Paula Sério; Carvalho, Félix; Bastos, Maria de Lourdes; Remião, Fernando

2013-01-01

268

Comparative production of human interleukin-2 fused with green fluorescent protein in several recombinant expression systems  

Microsoft Academic Search

The selection of an optimal recombinant expression system is important for successful protein production. Here, we compared production of human interleukin-2 (hIL-2)-green fluorescent protein (GFP) fusion proteins in several expression systems such as bacteria Escherichia coli, yeast Pichia pastoris, insect Spodoptera frugiperda Sf-9 cells, insect Tricoplusia ni larvae, and insect Drosophila melanogaster S2 cells. Due to the highly hydrophobic nature

Hyung Joon Cha; Hwa Sung Shin; Hye Jung Lim; Hye Sook Cho; Nimish N. Dalal; Minh Q. Pham; William E. Bentley

2005-01-01

269

Development of an expression system for eukarytoic proteins in methylotropic bacteria  

SciTech Connect

The objective of this project was to develop an expression vector for methylotrophic bacteria for use in the production of C{sup 13} and H{sup 2} labelled eukaryotic proteins by growing methylotrophic bacteria on labelled methanol or methylamine. The eukaryotic proteins calmodulin and troponin C were chosen as test cases. Genes encoding both proteins were cloned into different constructions and tested for expression. Moderate amounts of troponin C were found with one of the constructions.

Lidstrom, M.E. [California Inst. of Tech., Pasadena, CA (United States)

1996-09-01

270

Decreased expression of nucleophosmin/B23 increases drug sensitivity of adriamycin-resistant Molt-4 leukemia cells through mdr-1 regulation and Akt/mTOR signaling.  

PubMed

Nucleophosmin/B23 (NPM) is a nuclear protein with prosurvival and ribosomal RNA processing functions. However, the potential role of NPM involved in drug-resistance in leukemia has not been investigated clearly. In this study, we generated an adriamycin (ADM)-resistant lymphoblastic cell line Molt-4/ADR (MAR) by stepwise induction. Cell proliferation, sensitivity to chemotherapy agents and expressions of drug resistance related molecules were assessed. The IC50 of Molt-4 cells were 0.58±0.11?mol/L and MAR cells were 22.56±1.94?mol/L, meaning MAR cells were 38.63 fold resistant to Molt-4 cells. Furthermore, MAR cells gained an expression of mdr-1 (P-gp) and a higher expression of NPM compared to Molt-4 cells. Knockdown of NPM by RNA interference (RNAi) suppressed the viability of both Molt-4 and MAR cells. After NPM RNAi, the IC50 of MAR and Molt-4 cells were 3.83±0.38?mol/L and 0.19±0.02?mol/L respectively. Both of them revealed an increase of drug sensitivity with down-regulation of mdr-1 and Akt/mTOR signaling. Knockdown of mdr-1 could also reverse the drug resistance, with no change in NPM expression. It could be concluded that knockdown of NPM reversed the drug resistance by down-regulating P-gp and Akt/mTOR signal pathway, indicating that NPM may serve as a potential modulator in drug resistance. PMID:25457413

Wang, Lingyan; Chen, Buyuan; Lin, Minhui; Cao, Yanqin; Chen, Yingyu; Chen, Xinji; Liu, Tingbo; Hu, Jianda

2015-03-01

271

The pST44 polycistronic expression system for producing protein complexes in Escherichia coli  

Microsoft Academic Search

Protein complexes are responsible for key biological processes, but methods to produce recombinant protein complexes for biochemical and biophysical studies are limited. We have developed a second generation Escherichia coli polycistronic expression system which improves on the modularity of our original pST39 polycistronic system. This pST44 expression system simplifies the construction of polycis-tronic plasmids, particularly of variant plasmids expressing deletion

Song Tan; Ronald C. Kern; William Selleck

2005-01-01

272

Borrelia burgdorferi Proteins Whose Expression Is Similarly Affected by Culture Temperature and pH  

Microsoft Academic Search

Previously, we had demonstrated the upregulation in the expression of several proteins, including the lipoproteins OspC and P35, of Borrelia burgdorferi in the stationary growth phase. Since the expression of OspC is also known to be affected by culture temperature and pH, we examined the effects of both variables on the expression of the remaining stationary-phase-upregulated proteins. Our study revealed

RAMESH RAMAMOORTHY; DOROTHY SCHOLL-MEEKER

2001-01-01

273

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development  

Microsoft Academic Search

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma

Jessica S. Hoane; Vernon B. Carruthers; Boris Striepen; David P. Morrison; Rolf Entzeroth; Daniel K. Howe

2003-01-01

274

Differential Analysis of Protein Expression in RNA-Binding-Protein Transgenic and Parental Rice Seeds Cultivated under Salt Stress  

PubMed Central

Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in protein expression under salt stress, NT and RBP rice were cultured with or without 200 mM sodium chloride. Only two protein spots differed between NT and RBP rice seeds cultured under normal conditions, one of which was identified as a putative abscisic acid-induced protein. In NT rice seeds, 91 spots significantly differed between normal and salt-stress conditions. Two allergenic proteins of NT rice seeds, RAG1 and RAG2, were induced by high salt. In contrast, RBP rice seeds yielded seven spots and no allergen spots with significant differences in protein expression between normal and salt-stress conditions. Therefore, expression of fewer proteins was altered in RBP rice seeds by high salt than those in NT rice seeds. PMID:24410502

2015-01-01

275

Reciprocal influence of connexins and apical junction proteins on their expressions and functions  

PubMed Central

Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another. PMID:19046940

Derangeon, Mickaël; Spray, David C.; Bourmeyster, Nicolas; Sarrouilhe, Denis; Hervé, Jean-Claude

2009-01-01

276

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli  

PubMed Central

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Bruni, Renato

2014-01-01

277

Enhanced expression of rabies virus surface G-protein in Escherichia coli using SUMO fusion.  

PubMed

Fusion systems are known to increase the expression of difficult to express recombinant proteins in soluble form to facilitate their purification. Rabies glycoprotein was also tough to express at sufficient level in soluble form in both E. coli and plant. The present work was aimed to over-express and purify this membrane protein from soluble extract of E. coli. Fusion of Small Ubiqutin like Modifier (SUMO) with rabies glycoprotein increased ~1.5 fold higher expression and ~3.0 fold solubility in comparison to non-fused in E. coli. The SUMO fusion also simplified the purification process. Previously engineered rabies glycoprotein gene in tobacco plants provides complete protection to mice, but the expression was very low for purification. Our finding demonstrated that the SUMO-fusion was useful for enhancing expression and solubility of the membrane protein and again proves to be a good alternative technology for applications in biomedical and pharmaceutical research. PMID:22134654

Singh, Ankit; Yadav, Dinesh; Rai, Krishan Mohan; Srivastava, Meenal; Verma, Praveen C; Singh, Pradhyumna K; Tuli, Rakesh

2012-01-01

278

Protein Expression and PuriWcation 36 (2004) 207216 www.elsevier.com/locate/yprep  

E-print Network

Protein Expression and PuriWcation 36 (2004) 207­216 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.04.020 Protein from two other human membrane proteins were investigated. Peptide extensions with net negative charge 1

279

INCREASED LIVER PATHOLOGY IN HEPATITIS C VIRUS TRANSGENIC MICE EXPRESSING THE HEPATITIS B VIRUS X PROTEIN  

Technology Transfer Automated Retrieval System (TEKTRAN)

Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was id...

280

Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1?C expression strain  

PubMed Central

Background The non-pathogenic bacterium Mycobacterium smegmatis is widely used as a near-native expression host for the purification of Mycobacterium tuberculosis proteins. Unfortunately, the Hsp60 chaperone GroEL1, which is relatively highly expressed, is often co-purified with polyhistidine-tagged recombinant proteins as a major contaminant when using this expression system. This is likely due to a histidine-rich C-terminus in GroEL1. Results In order to improve purification efficiency and yield of polyhistidine-tagged mycobacterial target proteins, we created a mutant version of GroEL1 by removing the coding sequence for the histidine-rich C-terminus, termed GroEL1?C. GroEL1?C, which is a functional protein, is no longer able to bind nickel affinity beads. Using a selection of challenging test proteins, we show that GroEL1?C is no longer present in protein samples purified from the groEL1?C expression strain and demonstrate the feasibility and advantages of purifying and characterising proteins produced using this strain. Conclusions This novel Mycobacterium smegmatis expression strain allows efficient expression and purification of mycobacterial proteins while concomitantly removing the troublesome contaminant GroEL1 and consequently increasing the speed and efficiency of protein purification. PMID:21439037

2011-01-01

281

Altered brain protein expression profiles are associated with molecular neurological dysfunction in the PKU mouse model.  

PubMed

Phenylketonuria (PKU), if not detected and treated in newborns, causes severe neurological dysfunction and cognitive and behavioral deficiencies. Despite the biochemical characterization of PKU, the molecular mechanisms underlying PKU-associated brain dysfunction remain poorly understood. The aim of this study was to gain insights into the pathogenesis of this neurological damage by analyzing protein expression profiles in brain tissue of Black and Tan BRachyury-PahEnu2 mice (a mouse model of PKU). We compared the cerebral protein expression of homozygous PKU mice with that of their heterozygous counterparts using two-dimensional difference gel electrophoresis analysis, and identified 21 differentially expressed proteins, four of which were over-expressed and 17 under-expressed. An in silico bioinformatic approach indicated that protein under-expression was related to neuronal differentiation and dendritic growth, and to such neurological disorders as progressive motor neuropathy and movement disorders. Moreover, functional annotation analyses showed that some identified proteins were involved in oxidative metabolism. To further investigate the proteins involved in the neurological damage, we validated two of the proteins that were most strikingly under-expressed, namely, Syn2 and Dpysl2, which are involved in synaptic function and neurotransmission. We found that Glu2/3 and NR1 receptor subunits were over-expressed in PKU mouse brain. Our results indicate that differential expression of these proteins may be associated with the processes underlying PKU brain dysfunction, namely, decreased synaptic plasticity and impaired neurotransmission. We identified a set of proteins whose expression is affected by hyperphenylalaninemia. We think that phenylketonuria (PKU) brain dysfunction also depends on reduced Syn2 and Dpysl2 levels, increased Glu2/3 and NR1 levels, and decreased Pkm, Ckb, Pgam1 and Eno1 levels. These findings finally confirm that alteration in synaptic function, in transmission and in energy metabolism underlie brain damage provoked by hyperphenylalaninemias. PMID:24548049

Imperlini, Esther; Orrù, Stefania; Corbo, Claudia; Daniele, Aurora; Salvatore, Francesco

2014-06-01

282

Analysis of differentially expressed proteins in zebrafish (Danio rerio) embryos exposed to chlorpyrifos.  

PubMed

In this study, the protein expression profiles of zebrafish embryos under chlorpyrifos (CPF) stress were investigated. Zebrafish embryos were exposed to 0.25mg/L CPF, and embryo samples were collected until 24h post-fertilization (hpf). To gain a better understanding of the response of zebrafish embryos to CPF exposure, two-dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with mass spectrometry was employed to carry out a comparative proteomic analysis. Total proteins were extracted from the control and treated samples, separated by 2D PAGE, and visualized by silver staining. A total of 59 protein spots showed reproducible changes compared with the control. Of these 59 spots, 19 were selected and subjected to matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight mass spectrometry (TOF/TOF) analysis; 9 differentially expressed proteins were successfully identified, including 3 up-regulated proteins and 6 down-regulated proteins. The increased expression of 3 proteins associated with detoxification and stress response suggested that the activation of protective proteins was required in zebrafish embryos exposed to CPF. On the other hand, the decreased expression of 6 proteins is mainly involved in cytoskeleton structure, protein translation, signal transduction and lipoprotein metabolism. These data may help us understand the functions and the molecular mechanisms of these proteins in zebrafish embryos' response to CPF exposure. PMID:25445019

Liu, Lili; Xu, Yongxue; Xu, Lili; Wang, Jian; Wu, Wei; Xu, Lei; Yan, Yanchun

2014-10-28

283

The use of highly expressed FTH1 as carrier protein for cytosolic targeting in Hansenula polymorpha.  

PubMed

The iron storage protein ferritin is a member of the non-heme iron protein family. It can store and release iron, therefore it prevents the cell from damage caused by iron-dioxygen reactions as well as it provides iron for biological processing. To study whether the human ferritin heavy chain (FTH1) can be expressed in Hansenula polymorpha, we integrated an expression cassette for FTH1 and analyzed the protein expression. We found very efficient expression of FTH1 and obtained yields up to 1.9 g/L under non-optimized conditions. Based on this result we designed a FTH1-PTH fusion protein to successfully express the parathyroid hormone fragment 1-34 (PTH) for the first time intracellular in H. polymorpha. PMID:22212821

Eilert, Eva; Hollenberg, Cornelis P; Piontek, Michael; Suckow, Manfred

2012-06-15

284

Altered Expression of RNA Splicing Proteins in Alzheimer's Disease Patients: Evidence from Two Microarray Studies  

PubMed Central

Background/Aims Dysregulation of pre-mRNA splicing from an altered expression of RNA splice-regulatory proteins may act as the convergence point underlying aberrant gene expression changes in Alzheimer's disease (AD). Methods Two microarray datasets from a control/AD postmortem brain cohort of 31 subjects ? 9 controls and 22 AD subjects (National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database) – were used. Results Between the two microarray studies, the expression of six splice-regulatory protein genes showed concordant changes in AD. These genes were then correlated with gene expression changes of transcripts reported to be altered in AD. Amyloid beta (A4) precursor protein and tropomyosin receptor kinase B transcripts were found to correlate significantly with the same splice-regulatory proteins in the two studies. Conclusion This study highlights a susceptibility network that can potentially link a number of susceptibility genes. PMID:23637700

Wong, Jenny

2013-01-01

285

Protein expression profiles of intestinal epithelial co-cultures: effect of functionalised carbon nanotube exposure  

PubMed Central

To assess the biological effects of low level, water dispersible, functionalised carbon nanotube (f-CNT) exposure in an in vitro model simulating the digestive tract, cellular protein expression was quantified and compared using label-free quantitative mass spectrometry (LFQMS). Co-cultured cells were exposed to well-characterised SWCNT-COOH, MWCNT-COOH, and MWCNT-PVP. The relative expression of 2,282 unique proteins was compared across the dose groups. 428 proteins were found to be differentially expressed. At the high dose, the extent of differential protein expression was CNT-specific and directly related to CNT colloidal stability. Cells responded to low level MWCNT-PVP exposure with three-fold greater differential expression. Bioinformatic analysis indicated significant and f-CNT-specific effects on relevant molecular and cellular functions and canonical pathways, with little overlap across f-CNT type and in the absence of overt toxicity. PMID:24228069

Lai, Xianyin; Blazer-Yost, Bonnie L.; Clack, James W.; Fears, Sharry L.; Mitra, Somenath; Ntim, Susana Addo; Ringham, Heather N.

2013-01-01

286

Evolution of the correlation between expression divergence and protein divergence in mammals.  

PubMed

Divergence of protein sequences and gene expression patterns are two fundamental mechanisms that generate organismal diversity. Here, we have used genome and transcriptome data from eight mammals and one bird to study the positive correlation of these two processes throughout mammalian evolution. We demonstrate that the correlation is stable over time and most pronounced in neural tissues, which indicates that it is the result of strong negative selection. The correlation is not driven by genes with specific functions and may instead best be viewed as an evolutionary default state, which can nevertheless be evaded by certain gene types. In particular, genes with developmental and neural functions are skewed toward changes in gene expression, consistent with selection against pleiotropic effects associated with changes in protein sequences. Surprisingly, we find that the correlation between expression divergence and protein divergence is not explained by between-gene variation in expression level, tissue specificity, protein connectivity, or other investigated gene characteristics, suggesting that it arises independently of these gene traits. The selective constraints on protein sequences and gene expression patterns also fluctuate in a coordinate manner across phylogenetic branches: We find that gene-specific changes in the rate of protein evolution in a specific mammalian lineage tend to be accompanied by similar changes in the rate of expression evolution. Taken together, our findings highlight many new aspects of the correlation between protein divergence and expression divergence, and attest to its role as a fundamental property of mammalian genome evolution. PMID:23781097

Warnefors, Maria; Kaessmann, Henrik

2013-01-01

287

Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr.  

USGS Publications Warehouse

The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

Wiseman, Steve; Jorgensen, Even H.; Maule, Alec G.; Vijayan, Mathilakath M.

2011-01-01

288

Stochastic protein expression in individual cells at the single molecule level  

NASA Astrophysics Data System (ADS)

In a living cell, gene expression-the transcription of DNA to messenger RNA followed by translation to protein-occurs stochastically, as a consequence of the low copy number of DNA and mRNA molecules involved. These stochastic events of protein production are difficult to observe directly with measurements on large ensembles of cells owing to lack of synchronization among cells. Measurements so far on single cells lack the sensitivity to resolve individual events of protein production. Here we demonstrate a microfluidic-based assay that allows real-time observation of the expression of ?-galactosidase in living Escherichia coli cells with single molecule sensitivity. We observe that protein production occurs in bursts, with the number of molecules per burst following an exponential distribution. We show that the two key parameters of protein expression-the burst size and frequency-can be either determined directly from real-time monitoring of protein production or extracted from a measurement of the steady-state copy number distribution in a population of cells. Application of this assay to probe gene expression in individual budding yeast and mouse embryonic stem cells demonstrates its generality. Many important proteins are expressed at low levels, and are thus inaccessible by current genomic and proteomic techniques. This microfluidic single cell assay opens up possibilities for system-wide characterization of the expression of these low copy number proteins.

Cai, Long; Friedman, Nir; Xie, X. Sunney

2006-03-01

289

Efficient Expression of Acetylcholine-Binding Protein from Aplysia californica in Bac-to-Bac System  

PubMed Central

The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ?5?mg/L, which needs 55?h incubation after infection at the cell density of 2?×?106?cells/mL with an inoculum volume ratio of 1?:?100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies. PMID:25136612

Lin, Bo; Bing, Hui; Zhangsun, Dongting; Luo, Sulan

2014-01-01

290

TRAIL protein expression in breast cancer cells correlates with nuclear grade  

PubMed Central

Introduction TRAIL protein may serve as an escape mechanism for cancer cells from the immune response. The aim of the study was to assess whether the presence of TRAIL protein correlates with unfavourable prognostic factors in breast carcinoma. Material and methods The study group was composed of breast cancer patients treated surgically in the Department of Surgical Oncology, Medical University of Lodz, Poland, from January to December 2003. Inclusion criteria for the study were fulfilled by 117 women. The immunohistochemical study of TRAIL protein expression was performed in 118 breast carcinomas diagnosed in the study group. TRAIL protein expression was correlated with other variables: tumour size, lymph node status, grade, histological type of carcinoma, oestrogen and progesterone receptor status, HER2 expression, presence of lymphovascular invasion and age of the patient. Results Expression of TRAIL protein was present in 73% of breast carcinomas. The percentage of TRAIL-expressing breast carcinoma cells correlated with the nuclear grade (? = 0.26, p < 0.05; Tau Kendall test). The intensity of TRAIL expression (intensity of staining) in breast carcinoma cells correlated with the nuclear grade (? = 0.15, p < 0.05; Tau Kendall test). TRAIL expression in breast carcinoma did not correlate with other studied variables. Conclusions Our analysis revealed that expression of TRAIL protein in breast carcinoma cells correlates with nuclear grade of carcinoma. PMID:22371798

Bilski, Adam; Pasz-Walczak, Gra?yna; Kubiak, Robert; Sek, Piotr; Chalubinska, Justyna; Fendler, Wojciech; Wronski, Konrad; Piekarska, Anna; Pluta, Piotr; Potemski, Piotr; Jeziorski, Arkadiusz

2010-01-01

291

Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells  

PubMed Central

It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labour intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I ?/? HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal Enhanced Green Fluorescence Protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately three weeks. PMID:21925269

Chaudhary, Sarika; Pak, John E.; Pedersen, Bjørn P.; Bang, Lois J.; Zhang, Liye B.; Ngaw, Samantha M.M.; Green, Raissa G.; Sharma, Vinay; Stroud, Robert M.

2012-01-01

292

Expression and Characterization of the Mycobacterium tuberculosis Serine\\/Threonine Protein Kinase PknB  

Microsoft Academic Search

PknB is a member of the newly discovered eukaryotic-like protein serine\\/threonine kinase (PSTK) family of proteins. The pknB gene was cloned and expressed in Escherichia coli. The active recombinant protein was purified and shown to be reactive with antiphosphoserine antibodies, as well as with antibodies to the phosphorylated eukaryotic Ser\\/Thr kinases mitogen-activated protein kinase kinase 3 and 6, P38, and

YOSSEF AV-GAY; SARWAT JAMIL; STEVEN J. DREWS

1999-01-01

293

Viperin protein expression inhibits the late stage of respiratory syncytial virus morphogenesis.  

PubMed

We examined the effect of respiratory syncytial virus (RSV) infection on viperin protein expression in the permissive HEp2 and non-permissive RAW 264.7 macrophage cell lines. In RSV-infected HEp2 cells low levels of the viperin protein was localized to the virus-induced inclusion bodies and did not impair virus transmission in these cells. In contrast, RSV-infected RAW 264.7 cells increased expression of the STAT1 protein occurred at between 6 and 12h post-infection, which coincided with the appearance of P-STAT1. A relatively high level of viperin protein expression was detected in infected RAW 264.7 cells, and it was extensively localized throughout the cytoplasm of infected cells. The effect of early viperin protein expression on RSV infection in cells that are normally permissive to RSV cultivation was examined by using either transient transfected HEp2 cells or stable transfected HeLa cells that expressed the viperin protein. The early expression of viperin in HeLa cells did not prevent virus infection, and no significant inhibitory effect on either virus protein expression or targeting of virus proteins to the cell surface was noted. However, while inclusion body formation was not inhibited, early viperin protein expression was associated with the inhibition of virus filament formation and reduced cell-to-cell virus transmission. Inhibition of virus filament formation was also observed in HEp2 cells expressing viperin. Collectively our data suggested that viperin impaired RSV transmission by inhibiting virus filament formation, providing a basis for its anti-virus activity in RSV-infected cells. PMID:25433308

Jumat, Muhammad Raihan; Huong, Tra Ngyen; Ravi, Laxmi Iyer; Stanford, Rebecca; Tan, Boon Huan; Sugrue, Richard J

2015-02-01

294

Refolding of G protein ? subunits from inclusion bodies expressed in Escherichia coli  

PubMed Central

Heterotrimeric G proteins relay signals from G protein-coupled receptors (GPCRs) to the interior of the cell. The signaling cascades induced by G protein activation control a wide range of cellular processes. The ? subunit is believed to determine which G protein couples to each GPCR, and is the primary determinant of the type of signal transmitted. Several members of the G? family have been expressed in active form in E. coli. However, production levels of these proteins are limited: in most cases only ~10 % of total G? protein expressed is active; the rest accumulates in inclusion bodies. Although Gi? has been readily expressed in soluble form (to 10 mg/L), other ? subunits are minimally soluble, and many are exclusively expressed to inclusion bodies. Previous efforts to solubilize and refold G? from inclusion bodies have not been successful. Here we did a thorough study of the characteristics of G? subunits (human Gi?(1), human Gs? (short), human G11? and human Gt? (cone)), solubilized and purified from inclusion bodies. We find that we can obtain soluble protein both by on-column and rapid-dilution techniques. Comparison to native, soluble Gi? expressed from E. coli showed that although the refolded G? subunits were soluble and retained partial ?-helicity characteristic of the native, folded G? subunit, they did not bind GDP or GTP as effectively as native protein. We conclude that the refolded Gi? protein has a native-like secondary structure, but is predominately in a molten globular state. PMID:18249008

McCusker, Emily; Robinson, Anne Skaja

2008-01-01

295

Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium.  

PubMed

We constructed a series of expression vectors for purification of native proteins and protein complexes in Dictyostelium. Protein purification is achieved by either a C-terminal or N-terminal fusion of the protein of choice to the tandem affinity purification (TAP) tag. The TAP tag consists of a protein A tag and a calmodulin binding peptide (CBP) and has been successfully used for purification of native protein complexes from yeast and animal cells. Protein expression is driven by the constitutive actin 15 promoter and the vectors optionally carry additional green- or yellow fluorescent protein (GFP or YFP) tags for fusion at either a C- or N-terminal location. Tandem affinity purification of native Dictyostelium protein complexes was tested by using pArc-34, one of the members of the well characterized Dictyostelium Arp2/3 complex, as bait. After denaturation and SDS-PAGE separation of the pArc-34 associated proteins all members of the Arp2/3 complex could be identified. PMID:17296313

Meima, Marcel E; Weening, Karin E; Schaap, Pauline

2007-06-01

296

Tissue expression patterns of Schistosoma mansoni Venom Allergen-Like proteins 6 and 7.  

PubMed

The Schistosoma mansoni Venom Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-Coating Protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in host-pathogen interactions. Whole mount in situ hybridisation demonstrated a distinct expression pattern in oral and ventral suckers of adult worms for SmVAL6 and in the oesophageal gland for SmVAL7 transcripts, respectively. Additionally, immunocytochemistry analysis corroborated SmVAL7 expression in the oesophageal gland. Analysis of protein expression across the parasite's life cycle revealed that the SmVAL6 protein is upregulated in cercariae and adult male worms. Furthermore, SmVAL6 protein was identified by mass spectrometry in tegument fractions of adult worms. Finally, we speculate on possible functions of these two SmVALs at the host-parasite interface. PMID:22575701

Rofatto, Henrique K; Parker-Manuel, Sophia J; Barbosa, Tereza C; Tararam, Cibele A; Alan Wilson, R; Leite, Luciana C C; Farias, Leonardo P

2012-06-01

297

Evaluation of differential protein expression in Haliclona aquarius and sponge-associated microorganisms under cadmium stress.  

PubMed

A comparative proteomic approach was used to assess differentially expressed proteins in marine sponges after 36 h of exposure to cadmium (Cd). After separation performed by 2-D polyacrylamide gel electrophoresis, 46 protein spots indicated differential expression, and 17 of these proteins were identified by electrospray ionization quadrupole time-of-flight mass spectrometry. From the proteins identified, 76% were attributed to sponge-associated microorganisms (fungi and bacteria), and 24% were attributed to Haliclona aquarius. Some of the proteins that were identified may be related to cell proliferation and differentiation or processes of oxidative stress repair and energy procurement. An integrated evaluation based on spot expression levels and the postulated functions of these proteins allowed a more accurate evaluation of the stress caused to the sponge holobiont system by cadmium exposure. This study could provide new insights into the use of a proteomic approach in the marine sponge to assess the effects of Cd pollution in a marine environment. PMID:23545857

Wanick, Rodrigo Cunha; de Sousa Barbosa, Herbert; Frazão, Leonardo Revoredo; Santelli, Ricardo Erthal; Arruda, Marco Aurélio Zezzi; Coutinho, Cristiano Carvalho

2013-09-01

298

Expression of Bmi1 in Epidermis Enhances Cell Survival by Altering Cell Cycle Regulatory Protein Expression and Inhibiting Apoptosis  

Microsoft Academic Search

The polycomb group (PcG) genes are epigenetic suppressors of gene expression that play an important role in development. In this study, we examine the role of Bmi-1 (B-cell-specific Moloney murine leukemia virus integration site 1) as a regulator of human epidermal keratinocyte survival. We identify Bmi-1 mRNA and protein expression in epidermis and in cultured human keratinocytes. Bmi-1 is located

Kathy Lee; Gautam Adhikary; Sivaprakasam Balasubramanian; Ramamurthy Gopalakrishnan; Thomas McCormick; Goberdhan P Dimri; Richard L Eckert; Ellen A Rorke

2008-01-01

299

Apoptosis-related protein expression in rabbits with blast brain injury following early hyperbaric oxygen therapy?  

PubMed Central

We treated detonator-explosion-induced craniocerebral injury in rabbits with hyperbaric oxygen 1-24 hours post-injury. Expression of the apoptosis-regulating protein cytochrome c, the pro-apoptotic protein Bax and the apoptosis marker caspase-3 in the tissues surrounding the area of injury was significantly reduced, while that of the anti-apoptotic protein Bcl-2 was significantly increased. Our findings indicate that the curative effects of early hyperbaric oxygen on cortical cell apoptosis is associated with suppression of cytochrome c release from mitochondria. This mechanism underlies the observed reduction in Bax expression and upregulation of Bcl-2 expression.

Xu, Shaonian; Liu, Jiachuan; Zhang, Yongming; Wang, Chunlin; Wang, Jinbiao; Yang, Yanyan; Huo, Jian; Sun, Wenjiang

2012-01-01

300

RNA binding protein and binding site useful for expression of recombinant molecules  

DOEpatents

The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

Mayfield, Stephen (Cardiff, CA)

2000-01-01

301

Yeast PPR proteins, watchdogs of mitochondrial gene expression  

PubMed Central

PPR proteins are a family of ubiquitous RNA-binding factors, found in all the Eukaryotic lineages, and are particularly numerous in higher plants. According to recent bioinformatic analyses, yeast genomes encode from 10 (in S. pombe) to 15 (in S. cerevisiae) PPR proteins. All of these proteins are mitochondrial and very often interact with the mitochondrial membrane. Apart from the general factors, RNA polymerase and RNase P, most yeast PPR proteins are involved in the stability and/or translation of mitochondrially encoded RNAs. At present, some information concerning the target RNA(s) of most of these proteins is available, the next challenge will be to refine our understanding of the function of the proteins and to resolve the yeast PPR-RNA-binding code, which might differ significantly from the plant PPR code. PMID:24184848

Herbert, Christopher J; Golik, Pawel; Bonnefoy, Nathalie

2013-01-01

302

Identifying protein complexes based on the integration of PPI network and gene expression data.  

PubMed

Identification of protein complexes is crucial to understand principles of cellular organisation and predict protein functions. In this paper, a novel protein complex discovery algorithm IPCIPG is proposed based on the integration of Protein-Protein Interaction network (PPI network) and gene expression data. IPCIPG is a local search algorithm which has two versions: IPCIPG-n for identifying non-overlapping clusters and IPCIPG-o for detecting overlapping clusters. The experimental results on the yeast PPI network show that IPCIPG can identify protein complexes with specific biological meaning more effectively, precisely and comprehensively than six other algorithms: HUNTER, HC-PIN, CMC, SPICi, MOCDE and MCL. PMID:25667384

Chen, Weijie; Li, Min; Wu, Xuehong; Wang, Jianxin

2015-01-01

303

Expression of Nuclear Factor Erythroid 2 Protein in Malignant Cutaneous Tumors  

PubMed Central

Background Reactive oxygen species (ROS) damages cell molecules, and modifies cell signaling. The nuclear factor E2-related factor (Nrf2) is a critical transcription regulator, which protects cells against oxidative damage. Nrf2 expression is increased in a large number of cancers. However, little information has been reported regarding the expression of Nrf2 in skin cancers. Hence, we explored the expression of Nrf2 protein in skin cancers. Methods The Nrf2 protein expression in 24 specimens, including 6 malignant melanomas (MM), 6 squamous cell carcinomas (SCC), 6 basal cell carcinomas (BCC), and 6 normal skin tissues, was evaluated by western blotting. Immunohistochemical staining was performed. The expression of Kelch-like ECH-associated protein 1 (Keap1), the key regulator of Nrf2, was also analyzed by western blotting. Results Small interfering RNA transfection to the melanoma cell line G361 confirmed that an approximately 66 kDa band was the true Nrf2 band. The western blot revealed that the Nrf2 protein was definitely expressed in normal skin tissues, but the Nrf2 expression was decreased in MM, SCC, and BCC. Immunohistochemical examination showed that expression of Nrf2 was decreased in all skin cancer tissues compared to the normal skin tissues. Keap1 was not expressed in all malignant skin tumors and normal skin tissues by western blot. Conclusions ROS was increased in various types of cancers which proteins were highly expressed or underexpressed. This study demonstrated that the expression of Nrf2 protein was down-regulated in human malignant skin tumors. We suggest that decreased expression of Nrf2 is related to skin cancers. PMID:25396176

Choi, Chang Yong; Kim, Jin Young; Wee, Seo Yeong; Lee, Jang Hyun; Nam, Doo Hyun; Cho, Moon Kyun; Lee, Yoon Jin; Nam, Hae Seon; Lee, Sang Han; Cho, Sung Woo

2014-01-01

304

The HTLV Receptor Is a Widely Expressed Protein  

Microsoft Academic Search

The receptor for human T-cell leukemia virus type 1 (HTLV-1) was found to be expressed on a broad range of cell lines derived from multiple species. Receptor expression was assessed using human immunodeficiency virus type 1 particles, pseudotyped with the HTLV-1 envelope glycoprotein, and expressing luciferase under the control of an SV40 enhancer and promoter. Infection by pseudotyped virus was

Samuel R. Trejo; Lee Ratner

2000-01-01

305

Proteomic Analysis of Differentially Expressed Proteins in Fenneropenaeus chinensis Hemocytes upon White Spot Syndrome Virus Infection  

PubMed Central

To elucidate molecular responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection, two-dimensional gel electrophoresis was applied to investigate differentially expressed proteins in hemocytes of Chinese shrimp (Fenneropenaeus chinensis) at 24 h post infection (hpi). Approximately 580 protein spots were detected in hemocytes of healthy and WSSV-infected shrimps. Quantitative intensity analysis revealed 26 protein spots were significantly up-regulated, and 19 spots were significantly down-regulated. By mass spectrometry, small ubiquitin-like modifier (SUMO) 1, cytosolic MnSOD, triosephosphate isomerase, tubulin alpha-1 chain, microtubule-actin cross-linking factor 1, nuclear receptor E75 protein, vacuolar ATP synthase subunit B L form, inositol 1,4,5-trisphosphate receptor, arginine kinase, etc., amounting to 33 differentially modulated proteins were identified successfully. According to Gene Ontology annotation, the identified proteins were classified into nine categories, consisting of immune related proteins, stimulus response proteins, proteins involved in glucose metabolic process, cytoskeleton proteins, DNA or protein binding proteins, proteins involved in steroid hormone mediated signal pathway, ATP synthases, proteins involved in transmembrane transport and ungrouped proteins. Meanwhile, the expression profiles of three up-regulated proteins (SUMO, heat shock protein 70, and arginine kinase) and one down-regulated protein (prophenoloxidase) were further analyzed by real-time RT-PCR at the transcription level after WSSV infection. The results showed that SUMO and heat shock protein 70 were significantly up-regulated at each sampling time point, while arginine kinase was significantly up-regulated at 12 and 24 hpi. In contrast, prophenoloxidase was significantly down-regulated at each sampling time point. The results of this work provided preliminary data on proteins in shrimp hemocytes involved in WSSV infection. PMID:24587154

Li, Wei; Tang, Xiaoqian; Xing, Jing; Sheng, Xiuzhen; Zhan, Wenbin

2014-01-01

306

Expression, characterization, and immunoreactivities of a soluble hepatitis E virus putative capsid protein species expressed in insect cells.  

PubMed Central

The hepatitis E virus (HEV) open reading frame-2 (ORF-2) is predicted to encode a 71-kDa putative capsid protein involved in virus particle formation. When insect Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus containing the entire ORF-2 sequence, two types of recombinant proteins were produced; an insoluble protein of 73 kDa and a soluble protein of 62 kDa. The 62-kDa species was shown to be a proteolytic cleavage product of the 73-kDa protein. N-terminal sequence analysis of the 62-kDa protein indicated that it lacked the first 111 amino acids that are present in the full-length 73-kDa protein. A soluble 62-kDa protein was produced without the proteolytic processing by inserting the coding sequence of amino acids 112 to 660 of ORF-2 in a baculovirus expression vector and using the corresponding virus to infect Sf9 cells. The two recombinant 62-kDa proteins made by different mechanisms displayed immunoreactivities very compatible to each other. The 62-kDa proteins obtained by both proteolytic processing and reengineering demonstrated much higher sensitivities in detecting anti-HEV antibodies in human sera than the antigens made from bacteria, as measured by enzyme-linked immunosorbent assay. The data suggest that the soluble 62-kDa protein made from insect cells contains additional epitopes not present in recombinant proteins made from bacteria. Therefore, the 62-kDa protein may be useful for HEV diagnostic improvement and vaccine development. The reengineered construct allows for the consistent large-scale production of the soluble 62-kDa protein without proteolytic processing. PMID:9220158

Zhang, Y; McAtee, P; Yarbough, P O; Tam, A W; Fuerst, T

1997-01-01

307

The use of Group 3 LEA proteins as fusion partners in facilitating recombinant expression of recalcitrant proteins in E. coli.  

PubMed

Late embryogenesis abundant (LEA) proteins are intrinsically disordered proteins that accumulate in organisms during the development of dehydration stress tolerance and cold acclimation. Group 3 LEA proteins have been implicated in the prevention of cellular protein denaturation and membrane damage during desiccation and anhydrobiosis. We tested the ability of LEA proteins to facilitate recombinant expression of recalcitrant and intrinsic membrane proteins. Two Brassica napus Group 3 LEA proteins, BN115m and a truncated fragment of BNECP63, were fused to two target proteins identified as recalcitrant to overexpression in soluble form or outside of inclusion bodies. Fusion of a truncated peptide of BNECP63 is sufficient to provide soluble and high levels of recombinant overexpression of BNPsbS (an intrinsic membrane chlorophyll-binding protein of photosystem II light harvesting complex) and a peptide of the Hepatitis C viral polyprotein. Furthermore, fusion of the recombinant target proteins to BNECP63 or BN115 prevented irreversible heat- and freeze-induced precipitation. These experiments not only underscore the exploitation of LEA-type peptides in facilitating protein overexpression and protection, but also provide insights into the mechanism of LEA proteins in cellular protection. PMID:19364534

Singh, Jas; Whitwill, Steve; Lacroix, Geneviève; Douglas, Jennifer; Dubuc, Elyse; Allard, Ghislaine; Keller, Wilf; Schernthaner, Johann P

2009-09-01

308

Expression of the CUB domain containing protein 1 (CDCP1) gene in colorectal tumour cells.  

PubMed

Expression of CUB domain containing protein 1 (CDCP1) is upregulated in carcinoma cells. We quantitated CDCP1 gene expression in matched normal colon and tumour tissue and compared the level of expression to other genes upregulated in colorectal tumourigenesis. Furthermore, we show that the CDCP1 gene generates two transcripts which are co-expressed in normal and matched tumour tissue as well as in the majority of cell lines analysed. However, intracellular localisation studies revealed that only one of these transcripts encodes a protein that is localised to the cell surface. PMID:17335815

Perry, Sara E; Robinson, Philip; Melcher, Alan; Quirke, Philip; Bühring, Hans-Jörg; Cook, Graham P; Blair, G Eric

2007-03-20

309

Evidence of Prognostic Relevant Expression Profiles of Heat-Shock Proteins and Glucose-Regulated Proteins in Oesophageal Adenocarcinomas  

PubMed Central

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27(Ser15), p-HSP27(Ser78), p-HSP27(Ser82), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27(Ser15, Ser78, Ser82) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p?=?0.015) and multivariate analysis (p?=?0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies. PMID:22911792

Bauer, Karina; Wolff, Claudia; Malinowsky, Katharina; Bauer, Lukas; Drecoll, Enken; Bettstetter, Marcus; Feith, Marcus; Walch, Axel; Höfler, Heinz; Becker, Karl-Friedrich; Langer, Rupert

2012-01-01

310

Rift Valley Fever Virus Structural and Nonstructural Proteins: Recombinant Protein Expression and Immunoreactivity Against Antisera from Sheep  

PubMed Central

Abstract The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA). PMID:23962238

Faburay, Bonto; Wilson, William; McVey, D. Scott; Drolet, Barbara S.; Weingartl, Hana; Madden, Daniel; Young, Alan; Ma, Wenjun

2013-01-01

311

Poly C binding protein, a single-stranded DNA binding protein, regulates mouse mu-opioid receptor gene expression.  

PubMed

Previously, a single-stranded (ss) DNA element, polypyrimidine (PPy) element, was found to be important for the proximal promoter activity of mouse micro-opioid receptor (MOR) gene in a neuronal cell model. In this study, we identified the presence of unknown ssDNA binding proteins specifically bound to MOR ssPPy element in the mouse brain, implicating the physiological significance of these proteins. To identify the ssDNA binding proteins, yeast one-hybrid system with PPy element as the bait was used to screen a mouse brain cDNA library. The clone encoding poly C binding protein (PCBP) was obtained. Its full-length cDNA sequence and protein with molecular weight approximately 38 kDa were confirmed. Electrophoretic mobility shift analysis (EMSA) revealed that PCBP bound to ssPPy element, but not doubled-stranded, in a sequence-specific manner. EMSA with anti-PCBP antibody demonstrated the involvement of PCBP in MOR ssPPy/proteins complexes of mouse brain and MOR expressing neuroblastoma NMB cells. Functional analysis showed that PCBP trans-activated MOR promoter as well as a heterologous promoter containing MOR PPy element. Importantly, ectopic expression of PCBP in NMB cells up-regulated the expression level of endogenous MOR gene in vivo in a dose-dependent manner. Collectively, above results suggest that PCBP participates in neuronal MOR gene expression. PMID:15836633

Ko, Jane L; Loh, Horace H

2005-05-01

312

Differential expression of proteins in brain regions of Alzheimer's disease patients.  

PubMed

Alzheimer's disease (AD), a progressive neurodegenerative disorder and the most common form of dementia and cognitive impairment is usually characterized by neuritic amyloid plaques, cerebrovascular amyloidosis and neurofibrillary tangles. In order to find out the pathological protein expression, a quantitative proteome analysis of AD hippocampus, substantia nigra and cortex was performed and the extent of protein expression variation not only in contrast to age-matched controls but also among the understudied regions was analyzed. Expression alterations of 48 proteins were observed in each region along with significant co/contra regulation of malate dehydrogenase, lactate dehydrogenase B chain, aconitate hydratase, protein NipSnap homolog 2, actin cytoplasmic 1, creatine kinase U-type and glyceraldehyde-3-phosphate dehydrogenase. These differentially expressed proteins are mainly involved in energy metabolism, cytoskeleton integration, apoptosis and several other potent cellular/molecular processes. Interaction association network analysis further confirms the close interacting relationship between the co/contra regulated differentially expressed proteins among all the three regions. Elucidation of co/contra regulation of differentially expressed proteins will be helpful to understand disease progression and functional alterations associated with AD. PMID:24306222

Zahid, Saadia; Oellerich, Michael; Asif, Abdul R; Ahmed, Nikhat

2014-01-01

313

Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes.  

PubMed

Both Xenopus laevis oocytes and mammalian cells are widely used for heterologous expression of several classes of proteins, and membrane proteins especially, such as ion channels or receptors, have been extensively investigated in both cell types. A full characterization of a specific protein will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual-function vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels. PMID:11911656

Jespersen, T; Grunnet, M; Angelo, K; Klaerke, D A; Olesen, S P

2002-03-01

314

Fluorescent Protein-Expressing Neural Progenitor Cells as a Tool for Transplantation Studies  

PubMed Central

The purpose of this study was to generate quadruple fluorescent protein (QFP) transgenic mice as a source for QFP-expressing neural stem and progenitor cells (NSCs/NPCs) that could be utilized as a tool for transplantation research. When undifferentiated, these NSCs only express cyan fluorescent protein (CFP); however, upon neuronal differentiation, the cells express yellow fluorescent protein (YFP). During astrocytic differentiation, the cells express green fluorescent protein (GFP), and during oligodendrocytic differentiation, the cells express red fluorescent protein (DsRed). Using immunocytochemistry, immunoblotting, flow cytometry and electrophysiology, quadruple transgenic NPCs (Q-NPCs) and GFP-sorted NPCs were comprehensively characterized in vitro. Overall, the various transgenes did not significantly affect proliferation and differentiation of transgenic NPCs in comparison to wild-type NPCs. In contrast to a strong CFP and GFP expression in vitro, NPCs did not express YFP and dsRed either during proliferation or after differentiation in vitro. GFP-positive sorted NPCs, expressing GFP under the control of the human GFAP promoter, demonstrated a significant improvement in astroglial differentiation in comparison to GFP-negative sorted NPCs. In contrast to non-sorted and GFP-positive sorted NPCs, GFP-negative sorted NPCs demonstrated a high proportion of neuronal differentiation and proved to be functional in vitro. At 6 weeks after the intracerebroventricular transplantation of Q-NPCs into neonatal wild-type mice, CFP/DCX (doublecortin) double-positive transplanted cells were observed. The Q-NPCs did not express any other fluorescent proteins and did not mature into neuronal or glial cells. Although this model failed to visualize NPC differentiation in vivo, we determined that activation of the NPC glial fibrillary acid protein (GFAP) promoter, as indicated by GFP expression, can be used to separate neuronal and glial progenitors as a valuable tool for transplantation studies. PMID:24932758

Skardelly, Marco; Hempel, Eileen; Hirrlinger, Johannes; Wegner, Florian; Meixensberger, Jürgen; Milosevic, Javorina

2014-01-01

315

Fluorescent protein-expressing neural progenitor cells as a tool for transplantation studies.  

PubMed

The purpose of this study was to generate quadruple fluorescent protein (QFP) transgenic mice as a source for QFP-expressing neural stem and progenitor cells (NSCs/NPCs) that could be utilized as a tool for transplantation research. When undifferentiated, these NSCs only express cyan fluorescent protein (CFP); however, upon neuronal differentiation, the cells express yellow fluorescent protein (YFP). During astrocytic differentiation, the cells express green fluorescent protein (GFP), and during oligodendrocytic differentiation, the cells express red fluorescent protein (DsRed). Using immunocytochemistry, immunoblotting, flow cytometry and electrophysiology, quadruple transgenic NPCs (Q-NPCs) and GFP-sorted NPCs were comprehensively characterized in vitro. Overall, the various transgenes did not significantly affect proliferation and differentiation of transgenic NPCs in comparison to wild-type NPCs. In contrast to a strong CFP and GFP expression in vitro, NPCs did not express YFP and dsRed either during proliferation or after differentiation in vitro. GFP-positive sorted NPCs, expressing GFP under the control of the human GFAP promoter, demonstrated a significant improvement in astroglial differentiation in comparison to GFP-negative sorted NPCs. In contrast to non-sorted and GFP-positive sorted NPCs, GFP-negative sorted NPCs demonstrated a high proportion of neuronal differentiation and proved to be functional in vitro. At 6 weeks after the intracerebroventricular transplantation of Q-NPCs into neonatal wild-type mice, CFP/DCX (doublecortin) double-positive transplanted cells were observed. The Q-NPCs did not express any other fluorescent proteins and did not mature into neuronal or glial cells. Although this model failed to visualize NPC differentiation in vivo, we determined that activation of the NPC glial fibrillary acid protein (GFAP) promoter, as indicated by GFP expression, can be used to separate neuronal and glial progenitors as a valuable tool for transplantation studies. PMID:24932758

Skardelly, Marco; Hempel, Eileen; Hirrlinger, Johannes; Wegner, Florian; Meixensberger, Jürgen; Milosevic, Javorina

2014-01-01

316

Proteomic identification of differentially expressed proteins in sea cucumber Apostichopus japonicus coelomocytes after Vibrio splendidus infection.  

PubMed

Skin ulceration syndrome (SUS) was the main limitation in the development of Apostichopus japonicus culture industries. To better understand how Vibrio splendidus modulates SUS outbreak, the immune response of A. japonicus coelomocytes after the pathogen challenge were investigated through comparative proteomics approach, and differentially expressed proteins were screened and characterized in the present study. A total of 40 protein spots representing 30 entries were identified at 24, 72 and 96 h post-infection. Of these proteins, 32 were up-regulated and 8 were down-regulated in the V. splendidus challenged samples compared to those of control. These differentially expressed proteins were mainly classified into four categories by GO analysis, in which approximate 33% of proteins showed to be related to immunity response. The mRNA expression levels of 6 differentially expressed proteins were further validated by qRT-PCR. Similar protein-mRNA-level expression patterns were detected in genes of phospholipase (spot 4), G protein (spot 20), annexin (spot 30) and filamin (spot 31). Whilst the levels of ficolin (spot 12) and calumenin (spot 14) transcripts were not corresponded with those of their translation products. These data provide a new insight to understand the molecular immune mechanism of sea cucumber responsive towards pathogen infection. PMID:24468075

Zhang, Peng; Li, Chenghua; Li, Ye; Zhang, Pengjuan; Shao, Yina; Jin, Chunhua; Li, Taiwu

2014-06-01

317

Expression and purification of half-smooth tongue sole (Cynoglossus semilaevis) CSDAZL protein.  

PubMed

The csdazl gene is a sex related gene of half-smooth tongue sole (Cynoglossus semilaevis). Our research group have cloned full length cDNA of csdazl, and studied its expression pattern. To get the further information of csdazl, we constructed a prokaryotic expression plasmid, pET-32a-CSDAZL, expressed in Escherichia coli BL21 and purified the fusion protein by His Trap. In order to detect the biological activity of the fusion protein, we injected the protein with liposome into fish, and detected other sex-related genes' mRNA expression. The results showed that the expression levels of half-smooth tongue sole female-related genes Cyp19a and Foxl2 significantly decreased between 6 and 24 h; however, both genes' expressions returned to their normal levels 72 h after injection, indicating that recombinant CSDAZL protein could down-regulated the expression of female-related genes, Foxl2 and Cyp19a genes, implying that the fusion protein has biological activity and csdazl plays a role in sex differentiation by regulating sex related genes' expression. PMID:25064428

Wang, Kailin; Zhang, Hong; Hu, Qiaomu; Shao, Changwei; Chen, Songlin

2014-10-01

318

Pichia pastoris expressed EtMic2 protein as a potential vaccine against chicken coccidiosis.  

PubMed

Chicken coccidiosis caused by Eimeria species leads to tremendous economic losses to the avian industry worldwide. Identification of parasite life cycle specific antigens is a critical step in recombinant protein vaccine development against Eimeria infections. In the present study, we amplified and cloned the microneme-2 (EtMIC2) gene from Eimeria tenella wild type strain SD-01, and expressed the EtMic2 protein using Pichia pastoris and Escherichia coli expression systems, respectively. The EtMic2 proteins expressed by P. pastoris and E. coli were used as vaccines to immunize chickens and their protective efficacies were compared and evaluated. The results indicated that both P. pastoris and E. coli expressed EtMic2 proteins exhibited good immunogenicity in stimulating host immune responses and the Pichia expressed EtMic2 provided better protection than the E. coli expressed EtMic2 did by significantly increasing growth rate, inducing high specific antibody response, reducing the oocyst output and cecal lesions. Particularly, the Pichia expressed EtMic2 protein exhibited much better ability in inducing cell mediated immune response than the E. coli expressed EtMic2. PMID:25047705

Zhang, Jie; Chen, Peipei; Sun, Hui; Liu, Qing; Wang, Longjiang; Wang, Tiantian; Shi, Wenyan; Li, Hongmei; Xiao, Yihong; Wang, Pengfei; Wang, Fangkun; Zhao, Xiaomin

2014-09-15

319

Utility of proteomics techniques for assessing protein expression  

Technology Transfer Automated Retrieval System (TEKTRAN)

In recent years, proteomic technologies have been frequently used as an effective analytical tool for examining modifications of protein profiles for accessing the bio-safety of genetically modified crops (GMO). Understanding of natural variation of soybean seed proteins is critical for determining...

320

Staphylococcus aureus and Pseudomonas aeruginosa Express and Secrete Human Surfactant Proteins  

PubMed Central

Surfactant proteins (SP), originally known from human lung surfactant, are essential to proper respiratory function in that they lower the surface tension of the alveoli. They are also important components of the innate immune system. The functional significance of these proteins is currently reflected by a very large and growing number of publications. The objective goal of this study was to elucidate whether Staphylococcus aureus and Pseudomonas aeruginosa is able to express surfactant proteins. 10 different strains of S. aureus and P. aeruginosa were analyzed by means of RT-PCR, Western blot analysis, ELISA, immunofluorescence microscopy and immunoelectron microscopy. The unexpected and surprising finding revealed in this study is that different strains of S. aureus and P. aeruginosa express and secrete proteins that react with currently commercially available antibodies to known human surfactant proteins. Our results strongly suggest that the bacteria are either able to express ‘human-like’ surfactant proteins on their own or that commercially available primers and antibodies to human surfactant proteins detect identical bacterial proteins and genes. The results may reflect the existence of a new group of bacterial surfactant proteins and DNA currently lacking in the relevant sequence and structure databases. At any rate, our knowledge of human surfactant proteins obtained from immunological and molecular biological studies may have been falsified by the presence of bacterial proteins and DNA and therefore requires critical reassessment. PMID:23349731

Worlitzsch, Dieter; Bensel, Tobias; Sawers, R. Gary; Paulsen, Friedrich

2013-01-01

321

Expression of a Phytophthora sojae necrosis-inducing protein occurs during transition from biotrophy to necrotrophy.  

PubMed

Phytophthora sojae is an oomycete that causes stem and root rot on soybean plants. To discover pathogen factors that produce disease symptoms or activate plant defense responses, we identified putative secretory proteins from expressed sequence tags (ESTs) and tested selected candidates using a heterologous expression assay. From an analysis of 3035 ESTs originating from mycelium, zoospore, and infected soybean tissues, we identified 176 putative secreted proteins. A total of 16 different cDNAs predicted to encode secreted proteins ranging in size from 6 to 26 kDa were selected for expression analysis in Nicotiana benthamiana using an Agrobacterium tumefaciens binary potato virus X (PVX) vector. This resulted in the identification of a 25.6-kDa necrosis-inducing protein that is similar in sequence to other proteins from eukaryotic and prokaryotic species. The genomic region encoding the P. sojae necrosis-inducing protein was isolated and the expression pattern of the corresponding gene determined by RNA blot hybridization and by RT-PCR. The activity of this P. sojae protein was compared to proteins of similar sequence from Fusarium oxysporum, Bacillus halodurans, and Streptomyces coelicolor by PVX-based expression in N. benthamiana and by transient expression via particle bombardment in soybean tissues. The P. sojae protein was a powerful inducer of necrosis and cell death in both assays, whereas related proteins from other species varied in their activity. This study suggests that the P. sojae necrosis-inducing protein facilitates the colonization of host tissues during the necrotrophic phase of growth. PMID:12410814

Qutob, Dinah; Kamoun, Sophien; Gijzen, Mark

2002-11-01

322

Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. Black-Right-Pointing-Pointer Adipose lipin-1 expression is reduced in obesity. Black-Right-Pointing-Pointer Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. Black-Right-Pointing-Pointer Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-{kappa}B activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan) [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Yoshizaki, Takayuki [Innovation Center, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065 (Japan)] [Innovation Center, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065 (Japan); Hiranaka, Natsumi; Suzuki, Takeshi [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Kanazawa, Kaoru [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yoshida, Mika; Naito, Sumiyoshi [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Fujiya, Mikihiro; Kohgo, Yutaka [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan)] [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Ieko, Masahiro [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)

2011-11-11

323

Increase in the expression of a family of small guanosine triphosphate-binding proteins, rab proteins, during induced phagocyte differentiation.  

PubMed Central

Rab is a newly identified family of small G-proteins that share 35-70% homology with the yeast Sec4p and Ypt1p involved in the regulation of the secretory pathway. Mature phagocytes display functions requiring organized intracellular traffic and, for this reason, we questioned whether phagocyte differentiation could correlate with the increased expression of rab proteins. Rabbit antisera raised against the recombinant proteins rab1Ap, 2p, 4p, and 6p were able to detect the corresponding proteins in the human monoblast leukemic cell line U937. When these cells were induced to differentiate into monocyte/macrophage-like cells displaying functional characteristics of a normal phagocyte, rab1Ap, 2p, 4p, and 6p were increased and this correlated with an increase in the rab transcripts. Using a rab5 probe, we also observed an increased expression of the rab5 gene in differentiated cells. Similarly, differentiation of the human leukemic myeloblast HL60 cell line along either monocyte or granulocyte pathways induced an increased expression of the rab proteins. Rab proteins were also detected in human neutrophils and in guinea pig alveolar macrophages. As degranulation is one of the phagocyte functions acquired in the late stage of differentiation, we investigated whether rab proteins would be involved in this process. Although rab proteins were tightly membrane bound, none of them was detected in the specific or azurophil granules purified from human neutrophils. The increased expression of rab proteins in mature phagocytes suggests that they may promote functions highly developed in these cells. Images PMID:1847940

Maridonneau-Parini, I; Yang, C Z; Bornens, M; Goud, B

1991-01-01

324

Intraclonal Protein Expression Heterogeneity in Recombinant CHO Cells  

PubMed Central

Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise minimization may therefore be a novel strategy to reduce apparent expression instability and simplify cell line selection. PMID:20037651

Pilbrough, Warren; Munro, Trent P.; Gray, Peter

2009-01-01

325

Differential expression of proteins in red pear following fruit bagging treatment.  

PubMed

Fruit bagging is a very effective method for study of fruit qualities and anthocyanin synthesis. The characterization of differentially expressed proteins that were isolated from both bagged and normal fruit skin tissue is apparently an essential parameter for understanding the effect of shading on fruit qualities and to understand the mechanism of fruit coloring in Pyrus communis. Proteome maps of both bagged and normal P. communis 'Placer' fruit skin were obtained by performing two-dimensional electrophoresis analysis and compared to assess the extent to which protein distribution differed in pear skin. The comparative analysis showed 38 differentially expressed proteins between the two samples: with three protein spots up-regulated and 35 down-regulated in the bagged fruit. Differentially expressed protein spots were subjected to matrix-assisted laser desorption ionization time of flight (MALDI-TOF) analysis and the data compared to that of known proteins to deduce their possible functions. Of these, 21 protein spots were identified and classified into functional classes. These identified proteins were mainly involved in photosynthesis, signal transduction, energy pathway, protein folding and assembly, and carbohydrate and acidity metabolisms, and were under-expressed in bagged fruit skins. This work provides a first characterization of the proteome changes in response to fruit bagging treatment in red pears. PMID:21409615

Feng, Shouqian; Chen, Xuesen; Zhang, Yanmin; Wang, Yanling; Song, Yang; Chen, Xiao-liu; Li, Xiugen; Li, Min; Liu, Jin; Wang, Quanzheng; Liu, Meiyan

2011-03-01

326

Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.  

PubMed

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained. PMID:18256902

Yue, Chang-Wu; Zhang, Yi-Zheng

2009-03-01

327

In-Vivo Real-Time Control of Protein Expression from Endogenous and Synthetic Gene Networks  

E-print Network

We describe an innovative experimental and computational approach to control the expression of a protein in a population of yeast cells. We designed a simple control algorithm to automatically regulate the administration ...

Menolascina, Filippo

328

Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae  

SciTech Connect

Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.

Ito, Keisuke; Sugawara, Taishi [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan); Shiroishi, Mitsunori [Iwata Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501 (Japan); Tokuda, Natsuko [Department of Medical Chemistry, Kyoto University, Faculty of Medicine, Konoe-cho, Yoshida, Sakyo-Ku, Kyoto 606-8501 (Japan); Kurokawa, Azusa; Misaka, Takumi [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan); Makyio, Hisayoshi; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro [Iwata Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501 (Japan); Nomura, Norimichi; Murata, Takeshi [Iwata Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501 (Japan); Department of Medical Chemistry, Kyoto University, Faculty of Medicine, Konoe-cho, Yoshida, Sakyo-Ku, Kyoto 606-8501 (Japan); Abe, Keiko [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan); Iwata, So [Iwata Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501 (Japan); Department of Medical Chemistry, Kyoto University, Faculty of Medicine, Konoe-cho, Yoshida, Sakyo-Ku, Kyoto 606-8501 (Japan); RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ (United Kingdom)], E-mail: s.iwata@mfour.med.kyoto-u.ac.jp (and others)

2008-07-11

329

Searching limiting steps in the expression of chloroplast-encoded proteins: relations between gene copy number,  

E-print Network

copy number, transcription, transcript abundance and translation rate in the chloroplast expression is widespread in the prokaryote kingdom, mainly because translation essentially occurs co-transcriptionally number, transcription, transcript abundance and synthesis of photosynthetic proteins in the chloroplast

330

Exact protein distributions for stochastic models of gene expression using partitioning of Poisson processes  

E-print Network

Stochasticity in gene expression gives rise to fluctuations in protein levels across a population of genetically identical cells. Such fluctuations can lead to phenotypic variation in clonal populations, hence there is considerable interest in quantifying noise in gene expression using stochastic models. However, obtaining exact analytical results for protein distributions has been an intractable task for all but the simplest models. Here, we invoke the partitioning property of Poisson processes to develop a mapping that significantly simplifies the analysis of stochastic models of gene expression. The mapping leads to exact protein distributions using results for mRNA distributions in models with promoter-based regulation. Using this approach, we derive exact analytical results for steady-state and time-dependent distributions for the basic 2-stage model of gene expression. Furthermore, we show how the mapping leads to exact protein distributions for extensions of the basic model that include the effects of ...

Pendar, Hodjat; Kulkarni, Rahul V

2013-01-01

331

Context-dependent perturbation of neural systems in transgenic mice expressing a cytosolic prion protein  

E-print Network

We analyzed the relationship between pathogenic protein expression and perturbations to brain anatomy and physiology in a genetic model of prion disease. In this model, the mouse line 1D4, neuropathology is promoted by ...

Lindquist, Susan

332

VCU/Old Dominion University study finds CD97 gene expression and function correlate with WT1 protein expression  

Cancer.gov

Researchers at Virginia Commonwealth University Medical Center's VCU Massey Cancer Center and Harold F. Young Neurosurgical Center (Richmond, VA) and Old Dominion University (Norfolk, VA) have discovered that suppression of Wilms tumor 1 protein (WT1) results in downregulation of CD97 gene expression in three glioblastoma cell lines and reduces the characteristic invasiveness exhibited by glial tumor cells.

333

Expression and affinity purification of recombinant proteins from plants  

NASA Technical Reports Server (NTRS)

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

2002-01-01

334

Immobilization of foreign protein in BmNPV polyhedra by fusion expression with partial polyhedrin fragments.  

PubMed

Bombyx mori nucleopolyhedrovirus (BmNPV) produces large, proteinaceous crystal matrix named polyhedra, which occlude progeny virions which are produced during infection and protect virions from hostile environmental conditions. In this study, five overlapping N-terminal fragments of the BmNPV polyhedrin ORF were cloned and ligated with the foreign gene egfp, and five recombinant baculoviruses were constructed by BmNPV(Polh(+)) Bac-to-Bac baculovirus expression system was used to co-express the polyhedrin and fused protein. The results showed that the fusion proteins were highly expressed, and the foreign proteins fused with the 100aa fragment of polyhedrin could be embedded into polyhedra at a higher ratio. This study provides a new method for efficient preservation of useful proteins for the development of new biopesticide with toxin protein and delivery vector system of vaccines. PMID:24008009

Chen, Lin; Xiang, Xingwei; Yang, Rui; Hu, Xiaolong; Cao, Cuiping; Malik, Firdose Ahmad; Wu, Xiaofeng

2013-12-01

335

Co-expression of human agouti-related protein enhances expression and stability of human melanocortin-4 receptor.  

PubMed

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling proteins, and they are considered major targets of approximately half of all therapeutic agents. Human melanocortin-4 receptor (hMC4R) plays an important role in the control of energy homeostasis, and its mutants are directly related to severe human obesity. Here, we describe optimized protocols for the high-yield expression and purification of hMC4R that will accelerate structural study. Truncations of the N- and C-termini of hMC4R with T4 lysozyme (T4L) insertion increase the solubility as well as stability of the protein. Strikingly, co-expression of human mini-agouti-related protein (mini-AgRP) in Spodoptera frugiperda (Sf9) cells enables excellent stability of hMC4R. The protein yield in the human mini-AgRP co-expression system is increased by about 3-4times compared to that of hMC4R alone. Data from analytical size exclusion chromatography (aSEC) and thermostability assay show that hMC4R becomes homogeneous and stable with a melting temperature of 58°C in the presence of human mini-AgRP. PMID:25446108

Yun, Ji-Hye; Kim, Kuglae; Jung, Youngjin; Park, Jae-Hyun; Cho, Hyun-Soo; Lee, Weontae

2015-01-01

336

Over-expression of proteins using a modified pBAD24 vector in E. coli expression system.  

PubMed

A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence elements and a modified Shine-Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/10(6) c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/10(6) c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful prokaryotic expression system for efficient repression, modulation and elevated protein expression levels. PMID:19330488

Banerjee, Sampali; Salunkhe, Shardul S; Apte-Deshpande, Anjali D; Mandi, Naganath S; Mandal, Goutam; Padmanabhan, Sriram

2009-07-01

337

Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli  

Microsoft Academic Search

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a\\u000a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several

Chang-Wu Yue; Yi-Zheng Zhang

2009-01-01

338

Growth and recombinant protein expression with Escherichia coli in different batch cultivation media  

Microsoft Academic Search

Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple\\u000a batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently\\u000a used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH)

Ralf Hortsch; Dirk Weuster-Botz

2011-01-01

339

Prognostic value of NDRG1 and SPARC protein expression in breast cancer patients  

Microsoft Academic Search

An increasing number of studies have shown altered expression of secreted protein acidic and rich in cysteine (SPARC) and\\u000a N-myc down-regulated gene (NDRG1) in several malignancies, including breast carcinoma; however, the role of these potential\\u000a biomarkers in tumor development and progression is controversial. In this study, NDRG1 and SPARC protein expression was evaluated\\u000a by immunohistochemistry on tissue microarrays containing breast

Maria Aparecida Nagai; Renê Gerhard; José Humberto T. G. Fregnani; Suely Nonogaki; Regina Barbosa Rierger; Mário Mourão Netto; Fernando A. Soares

2011-01-01

340

Increased Tolerance to Two Oomycete Pathogens in Transgenic Tobacco Expressing Pathogenesis-Related Protein 1a  

Microsoft Academic Search

Expression of pathogenesis-related protein 1a (PR-1a), a protein of unknown biochemical function, is induced to high levels in tobacco in response to pathogen infection. The induction of PR-1a expression is tightly correlated with the onset of systemic acquired resistance (SAR), a defense response effective against a variety of fungal, viral, and bacterial pathogens. While PR-1a has been postulated to be

Danny Alexander; Robert M. Goodman; Manuela Gut-Rella; Christopher Glascock; Kristianna Weymann; Leslie Friedrich; Daryl Maddox; Patricia Ahl-Goy; Tom Luntz; Eric Ward; John Ryals

1993-01-01

341

YKL-40 protein expression in normal adult human tissues – an immunohistochemical study  

Microsoft Academic Search

YKL-40, a 40 kDa plasma protein, is secreted by macrophages, neutrophils, chondrocytes, vascular smooth muscle cells and cancer\\u000a cells. High plasma YKL-40 is found in patients with inflammatory diseases and cancer, but it is not known how the protein\\u000a is expressed in tissues. This immunohistochemical study was carried out with the purpose of mapping and grading cytoplasmic\\u000a expression of YKL-40 in

Merete Ringsholt; Estrid V. S. Høgdall; Julia S. Johansen; Paul A. Price; Lise H. Christensen

2007-01-01

342

Purification and expression of fatty acid binding proteins in chicken liver and intestine  

E-print Network

PURIFICATION AND EXPRESSION OF FATTY ACID BINDING PROTEINS IN CHICKEN LIVER AND INTESTINE A Thesis by JULIA ELLEN SEWELL Submitted to the Graduate College of Texas A&M University in partial fulfillment of the requirements for the degree... of MASTER OF SCIENCE August 1988 Major subject: Nutrition PURIFICATION AND EXPRESSION OF FATTY ACID BINDING PROTEINS IN CHICKEN LIVER AND INTESTINE A Thesis by JULIA ELLEN SEWELL Approved as to style and content by: Pamela S. Hargi (Chair of Committ...

Sewell, Julia Ellen

1988-01-01

343

Regional Expression of Key Cell Cycle Proteins in Brain from Subjects with Amnestic Mild Cognitive Impairment  

Microsoft Academic Search

Mild cognitive impairment (MCI) is regarded as a transition stage between the cognitive changes of normal aging and the more\\u000a serious problems caused by Alzheimer’s disease (AD). Previous studies had demonstrated increased expression of cell cycle\\u000a proteins in AD brain. In the present study, we have analyzed the expression of the cell cycle proteins, CDK2, CDK5 and cyclin\\u000a G1 in

Rukhsana Sultana; D. Allan Butterfield

2007-01-01

344

Expression of the gene encoding the PR-like protein PRms in germinating maize embryos  

Microsoft Academic Search

The PRms protein is a pathogenesis-related (PR)-like protein whose mRNA accumulates during germination of maize seeds. Expression of the PRms gene is induced after infection of maize seeds with the fungus Fusarium moniliforme. To further our investigations on the expression of the PRms gene we examined the accumulation of PRms mRNA in different tissues of maize seedlings infected with E.

Josep M. Casacuberta; Dora Raventós; Pere Puigdoménech; Blanca San Segundo

1992-01-01

345

Protein Expression Analysis in Temparature-adaptated Mutant Strains of Moderately thermophile Geobacillus stearothermophilus  

Microsoft Academic Search

Optimum growth of moderately thermophile Geobacillus stearothermophilus (Bst) has been observed at 55ºC, and suppressed by the downshift to lower temperature. In order to understand the relationship between growth temperature and protein expression, we made Bst mutant strains which were adapted to lower temperature (30ºC) compared with wild type strain, and then we examined their protein expression under various growth

Hitomi Kyogoku; Yasuaki Yamaguchi; Momoyo Miyano-Ono; Ayano Yamaguchi; Takanori Satoh

346

Membrane-Expressed and Extracellular Stress Proteins in Infectious Disease  

Microsoft Academic Search

\\u000a This chapter focuses on the dichotomous effects of eukaryotic and microbial heat shock proteins (HSPs) with molecular weights\\u000a of 60, 70, and 90 kDa on the host’s immune system. It has previously been shown that membrane-bound and extracellular heat\\u000a shock proteins elicit potent anti-cancer immune responses. Herein are discussed the immunostimulatory and immunosuppressive\\u000a properties of heat shock proteins in bacterial and

Gabriele Multhoff

347

NF-?B1 inhibits c-Myc protein degradation through suppression of FBW7 expression  

PubMed Central

NF-?B is a well-known transcription factor in regulation of multiple gene transcription and biological processes, and most of them are relied on its transcriptional activity of the p65/RelA subunit, while biological function of another ubiquitously expressed subunit NF-?B1 (p50) remains largely unknown due to lack transcriptional activation domain. Here we discovered a novel biological function of p50 as a regulator of oncogenic c-Myc protein degradation upon arsenite treatment in a NF-?B transcriptional-independent mechanism. Our results found that p50 was crucial for c-Myc protein induction following arsenite treatment by using specific knockdown and deletion of p50 in its normal expressed cells as well as reconstituting expression of p50 in its deficient cells. Subsequently we showed that p50 upregulated c-Myc protein expression mainly through inhibiting its degradation. We also identified that p50 exhibited this novel property by suppression of FBW7 expression. FBW7 was profoundly upregulated in p50-defecient cells in comparison to that in p50 intact cells, whereas knockdown of FBW7 in p50-/- cells restored arsenite-induced c-Myc protein accumulation, assuring that FBW7 up-regulation was responsible for defect of c-Myc protein expression in p50-/- cells. In addition, we discovered that p50 suppressed fbw7 gene transcription via inhibiting transcription factor E2F1 transactivation. Collectively, our studies demonstrated a novel function of p50 as a regulator of c-Myc protein degradation, contributing to our notion that p50-regulated protein expression through multiple levels at protein translation and degradation, further providing a significant insight into the understanding of biomedical significance of p50 protein. PMID:24457827

Huang, Haishan; Ma, Li; Li, Jingxia; Yu, Yonghui; Zhang, Dongyun; Wei, Jinlong; Jin, Honglei; Xu, Derek; Gao, Jimin; Huang, Chuanshu

2014-01-01

348

Changes in HSP gene and protein expression in natural scrapie with brain damage  

PubMed Central

Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease. PMID:21314976

2011-01-01

349

Suppression of polyglutamine protein toxicity by co-expression of a heat-shock protein 40 and a heat-shock protein 110  

PubMed Central

A network of heat-shock proteins mediates cellular protein homeostasis, and has a fundamental role in preventing aggregation-associated neurodegenerative diseases. In a Drosophila model of polyglutamine (polyQ) disease, the HSP40 family protein, DNAJ-1, is a superior suppressor of toxicity caused by the aggregation of polyQ containing proteins. Here, we demonstrate that one specific HSP110 protein, 70?kDa heat-shock cognate protein cb (HSC70cb), interacts physically and genetically with DNAJ-1 in vivo, and that HSC70cb is necessary for DNAJ-1 to suppress polyglutamine-induced cell death in Drosophila. Expression of HSC70cb together with DNAJ-1 significantly enhanced the suppressive effects of DNAJ-1 on polyQ-induced neurodegeneration, whereas expression of HSC70cb alone did not suppress neurodegeneration in Drosophila models of either general polyQ disease or Huntington's disease. Furthermore, expression of a human HSP40, DNAJB1, together with a human HSP110, APG-1, protected cells from polyQ-induced neural degeneration in flies, whereas expression of either component alone had little effect. Our data provide a functional link between HSP40 and HSP110 in suppressing the cytotoxicity of aggregation-prone proteins, and suggest that HSP40 and HSP110 function together in protein homeostasis control. PMID:24091676

Kuo, Y; Ren, S; Lao, U; Edgar, B A; Wang, T

2013-01-01

350

Expression of proteins involved in epigenetic regulation in human cutaneous melanoma and peritumoral skin.  

PubMed

Epigenetic processes play a critical role in melanoma development. However, little is known about proteins responsible for epigenetic transformations in melanoma cells. The processes in the peritumoral skin within the excision margin are almost unstudied. We studied the changes in expression of 112 proteins involved in epigenetic regulation of gene expression in the human cutaneous melanoma and its peritumoral zone using "The Proteomic Antibody Microarrays" (GRAA2, Sigma-Aldrich). Dimethylated histone H3 at lysines 4 and 9 as well as proteins involved in the regulation of transcription (histone deacetylases HDAC-1 and HDAC-11, DNA methyl-binding protein Kaiso), cell cycle control (protein kinases Aurora-? and PKR, chromosome protein CENP-E , and phosphorylated and acetylated histone H3), DNA repair (phosphorylated histone H2AX), and nuclear protein import (importins ?3 and ?5/7) were over-expressed in the melanoma tissue as compared with normal skin. At the same time, HDAC-10 and proliferating cell nuclear antigen PCNA were downregulated. In the peritumoral skin, at the excision margin (1-2 cm from the melanoma edge), we observed similar changes in expression of these proteins and, additionally, over-expression of arginine methyltransferases PRMT5 and NAD-dependent histone deacetylase SIR2. Histone methyltransferase G9a and metastasis-associated protein 2 were downregulated. Therefore, epigenetic regulation that requires histone modifications and expression of some regulatory proteins is of importance for melanoma development and propagation. The observed changes in the peritumoral skin may indicate the epigenetic pre-tuning in this zone possibly involved in malignant transformation. These results can be potentially useful for melanoma diagnostics and targeted therapy. PMID:24850177

Uzdensky, Anatoly; Demyanenko, Svetlana; Bibov, Mikhail; Sharifulina, Svetlana; Kit, Oleg; Przhedetski, Yury; Pozdnyakova, Viktoria

2014-08-01

351

Brain Expressed and X-Linked (Bex) Proteins Are Intrinsically Disordered Proteins (IDPs) and Form New Signaling Hubs  

PubMed Central

Intrinsically disordered proteins (IDPs) are abundant in complex organisms. Due to their promiscuous nature and their ability to adopt several conformations IDPs constitute important points of network regulation. The family of Brain Expressed and X-linked (Bex) proteins consists of 5 members in humans (Bex1-5). Recent reports have implicated Bex proteins in transcriptional regulation and signaling pathways involved in neurodegeneration, cancer, cell cycle and tumor growth. However, structural and biophysical data for this protein family is almost non-existent. We used bioinformatics analyses to show that Bex proteins contain long regions of intrinsic disorder which are conserved across all members. Moreover, we confirmed the intrinsic disorder by circular dichroism spectroscopy of Bex1 after expression and purification in E. coli. These observations strongly suggest that Bex proteins constitute a new group of IDPs. Based on these findings, together with the demonstrated promiscuity of Bex proteins and their involvement in different signaling pathways, we propose that Bex family members play important roles in the formation of protein network hubs. PMID:25612294

Fernandez, Eva M.; Díaz-Ceso, María D.; Vilar, Marçal

2015-01-01

352

High-throughput expression and purification of membrane proteins  

PubMed Central

High-throughput (HT) methodologies have had a tremendous impact on structural biology of soluble proteins. High-resolution structure determination relies on the ability of the macromolecule to form ordered crystals that diffract X-rays. While crystallization remains somewhat empirical, for a given protein, success is proportional to the number of conditions screened and to the number of variants trialed. HT techniques have greatly increased the number of targets that can be trialed and the rate at which these can be produced. In terms of number of structures solved, membrane proteins appear to be lagging many years behind their soluble counterparts. Likewise, HT methodologies for production and characterization of these hydrophobic macromolecules are only now emerging. Presented here is an HT platform designed exclusively for membrane proteins that has processed over 5000 targets. PMID:20394823

Mancia, Filippo; Love, James

2010-01-01

353

Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans.  

PubMed

High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack post-translational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work, we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with (15)N,(13)C by providing them with isotopically labeled bacteria. (2)H labeling also was achieved by growing C. elegans in the presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the "test" GPCR to demonstrate the viability of this approach. Although the worms' cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization. PMID:25461480

Salom, David; Cao, Pengxiu; Yuan, Yiyuan; Miyagi, Masaru; Feng, Zhaoyang; Palczewski, Krzysztof

2015-03-01

354

Expression of fas protein on CD4+T cells irradiated by low level He-Ne  

NASA Astrophysics Data System (ADS)

Objective: To investigate the influence on the Expression of Fas protein on CD4+ T cells irradiated by low level He-Ne laser in the cases of psoriasis. Methods:the expression of CD4+ T Fas protein was determined in the casee of psoriasis(n=5) pre and post-low level laser irradiation(30 min?60min and 120min)by flow cytometry as compared withthe control(n=5). Results:In the cases of psoriasis,the expression of CD4+T FAS protein 21.4+/-3.1% was increased significantly than that of control group 16.8+/-2.1% pre-irradiation, p<0.05in the control,there is no difference between pre and post- irradiation,p>0.05in the cases , the expression of CD4+T Fas protein wae positively corelated to the irradiation times, when the energy density arrived to 22.92J/cm2?60 minutes?and 45.84J/cm2?120minutes?, the expression of CD4+ T Fas protein was increased significantly as compared with pre-irradiation,p<0.05.Conclusion: The expression of CD4+T Fas protein may be increased by low level He-Ne laser irradiation ,the uncontrolled status of apoptosis could be corrected.

Nie, Fan; Zhu, Jing; Zhang, Hui-Guo

2005-07-01

355

Characterization of the expression of MHC proteins in human embryonic stem cells  

PubMed Central

Human embryonic stem (ES) cells are pluripotent cells that may be used in transplantation medicine. These cells can be induced to differentiate into cells from the three embryonic germ layers both in vivo and in vitro. To determine whether human ES cells might be rejected after transplantation, we examined cell surface expression of the MHC proteins in these cells. Our results show very low expression levels of MHC class I (MHC-I) proteins on the surface of human ES cells that moderately increase on in vitro or in vivo differentiation. A dramatic induction of MHC-I proteins was observed when the cells were treated with IFN-? but not with IFN-? or -?. However, all three IFNs induced expression of MHC-I proteins in differentiated human ES cells. MHC-II proteins and HLA-G were not expressed on the surface of undifferentiated or differentiated cells. Ligands for natural killer cell receptors were either absent or expressed in very low levels in human ES cells and in their differentiated derivatives. In accordance, natural killer cytotoxic assays demonstrated only limited lysis of both undifferentiated and differentiated cells. To initiate a histocompatibility databank of human ES cells, we have isotyped several of the published ES cell lines for their human leukocyte antigens. In conclusion, our results demonstrate that human ES cells can express high levels of MHC-I proteins and thus may be rejected on transplantation. PMID:12114532

Drukker, Micha; Katz, Gil; Urbach, Achia; Schuldiner, Maya; Markel, Gal; Itskovitz-Eldor, Joseph; Reubinoff, Benjamin; Mandelboim, Ofer; Benvenisty, Nissim

2002-01-01

356

Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystropy  

SciTech Connect

The myotonic dystrophy mutation has recently been identified; however, the molecular mechanism of the disease is still unknown. The sequence of the myotonin-protein kinase gene was determined, and messenger RNA spliced forms were identified in various tissues. Antisera were developed for analytical studies. Quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to demonstrate that decreased levels of the messenger RNA and protein expression are associated with adult form of myotonic dystropy. 12 refs., 5 figs.

Yinghui Fu; Friedman, D.L.; Richards, S.; Pearlman, J.A.; Gibbs, R.A.; Pizzuti, A.; Perryman, M.B.; Fenwick, R.G. Jr.; Caskey, C.T. (Baylor College of Medicine, Houston, TX (United States)); Ashizawa, Tetsuo (Taylor College of Medicine, Houston, TX (United States) Veterans Administration Medical Center, Houston, TX (United States)); Scarlato, G. (Universita di Milano, Milan (Italy))

1993-04-09

357

Parasitization by Scleroderma guani influences protein expression in Tenebrio molitor pupae.  

PubMed

Ectoparasitoid wasps deposit their eggs onto the surface and inject venom into their hosts. Venoms are chemically complex and they exert substantial impact on hosts, including permanent or temporary paralysis and developmental arrest. These visible venom effects are due to changes in expression of genes encoding physiologically relevant proteins. While the influence of parasitization on gene expression in several lepidopterans has been reported, the molecular details of parasitoid/beetle relationships remain mostly unknown. This shortcoming led us to pose the hypothesis that envenomation by the ectoparasitic ant-like bethylid wasp Scleroderma guani leads to changes in protein expression in the yellow mealworm beetle Tenebrio molitor. We tested our hypothesis by comparing the proteomes of non-parasitized and parasitized host pupae using iTRAQ-based proteomics. We identified 41 proteins that were differentially expressed (32?- and 9?-regulated) in parasitized pupae. We assigned these proteins to functional categories, including immunity, stress and detoxification, energy metabolism, development, cytoskeleton, signaling and others. We recorded parallel changes in mRNA levels and protein abundance in 14 selected proteins following parasitization. Our findings support our hypothesis by documenting changes in protein expression in parasitized hosts. PMID:24852673

Zhu, Jia-Ying; Wu, Guo-Xing; Ze, Sang-Zi; Stanley, David W; Yang, Bin

2014-07-01

358

Fatty acids increase hepatitis B virus X protein stabilization and HBx-induced inflammatory gene expression.  

PubMed

The protein level of human hepatitis B virus (HBV) in infection is variable, depending on patient context. We previously reported that HBV X protein (HBx) induces hepatic lipid accumulation and inflammation. Here, we show that abnormal levels of hepatic fatty acids increase HBx protein stability during HBV expression, resulting in the potentiation of HBx-induced inflammation. Reactive oxygen species, Ca(2+) signaling and expression levels of various lipid metabolic genes were investigated in HBx-expressing cells and in HBx transgenic mice. Fatty acids, including palmitate, stearate and oleate, increased HBx protein stability by preventing proteasome-dependent degradation. Hepatic inflammation induced by a high fat diet (HFD) and HBx was measured based on the expression of interleukin-6 and tumor necrosis factor ?. In addition, the protein level of HBx increased in HFD-HBx transgenic mice. Reactive oxygen species production and intracellular Ca(2+) signal activation play critical roles in fatty-acid-induced HBx stabilization. Abnormal levels of hepatic fatty acids resulted in synergistic induction of HBx protein and liver inflammatory gene expression through HBx protein stabilization. These results indicate that different fatty acid levels in the liver might affect HBV-induced pathogenesis. PMID:24612645

Cho, Hyun Kook; Kim, So Young; Yoo, Seong Keun; Choi, Yung Hyun; Cheong, Jaehun

2014-05-01

359

Pooled ORF Expression Technology (POET) USING PROTEOMICS TO SCREEN POOLS OF OPEN READING FRAMES FOR PROTEIN EXPRESSION*S  

E-print Network

Pooled ORF Expression Technology (POET) USING PROTEOMICS TO SCREEN POOLS OF OPEN READING FRAMES developed a pooled ORF expression technology, POET, that uses recombinational cloning and proteomic methods called pooled ORF expression technology (POET)1 that avoids many of the logistical issues associated

360

Evaluation of Affinity-Tagged Protein Expression Strategies using Local and Global Isotope Ratio Measurements  

SciTech Connect

Protein enrichments of engineered, affinity-tagged (or bait ) fusion proteins with interaction partners are often laden with background, non-specific proteins, due to interactions that occur in vitro as an artifact of the technique. Furthermore, the in vivo expression of the bait protein may itself affect physiology or metabolism. In this study, intrinsic affinity purification challenges were investigated in a model protein complex, DNA-dependent RNA polymerase (RNAP), encompassing chromosome- and plasmid-encoding strategies for bait proteins in two different microbial species: Escherichia coli and Rhodopseudomonas palustris. Isotope ratio measurements of bait protein expression strains relative to native, wild-type strains were performed by liquid chromatography tandem mass spectrometry (LC-MS-MS) to assess bait protein expression strategies in each species. Authentic interacting proteins of RNAP were successfully discerned from artifactual co-isolating proteins by the isotopic differentiation of interactions as random or targeted (I-DIRT) method (A. J. Tackett et al. J. Proteome Res. 2005, 4 (5), 1752-1756). To investigate broader effects of bait protein production in the bacteria, we compared proteomes from strains harboring a plasmid that encodes an affinity-tagged subunit (RpoA) of the RNAP complex with the corresponding wild-type strains using stable isotope metabolic labeling. The ratio of RpoA abundance in plasmid strains versus wild type was 0.8 for R. palustris and 1.7 for E. coli. While most other proteins showed no appreciable difference, proteins significantly increased in abundance in plasmid-encoded bait-expressing strains of both species included the plasmid encoded antibiotic resistance protein, GenR and proteins involved in amino acid biosynthesis. Together, these local, complex-specific and more global, whole proteome isotopic abundance ratio measurements provided a tool for evaluating both in vivo and in vitro effects of plasmid-encoding strategies for bait protein expression. This approach has the potential for enabling discovery of protein-protein interactions among the growing number of sequenced microbial species without the need for development of chromosomal insertion systems.

Hervey, IV, William Judson [ORNL; Khalsa-Moyers, Gurusahai K [ORNL; Lankford, Patricia K [ORNL; Owens, Elizabeth T [ORNL; McKeown, Catherine K [ORNL; Lu, Tse-Yuan S [ORNL; Foote, Linda J [ORNL; Morrell-Falvey, Jennifer L [ORNL; McDonald, W Hayes [ORNL; Pelletier, Dale A [ORNL; Hurst, Gregory {Greg} B [ORNL

2009-01-01

361

Expression, Purification, and Functional Analysis of the TyrR Protein of Haemophilus influenzae  

Microsoft Academic Search

The gene that was inferred to encode the TyrR protein ofHaemophilus influenzaeRd was synthesized by polymerase chain reaction and inserted into a T7-based expression vector. Methods were developed to overexpress the TyrR protein ofH. influenzaeinEscherichia coliand to purify the protein on a large scale. Bothin vitroandin vivofunctional comparisons of theH. influenzaeandE. coliTyrR proteins were carried out. The TyrR protein ofH.

Qin Zhu; Shimin Zhao; Ronald L. Somerville

1997-01-01

362

Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection.  

PubMed

Cocktail of polyclonal antibodies (PAb) were produced that will help in multiple virus detection and overcome the limitation of individual virus purification, protein expression and purification as well as immunization in multiple rabbits. A dual fusion construct was developed using conserved coat protein (CP) sequences of Cucumber mosaic virus (CMV) and Papaya ringspot virus (PRSV) in an expression vector, pET-28a(+). The fusion protein (?40kDa) was expressed in Escherichia coli and purified. Likewise, a triple fusion construct was developed by fusing conserved CP sequences of CMV and PRSV with conserved nucleocapsid protein (N) sequence of Groundnut bud necrosis virus (GBNV) and expressed as a fusion protein (?50kDa) in pET-28a(+). PAb made separately to each of these three viruses recognized the double and triple fusion proteins in Western blot indicating retention of desired epitopes for binding with target antibodies. The fusion proteins (?40kDa and ?50kDa) were used to produce cocktail of PAb by immunizing rabbits, which simultaneously detected natural infection of CMV and PRSV or CMV, PRSV and GBNV in Cucurbitaceous, Solanaceous and other hosts in DAC-ELISA. This is the first report on production of a cocktail of PAb to recombinant fusion protein of two or three distinct viruses. PMID:24161814

Kapoor, Reetika; Mandal, Bikash; Paul, Prabir Kumar; Chigurupati, Phaneendra; Jain, Rakesh Kumar

2014-02-01

363

Epitope expression and hyperphosphorylation of tau protein in corticobasal degeneration: differentiation from progressive supranuclear palsy  

Microsoft Academic Search

Corticobasal degeneration (CBD) is a rare, progressive neurological disorder characterized by widespread neuronal and glial accumulation of abnormal tau protein. Using immunohistochemistry we analyzed tau epitope expression and phosphorylation state in CBD and compared them to cytoskeletal changes in Alzheimer's disease (AD) and progressive supranuclear palsy (PSP). Epitopes spanning the entire length of the tau protein were present in CBD

M. B. Feany; H. Ksiezak-Reding; W.-K. Liu; I. Vincent; S.-H. C. Yen; D. W. Dickson

1995-01-01

364

Papillomavirus Capsid Protein Expression in Escherichia coli: Purification and Assembly of HPV11  

E-print Network

Papillomavirus Capsid Protein Expression in Escherichia coli: Purification and Assembly of HPV11 proteins of human papillomavirus (HPV) types 11 and 16 were puri®ed and analyzed for structural integrity for vaccine design. # 2001 Academic Press Keywords: human papillomavirus; virus assembly; virus capsids

Harrison, Stephen C.

365

Heat shock protein expression in thermotolerant and thermosensitive lines of cotton  

Microsoft Academic Search

The expression of heat shock proteins (HSPs) was compared between genetically characterized heat tolerant and heat sensitive lines of cotton (Gossypium hirsutum andG. barbadense) using electrophoretic analysis ofin vivo labelled proteins. No differences were observed between the two lines with regard to: the temperature at which HSP synthesis was induced (37°C); the temperature at which HSP synthesis was maximal (45°C);

Susan E. Fender; Mary A. O'Connell

1989-01-01

366

Differences in expression of retinal proteins between diabetic and normal rats  

PubMed Central

AIM: To compare and identify the differences in expression of retinal proteins between normal and diabetic rats, and to analyze the molecular pathogenetic mechanisms of retinal diseases caused by diabetes. METHODS: Changes in protein expression of retinal tissues from diabetic and normal rats were observed using 2-dimensional polyacrylamide gel electrophoresis (2-DE). Some protein spots exhibiting statistically significant variations (P < 0.05) were selected randomly and identified by tandem mass spectrometry and analyzed by bioinformatics. RESULTS: 2-DE showed that the expression was up-regulated in 5 retinal proteins, down-regulated in 23 retinal proteins, and disappeared in 8 retinal proteins. Eight spots were identified from the 36 spots by tandem mass spectrometry (MS/MS) and analyzed by bioinformatics. Guanylate kinase 1, triosephosphate isomerase 1, ATP synthase subunit d, albumin and dimethylarginine dimethylaminohydrolase 2 played an important role in signal transduction. Triosephosphate isomerase 1, crystallin alpha B, ATP synthase subunit d and peroxiredoxin 6 were involved in energy metabolism of retinal tissues. Guanylate kinase 1 played an important role in photoexcitation of retinal rod photoreceptor cells. Whether crystallin beta A1 plays a role in diabetic retinas is unknown so far. CONCLUSION: There are differences in expression of retinal proteins between diabetic and normal rats. These proteins may be involved in the mechanisms and prognosis of retinal diseases caused by diabetes. PMID:17465459

Liu, Shang-Qing; Kang, Jian; Li, Cheng-Jun; Tang, En-Jie; Wen, Bin; Cai, Rong; Yang, Hui-Jun

2007-01-01

367

Sistemas de expresión para proteínas terapéuticas recombinantes Expression Systems for Therapeutic Recombinant Proteins  

Microsoft Academic Search

Several biopharmaceutical products based on recombinant proteins are manufactured at present by biopharma- ceutical industry. Expression systems to obtain biopharmaceuticals are based on cloning vectors of genes coding for the protein of interest into a suitable host cell such as bacteria, yeast and cellular lines derived from mammals. Several biopharmaceuticals such as clotting factors, hormones, cytokines and enzymes have been

Maria Elisa Drago Serrano

368

Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement  

NASA Astrophysics Data System (ADS)

Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

Zhang, Yuanxing

369

Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein  

Microsoft Academic Search

Background & Aims:The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood. Indirect evidence suggests that oxidative stress and mitochondrial injury play a role. The aim of this study was to determine if the HCV core protein itself alters mitochondrial function and contributes to oxidative stress.Methods:HCV core protein was expressed in 3 different cell lines,

Michiari Okuda; Kui Li; Michael R. Beard; Lori A. Showalter; Frank Scholle; Stanley M. Lemon; Steven A. Weinman

2002-01-01

370

Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens  

PubMed Central

Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens. PMID:24948937

Zanetti, Flavia Adriana; Grand, María Daniela Conte; Mitarotonda, Romina Cristina; Taboga, Oscar Alberto; Calamante, Gabriela

2014-01-01

371

Identification of a Novel Type of Silk Protein and Regulation of Its Expression*  

E-print Network

Identification of a Novel Type of Silk Protein and Regulation of Its Expression* (Received The silk of lepidopteran insects has been studied ex- tensively as proteins of two categories: the fibroins, which are produced in the posterior section of silk glands, and the sericins, which are secreted

Â?urovec, Michal

372

EEffects of cardiopulmonary bypass on tight junction protein expressions in intestinal mucosa of rats  

Microsoft Academic Search

AIM: To investigate the tight junction protein expressions of intestinal mucosa in an experimental model of cardiopulmonary bypass (CPB) in rats. METHODS: Thirty anesthetized rats were randomly divided into two groups: Group S (n = 10) served as sham operation and group C (n = 20) served as CPB which underwent CPB for 1 h. Expression of occludin and zonula

Ying-Jie Sun; Wei-Min Chen; Tie-Zheng Zhang; Hui-Juan Cao; Jin Zhou

2008-01-01

373

Human ?-Defensin Expression Is Not Dependent on CCAAT/Enhancer Binding Protein-? in a Murine Model  

PubMed Central

Specific granule deficiency (SGD) is a rare congenital disorder characterized by recurrent infections. The disease is caused by inactivating mutations of the CCAAT/enhancer binding protein-? (C/EBP-?) gene. As a consequence, specific and gelatinase granules lack most matrix proteins. Furthermore, azurophil granules contain diminished amounts of their most abundant proteins, ?-defensins, also known as human neutrophil peptides (HNPs). In accordance with this, in vitro models have demonstrated induction of HNPs by C/EBP-?. Since mice do not express myeloid defensins, they cannot per se be used to characterize the role of C/EBP-? in controlling HNP expression in vivo. We therefore crossed a transgenic HNP-1-expressing mouse with the Cebpe-/- mouse to study the in vivo significance of C/EBP-? for HNP-1 transcription and expression. Surprisingly, neither expression nor processing of HNP-1 was affected by lack of C/EBP-? in these mice. Transduction of C/EBP-? into primary bone marrow cells from HNP-1 mice induced some HNP-1 expression, but not to levels comparable to expression human cells. Taken together, our data infer that the HNP-1 of the transgenic mouse does not show an expression pattern equivalent to endogenous secondary granule proteins. This limits the use of these transgenic mice as a model for human conditions. PMID:24658030

Glenthøj, Andreas; Dahl, Sara; Larsen, Maria T.; Cowland, Jack B.; Borregaard, Niels

2014-01-01

374

Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids  

E-print Network

Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids Amir. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing

375

Phosphate stimulates Matrix Gla Protein expression in chondrocytes through the ERK signaling pathway  

E-print Network

1 Phosphate stimulates Matrix Gla Protein expression in chondrocytes through the ERK signaling. Email: Jerome.guicheux@nantes.inserm.fr Short title Phosphate stimulates MGP expression via ERK-regulated kinase 1 and 2 (ERK1/2) was performed in rib organ cultures from newborn mice. Results indicated that Pi

Boyer, Edmond

376

Adipose Triglyceride Lipase and Hormone-Sensitive Lipase Protein Expression Is Decreased in the Obese  

E-print Network

Adipose Triglyceride Lipase and Hormone-Sensitive Lipase Protein Expression Is Decreased discovered adipose triglyceride lipase (ATGL). The aim of the present study was to examine whether ATGL non- HSL lipase adipose triglyceride lipase (ATGL), being pre- dominantly expressed in adipose tissue

Paris-Sud XI, Université de

377

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes.  

PubMed

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co-localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post-meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2-GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post-meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage-specific spermatogenic cell proteins and developmental events. genesis 52:976-984, 2014. © 2014 Wiley Periodicals, Inc. PMID:25293348

Brown, Paula R; Odet, Fanny; Bortner, Carl D; Eddy, Edward M

2014-12-01

378

A novel copper-regulated promoter system for expression of heterologous proteins in Schizosaccharomyces pombe  

E-print Network

A novel copper-regulated promoter system for expression of heterologous proteins the promoter of the ctr41 copper transporter gene from S. pombe, we created a series of vectors, named pctr41 -X, which regulate the expression of heterologous genes as a function of copper availability

Labbé, Simon

379

The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function  

Microsoft Academic Search

Two methods are described for using the jellyfish green fluorescent protein (GFP) as a reporter gene for ion channel expression. GFP fluorescence can be used to identify the transfected cells, and to estimate the relative levels of ion channel expression, in cotransfection experiments. A GFP-NMDAR1 chimera can be constructed that produces a functional, fluorescent receptor subunit. These methods should facilitate

John Marshall; Raymond Molloy; Guy W. J Moss; James R Howe; Thomas E Hughes

1995-01-01

380

Altered expression of cell cycle and apoptotic proteins in chronic hepatitis C virus infection  

PubMed Central

Background A disrupted cell cycle progression of hepatocytes was reported in chronic hepatitis C virus (HCV) infection, which can contribute significantly in the associated pathogenesis. The present study aimed to further elaborate these disruptions by evaluating the expression of key cell cycle and apoptotic proteins in chronic HCV infection with particular reference to genotype 3. Archival liver biopsy specimens of chronic HCV-infection (n = 46) and normal histology (n = 5) were analyzed by immunohistochemistry using antibodies against proliferation marker Mcm-2, G1 phase marker Cyclin D1, S phase marker Cyclin A, cell cycle regulators p21 (CDK inhibitor) and p53 (tumor suppressor protein), apoptotic protein Caspase-3 and anti-apoptotic protein Bcl-2. Results Elevated Mcm-2 expression was observed in hepatocytes in chronic HCV infection, indicating increased cell cycle entry. Cyclin D1 expression was higher than cyclin A, which suggests a slow progression through the G1 phase. Expression of cell cycle regulators p21 and p53 was elevated, with no concordance between their expressions. The Mcm-2 and p21 expressions were associated with the fibrosis stage (p = 0.0001 and 0.001 respectively) and that of p53 with the inflammation grade (p = 0.051). Apoptotic marker, Caspase-3, was mostly confined to sinusoidal lining cells with little expression in hepatocytes. Anti-apoptotic protein, Bcl-2, was negligible in hepatocytes and detected principally in infiltrating lymphocytes. Expression of all these proteins was unrelated to the HCV genotype and were detected only rarely in the hepatocytes of normal liver. Conclusion The results showed an arrested cell cycle state in the hepatocytes of chronic HCV infection, regardless of any association with genotype 3. Cell cycle arrest is characterized by an increased expression of p21, in relation to fibrosis, and of p53 in relation to inflammation. Furthermore, expression of p21 was independent of the p53 expression and coincided with the reduced expression of apoptotic protein Caspase-3 in hepatocytes. The altered expression of these cell cycle proteins in hepatocytes is suggestive of an impaired cell cycle progression that could limit the regenerative response of the liver to ongoing injury, leading to the progression of disease. PMID:18680610

Sarfraz, Saira; Hamid, Saeed; Siddiqui, Anwar; Hussain, Snawar; Pervez, Shahid; Alexander, Graeme

2008-01-01