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1

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

PubMed

P-Glycoprotein (P-gp) plays a major role in drug-drug and herb-drug interactions. Mulberroside A (Mul A) is one of the main bioactive constituents of Sangbaipi, the dried root-bark of Morus alba L. (white mulberry), which is officially listed in the Chinese Pharmacopoeia. In the present study, we investigated the effect of Mul A treatment on mRNA expression and protein expression of P-gp in the Caco-2 cells by real-time qPCR and Western blot analysis. The effect of Mul A treatment on the function of P-gp in vitro and in vivo was assessed by Rho123 transport assay and a pharmacokinetic study. The potential roles of protein kinase C (PKC) and nuclear factor kappa B (NF-?B) in the expression regulation of P-gp after Mul A treatment were also investigated. The results revealed that Mul A treatment significantly decreased the mRNA and protein expression of P-gp in Caco-2 cells after treatment with Mul A (5-20?M). Furthermore, Mul A treatment displayed apparently inhibitory effect on the function of P-gp both in vitro and in vivo. In addition, activation of PKC activity and NF-?B nuclear translocation were observed in the presence of Mul A, which suggested that PKC and NF-?B might play crucial roles in Mul A-induced suppression of P-gp. Our study demonstrated that Mul A treatment could down-regulate P-gp expression and function accompanied by the activation of PKC and NF-?B, and this should be taken into consideration in potential herb-drug interactions when Mul A or M. alba are co-administered with other drugs transported by P-gp. PMID:24530447

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

2014-04-25

2

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

PubMed Central

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.

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

2013-01-01

3

Dietary regulation of P-gp function and expression.  

PubMed

Food-drug interactions have been associated with clinically important pharmacokinetic and pharmacodynamic changes of a drug. The aim of this paper is to review the regulation of P-glycoprotein (P-gp) by dietary components and to correlate the changes in cellular P-gp function and expression with drug bioavailability. In summary, the published literature has provided extensive data supporting the modulation of drug bioavailability through P-gp regulation by components in food groups such as fruit juices, spices, herbs, cruciferous vegetables and green tea. Most of these data were, however, derived from in vitro cell models and, except for the St John's wort, the clinical significance of most reported interactions remains to be clarified. Studies on piperine and capsaicin have underscored an often poor correlation between in vivo and in vitro data, whereas experiments involving curcumin highlighted differences between acute and chronic consumption of a dietary component on P-gp function and expression in vivo. A better understanding of the pharmacokinetic and pharmacodynamic profiles of the dietary components will aid in addressing these knowledge gaps. PMID:19545213

Zhang, Wenxia; Han, Yi; Lim, Siok Lam; Lim, Lee Yong

2009-07-01

4

Expression of Chlamydia muridarum plasmid genes and immunogenicity of pGP3 and pGP4 in different mouse strains.  

PubMed

Chlamydia muridarum carries a cryptic plasmid (pMoPn) of 7.5kb, which encodes seven genes. Our aims were to describe the transcriptional pattern of the pMoPn genes in C. muridarum-infected mice and to evaluate the host immune responses against pGP3 and pGP4 proteins. BALB/c and C57BL/6N female mice were inoculated intranasally with C. muridarum and sacrificed at different time points, and the total RNA was extracted from the lung suspensions to determine the levels of expression of the different plasmid genes by RT qPCR. The supernatants of the lungs were subjected to the quantitation of recoverable C. muridarum. TCA04 and TCA05, which encode pGP3 and pGP4, respectively, were amplified by PCR and cloned into the pET vector. The proteins were overexpressed in E. coli HB101 and purified. Selected groups of BALB/c and C57BL/6N mice were infected with C. muridarum 1-3 times. The humoral immune responses in the sera of the mice to the proteins encoded by TCA04 and TCA05 were tested by Western blotting, and the cellular immune responses were assessed in lymphocyte proliferation assays. The proteins recognized by the mouse sera were further analysed by a LC/MSMS technique. The kinetics of C. muridarum growth were similar in the mouse strains used, but the pathogen burden was higher in the BALB/c mice in the late phase of infection. All the plasmid genes in the BALB/c mice showed an increased level of expression on day 7, whereas the expression of the same genes did not change on day 7 in the C57BL/6N mice. The levels of expression of the plasmid genes were higher in the C57BL/6N mice at later time points. In Western blot assays, the sera of the singly infected C57BL/6N mice reacted with the monomeric form of pGP3, whereas the sera of the singly infected BALB/c mice reacted with the trimeric form of pGP3. The sera of the multiply infected C57BL/6N mice also recognized pGP4. Similarly to the humoral immune response, cellular immune responses to pGP3 and pGP4 were detected in the C. muridarum-infected C57BL/6N mice, but the spleen cells of BALB/c mice responded with proliferation only to the pGP3 protein. These results suggest that the proteins encoded by pMoPn genes may modulate the host immune response during C. muridarum infection, and that the evolved immune response against plasmid proteins, similarly to that against other chlamydial proteins, depends on the genetic background of the host. PMID:24631212

Mosolygó, Tímea; Faludi, Ildikó; Balogh, Emese P; Szabó, Agnes M; Karai, Adrienn; Kerekes, Fanni; Virók, Dezs? P; Endrész, Valéria; Burián, Katalin

2014-05-01

5

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

6

Relationship between Permeability Glycoprotein (P-gp) Gene Expression and Enrofloxacin Metabolism in Nile Tilapia.  

PubMed

Abstract The aim of this study was to analyze the influence of permeability glycoprotein (P-gp) gene expression on enrofloxacin (ENR) metabolism in aquatic animals. Nile Tilapia Oreochomis niloticus were fed different doses of ENR ranging from 0 to 80 mg/kg. The P-gp gene expression levels were determined by quantitative real-time PCR (qRT-PCR) at indicated time points after drug administration. Drug metabolism was determined by HPLC. The P-gp gene expression in liver and kidney was greatly enhanced 30 min after ENR administration at 40 mg/kg, peaked 3 h after drug administration, and then gradually decreased. Thirty minutes after a single oral administration of ENR (0, 20, 40, or 80 mg/kg), the P-gp gene expression increased in a dose-dependent manner. The P-gp gene expression levels in the kidney were significantly higher than those in the liver. Additionally, the metabolic rate of ENR in kidney was more rapid than that in liver. Furthermore, a close correlation was found between P-gp gene expression and ENR concentrations. These results suggest that P-gp may be involved in the ENR metabolism process in Nile Tilapia, providing a novel model for the potential utility of gene expression and drug metabolism studies in aquatic animals. Received July 9, 2013; accepted October 17, 2013. PMID:24895858

Hu, Kun; Cheng, Gang; Zhang, Haixin; Wang, Huicong; Ruan, Jiming; Chen, Li; Fang, Wenhong; Yang, Xianle

2014-06-01

7

P-glycoprotein (P-gp) in the monogonont rotifer, Brachionus koreanus: molecular characterization and expression in response to pharmaceuticals.  

PubMed

P-glycoprotein is involved in the efflux of diverse chemicals, including hydrophobic compounds and pharmaceuticals as a first line of defense. Here, we firstly identified and characterized the P-gp (Bk-P-gp) gene in the rotifer, Brachionus koreanus. Bk-P-gp was highly conserved in genomic organization compared to the human P-gp gene. Messenger RNA expression of Bk-P-gp revealed that it would be regulated by temperature change via 14 heat shock response elements in its promoter region. Bk-P-gp showed a high similarity of motifs/domains compared to those of vertebrates in its amino acid sequences. To check whether Bk-P-gp would be inducible, we exposed B. koreanus to six pharmaceuticals including antibiotics for use in aquaculture and observed dose- and time-dependency on transcripts of Bk-P-gp for 24h over a wide range of concentration. Efflux assay and membrane topology supported its conserved function for transportation of a number of chemicals upon cellular damage. To reveal the effect of pharmaceuticals on the rotifer, we measured survival rate and population growth rate after exposure to six pharmaceuticals. In an acute toxicity test, both NOEC and LC?? values for all the pharmaceuticals were high for 24 h. ATP, CBZ, SMX, and TMP markedly inhibited the population growth of B. koreanus after exposure up to 100 mg/L for 10 days. In this paper, we demonstrated that various pharmaceuticals can retard growth rate with up-regulation of the P-gp gene as a cellular defense system. This finding provides a better understanding of molecular mechanisms involved in pharmaceutical-mediated cellular damage in B. koreanus. PMID:22446822

Rhee, Jae-Sung; Jeong, Chang-Bum; Kim, Bo-Mi; Lee, Jae-Seong

2012-06-15

8

ABC Transporter (P-gp/ABCB1, MRP1/ABCC1, BCRP/ABCG2) Expression in the Developing Human CNS  

PubMed Central

P-glycoprotein (P-gp/ABCB1), multidrug resistance protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) are plasma membrane efflux pumps that limit the intracellular uptake and retention of numerous lipophilic, amphipathic xeno- and endobiotics. Little is known about the neonatal and developmental expression of P-gp/ABCB1, MRP1/ABCC1, and BCRP/ABCG2 in the human central nervous system (CNS), therefore postmortem CNS tissue from infants born 220/7- 420/7 week gestation and adults was immunostained to determine their ontogeny and cellular localization. P-gp/ABCB1 imunostaining was observed in microvessel endothelial cells as early as 220/7 weeks, increasing in prevalence and intensity with maturation, and later in gestation in large pyramidal neurons. MRP1/ABCC1 immunostaining was prominent early in the choroid plexus and ventricular ependyma, and noted later in large pyramidal neurons. BCRP/ABCG2 expression was limited to microvessel endothelial cells. P-gp/ABCB1, MRP1/ABCC1 and BCRP/ABCG2 in adult brain matched term newborn CNS but with more intense immunostaining. We conclude that P-gp/ABCB1, MRP1/ABCC1, and BCRP/ABCG2 are expressed in a developmental, cell specific, fashion in the human CNS. The complementary pattern of P-gp/ABCB1 and BCRP/ABCG2 at the blood-brain with MRP1/ABCC1 at the blood-CSF barriers may limit CNS uptake and retention of drugs and toxins in neonates.

Daood, Monica J.; Tsai, Cathy; Ahdab-Barmada, Mamdouha; Watchko, Jon F.

2010-01-01

9

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

PubMed Central

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.

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

2013-01-01

10

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

PubMed

There is a controversy over the link between phenotypes of multidrug resistance (MDR) and clinical outcome in leukemia/lymphoma patients. This may be because the process behind the induction and loss of expression of genotypes and phenotypes by which MDR develops and the role of MDR in fresh cells of human leukemia/lymphoma are not clearly defined. P-glycoprotein (P-gp) increased and decreased along with mdr-1 expression in three cell lines out of five vincristine (VCR)-resistant cell lines. MRP appeared with increased mrp expression in the other two cell lines. After the drug was removed from the culture system, mdr-1/P-gp changed in parallel with the level of VCR resistance, although mrp and MRP did not. It was concluded that P-gp is directly derived from mdr-1 and that mdr-1/P-gp supports the VCR-resistance but mrp/MRP is not directly linked to the VCR-resistance. These results should contribute to a better understanding of MDR phenomenon in cancer. PMID:19414348

Hirose, Masao

2009-04-01

11

Increased malignant behavior in neuroblastoma cells with acquired multi-drug resistance does not depend on P-gp expression.  

PubMed

Acquisition of P-gp-mediated multidrug-resistance does not always correlate with observed malignant behavior of NB. To characterize alterations accompanying development of multidrug-resistance in NB we established two neuroblastoma cell sublines resistant to vincristine (UKF-NB-3rVCR10) and doxorubicin (UKF-NB-3rDOX20). UKF-NB-3rVCR10 and UKF-NB-3rDOX20 overexpressed functional P-gp and developed an increased malignant phenotype: presented constitutive phosphorylation of AKT, resistance to gamma-irradiation, and had increased survival in serum-free medium. Inhibition of P-gp restored chemosensitivity but did not affect increased survival in serum-free medium and sensitivity to gamma-irradiation. Inhibition of AKT had no influence on chemoresistance but restored sensitivity to serum starvation. Both resistant cell lines acquired additional chromosomal changes. UKF-NB-3rVCR10 cells acquired a missense P53 mutation in exon 5, an increased MYCN amplification, an enhanced adhesion to endothelium, a decreased NCAM expression, a distinctly higher clonogenicity, and an increased in vivo tumorigenicity. We conclude that acquisition of increased malignant behavior in neuroblastoma occurs concomitantly with multidrug-resistance and is P-gp-independent. PMID:16142320

Kotchetkov, Rouslan; Driever, Pablo Hernáiz; Cinatl, Jaroslav; Michaelis, Martin; Karaskova, Jana; Blaheta, Roman; Squire, Jeremy A; Von Deimling, Andreas; Moog, Jussi; Cinatl, Jindrich

2005-10-01

12

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

PubMed

P-glycoprotein (P-gp)-mediated efflux system plays an important role to maintain chemical balance in mammalian cells for endogenous and exogenous chemical compounds. However, despite the extensive characterisation of P-gp potential interaction with drug-like molecules, the interaction of carbon nanoparticles with this type of protein molecule is poorly understood. Thus, carbon nanoparticles were analysed, such as buckminsterfullerenes (C20, C60, C70), capped armchair single-walled carbon nanotube (SWCNT or C168), and P-gp interactions using different molecular docking techniques, such as gradient optimisation algorithm (ADVina), Lamarckian genetic algorithm (FastDock), and shape-based approach (PatchDock) to estimate the binding affinities between these structures. The theoretical results represented in this work show that fullerenes might be P-gp binders because of low levels of Gibbs free energy of binding (?G) and potential of mean force (PMF) values. Furthermore, the SWCNT binding is energetically unfavourable, leading to a total decrease in binding affinity by elevation of the residual area (Ares), which also affects the ?-? stacking mechanisms. Further, the obtained data could potentially call experimental studies using carbon nanostructures, such as SWCNT for development of drug delivery vehicles, to administer and assess drug-like chemical compounds to the target cells since organisms probably did not develop molecular sensing elements to detect these types of carbon molecules. PMID:24088267

Shityakov, Sergey; Förster, Carola

2013-01-01

13

Characterization of Human Colorectal Cancer MDR1/P-gp Fab Antibody  

PubMed Central

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

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

2013-01-01

14

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

?-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

Ma, Hongye; Cheng, Lei; Hao, Keji; Li, Yanping; Song, Xiaobo; Zhou, Huimin; Jia, Li

2014-01-01

15

A structural model for the mass action kinetic analysis of P-gp mediated transport through confluent cell monolayers.  

PubMed

The structural model for P-gp mediated transport across confluent cell monolayers uses the generally accepted mass action reactions for P-gp binding and efflux, together with the known structural parameters for P-gp (large substrate binding site accessible from the membrane) and the apical plasma membrane in which it resides (lipid bilayer partition coefficient of substrate and volume of apical plasma membrane allow estimation of substrate concentration at binding site). The model considers binding of substrate to P-gp from within the inner leaflet of the apical membrane, with an on rate constant, k 1 (M(-1)s(-1)), and off rate constant k r (s(-1)), as well as an efflux rate constant from P-gp into the apical chamber, k 2 (s(-1)). The model also explicitly estimates the active P-gp protein level, known as P-gp efflux active surface density T(0). For each new drug, fitting these parameters requires use of multiple initial drug concentrations and multiple time points at each concentration, until steady state is reached between P-gp-mediated efflux into the apical chamber and passive permeability from apical chamber back into the cytosol. Although this model optimally requires a larger than usual dataset for analysis, it does provide important mechanistic information through estimates of these on, off and efflux rate constants, as well as efflux active P-gp surface density. This more detailed description of efflux from polarized confluent cell monolayers has (1) provided insight into the unexpected relationship between P-gp IC50 and K i in this system, (2) highlighted the kinetic need for GF120918 inhibitable apical and basolateral uptake transporters for digoxin, and (3) provided possible explanations for the extreme lab-to-lab variability in P-gp IC50 values observed for inhibition of digoxin transport. This model can also be used to distinguish between efflux active P-gp and total apical plasma membrane P-gp, which may be important when P-gp is expressed in a microvillous membrane. PMID:24523118

Bentz, Joe; Ellens, Harma

2014-01-01

16

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

PubMed Central

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

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

2012-01-01

17

Involvement of AP-1 and NF-?B in the up-regulation of P-gp in vinblastine resistant Caco-2 cells.  

PubMed

Caco-2 is a widely used cell model in drug absorption and P-glycoprotein (P-gp, MDR1) substrate identification. Long-term vinblastine treatment of Caco-2 cells could increase the expression of P-gp; thus, the vinblastine resistant Caco-2 (Caco-2 vbl) cells can be used as a rapid and sensitive alternative model in identifying P-gp substrates. The mechanism of P-gp induction in this model is not clear; this study was therefore intended to clarify the possible factors involved in P-gp up-regulation in Caco-2 vbl cells. Since vinblastine is the inducer of both activator protein-1 (AP-1) and nuclear factor kappa B (NF-?B), we investigated the role of AP-1 and NF-?B in the regulation of MDR1 gene expression. Our results indicated that the AP-1 and NF-?B luciferase activity was higher in Caco-2 vbl cells than that in Caco-2 cells according to reporter gene assay. The mRNA expression of AP-1 subunit c-Jun and NF-?B was increased in Caco-2 vbl cells. The c-Jun inhibitor SP600125 and NF-?B inhibitor pyrrolidine dithiocarbamate (PDTC) suppressed the expression of MDR1 mRNA in Caco-2 vbl cells. In conclusion, this study provides the evidence that AP-1 and NF-?B are involved in the P-gp induction in Caco-2 vbl cells. PMID:24088727

Chen, Qiuxia; Bian, Yicong; Zeng, Su

2014-01-01

18

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

PubMed Central

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

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

2013-01-01

19

A multi-system approach assessing the interaction of anticonvulsants with P-gp.  

PubMed

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

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

2013-01-01

20

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

21

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

22

Expression of P-glycoprotein in southeastern oysters, Crassostrea virginica.  

PubMed

These studies provide important fundamental information regarding the expression of P-glycoprotein (p-gp) in southeastern oysters (Crassostrea virginica). Using rhodamine transport studies, p-gp activity was detected in newly fertilized embryos. A monoclonal antibody (C219) was used to evaluate p-gp expression in oyster tissues. On the basis of laboratory studies, p-gp expression tended to be higher in gill tissues than mantle tissues, and was generally not related to salinity differences. Seasonal studies were conducted with oysters collected monthly for 1 year from Lighthouse Creek, an unpolluted site. There was a general pattern of higher p-gp expression in the warmer months and lower expression in the colder months. In contrast, total gill protein concentrations decreased during the warmer months and increased during the colder months. These studies indicate that there are seasonal patterns in p-gp expression which may represent an adaptive response to natural stressors associated with summer conditions. PMID:11488357

Keppler, C J; Ringwood, A H

2001-07-01

23

Colchicine effect on P-glycoprotein expression and activity: In silico and in vitro studies.  

PubMed

Colchicine is a P-glycoprotein (P-gp) substrate that induces its expression, thus increasing the risk for unexpected pharmacokinetic interactions with this drug. Because increased P-gp expression does not always correlate with increased activity of this efflux pump, we evaluated the changes in both P-gp expression and activity induced by colchicine using an in vitro model. Caco-2 cells were incubated with 0.1-100?M colchicine up to 96h. Cytotoxicity was evaluated by the MTT and LDH leakage assays, P-gp expression and activity were evaluated by flow cytometry and P-gp ATPase activity was measured in MDR1-Sf9 membrane vesicles. Furthermore, colchicine fitting in P-gp induction and competitive inhibition pharmacophore hypothesis, and docking studies evaluating the interaction between colchicine and P-gp drug binding pocket were tested in silico. Significant cytotoxicity was noted after 48h. At 24h a significant increase in P-gp expression was observed, which was not accompanied by an increase in transport activity. Moreover, colchicine significantly increased P-gp ATPase activity, demonstrating to be actively transported by the pump. New pharmacophores were constructed to predict P-gp modulatory activity. Colchicine fitted both the P-gp induction and competitive inhibition models. In silico, colchicine was predicted to bind to the P-gp drug-binding pocket suggesting a competitive mechanism of transport. These results show that colchicine induced P-gp expression in Caco-2 cells but the activity of the protein remained unchanged, highlighting the need to simultaneously evaluate P-gp expression and activity. With the newly constructed pharmacophores, new drugs can be initially screened in silico to predict such potential pharmacokinetic interactions. PMID:24759273

Silva, Renata; Carmo, Helena; Vilas-Boas, Vânia; Barbosa, Daniel José; Palmeira, Andreia; Sousa, Emília; Carvalho, Félix; Bastos, Maria de Lourdes; Remião, Fernando

2014-07-25

24

Increased expression of sorcin is associated with multidrug resistance in leukemia cells via up-regulation of MDR1 expression through cAMP response element-binding protein.  

PubMed

Sorcin, a 22kDa Ca(2+) binding protein, was first identified in a vincristine-resistant Chinese hamster lung cell line, and was later demonstrated to be involved in the development of multidrug-resistance (MDR) phenotypes in a variety of human cancer cell lines. However, the exact role of sorcin in MDR cells is yet to be fully elucidated. Here we explored the role of sorcin in the development of MDR in leukemia cells, and revealed that the expression level of sorcin was directly correlated to the expression of MDR1/P-glycoprotein (P-gp). In addition, it was shown that sorcin induced the expression of MDR1/P-gp through a cAMP response element (CRE) between -716 and -709bp of the mdr1/p-gp gene. Furthermore, overexpression of sorcin increased the phosphorylation of CREB1 and the binding of CREB1 to the CRE sequence of mdr1/p-gp promoter, and induced the expression of MDR1/P-gp. These findings suggested that sorcin induces MDR1/P-gp expression markedly through activation of the CREB pathway and is associated with the MDR phenotype. The new findings may be helpful for understanding the mechanisms of MDR in human cancer cells, prompting its further investigation as a molecular target to overcome MDR. PMID:24796664

Yamagishi, Nobuyuki; Nakao, Ryota; Kondo, Rumi; Nishitsuji, Mai; Saito, Youhei; Kuga, Takahisa; Hatayama, Takumi; Nakayama, Yuji

2014-06-13

25

The role of inter-segmental differences in P-glycoprotein expression and activity along the rat small intestine in causing the double-peak phenomenon of substrate plasma concentration.  

PubMed

  Conflicting results have been reported on segmental differences in expression of P-glycoprotein (P-gp) along the small intestine of animals and humans. In this study, we investigated P-gp mRNA and protein levels within each of nine segments of rat small intestine. In addition, P-gp activity in each segment was evaluated in terms of permeability of rhodamine123 (Rho123), a typical P-gp substrate, using the serial intestinal non-everted sac method. The P-gp mRNA levels tended to increase from the duodenum to the ileum, with peaks in the upper and lower ileum, while P-gp protein level reached its maximum in the middle ileum. The activity of P-gp was also the highest in the middle ileum, and was highly correlated with P-gp protein level. The double-peaked plasma concentration profile that was observed following oral administration of Rho123 to rats could be well reproduced by an intestinal compartmental kinetic model incorporating inter-segmental differences of absorption and excretion rate constants. Our results suggest that the heterogeneous distribution of P-gp along the small intestine plays a key role in causing the double-peak of plasma concentration of P-gp substrates following oral administration to rats. PMID:22850759

Wada, Sho; Kano, Takashi; Mita, Suzune; Idota, Yoko; Morimoto, Kaori; Yamashita, Fumiyoshi; Ogihara, Takuo

2013-01-01

26

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function.  

PubMed

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs. PMID:21186402

Chai, Lilly; McLaren, Rajashree P; Byrne, Ann; Chuang, Wei-Lien; Huang, Yinyin; Dufault, Michael R; Pacheco, Joshua; Madhiwalla, Shruti; Zhang, Xiaokui; Zhang, Mindy; Teicher, Beverly A; Carter, Kara; Cheng, Seng H; Leonard, John P; Xiang, Yibin; Vasconcelles, Michael; Goldberg, Mark A; Copeland, Diane P; Klinger, Katherine W; Lillie, James; Madden, Stephen L; Jiang, Yide A

2011-03-01

27

Differential expression of DNA topoisomerase II alpha and -beta in P-gp and MRP-negative VM26, mAMSA and mitoxantrone-resistant sublines of the human SCLC cell line GLC4.  

PubMed Central

Sublines of the human small-cell lung carcinoma (SCLC) cell line GLC4 with acquired resistance to teniposide, amsacrine and mitoxantrone (GLC4/VM20x, GLC4/AM3x and GLC4/MIT60x, respectively) were derived to study the contribution of DNA topoisomerase II alpha and -beta (TopoII alpha and -beta) to resistance for TopoII-targeting drugs. The cell lines did not overexpress P-glycoprotein or the multidrug resistance-associated protein but were cross-resistant to other TopoII drugs. GLC4/VM20x showed a major decrease in TopoII alpha protein (54%; for all assays presented in this paper the GLC4 level was defined to be 100%) without reduction in TopoII beta protein; GLC4/AM3x showed only a major decrease in TopoII beta protein (to 18%) and not in TopoII alpha. In GLC4/MIT60x, the TopoII alpha and -beta protein levels were both decreased (TopoII alpha to 31%; TopoII beta protein was undetectable). The decrease in TopoII alpha protein in GLC4/VM20x and GLC4/MIT60x, was mediated by decreased TopoII alpha mRNA levels. Loss of TopoII alpha gene copies contributed to the mRNA decrease in these cell lines. Only in the GLC4/MIT60x cell line was an accumulation defect observed for the drug against which the cell line was made resistant. In conclusion, TopoII alpha and -beta levels were decreased differentially in the resistant cell lines, suggesting that resistance to these drugs may be mediated by a decrease in a specific isozyme. Images Figure 1 Figure 3

Withoff, S.; de Vries, E. G.; Keith, W. N.; Nienhuis, E. F.; van der Graaf, W. T.; Uges, D. R.; Mulder, N. H.

1996-01-01

28

MDR1 synonymous polymorphisms alter transporter specificity and protein stability in a stable epithelial monolayer.  

PubMed

The drug efflux function of P-glycoprotein (P-gp) encoded by MDR1 can be influenced by genetic polymorphisms, including two synonymous changes in the coding region of MDR1. Here we report that the conformation of P-gp and its drug efflux activity can be altered by synonymous polymorphisms in stable epithelial monolayers expressing P-gp. Several cell lines with similar MDR1 DNA copy number were developed and termed LLC-MDR1-WT (expresses wild-type P-gp), LLC-MDR1-3H (expresses common haplotype P-gp), and LLC-MDR1-3HA (a mutant that carries a different valine codon in position 3435). These cell lines express similar levels of recombinant mRNA and protein. P-gp in each case is localized on the apical surface of polarized cells. However, the haplotype and its mutant P-gps fold differently from the wild-type, as determined by UIC2 antibody shift assays and limited proteolysis assays. Surface biotinylation experiments suggest that the non-wild-type P-gps have longer recycling times. Drug transport assays show that wild-type and haplotype P-gp respond differently to P-gp inhibitors that block efflux of rhodamine 123 or mitoxantrone. In addition, cytotoxicity assays show that the LLC-MDR1-3H cells are more resistant to mitoxantrone than the LLC-MDR1-WT cells after being treated with a P-gp inhibitor. Expression of polymorphic P-gp, however, does not affect the host cell's morphology, growth rate, or monolayer formation. Also, ATPase activity assays indicate that neither basal nor drug-stimulated ATPase activities are affected in the variant P-gps. Taken together, our findings indicate that "silent" polymorphisms significantly change P-gp function, which would be expected to affect interindividual drug disposition and response. PMID:24305879

Fung, King Leung; Pan, James; Ohnuma, Shinobu; Lund, Paul E; Pixley, Jessica N; Kimchi-Sarfaty, Chava; Ambudkar, Suresh V; Gottesman, Michael M

2014-01-15

29

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

PubMed

Primary objective of this investigation was to delineate the differential impact of efflux transporters P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) on brain disposition and plasma pharmacokinetics of pazopanib. In addition, this research investigated whether inhibition of these efflux transporters with clinically relevant efflux modulators canertinib or erlotinib could be a viable strategy for improving pazopanib brain delivery. In vitro assays with MDCKII cell monolayers suggested that pazopanib is a high affinity substrate for Bcrp1 and a moderate substrate for P-gp. Co-incubation with specific transport inhibitors restored cell accumulation and completely abolished the directionality of pazopanib flux. Brain and plasma pharmacokinetic studies were conducted in FVB wild type mice in the absence and presence of specific transport inhibitors. Drug levels in plasma and brain were determined using a validated high performance liquid chromatography method using vandetanib as an internal standard. In vivo studies indicated that specific inhibition of either P-gp (by zosuquidar or LY335979) or Bcrp1 (by Ko143) alone did not significantly alter pazopanib brain accumulation. However, dual P-gp/Bcrp1 inhibition by elacridar (GF120918), significantly enhanced pazopanib brain penetration by ~5-fold without altering its plasma concentrations. Thus, even though Bcrp1 showed higher affinity towards pazopanib in vitro, in vivo at the mouse BBB both P-gp and Bcrp1 act in concert to limit brain accumulation of pazopanib. Furthermore, erlotinib and canertinib as clinically relevant efflux modulators efficiently abrogated directionality in pazopanib efflux in vitro and their co-administration resulted in 2-2.5-fold increase in pazopanib brain accumulation in vivo. Further pre-clinical and clinical investigations are warranted as erlotinib or canertinib may have a synergistic pharmacological effect in addition to their primary role of pazopanib efflux modulation as a combination regimen for the treatment of recurrent brain tumors. PMID:22688250

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

2012-10-15

30

Crystal Structure of an EAL Domain in Complex with Reaction Product 5?-pGpG  

PubMed Central

FimX is a large multidomain protein containing an EAL domain and involved in twitching motility in Pseudomonas aeruginosa. We present here two crystallographic structures of the EAL domain of FimX (residues 438–686): one of the apo form and the other of a complex with 5?-pGpG, the reaction product of the hydrolysis of c-di-GMP. In both crystal forms, the EAL domains form a dimer delimiting a large cavity encompassing the catalytic pockets. The ligand is trapped in this cavity by its sugar phosphate moiety. We confirmed by NMR that the guanine bases are not involved in the interaction in solution. We solved here the first structure of an EAL domain bound to the reaction product 5?-pGpG. Though isolated FimX EAL domain has a very low catalytic activity, which would not be significant compared to other catalytic EAL domains, the structure with the product of the reaction can provides some hints in the mechanism of hydrolysis of the c-di-GMP by EAL domains.

Robert-Paganin, Julien; Nonin-Lecomte, Sylvie; Rety, Stephane

2012-01-01

31

Use of Chemical Chaperones in the Yeast Saccharomyces cerevisiae to Enhance Heterologous Membrane Protein Expression: High-Yield Expression and Purification of Human P-Glycoprotein  

Microsoft Academic Search

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? 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

Robert A. Figler; Hiroshi Omote; Robert K. Nakamoto; Marwan K. Al-Shawi

2000-01-01

32

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

33

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

PubMed

Bestatin is an immunomodulator with antitumor activity. This study was performed to investigate the effect of P-gp on the intestinal absorption and antiproliferative activity of bestatin. Our results showed that P-gp inhibitors significantly increased rat intestinal absorption of bestatin in vivo and in vitro. The net efflux ratio of bestatin was 2.2 across mock-/MDR1-MDCK cell monolayers and was decreased by P-gp inhibitors, indicating bestatin was a substrate of P-gp. Furthermore, the IC50 values of bestatin on U937 and K562 cells were decreased dramatically and the intracellular concentrations of bestatin were increased by incubation of cells with verapamil or Cyclosporin A. K562/ADR cells exhibited a higher IC50 value and a lower intracellular level of bestatin. The bestatin level in K562/ADR cells was partially restored by incubation with doxorubicin. However, P-gp and APN mRNA levels were not changed by bestatin. These results suggested that the intestinal absorption and accumulation in cancer cells for bestatin were limited by P-gp-mediated efflux. Additional attention should be paid to the alternative exposure of bestatin when bestatin was coadministered with drugs as P-gp substrates in clinic. PMID:23981338

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

2013-11-20

34

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.

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

2013-01-01

35

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

PubMed

Modulation of drug transporters via herbal medicines which have been widely used in combination with conventional prescription drugs may result in herb-drug interactions in clinical practice. The present study was designed to investigate the inhibitory effects of 50 major herbal constituents on P-glycoprotein (P-gp) in vitro and in vivo as well as related inhibitory mechanisms. Among these herbal medicines, four constituents, including emodin, 18?-glycyrrhetic acid (18?-GA), dehydroandrographolide (DAG), and 20(S)-ginsenoside F? [20(S)-GF?] exhibited significant inhibition (>50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC??=9.42 ?M), followed by 18?-GA (IC??=21.78 ?M), 20(S)-GF? (IC??=76.08 ?M) and DAG (IC??=77.80 ?M). P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with Km and Vmax values of 48.61, 29.09 ?M and 71.29, 38.45 nmol/min/mg protein, respectively. However, 18?-GA and 20(S)-GF? exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Molecular docking analysis (CDOCKER) further elucidated the mechanism for structure-inhibition relationships of herbal constituents with P-gp. When digoxin was co-administered to male SD rats with emodin or 18?-GA, the AUC(??t) and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18?-GA, DAG, 20(S)-GF? and Rh? at 10 ?M significantly inhibited CYP3A4/5 activity, while emodin activated the metabolism of midazolam in human liver microsomes. In conclusion, four herbal constituents demonstrated inhibition of P-gp to specific extents in vitro and in vivo. Taken together, our findings provided the basis for the reliable assessment of the potential risks of herb-drug interactions in humans. PMID:24380838

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

2014-03-01

36

Contribution of radixin to P-glycoprotein expression and transport activity in mouse small intestine in vivo.  

PubMed

The ERM proteins, ezrin, radixin, and moesin, are membrane-cytoskeleton cross-linkers with multiple physiological functions. We previously showed that radixin is involved in posttranslational regulation of P-glycoprotein (P-gp) in human hepatoblastoma HepG2 cells. Here, we investigated the physiological role of radixin in regulating P-gp expression and activity in the small intestine by comparing wild-type- and radixin knockout (Rdx) mice. In intestinal tissue homogenates, P-gp protein levels increased markedly from the upper part to the lower part of the small intestine in both wild-type- and Rdx(-/-) mice. In the membrane fractions, a similar pattern was seen in wild-type mice. However, the membrane expression of P-gp protein remained at the same level from the upper to the lower part of the small intestine in Rdx(-/-) mice. When rhodamine123 (Rho123), a substrate of P-gp, was orally administered to Rdx(-/-) and wild-type mice, the absorption phase of Rho123 was greater in Rdx(-/-) than in wild-type mice, whereas the elimination phase in Rdx(-/-) mice was not different from that of wild-type mice. Our results indicate that radixin plays an important role in regulating P-gp localization and P-gp functional activity at the intestinal membrane. PMID:23754525

Yano, Kentaro; Tomono, Takumi; Sakai, Riyo; Kano, Takashi; Morimoto, Kaori; Kato, Yukio; Ogihara, Takuo

2013-08-01

37

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.

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

2012-01-01

38

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

PubMed

Bivalves naturally exposed to toxic algae have mechanisms to prevent from harmful effects of diarrhetic shellfish poisoning (DSP) toxins. However, quite few studies have examined the mechanisms associated, and the information currently available is still insufficient. Multixenobiotic resistance (MXR) is ubiquitous in aquatic invertebrates and plays an important role in defense against xenobiotics. Here, to explore the roles of P-glycoprotein (P-gp) in the DSP toxins resistance in shellfish, complete cDNA of P-gp gene in the mussel Perna viridis was cloned and analyzed. The accumulation of okadaic acid (OA), a main component of DSP toxins, MXR activity and expression of P-gp in gills of P. viridis were detected after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins in the presence or absence of P-gp inhibitors PGP-4008, verapamil (VER) and cyclosporin A (CsA). The mussel P. viridis P-gp closely matches MDR/P-gp/ABCB protein from various organisms, having a typical sequence organization as full transporters from the ABCB family. After exposure to P. lima, OA accumulation, MXR activity and P-gp expression significantly increased in gills of P. viridis. The addition of P-gp-specific inhibitors PGP-4008 and VER decreased MXR activity induced by P. lima, but had no effect on the OA accumulation in gills of P. viridis. However, CsA, a broad-spectrum inhibitor of ABC transporter not only decreased MXR activity, but also increased OA accumulation in gills of P. viridis. Together with the ubiquitous presence of other ABC transporters such as MRP/ABCC in bivalves and potential compensatory mechanism in P-gp and MRP-mediated resistance, we speculated that besides P-gp, other ABC transporters, especially MRP might be involved in the resistance mechanisms to DSP toxins. PMID:24811006

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

2014-08-01

39

Reciprocal competition between lipid nanocapsules and P-gp for paclitaxel transport across Caco-2 cells.  

PubMed

Lipid nanocapsules (LNCs) have been shown to improve paclitaxel (Ptx) bioavailability and transport across an intestinal barrier model. In the present study, the interaction between P-glycoprotein (P-gp) and LNC transport across Caco-2 cells are investigated. Transport experiments have been performed on Caco-2 cells displaying different P-gp activities (early and later cell passages). The permeability of Ptx encapsulated in LNCs has been studied in the presence of P-gp inhibitors (verapamil and vinblastin) or unloaded LNCs. The uptake of dye-labelled LNCs was also observed in the presence of the same inhibitors. It was found that the permeability of Ptx varied depending on the passages with later ones showing higher absolute values (5.74+/-1.21 cms(-1) vs 133.41+/-5.74 cms(-1)). P-gp inhibition obtained with verapamil or vinblastin improved Ptx transport up to 98%. LNCs have also demonstrated their capacity to increase their own transport. Experiments performed with dye-labelled LNCs demonstrated an enhancement of the uptake of dye (Nile red), only in the presence of verapamil. These results demonstrated an effect of P-gp on the transport of Ptx when loaded in LNCs and support a direct effect of P-gp on their endocytosis in Caco-2 cells. These finding may assist in the development of new nanomedicine for oral administration. PMID:20438839

Roger, E; Lagarce, F; Garcion, E; Benoit, J-P

2010-08-11

40

Cross-resistance to antifolates in multidrug resistant cell lines with P-glycoprotein or multidrug resistance protein expression  

Microsoft Academic Search

Resistance to some (lipophilic) antifolates has been associated with P-glycoprotein (P-gp)mediated multidrug resistance (MDR). A possible relationship with non-P-gp MDR has not been established. We studied resistance to antifolates in SW-1573 human lung carcinoma cells, a P-gp overexpressing variant SW-1573\\/2R160 and a multidrug resistance protein (MRP) overexpressing variant SW-1573\\/2R120. In this study, thymidylate synthase (TS) inhibitors with different properties concerning

Baukelien van Triest; Herbert M. Pinedo; Frank Telleman; Clasina L. van der Wilt; Gerrit Jansen; Godefridus J. Peters

1997-01-01

41

Extracellular high mobility group box chromosomal protein 1 promotes drug resistance by increasing the expression of P?glycoprotein expression in gastric adenocarcinoma cells.  

PubMed

It has previously been reported that high mobility group box chromosomal protein 1 (HMGB1) is overexpressed in the majority of gastric adenocarcinoma cell types, and that HMGB1 can be released into the extracellular matrix from stressed or necrotic cancer cells. HMGB1 is considered to promote cell proliferation and invasion in gastric adenocarcinoma cells. Furthermore, in a number of cancer cell types, HMGB1 has been reported to promote autophagy and inhibit anticancer drug?induced apoptosis, which has been identified as an important mechanism in the development of multidrug resistance (MDR). However, there have been no studies on the effects of HMGB1 on expression of the MDR?related transporter proteins in gastric adenocarcinoma. In the present study, extracellular HMGB1 increased the expression levels of P-glycoprotein (P-gp) at the pre-transcriptional and post?transcriptional levels in the human gastric adenocarcinoma cell lines, SGC7901, MKN28 and AGS, as detected by quantitative polymerase chain reaction and western blot assays. MTT and apoptosis assays were also performed and it was demonstrated that extracellular HMGB1 subsequently enhanced resistance to the P?gp?related drugs, adriamycin and vincristine. In brief, this study demonstrated that extracellular HMGB1 may promote drug resistance to adriamycin and vincristine by upregulating P?gp in human gastric adenocarcinoma cells. PMID:24549588

Yin, Yuping; Li, Wei; Deng, Meizhou; Zhang, Peng; Shen, Qian; Wang, Guobing; Tao, Kaixiong

2014-04-01

42

Functional Correlation of P-Glycoprotein Expression and Genotype with Expression of the Human Immunodeficiency Virus Type 1 Coreceptor CXCR4  

Microsoft Academic Search

The aim of this study was to investigate the relationship between lymphocyte P-glycoprotein (P-gp) expres- sion and genotype in vivo and the expression of lymphocyte receptors critical in the life cycle of human immunodeficiency virus type 1 (HIV-1), i.e., CD4, CCR5, and CXCR4. Using flow cytometry to quantify each membrane receptor\\/transporter, we demonstrate a highly significant correlation between P-gp protein

Andrew Owen; Becky Chandler; Patrick G. Bray; Stephen A. Ward; C. Anthony Hart; David J. Back; Saye H. Khoo

2004-01-01

43

Characterization of SAGE Mdr1a (P-gp), Bcrp, and Mrp2 knockout rats using loperamide, paclitaxel, sulfasalazine, and carboxydichlorofluorescein pharmacokinetics.  

PubMed

Transporter gene knockout rats are practically advantageous over murine models for pharmacokinetic and excretion studies, but their phenotypic characterization is lacking. At present, relevant aspects of pharmacokinetics, metabolism, distribution, and excretion of transporter probes [P-glycoprotein (P-gp): loperamide and paclitaxel; breast cancer resistance protein (Bcrp): sulfasalazine; and multidrug resistance-associated protein 2 (Mrp2): carboxydichlorofluorescein] were studied systematically across SAGE P-gp, Bcrp, and Mrp2 knockout rats. In Mdr1a knockout rats, loperamide and paclitaxel oral bioavailability was 2- and 4-fold increased, respectively, whereas clearance was significantly reduced (40-42%), consistent with the expected 10- to 20-fold reduction in paclitaxel excretion. N-Desmethyl-loperamide pharmacokinetics were not altered in any of the three knockouts after oral loperamide. In rats lacking P-gp, paclitaxel brain partitioning was significantly increased (4-fold). This finding is consistent with observations of loperamide central nervous system opioid pharmacology in Mdr1a knockout rats. Sulfasalazine oral bioavailability was markedly increased 21-fold in Bcrp knockouts and, as expected, was also 2- to 3-fold higher in P-gp and Mrp2 knockout rats. The sulfapyridine metabolite/parent ratio was decreased 10-fold in rats lacking Bcrp after oral, but not intravenous, sulfasalazine administration. Carboxydichlorofluorescein biliary excretion was obliterated in Mrp2 knockout rats, resulting in 25% decreased systemic clearance and 35% increased half-life. In contrast, carboxydichlorofluorescein renal clearance was not impaired in the absence of Mrp2, Bcrp, or P-gp. In conclusion, SAGE Mdr1a, Bcrp, and Mrp2 knockout rats generally demonstrated the expected phenotypes with respect to alterations in pharmacokinetics of relevant probe substrates; therefore, these knockout rats can be used as an alternative to murine models whenever a larger species is practically advantageous or more relevant to the drug discovery/development program. PMID:22711747

Zamek-Gliszczynski, Maciej J; Bedwell, David W; Bao, Jing Q; Higgins, J William

2012-09-01

44

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

45

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

PubMed

The deleterious impacts of marine pollutants on reef corals and their symbiotic algae are an important element of global coral reef decline. In the current study we examined the impacts of toxicants on the reef coral Montastraea franksi by analysing the expression of three stress-related genes belonging to the coral host, using Taqman real-time quantitative reverse transcription-PCR. Gene expression profiles of P-glycoprotein (or multi-xenobiotic resistance protein) (P-gp); heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) were examined following 4 and 8h exposures to the heavy metal copper (3, 10, 30 and 100 microgL(-1)) or the third generation oil dispersant Corexit9527 (1, 5, 10 and 50 ppm). Additionally, the expression of P-gp was examined following exposure to 0.5 and 5 microM concentrations of the chemotherapeutic drug vinblastine, a classic substrate of P-gp. The expression of P-gp increased significantly in corals treated with vinblastine and also increased following exposure to copper and Corexit9527. Hsp70, and to a lesser extent Hsp90 expression increased following exposure to copper and Corexit9527 indicating a general cellular stress response. Densities of symbiotic algae in the tissues of the corals did not change significantly during the experiments, nor was any loss or paling of coral tissues observed. These findings provide important insight into how corals defend themselves against pollution and complement ongoing initiatives developing molecular biomarkers of stress in reef-building corals. PMID:19501419

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

2009-07-26

46

Expression of P-glycoprotein in adult T-cell leukemia cells  

Microsoft Academic Search

We have examined the expression of P-glycoprotein (P-gp) in adult T-cell leukemia (ATL) samples from 25 patients. Based on immunoblotting with a monoclonal antibody against P-gp, C219, 8 of 20 ATL patients were P-gp positive at the initial presentation. All 6 patients at the relapsed stage were P-gp positive, and refractory to chemotherapy. The expression of MDR1 mRNA in P-gp-positive

Yasuo Kuwazuru; Shuichi Hanada; Tatsuhiko Furukawa; Akihiko Yoshimura; Tomoyuki Sumizawa; Atae Utsunomiya; Kazuaki Ishibashi; Takeshi Saito; Kimiharu Uozumi; Masao Maruyama

1990-01-01

47

Anti-inflammatory properties of anthraquinones and their relationship with the regulation of P-glycoprotein function and expression.  

PubMed

There is a growing interest in natural products that potentially have anti-inflammatory properties and inhibit P-glycoprotein (P-gp) function. In this report, we assessed the effects of anthraquinone derivatives from rhubarb on LPS-induced RAW 264.7 macrophages to determine their anti-inflammatory potential. The derivatives were also tested in Caco-2 cell lines to evaluate the inhibition of the drug efflux function of P-gp. The transport abilities were examined and the cellular accumulation of rhodamine-123 (R-123) was also measured. Electorphoretic mobility shift assay (EMSA) was performed to check the activator protein-1 (AP-1) DNA binding affinity. Five anthraquinones were tested to determine their inhibitory activities on NO production and the protein and mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, the level of prostaglandin E(2) (PGE(2)) was determined in LPS-induced RAW264.7 macrophages. Emodin was found to be the most potent inhibitor, and it also reduced paw swelling in the mouse model of carrageenan-induced paw edema. In Caco-2 cells, emodin elevated the accumulation of R-123 and decreased the efflux ratio of R-123, which indicates the inhibition of P-gp function. The inhibition of COX-2 protein by emodin paralleled the decrease in P-gp expression. In addition, mitogen-activated protein kinase (MAPK) expression was decreased through the prevention of AP-1 DNA binding, which leads to downregulation in the expression of P-gp. Our data indicate that the decrease of P-gp expression is caused by the decreased expression of COX-2 through the MAPK/AP-1 pathway. Based on our results, we suggest that anti-inflammatory drugs with COX-2 inhibitory activity might be used to modulate P-gp function and expression. PMID:23174748

Choi, Ran Joo; Ngoc, Tran Minh; Bae, Kihwan; Cho, Hyun-Jong; Kim, Dae-Duk; Chun, Jaemoo; Khan, Salman; Kim, Yeong Shik

2013-01-23

48

P-glycoprotein expression induced by glucose depletion enhanced the chemosensitivity in human hepatocellular carcinoma cell-lines.  

PubMed

Chemoresistance in cancer cells is frequently associated with an over-expression of the P-glycoprotein (P-gp). The expression of P-gp can be regulated as the cells encounter a number of chemical, physical or environmental stimuli. In this study, P-gp was found gradually expressed in a human hepatocellular carcinoma (HCC) QGY-7703 cells after 48 h of culturing in glucose-free medium. This phenomenon disappeared after the removal of glucose deprivation culture conditions. Mdr1-cDNA isolated from the cell line cultured in glucose-free conditions (namely QGY-7703G), was transiently transformed into the parent QGY-7703 cells, and multi-drug resistance was eventually induced. Results from XTT cytotoxicity assays indicated that the mdr1 gene was functional and the P-gp could restore the QGY-7703 cell's ability to withstand high concentrations of a number of chemotherapeutic agents. A P-gp inhibitor, verapamil, could completely reverse the cellular drug resistance when applied to the QGY-7703G cells. Our results indicated that an alteration of a specific state in cells caused by an external stimulus in vitro may lead to an expression of stress proteins (e.g. P-gp), which may enhance the cells' survival in adverse conditions. The expressed P-gp induced by glucose deprivation has a functional role in affecting the chemosensitivity in HCC QGY-7703G cells. Inhibition of P-gp activity may enhance the effect of the cancer cells towards cancer chemotherapy. PMID:15914037

Cheng, Samuel Chak-Sum; Zhou, Jing; Xie, Yong

2005-04-01

49

Expression and significance of hypoxia-inducible factor-1 alpha and MDR1/P-glycoprotein in human colon carcinoma tissue and cells  

PubMed Central

Purpose Hypoxia in tumors is generally associated with chemoresistance and radioresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance (MDR1) gene/transporter P-glycoprotein (P-gp) has not been clearly investigated. This study aims at examining the expression levels of HIF-1? and MDR1/P-gp in human colon carcinoma tissues and cell lines (HCT-116, HT-29, LoVo, and SW480) and ascertaining whether HIF-1? plays an important role in tumor multidrug resistance with MDR1/P-gp. Methods The expression and distribution of HIF-1? and P-gp proteins were detected in human colon carcinoma tissues and cell lines by immunohistochemistry and immunocytochemistry using streptavidin/peroxidase (SP) and double-label immunofluorescence methods. HIF-1? and MDR1 mRNA expression levels in cell lines were analyzed using RT-PCR under normoxic and hypoxic conditions, respectively. Results The immunohistochemical method shows that HIF-1? and P-gp expression were not correlated with gender, age, location, and differentiation degree (P > 0.05). However, the expression of HIF-1? and P-gp at different Dukes’ stages and whether involved in lymphatic invasion shows a significant difference (P < 0.05). Correlation analysis displays that HIF-1? protein expression was correlated significantly with P-gp expression (P < 0.01). Double-label immunofluorescence demonstrates that coexpression of HIF-1? and P-gp does exist in human colon carcinoma tissues. The mRNA expression of HIF-1? and MDR1 was detected in the four human colon carcinoma cell lines under both normoxia and hypoxia. Optical density values representing mRNA expression levels of HIF-1? and MDR1 were found to be significantly higher in the same type cells under hypoxic conditions than that under normoxic conditions, respectively (P < 0.01). However, no significant differences of HIF-1? or MDR1 mRNA expression were found among these cell lines, which exposed under the same PaO2 cultural conditions (P > 0.05). And the immunocytochemistry results were corresponding with the analysis of mRNA expression. Conclusions These results suggest that hypoxia induce the expression of HIF-1? and MDR1/P-gp in colon carcinoma and HIF-1? expression may be associated with the gene MDR1 (P-gp) and interactively involved in the occurrence of tumor multidrug resistance.

Ding, Zhenyu; Yang, Li; Xie, Xiaodong; Xie, Fangwei; Pan, Feng; Li, Jianjun; He, Jianming

2010-01-01

50

ABCB1 haplotypes are associated with P-gp activity and affect a major molecular response in chronic myeloid leukemia patients treated with a standard dose of imatinib  

PubMed Central

Despite the high efficacy of imatinib mesylate (IM) treatment for chronic myeloid leukemia (CML) patients, some individuals develop resistance due to impaired bioavailability. It has been previously demonstrated that the haplotypes for ATP-binding cassette subfamily B member 1 (ABCB1)with c.1236C>T, c.3435C>T and c.2677G>T/A polymorphisms markedly affect the secondary structure of ABCB1 mRNA and its activity. These modifications may affect efflux transporter activity and response to treatment with IM. The aim of the present study was to investigate the influence of ABCB1 haplotypes on P-glycoprotein (P-gp) activity, IM plasma levels and IM response. In total, 28 chronic-phase CML patients treated with a standard dose of IM (400 mg/day) were studied. The patients were selected according to the haplotypes of ABCB1, with c.1236C>T, c.3435C>T and c.2677G>T polymorphisms, and were classified into two groups based on the presence of the mutated allele in each genotype for the three ABCB1 polymorphisms. In addition, expression of P-gp and breakpoint cluster region-abelson 1 (BCR-ABL1), ABCB1 and solute carrier family 22 member 1 (SLC22A1) mRNA were evaluated. The P-gp activity in the wild-type group was found to be higher than that in the mutated group (59.1 vs. 38.3%; P=0.001). Furthermore, the patients who did not achieve major molecular response (MMR) showed a higher rate of efflux mediated by P-gp when compared with individuals who achieved MMR (64.7 vs. 45.7%; P=0.001). All patients without MMR demonstrated effluxes of >60%. In addition, patients without MMR exhibited lower plasma concentrations of IM compared with those with MMR (0.51 vs. 1.42 ?g/ml; P=0.001). Higher levels of SLC22A1 mRNA were observed in patients who achieved MMR and complete molecular response (P<0.05). In conclusion, the ABCB1 1236CT/3435CT/2677GT and 1236TT/3435TT/2677TT haplotypes are associated with reduced P-gp activity and MMR in chronic-phase CML patients treated with a standard dose of IM.

VIVONA, DOUGLAS; LIMA, LUCIENE TEREZINA; RODRIGUES, ALICE CRISTINA; BUENO, CAROLINA TOSIN; ALCANTARA, GREYCE KELLY STEINHORST; BARROS, LUIZA SALDANHA RIBEIRO; DE MORAES HUNGRIA, VANIA TIESTSCHE; CHIATTONE, CARLOS SERGIO; DE LOURDES LOPES FERRARI CHAUFFAILLE, MARIA; GUERRA-SHINOHARA, ELVIRA MARIA

2014-01-01

51

In vitro modulation of ABCB1/P-glycoprotein expression by polyphenols from Mangifera indica.  

PubMed

Many plant compounds are able to modulate the activity and/or the expression of the major multidrug transporter ABCB1/P-glycoprotein (P-gp). In this study, mango (Mangifera indica L.) stem bark extract (MSBE), its main polyphenol mangiferin and the mangiferin aglycone derivative norathyriol, as well as catechin, gallic acid and quercetin, were investigated for their potential ability to influence ABCB1 gene and P-gp expression in HK-2 cells, a proximal tubule line constitutively expressing this transporter. Western blot analysis demonstrated a concentration-dependent decrease in P-gp in cells cultured in the presence of MSBE for 72 h. Gallic acid and quercetin also decreased the levels of P-gp at all studied concentrations, whereas catechin was almost ineffective. However, in cells exposed to mangiferin (10-200 microM), the P-gp amount showed a concentration- and time-dependent increase, being 2-fold higher than the controls after 72 h. Norathyriol (5 microM) induced P-gp, but the effect decreased at higher concentrations. The changes in the P-gp protein amount were correlated with relative changes in the ABCB1 mRNA content and with the efflux activity of the transporter. The transcriptional inhibitor 1-d-ribofuranosylbenzimidazole (DRB) contrasted the increased expression of ABCB1 by mangiferin, suggesting that the increase could be due to transcriptional up-regulation of ABCB1 mRNA. Mangiferin-treated cells overexpressing the transporter were protected against the cytotoxicity of the known P-gp substrate cyclosporine A. However, the opposite effect was not observed in cells pretreated with MSBE. These results demonstrate that MSBE and mango polyphenols, already shown in our previous studies to influence P-gp activity, may also interact with ABCB1/P-gp at the expression level. In particular, we show for the first time that the main mango polyphenol mangiferin up-regulates this multidrug transporter. The molecular mechanisms and the consequences of these effects, including the possibility of interactions with conventional drugs or other herbal constituents, remain to be elucidated. PMID:20513373

Chieli, Elisabetta; Romiti, Nadia; Rodeiro, Idania; Garrido, Gabino

2010-08-01

52

Deactivation of Signal Transducer and Activator of Transcription 3 Reverses Chemotherapeutics Resistance of Leukemia Cells via Down-Regulating P-gp  

PubMed Central

Multidrug resistance (MDR) caused by overexpression of p-glycoprotein is a major obstacle in chemotherapy of malignant cancer, which usually is characterized by constitutive activation of signal transducer and activator of transcription 3 (STAT3), but their relation between MDR and STAT3 remains unclear. Here, we showed that STAT3 was overexpressed and highly activated in adriamycin-resistant K562/A02 cells compared with its parental K562 cells. Blockade of activation of STAT3 by STAT3 decoy oligodeoxynucleotide (ODN) promoted the accumulation and increased their sensitivity to adriamycin by down-regulating transcription of mdr1 and expression of P-gp, which were further confirmed by using STAT3-specific inhibitor JSI-124. Inhibition of STAT3 could also decrease mdr1 promoter mediated luciferase expression by using mdr1 promoter luciferase reporter construct. Otherwise, activation of STAT3 by STAT3C improved mdr1 transcription and P-gp expression. The ChIP results demonstrated that STAT3 could bind to the potential promoter region of mdr1, and STAT3 decoy depressed the binding. Further mutation assay show +64?+72 region could be the STAT3 binding site. Our data demonstrate a role of STAT3 in regulation of mdr1 gene expression in myeloid leukemia and suggest that STAT3 may be a promising therapeutic target for overcoming MDR resistance in myeloid leukemia.

Zhang, Xulong; Xiao, Weihua; Wang, Lihua; Tian, Zhigang; Zhang, Jian

2011-01-01

53

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

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

2012-05-01

54

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

NASA Astrophysics Data System (ADS)

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

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

2012-06-01

55

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

PubMed

Magnetic nanoparticles (NP) were developed for the active brain targeting of water-soluble P-glycoprotein (P-gp) substrate rhodamine 123 (Rh123). The NP matrix of poly(lactide-co-glycolide) (PLGA) and methoxy poly(ethyleneglycol)-poly(lactic acid) (M-PEG-PLA) was prepared by single emulsion solvent evaporation of polymers with oleic acid-coated magnetic nanoparticles (OAMNP) and Rh123. All formulations were characterized in terms of morphology, particle size, magnetic content and Rh123 encapsulation efficiency. The maximum encapsulation efficiency of Rh123 was 45 ± 3% and of OAMNP was 42 ± 4%. The brain targeting and biodistribution study was performed on Sprague Dawley rats (3 groups, n = 6). Rh123 (0.4 mg kg(-1)) was administered in saline form, NP containing Rh123, and NP containing Rh123 in the presence of a magnetic field (0.8 T). The fluorimetric analysis of brain homogenates revealed a significant uptake (p < 0.05) of Rh123 in the magnetically targeted group relative to controls. These results were supported by fluorescence microscopy. This study reveals the ability of magnetically targeted nanoparticles to deliver substances to the brain, the permeation of which would otherwise be inhibited by the P-gp system. PMID:22652439

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

2012-06-29

56

The impact of P-gp functionality on non-steady state relationships between CSF and brain extracellular fluid.  

PubMed

In the development of central nervous system (CNS)-targeted drugs, the prediction of human CNS target exposure is a big challenge. Cerebrospinal fluid (CSF) concentrations have often been suggested as a 'good enough' surrogate for brain extracellular fluid (brainECF, brain target site) concentrations in humans. However, brain anatomy and physiology indicates prudence. We have applied a multiple microdialysis probe approach in rats, for continuous measurement and direct comparison of quinidine kinetics in brainECF, CSF, and plasma. The data obtained indicated important differences between brainECF and CSF kinetics, with brainECF kinetics being most sensitive to P-gp inhibition. To describe the data we developed a systems-based pharmacokinetic model. Our findings indicated that: (1) brainECF- and CSF-to-unbound plasma AUC0-360 ratios were all over 100 %; (2) P-gp also restricts brain intracellular exposure; (3) a direct transport route of quinidine from plasma to brain cells exists; (4) P-gp-mediated efflux of quinidine at the blood-brain barrier seems to result of combined efflux enhancement and influx hindrance; (5) P-gp at the blood-CSF barrier either functions as an efflux transporter or is not functioning at all. It is concluded that in parallel obtained data on unbound brainECF, CSF and plasma concentrations, under dynamic conditions, is a complex but most valid approach to reveal the mechanisms underlying the relationship between brainECF and CSF concentrations. This relationship is significantly influenced by activity of P-gp. Therefore, information on functionality of P-gp is required for the prediction of human brain target site concentrations of P-gp substrates on the basis of human CSF concentrations. PMID:23539188

Westerhout, Joost; Smeets, Jean; Danhof, Meindert; de Lange, Elizabeth C M

2013-06-01

57

Effect of Repeated Oral Treatment with Etoposide on the Expression of Intestinal P-glycoprotein and Oral Morphine Analgesia.  

PubMed

  Currently, the World Health Organization recommends oral administration of opioid analgesics for patients with cancer to treat cancer-related pain from the initial stage of treatment. Furthermore, many anticancer drugs have been newly-developed and approved as oral form. Because of this trend, the chances of drug-drug interactions between anticancer drugs and opioid analgesics during absorption process from the intestine are likely to increase. To investigate these possible drug-drug interactions, we have focused on intestinal P-glycoprotein (P-gp) which regulates the absorption of various substrate drugs administered orally. Previously, we have found that repeated oral treatment with etoposide (ETP), an anticancer drug, attenuates analgesia of oral morphine, a substrate drug for P-gp, by increasing the expression and activity of intestinal P-gp. However, the mechanism by which ETP treatment increases the intestinal P-gp expression and decreases oral morphine analgesia remains unclear. RhoA, a small G-protein, and ROCK, an effector of RhoA, pathway has been attracted attention with regard to their involvement in the regulatory mechanism of the expression and activity of P-gp. Interestingly, this pathway is activated in response to various signaling induced by some anticancer drugs. Furthermore, it has been reported that ezrin/radixin/moesin (ERM) play a key role in the plasma membrane localization of P-gp, and that RhoA/ROCK pathway regulates the activation process of ERM. This review article introduces the result of our previous research as well as recent findings on the involvement of ERM via activation of RhoA/ROCK in the increased expression of intestinal P-gp and decreased oral morphine analgesia induced by repeated oral treatment with ETP. PMID:24882643

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

2014-01-01

58

?Np73? regulates MDR1 expression by inhibiting p53 function  

PubMed Central

The p73 protein is a transcription factor and member of the p53 protein family that expresses as a complex variety of isoforms. ?Np73? is an N-terminally truncated isoform of p73. We found that ?Np73 protein is upregulated in human gastric carcinoma suggesting that ?Np73 may play an oncogenic role in these tumors. Although it has been shown that ?Np73? inhibits apoptosis and counteracts the effect of chemotherapeutic drugs, the underlying mechanism by which this p73 isoform contributes to chemotherapeutic drug response remains to be explored. We found that ?Np73? upregulates MDR1 mRNA and p-glycoprotein (p-gp), which is involved in chemotherapeutic drug transport. This p-gp upregulation was accompanied by increased p-gp functional activity in gastric cancer cells. Our data suggest that upregulation of MDR1 by ?Np73? is mediated by interaction with p53 at the MDR1 promoter.

Vilgelm, A; Wei, JX; Piazuelo, MB; Washington, MK; Prassolov, V; El-Rifai, W; Zaika, A

2014-01-01

59

Expression and significance of hypoxia-inducible factor-1? and MDR1/P-glycoprotein in laryngeal carcinoma tissue and hypoxic Hep-2 cells  

PubMed Central

The present study aimed to evaluate the expression of hypoxia-inducible factor-1? (HIF-1?) and MDR1/P-glycoprotein (P-gp) in human laryngeal squamous cell carcinoma (LSCC) tissues, and also to investigate the regulation of MDR1 gene expression by HIF-1? in Hep-2 cells under hypoxic conditions. The expression of HIF-1? and MDR1/P-gp in human LSCC tissues was examined using immunohistochemistry. The HIF-1? and MDR1 gene expression in the Hep-2 cells was detected using real-time quantitative reverse transcription (QRT)-PCR and western blot analysis under normoxic and hypoxic conditions. In hypoxia, HIF-1? expression was inhibited by RNA interference. HIF-1? and MDR1/P-gp expression was high in the LSCC tissues and was associated with the clinical stage and lymph node metastasis (P<0.05). HIF-1? expression was positively correlated with MDR1/P-gp expression (P<0.01). In the Hep-2 cells, HIF-1? and MDR1/P-gp expression significantly increased in response to hypoxia. The inhibition of HIF-1? expression synergistically downregulated the expression of the MDR1 gene in hypoxic Hep-2 cells. HIF-1? expression is positively correlated with MDR1/P-gp expression in LSCC, and the two proteins may be able to serve as potential biomarkers for predicting the malignant progression and metastasis of LSCC. HIF-1? may be critical for the upregulation of MDR1 gene expression induced by hypoxia in Hep-2 cells.

XIE, JIN; LI, DA-WEI; CHEN, XIN-WEI; WANG, FEI; DONG, PIN

2013-01-01

60

Down-regulation of the HGF/MET autocrine loop induced by celecoxib and mediated by P-gp in MDR-positive human hepatocellular carcinoma cell line.  

PubMed

Many tumors are resistant to drug-induced cell-cycle arrest and apoptosis. We have reported that apoptosis can be restored in human multidrug-resistant (MDR) hepatocellular carcinoma cell lines by celecoxib. Here we show that P-glycoprotein (P-gp) mediates cell-cycle arrest and autophagy induced by celecoxib in human MDR overexpressing hepatocellular carcinoma cell line by down-regulation of the HGF/MET autocrine loop and Bcl-2 expression. Exposure of cells to a low concentration of celecoxib down-regulated the expression of mTOR and caused G1 arrest and autophagy, while higher concentration triggered apoptosis. Cell growth inhibition and autophagy were associated with up-regulation of the expression of TGFbeta1, p16(INK4b), p21(Cip1) and p27(Kip1) and down-regulation of cyclin D1, cyclin E, pRb and E2F. The role of P-glycoprotein expression in resistance of MDR cell clone to cell-cycle arrest, autophagy and apoptosis was shown in cells transfected with MDR1 small interfering RNA. These findings demonstrate that the constitutive expression of P-gp is involved in the HGF/MET autocrine loop that leads to increased expression of Bcl-2 and mTor, inhibition of eIF2alpha expression, resistance to autophagy/apoptosis and progression in the cell-cycle. Since mTor inhibitors have been proposed in treatment of "drug resistant" cancer, these data may help explain the reversing effect of mTor inhibitors. PMID:19447220

Mazzanti, Roberto; Platini, Francesca; Bottini, Consuelo; Fantappiè, Ornella; Solazzo, Michela; Tessitore, Luciana

2009-07-01

61

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

62

Prognostic significance of the expressions of metallothionein, glutathione-S-transferase-pi, and P-glycoprotein in curatively resected gastric cancer.  

PubMed

Although experimental studies indicate that overexpression of metallothionein (MT), glutathione-S-transferase-pi (GST-pi), or P-glycoprotein (P-GP) is related to the drug resistance of cancer cells, the clinical significance of the overexpression remains to be elucidated. The expressions of MT, GST-pi, and P-GP wre evaluated immunohistochemically in 74 specimens of gastric adenocarcinoma in T1-3N1-2 stages which were resected with curative intent. Fluorinated pyrimidines, mitomycin C, and Adriamycin were prescribed in 73, 54, and 2 patients, respectively. The staining characteristics were investigated in relation to the clinical results. The cell-proliferative activity was studied with anti-proliferating cell nuclear antigen antibody. Expressions of GST-pi and P-GP correlated with the staining intensity of normal mucosa. Five-year disease-free survival rates (DFSRs) of GST-pi-negative and GST-pi-positive groups were 75.0 and 49.0%. The 5-year DFSRs of P-GP-negative and P-GP-positive groups were 68.2 and 38.6%. Concurrent expression among the three proteins was associated with the survival: 5-year DFSR of no- or one-protein-positive group was 75.0%, while those of 2- and 3-protein-positive groups were 56.0 and 38.9%, respectively. Tumors concurrently expressing 2 or 3 proteins have a high proliferative activity. Expressions of MT, GST-pi, and P-GP by the tumor are associated with a poorer prognosis of the patients. PMID:9260601

Monden, N; Abe, S; Sutoh, I; Hishikawa, Y; Kinugasa, S; Nagasue, N

1997-01-01

63

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

PubMed

Abstract 1.?The promising anticancer agent SNX-2112 (a novel Hsp90 inhibitor) is poorly bioavailable after oral administration. Here, we aim to determine the role of P-glycoprotein (P-gp) in the intestinal absorption of SNX-2112. 2.?We found that SNX-2112 significantly stimulated P-gp ATPase activity in in vitro ATPase assay with a small EC50 (the half-maximal effective concentration) value of 0.32?µM. 3.?In the single-pass perfused rat intestine model, absorption of SNX-2112 was not favored in the small intestine with a [Formula: see text] (the wall permeability) value of 0.38-0.64. By contrast, the compound was well absorbed in the colon with a [Formula: see text] value of 1.19. The P-gp inhibitors cyclosporine and elacridar (i.e. GF120918A) markedly enhanced SNX-2112 absorption in all four intestinal segments (i.e. duodenum, jejunum, ileum and colon) and the fold change ranged from 3.1 to 14.1. Pharmacokinetic study revealed that cyclosporine increased the systemic exposure of SNX-2112 by a 2.5-fold after oral administration. 4.?This is the first report that P-gp-mediated efflux is a limiting factor for intestinal absorption of SNX-2112 in rats. PMID:24555822

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

2014-08-01

64

[(11)C-carbonyl]CEP-32496: Radiosynthesis, biodistribution and PET study of brain uptake in P-gp/BCRP knockout mice.  

PubMed

CEP-32496 is a novel, orally active serine/threonine-protein kinase B-raf (BRAF) (V600E) kinase inhibitor that is being investigated in clinical trials for the treatment of some cancers in patients. In this study, we developed [(11)C-carbonyl]CEP-32496 as a novel positron emission tomography (PET) probe to study its biodistribution in the whole bodies of mice. [(11)C]CEP-32496 was synthesized by the reaction of 5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-amine hydrochloride (1·HCl) with [(11)C]phosgene, followed by treatment with 3-(6,7-dimethoxyquinozolin-4-yloxy)aniline (2). Small-animal PET studies with [(11)C]CEP-32496 indicated that radioactivity levels (AUC0-90min, SUV×min) accumulated in the brains of P-gp/BCRP knockout mice at a 8-fold higher rate than in the brains of wild-type mice. PMID:24930831

Shimoda, Yoko; Yui, Joji; Fujinaga, Masayuki; Xie, Lin; Kumata, Katsushi; Ogawa, Masanao; Yamasaki, Tomoteru; Hatori, Akiko; Kawamura, Kazunori; Zhang, Ming-Rong

2014-08-01

65

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

PubMed

Over-expression of P-glycoprotein (P-gp), a primary multidrug transporter which is located in plasma membranes, plays a major role in the multidrug resistance (MDR) of cytotoxic chemotherapy. Naamidines are a class of marine imidazole alkaloids isolated from Leucetta and Clathrina sponges, possessing a Y-shaped scaffold. Based on the results previously obtained from the third-generation MDR modulator ONT-093 and other modulators developed in our group, we designed and synthesized a series of novel 4,5-di-substituted benzyl-1-methyl-1H-imidazol-2-substituted amines using the Naamidine scaffold as the structure template. Subsequently, their reversing activity for Taxol resistance has been evaluated in P-gp-mediated multidrug resistance breast cancer cell line MDA435/LCC6MDR. Compounds 12c with a Y-shaped scaffold, and compound 17c which is 'X-shaped' scaffold and possesses a 4-diethylamino group at aryl ring B, turned out to be the most potent P-gp modulators. It appears that compounds 12c and 17c at 1 ?M concentration can sensitize LCC6MDR cells toward Taxol by 26.4 and 24.5 folds, with an EC50 212.5 and 210.5 nM, respectively. These two compounds are about 5-6 folds more potent than verapamil (RF = 4.5). Moreover, compounds 12c and 17c did not exhibit obvious cytotoxicity in either cancer cell lines or normal mouse fibroblast cell lines. This study has demonstrated that the synthetic Naamidine analogues can be potentially employed as effective, safe modulators for the P-gp-mediated drug resistance cancer cells. PMID:24952376

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

2014-08-18

66

Expression of multidrug resistance 1 and multidrug resistance-related protein 1 in C57BL/6 mice treated with benzene.  

PubMed

ATP-binding cassette super family (ABC) proteins are considered key to oncology and pharmacology studies. We examined the effect of benzene on ABC pump protein levels in C57BL/6 mouse bone marrow mononuclear cells. After a 2-week gavage (200 mg/kg, 5 days per week), the number of peripheral leukocytes, lymphocytes and basophils dropped significantly; there was also a significant decrease in MDR1 and MRP1 gene expression. A significant reduction in expression of P-gp was found; however, there was no significant decrease in the expression of MRP1 and NF-?B p65. We conclude that regulation of membrane efflux transport protein could be a factor in benzene hematotoxicity. PMID:24301953

Huang, J-S; Zhao, M-D; Shi, J-M; Zhang, J-H; Li, B; Fan, W; Zhou, Y-L

2013-01-01

67

Intact lipid rafts regulate HIV-1 Tat protein-induced activation of the Rho signaling and upregulation of P-glycoprotein in brain endothelial cells  

PubMed Central

The Rho signaling has an essential function in human immunodeficiency virus (HIV)-1-mediated disruption of the integrity of the blood–brain barrier (BBB). However, it is unknown how membrane domains, such as lipid rafts, can influence HIV-1-mediated activation of the Rho pathway and how these processes can affect the expression of the efflux transporters at the BBB level. This study is focused on the function of HIV-1 protein Tat in activation of the Rho signaling and upregulation of P-glycoprotein (P-gp) in human brain endothelial cells. Treatment with Tat markedly elevated GTP-RhoA levels and the potential downstream effectors, such as myosin phosphatase target subunit 1 and myosin light chain. In addition, Tat upregulated expression and promoter activity of P-gp as well as its efflux function. Inhibition of the Rho signaling cascade effectively blocked P-gp overexpression at the level of promoter activity. Disruption of lipid rafts by depletion of membrane cholesterol by methyl-beta-cyclodextrin, but not caveolin-1 silencing, also abolished Tat-mediated RhoA activation and P-gp upregulation. The present data indicate the critical function of intact lipid rafts and the Rho signaling in HIV-1-mediated upregulation of P-gp and potential development of drug resistance in brain endothelial cells.

Zhong, Yu; Hennig, Bernhard; Toborek, Michal

2010-01-01

68

[11C]phenytoin revisited: synthesis by [11C]CO carbonylation and first evaluation as a P-gp tracer in rats  

PubMed Central

Background At present, several positron emission tomography (PET) tracers are in use for imaging P-glycoprotein (P-gp) function in man. At baseline, substrate tracers such as R-[11C]verapamil display low brain concentrations with a distribution volume of around 1. [11C]phenytoin is supposed to be a weaker P-gp substrate, which may lead to higher brain concentrations at baseline. This could facilitate assessment of P-gp function when P-gp is upregulated. The purpose of this study was to synthesize [11C]phenytoin and to characterize its properties as a P-gp tracer. Methods [11C]CO was used to synthesize [11C]phenytoin by rhodium-mediated carbonylation. Metabolism and, using PET, brain pharmacokinetics of [11C]phenytoin were studied in rats. Effects of P-gp function on [11C]phenytoin uptake were assessed using predosing with tariquidar. Results [11C]phenytoin was synthesized via [11C]CO in an overall decay-corrected yield of 22?±?4%. At 45 min after administration, 19% and 83% of radioactivity represented intact [11C]phenytoin in the plasma and brain, respectively. Compared with baseline, tariquidar predosing resulted in a 45% increase in the cerebral distribution volume of [11C]phenytoin. Conclusions Using [11C]CO, the radiosynthesis of [11C]phenytoin could be improved. [11C]phenytoin appeared to be a rather weak P-gp substrate.

2012-01-01

69

Cyclosporine A (CsA) affects the pharmacodynamics and pharmacokinetics of the atypical antipsychotic amisulpride probably via inhibition of P-glycoprotein (P-gp)  

Microsoft Academic Search

Summary.  The importance of P-glycoprotein (P-gp) in the pharmacokinetics of amisulpride and the effects of a P-gp inhibitor cyclosporine\\u000a A (CsA) was investigated both, in vitro and in vivo.\\u000a \\u000a In vitro and in vivo results indicated amisulpride as a substrate of P-gp. Amisulpride was not metabolized by rat liver microsomes. Open field\\u000a behavior showed time dependent abolishment in locomotion by amisulpride

U. Schmitt; A. Abou El-Ela; L. J. Guo; H. Glavinas; P. Krajcsi; J. M. Baron; C. Tillmann; C. Hiemke; P. Langguth; S. Härtter

2006-01-01

70

Expression of P-glycoprotein in adult T-cell leukemia cells  

SciTech Connect

We have examined the expression of P-glycoprotein (P-gp) in adult T-cell leukemia (ATL) samples from 25 patients. Based on immunoblotting with a monoclonal antibody against P-gp, C219, 8 of 20 ATL patients were P-gp positive at the initial presentation. All 6 patients at the relapsed stage were P-gp positive, and refractory to chemotherapy. The expression of MDR1 mRNA in P-gp-positive ATL cells was increased at the relapsed stage of one patient. P-gp of this patient was photolabeled with (3H)azidopine and the labeling was inhibited with nimodipine, vinblastine and progesterone. These results suggest that P-gp expressed in ATL cells from patients at relapsed stage has the same binding site(s) for the drugs as that in multidrug resistant cells, and is correlated with the refractory nature of the cells to chemotherapy.

Kuwazuru, Y.; Hanada, S.; Furukawa, T.; Yoshimura, A.; Sumizawa, T.; Utsunomiya, A.; Ishibashi, K.; Saito, T.; Uozumi, K.; Maruyama, M. (Kagoshima Univ. (Japan))

1990-11-15

71

Breast Cancer-Derived Microparticles Display Tissue Selectivity in the Transfer of Resistance Proteins to Cells  

PubMed Central

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.

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

2013-01-01

72

Downregulation of mdr1a expression in the brain and liver during CNS inflammation alters the in vivo disposition of digoxin  

PubMed Central

Inflammation is a pathophysiological event that has relevance for altered drug disposition in humans. Two functions of P-glycoprotein (P-gp) are hepatic drug elimination and prevention of drug entry into the central nervous system (CNS). Our objective was to investigate if localized CNS inflammation induced by Escherichia coli lipopolysaccharide (LPS) would modify mdr1a/P-gp expression and function in the brain and liver. Our major finding was that the CNS inflammation in male rats produced a loss in the expression of mdr1a mRNA in the brain and liver that was maximal 6 h after intracranial ventricle (i.c.v.) administration of LPS. When 3H-digoxin was used at discrete time points, as a probe for P-gp function in vivo, an increase in brain and liver 3H-radioactivity and plasma level of parent digoxin was produced 6 and 24 h following LPS treatment compared to the saline controls. Digoxin disposition was similarly altered in mdr1a+/+ mice but not in mdr1a?/? mice 24 h after administering LPS i.c.v. In male rats, the biliary elimination of parent digoxin was reduced at 24 h (60%) and 48 h (40%) after LPS treatment and was blocked by the P-gp substrate cyclosporin A. An observed loss in CYP3A1/2 protein and organic anion transporting polypeptide 2 mRNA in the liver may make a minor contribution to digoxin elimination in male rats after LPS treatment. Conditions which impose inflammation in the CNS produce dynamic changes in mdr1a/P-gp expression/function that may alter hepatic drug elimination and the movement of drugs between the brain and the periphery. The use of experimental models of brain inflammation may provide novel insight into the regulation of P-gp function in that organ.

Goralski, Kerry B; Hartmann, Georgy; Piquette-Miller, Micheline; Renton, Kenneth W

2003-01-01

73

Downregulation of mdr1a expression in the brain and liver during CNS inflammation alters the in vivo disposition of digoxin.  

PubMed

1. Inflammation is a pathophysiological event that has relevance for altered drug disposition in humans. Two functions of P-glycoprotein (P-gp) are hepatic drug elimination and prevention of drug entry into the central nervous system (CNS). Our objective was to investigate if localized CNS inflammation induced by Escherichia coli lipopolysaccharide (LPS) would modify mdr1a/P-gp expression and function in the brain and liver. 2. Our major finding was that the CNS inflammation in male rats produced a loss in the expression of mdr1a mRNA in the brain and liver that was maximal 6 h after intracranial ventricle (i.c.v.) administration of LPS. When (3)H-digoxin was used at discrete time points, as a probe for P-gp function in vivo, an increase in brain and liver (3)H-radioactivity and plasma level of parent digoxin was produced 6 and 24 h following LPS treatment compared to the saline controls. Digoxin disposition was similarly altered in mdr1a(+/+) mice but not in mdr1a(-/-) mice 24 h after administering LPS i.c.v. 3. In male rats, the biliary elimination of parent digoxin was reduced at 24 h (60%) and 48 h (40%) after LPS treatment and was blocked by the P-gp substrate cyclosporin A. An observed loss in CYP3A1/2 protein and organic anion transporting polypeptide 2 mRNA in the liver may make a minor contribution to digoxin elimination in male rats after LPS treatment. 4. Conditions which impose inflammation in the CNS produce dynamic changes in mdr1a/P-gp expression/function that may alter hepatic drug elimination and the movement of drugs between the brain and the periphery. The use of experimental models of brain inflammation may provide novel insight into the regulation of P-gp function in that organ. PMID:12746221

Goralski, Kerry B; Hartmann, Georgy; Piquette-Miller, Micheline; Renton, Kenneth W

2003-05-01

74

Validation of quinidine as a probe substrate for the in vitro P-gp inhibition assay in Caco-2 cell monolayer  

Microsoft Academic Search

Although quinidine has been recommended as a probe substrate for the P-gp inhibition assay using Caco-2 cell monolayer, it\\u000a has not been studied widely in the in vitro system. In the present investigation, in vitro permeability studies using Caco-2\\u000a cell monolayer were carried out in order to optimize and validate quinidine as a P-gp probe substrate. In bi-directional Caco-2\\u000a assay

Anand G. Patil; Russell D’Souza; Neeta Dixit; Anagha Damre

75

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.

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

2014-01-01

76

Clinicopathological Parameters and Expression of P-Glycoprotein and MRP-1 in Retinoblastoma  

Microsoft Academic Search

Purpose: The immunohistochemical expressions of two multidrug resistance proteins, P-glycoprotein (P-gp) and multidrug resistance-related protein-1 (MRP-1), were studied in retinoblastoma and the correlations with the clinicopathological parameters were assessed. Method: Sixty-five enucleated eyes containing retinoblastoma were included in the study. Following hematoxylin-eosin staining, tumor differentiation, presence of choroidal invasion, optic nerve invasion, retinal invasion, necrosis and presence of calcification were

Hayyam Kiratli; Sevgül Bilgiç

2007-01-01

77

Development of a quantitative LC-MS/MS analytical method coupled with turbulent flow chromatography for digoxin for the in vitro P-gp inhibition assay.  

PubMed

Caco-2 cells, the human colon carcinoma cells, are typically used for screening compounds for their permeability characteristics and P-glycoprotein (P-gp) interaction potential during discovery and development. The P-gp inhibition of test compounds is assessed by performing bi-directional permeability studies with digoxin, a well established P-gp substrate probe. Studies performed with digoxin alone as well as digoxin in presence of test compounds as putative inhibitors constitute the P-gp inhibition assay used to assess the potential liability of discovery compounds. Radiolabeled (3)H-digoxin is commonly used in such studies followed by liquid scintillation counting. This manuscript describes the development of a sensitive, accurate, and reproducible LC-MS/MS method for analysis of digoxin and its internal standard digitoxin using an on-line extraction turbulent flow chromatography coupled to tandem mass spectrometric detection that is amendable to high throughput with use of 96-well plates. The standard curve for digoxin was linear between 10 nM and 5000 nM with regression coefficient (R(2)) of 0.99. The applicability and reliability of the analysis method was evaluated by successful demonstration of efflux ratio (permeability B to A over permeability A to B) greater than 10 for digoxin in Caco-2 cells. Additional evaluations were performed on 13 marketed compounds by conducting inhibition studies in Caco-2 cells using classical P-gp inhibitors (ketoconazole, cyclosporin, verapamil, quinidine, saquinavir etc.) and comparing the results to historical data with (3)H-digoxin studies. Similarly, P-gp inhibition studies with LC-MS/MS analytical method for digoxin were also performed for 21 additional test compounds classified as negative, moderate, and potent P-gp inhibitors spanning multiple chemo types and results compared with the historical P-gp inhibition data from the (3)H-digoxin studies. A very good correlation coefficient (R(2)) of 0.89 between the results from the two analytical methods affords an attractive LC-MS/MS analytical option for labs that need to conduct the P-gp inhibition assay without using radiolabeled compounds. PMID:17524973

Smalley, James; Marino, Anthony M; Xin, Baomin; Olah, Timothy; Balimane, Praveen V

2007-07-01

78

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

PubMed Central

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

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

2013-01-01

79

Multidrug resistance markers P-glycoprotein, multidrug resistance protein 1, and lung resistance protein in non-small cell lung cancer: prognostic implications  

Microsoft Academic Search

Purpose: The aim of this retrospective study was to comparatively investigate the expression of the three drug-resistance genes P-glycoprotein (P-gp), mul- tidrug-resistance protein 1 (MRP1), and lung resistance protein (LRP), in non-small cell lung cancer (NSCLC) tissues, and to assess possible associations with clinico- pathologic features. Methods: Tumor specimens from 126 patients were analyzed by immunohistochemistry and, in selected cases,

Walter Berger; Ulrike Setinek; Peter Hollaus; Thomas Zidek; Elisabeth Steiner; L. Elbling; H. Cantonati; Johannes Attems; Andrea Gsur; Michael Micksche

2005-01-01

80

Expression of membrane drug efflux transporters in the sigmoid colon of HIV-infected and uninfected men.  

PubMed

The use of antiretroviral therapy (ART) as pre-exposure prophylaxis (PrEP) has gained global attention as a promising HIV prevention strategy in men who have sex with men. Permeability of these agents in the rectal mucosa may be partially regulated by interactions with drug efflux transporters, P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) and/or breast cancer resistance protein (BCRP). The objective of this work was to investigate the expression of drug efflux transporters in recto-sigmoid colon tissues of HIV-infected and uninfected men, and evaluate the association of ART and/or HIV infection with drug transporter expression. MDR1/P-gp, MRPs (1-4) and BCRP mRNA and protein expression were detected in sigmoid colon biopsies of HIV-uninfected individuals. Biopsies from HIV-infected, ART-naïve participants revealed a significant downregulation of P-gp and MRP2 protein levels compared to HIV-uninfected individuals. Biopsies from HIV-infected ART-treated patients showed 1.9-fold higher P-gp protein expression and 1.5-fold higher MRP2 protein expression compared to the ones obtained from the HIV-infected ART-naïve patients. This is a first report demonstrating that HIV infection or ART could alter expression of drug efflux transporters in gut mucosa which in turn could affect the permeability of PrEP antiretroviral agents across this barrier, a highly vulnerable site of HIV transmission. PMID:23856938

De Rosa, María Fabiana; Robillard, Kevin R; Kim, Connie J; Hoque, Md Tozammel; Kandel, Gabor; Kovacs, Colin; Kaul, Rupert; Bendayan, Reina

2013-09-01

81

Data Mining for Expressivity of Recombinant Protein Expression  

NASA Astrophysics Data System (ADS)

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

Kira, Satoshi; Isoai, Atsushi; Yamamura, Masayuki

82

Calcein assay: a high-throughput method to assess P-gp inhibition.  

PubMed

Transporter mediated drug-drug interactions (tDDI) mediated by ABCB1 have been shown to be clinically relevant. Hence, the assessment of the ABCB1 tDDI potential early in the drug development process has gained interest. We have evaluated the Calcein assay as a means of assessing the ABCB1 tDDI that is amenable to high throughout and compared it with the monolayer efflux assay. We found the Calcein assay, when performed in K562MDR cells using the protocol originally published more sensitive than digoxin transport inhibition in MDCKII-MDR1 cells. Application of the Calcein assay to cell lines containing different amounts of ABCB1, yielded IC(50) values that varied 10-100-fold. The differences observed for IC(50) values for the same compounds were in the following rank order: IC(50, MDCKII-MDR1) >IC(50, K562MDR)>IC(50, hCMEC/D3). Higher IC(50) values were obtained in cells with higher ABCB1 expression. The Calcein assay is a high-throughput alternative to digoxin transport inhibition as it appears to have a comparable selectivity but higher sensitivity than previously published digoxin transport inhibition in MDCKII-MDR1 cells. In addition, it can be performed in a barrier-specific manner highlighting the dependence of ABCB1 IC(50) values on different ABCB1 expression levels. PMID:21657832

Glavinas, H; von Richter, O; Vojnits, K; Mehn, D; Wilhelm, I; Nagy, T; Janossy, J; Krizbai, I; Couraud, P; Krajcsi, P

2011-08-01

83

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

84

Expression of P-glycoprotein in HeLa cells confers resistance to ceramide cytotoxicity.  

PubMed

The role of glucosylceramide synthase (GCS) in regulating ceramide-induced apoptosis has been widely studied. The purpose of this investigation was to evaluate the role of P-glycoprotein (P-gp) in regulating ceramide cytotoxicity by using C6-ceramide. To accomplish this, we employed HeLa cells with conditional expression of the multidrug resistance gene 1/P-gp. HeLa cells expressing P-gp (P-gp/on cells) challenged with [14C]C6-ceramide (6 µM), synthesized 4.5-fold the amount of C6-glucosylceramide (GC) compared to HeLa cells with suppressed expression of P-gp (P-gp/off cells), whereas the generated levels of C6-sphingomyelin were almost equal (33 and 29% of intracellular 14C, respectively). Tamoxifen, a P-gp antagonist, decreased the C6-GC levels from 3.5-1.0% in the P-gp/off and from 17-2.8% of the total lipid 14C levels in the P-gp/on cells. Tamoxifen did not inhibit cell-free C6-GC synthesis in the P-gp/off or P-gp/on homogenates. However, a specific GCS inhibitor, ethylenedioxy-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (ethylenedioxy-P4), blocked synthesis by 90%. In the cytotoxicity assays, the P-gp/off cells were sensitive to C6-ceramide and the P-gp/on cells were resistant. Resistance to C6-ceramide in the P-gp/on cells was reversed by tamoxifen but not by ethylenedioxy-P4. Experiments in another cervical cancer model showed that multidrug-resistant P-gp-rich KB-V1 cells synthesized 3-fold more C6-GC from C6-ceramide than the parental, P-gp-poor KB-3-1 cells, and whereas tamoxifen had no effect on the C6-GC synthesis in the KB-3-1 cells, it inhibited synthesis by 70% in the KB-V1 cells. This study demonstrates that P-gp potentiates C6-ceramide glycosylation and if antagonized augments C6-ceramide sensitivity, both features previously ascribed to GCS. We propose that P-gp can be an effective target for enhancing short-chain ceramide cytotoxicity in the treatment of drug-resistant cancer. PMID:21042729

Chapman, Jacqueline V; Gouazé-Andersson, Valérie; Cabot, Myles C

2010-12-01

85

Dynamic and intracellular trafficking of P-glycoprotein-EGFP fusion protein: Implications in multidrug resistance in cancer.  

PubMed

In our present study, a P-glycoprotein-EGFP (P-gp-EGFP) fusion plasmid was constructed and functionally expressed in HeLa cells to investigate the intracellular localization and trafficking of P-glycoprotein (P-gp). Using immunocytochemistry and fluorescent confocal microscopy techniques, colocalization studies showed that after transfection, P-gp-EGFP was progressively transported from the endoplasmic reticulum (ER) to the Golgi and finally to the plasma membrane within 12-48 hr. The degree of intracellular accumulation of daunorubicin was related to the particular localization of P-gp-EGFP. Significant daunorubicin accumulation occurred in transfected cells when P-gp-EGFP was localized predominantly within the ER, and accumulation remained high when P-gp-EGFP was mainly localized in the Golgi. However, there was little or no intracellular accumulation of daunorubicin when P-gp-EGFP was localized predominantly on the plasma membrane. Blocking the intracellular trafficking of P-gp-EGFP with brefeldin A (BFA) and monensin resulted in inhibition of traffic of P-gp-EGFP and retention of P-gp-EGFP intracellularly. Intracellular accumulation of daunorubicin also increased in the presence of BFA or monensin. Our study shows that P-gp-EGFP can be used to define the dynamics of P-gp traffic in a transient expression system, and demonstrates that localization of P-gp on the plasma membrane is associated with the highest level of resistance to daunorubicin accumulation in cells. Modulation of intracellular localization of P-gp with agents designed to selectively modify its traffic may provide a new strategy for overcoming multidrug resistance in cancer cells. PMID:14750166

Fu, Dong; Bebawy, Mary; Kable, Eleanor P W; Roufogalis, Basil D

2004-03-20

86

Mucoadhesive properties and interaction with P-glycoprotein (P-gp) of thiolated-chitosans and -glycol chitosans and corresponding parent polymers: a comparative study.  

PubMed

The aim of the present work was to compare the mucoadhesive and efflux pump P-glycoprotein (P-gp) interacting properties of chitosan (CS)- and glycolchitosan (GCS)-based thiomers and corresponding unmodified parent polymers. For this purpose, the glycol chitosan-N-acetyl-cysteine (GCS-NAC) and glycol chitosan-glutathione (GCS-GSH) thiomers were prepared under simple and mild conditions. Their mucoadhesive characteristics were studied by turbidimetric and zeta potential measurements. The P-gp interacting properties were evaluated measuring the effects of thiolated- and unmodified-polymers on the bidirectional transport (BA/AB) of rhodamine-123 across Caco-2 cells as well as in the calcein-AM and ATPase activity assays. Although all the thiomers and unmodified polymers showed optimal-excellent mucoadhesive properties, the best mucoadhesive performances have been obtained by CS and CS-based thiomers. Moreover, it was found that the pretreatment of Caco-2 cell monolayer with GCS-NAC or GCS restores Rho-123 cell entrance by inhibiting P-gp activity. Hence, GCS-NAC and GCS may constitute new biomaterials useful for improving the bioavailability of P-gp substrates. PMID:24521085

Trapani, Adriana; Palazzo, Claudio; Contino, Marialessandra; Perrone, Maria Grazia; Cioffi, Nicola; Ditaranto, Nicoletta; Colabufo, Nicola Antonio; Conese, Massimo; Trapani, Giuseppe; Puglisi, Giovanni

2014-03-10

87

C-di-GMP Hydrolysis by Pseudomonas aeruginosa HD-GYP Phosphodiesterases: Analysis of the Reaction Mechanism and Novel Roles for pGpG  

PubMed Central

In biofilms, the bacterial community optimizes the strategies to sense the environment and to communicate from cell to cell. A key player in the development of a bacterial biofilm is the second messenger c-di-GMP, whose intracellular levels are modulated by the opposite activity of diguanylate cyclases and phosphodiesterases. Given the huge impact of bacterial biofilms on human health, understanding the molecular details of c-di-GMP metabolism represents a critical step in the development of novel therapeutic approaches against biofilms. In this study, we present a detailed biochemical characterization of two c-di-GMP phosphodiesterases of the HD-GYP subtype from the human pathogen Pseudomonas aeruginosa, namely PA4781 and PA4108. Upstream of the catalytic HD-GYP domain, PA4781 contains a REC domain typical of two-component systems, while PA4108 contains an uncharacterized domain of unknown function. Our findings shed light on the activity and catalytic mechanism of these phosphodiesterases. We show that both enzymes hydrolyse c-di-GMP in a two-step reaction via the linear intermediate pGpG and that they produce GMP in vitro at a surprisingly low rate. In addition, our data indicate that the non-phosphorylated REC domain of PA4781 prevents accessibility of c-di-GMP to the active site. Both PA4108 and phosphorylated PA4781 are also capable to use pGpG as an alternative substrate and to hydrolyse it into GMP; the affinity of PA4781 for pGpG is one order of magnitude higher than that for c-di-GMP. These results suggest that these enzymes may not work (primarily) as genuine phosphodiesterases. Moreover, the unexpected affinity of PA4781 for pGpG may indicate that pGpG could also act as a signal molecule in its own right, thus further widening the c-di-GMP-related signalling scenario.

Stelitano, Valentina; Giardina, Giorgio; Paiardini, Alessandro; Castiglione, Nicoletta; Cutruzzola, Francesca; Rinaldo, Serena

2013-01-01

88

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

89

Dual approach utilizing self microemulsifying technique and novel P-gp inhibitor for effective delivery of taxanes.  

PubMed

In the present work, concomitant use of self-microemulsifying drug delivery systems (SMEDDS) and a novel third-generation P-gp inhibitor, GF120918 (elacridar), for the effective transport of taxanes (paclitaxel and docetaxel) across an in vitro model of the intestinal epithelium and uptake into tumor cells were investigated. On the basis of solubility studies and ternary phase diagrams, different SMEDDS formulations of taxanes were prepared and characterized. In caco-2 cell permeation study, paclitaxel-loaded SMEDDS along with GF120918 showed a four-fold increase in apparent permeability, while docetaxel-loaded SMEDDS in combination with GF120918 showed a nine-fold increase in permeability, as compared to plain drug solution. Cell uptake studies on A549 cells were performed with microemulsions formed from both SMEDDS formulations loaded with rhodamine 123 dye and showed good uptake than plain dye solution. Confocal laser scanning microscopic images further confirmed the higher uptake of both SMEDDS formulations in the presence of GF120918. PMID:22439872

Chaurasiya, Akash; Singh, Ajeet K; Jain, Gaurav K; Warsi, Musarrat H; Sublet, Emmanuelle; Ahmad, Farhan J; Borchard, Gerrit; Khar, Roop K

2012-01-01

90

P-glycoprotein of blood brain barrier: cross-reactivity of MAb C219 with a 190 kDa protein in bovine and rat isolated brain capillaries  

Microsoft Academic Search

P-glycoprotein (P-gp), an active efflux pump of antitumor drugs, is strongly expressed in endothelial cells of the blood brain barrier (BBB). Two proteins (155 and 190 kDa) were detected by Western blot analysis of beef and rat capillaries with the monoclonal antibody (MAb) C219. In order to characterize the nature of these proteins, their profile of solubilization by different detergents

Edith Beaulieu; Michel Demeule; Jean-François Pouliot; Diana A. Averill-Bates; Gérard F. Murphy; Richard Béliveau

1995-01-01

91

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.

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

2014-01-01

92

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

93

P-Glycoprotein Expression and DNA Topoisomerase I and II Activity in Benign Tumors of the Ovary and in Malignant Tumors of the Ovary, before and after Platinum\\/Cyclophosphamide Chemotherapy1  

Microsoft Academic Search

P-glycoprotein (P-gp) expression and DNA topoisomerase (Topo) II are important variables in multidrug resistant tumor cell lines. The aim of this study was to evaluate P-gp expression and Topo I and II activity in benign and malignant epithelial ovarian tumors. P-gp expression was analyzed immunohistochemically in cryostat sections of fresh tumor specimens. In the same specimens Topo I and II

Steven de Jong; Henk Boonstra; Annette Gouw; Pax H. B. Willemse; Jan G. Zijlstra; Elisabeth G. E. de Vries

1991-01-01

94

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

95

Regulatory Protein Coordinating Gene Expression  

NSDL National Science Digital Library

The action of the glucocorticoid receptor is illustrated. On the left is shown a series of genes, each of which has various gene activator proteins bound to its regulatory region. However, these bound proteins are not sufficient on their own to activate transcription efficiently. On the right is shown the effects of adding an additional gene regulatory protein

BEGIN:VCARD VERSION:2.1 FN:Bruce Alberts N:Alberts;Bruce REV:2005-04-16 END:VCARD; BEGIN:VCARD VERSION:2.1 FN:Dennis Bray N:Bray;Dennis REV:2005-04-16 END:VCARD; BEGIN:VCARD VERSION:2.1 FN:Alexander Johnson N:Johnson;Alexander REV:2005-04-16 END:VCARD; BEGIN:VCARD VERSION:2.1 FN:Julian Lewis N:Lewis;Julian REV:2005-04-16 END:VCARD; BEGIN:VCARD VERSION:2.1 FN:Martin Raff N:Raff;Martin REV:2005-04-16 END:VCARD; BEGIN:VCARD VERSION:2.1 FN:Keith Roberts N:Roberts;Keith REV:2005-04-16 END:VCARD

1998-07-01

96

Expression of Human Milk Proteins in Plants  

Microsoft Academic Search

Human milk proteins are believed to have a multitude of biological activities benefiting the newborn infant. Such functions include antibacterial and antiviral activities, enhancement of the immune system and increased nutrient absorption. To date, only breast-fed infants have been exposed to these proteins. However, by using genetic engineering it is now possible to express these proteins in plants, such as

Bo Lonnerdal

97

Heat shock protein expression in fish  

Microsoft Academic Search

Heat shock proteins (HSP) are a family of proteins expressed in response to a wide range of biotic and abiotic stressors. They are thus also referred to as stress proteins. Their extraordinarily high degree of identity at the amino acid sequence level and the fact that this cellular stress response has been described in nearly all organisms studied, make this

George K. Iwama; Philip T. Thomas; Robert B. Forsyth; Mathilakath M. Vijayan

1998-01-01

98

Irradiation of rat brain reduces P-glycoprotein expression and function  

PubMed Central

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

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

2007-01-01

99

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

100

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.

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

2014-01-01

101

Protein Expression Regulation under Oxidative Stress*  

PubMed Central

Oxidative stress is known to affect both translation and protein turnover, but very few large scale studies describe protein expression under stress. We measure protein concentrations in Saccharomyces cerevisiae over the course of 2 h in response to a mild oxidative stress induced by diamide, providing detailed time-resolved information for 815 proteins, with additional data for another ?1,100 proteins. For the majority of proteins, we discover major differences between the global transcript and protein response. Although mRNA levels often return to baseline 1 h after treatment, protein concentrations continue to change. Integrating our data with features of translation and protein degradation, we are able to predict expression patterns for 41% of the proteins in the core data set. Predictive features include, among others, targeting by RNA-binding proteins (Lhp1 and Khd1), RNA secondary structures, RNA half-life, and translation efficiency under unperturbed conditions and in response to oxidative reagents, but not chaperone binding. We are able to both describe general dynamics of protein concentration changes and suggest possible regulatory mechanisms for individual proteins.

Vogel, Christine; Silva, Gustavo Monteiro; Marcotte, Edward M.

2011-01-01

102

Expressed protein ligation for protein semisynthesis and engineering.  

PubMed

Over the past decade, a significant methodological development in peptide ligation strategies has been elaborated that now permits the assembly of peptides and proteins. Native chemical ligation (NCL) has been introduced to join synthetic unprotected peptides by using the chemoselective reaction between a C-terminal thioester and an N-terminal cysteine residue to result in a native peptide bond. Although this method has been applied to obtain peptides, small proteins, or protein domains (up to approx 150 residues), larger proteins could not been easily received because of the limited size of the ligated fragments. Intein technologies benefit from the opportunity to participate in the production of polypeptides with the reactive groups necessary for NCL aside from the rapid isolation of highly pure recombinant proteins. Expressed protein ligation extends the scope of NCL by overcoming the size limitation of target proteins accessible to synthesis. The intein splicing and EPL have been already proven to be useful for protein semisynthesis and for various investigations, including the studies of protein-protein interactions, segmental isotopic labeling for protein structure determination, synthesis of cytotoxic proteins, protein cyclization, and site-specific incorporation of noncanonical amino acids and biophysical probes into a protein sequence. PMID:16044543

Machova, Zuzana; Beck-Sickinger, Annette G

2005-01-01

103

Biotechnology Protein Expression and Purification Facility  

NASA Technical Reports Server (NTRS)

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

2003-01-01

104

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

PubMed

Treatment of carcinoma commonly fails due to chemoresistance. Studies have shown that endothelial cells acquire resistance via the tumor microenvironment. Microvesicle (MV) shedding from the cell membrane to the microenvironment plays an important role in communication between cells. The aim of the present study was to determine whether MCF-7 adriamycin-resistant cells (MCF-7/ADM) shed MVs that alter the characteristics of human microvessel endothelial cells (HMECs). MVs from tumor cells transferred a Ca(2+)-permeable channel TrpC5 to HMECs, inducing the expression of P-glycoprotein (P-gp) by activation of the transcription factor NFATc3 (nuclear factor of activated T cells isoform c3). Expression of the mdr1 gene was blocked by the TrpC5-blocking antibody T5E3, and the production of P-gp in HMECs was reduced by blockade of TrpC5. Thus, we postulate that endothelial cells acquire the resistant protein upon exposure to TrpC5-containg MVs in the microenvironment, and express P-gp in the TrpC5-NFATc3 signal pathway. PMID:24582564

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

2014-03-28

105

Enhanced expression of adenovirus transforming proteins.  

PubMed Central

Proteins encoded in regions EIA and EIB of human adenoviruses cause transformation of rodent cells. One protein from EIA also stimulates transcription of other early regions at early times in a productive infection. In the past, direct analysis of these proteins synthesized in vivo has been difficult because of the low levels produced in both transformed cells and productively infected cells. We present a simple method which leads to expression of EIA and EIB mRNAs and proteins at 30-fold greater levels than those observed during the early phase of a standard productive infection. Under these conditions, these proteins are among the most prominent translation products of infected cells. This allowed direct visualization of EIA and EIB proteins on two-dimensional gels of pulse-labeled total cell protein. Experiments with EIA and EIB mutants confirm that the identified proteins are indeed encoded in these regions. Two EIA proteins are observed, one translated from each of the major early EIA mRNAs. Both of these EIA proteins are phosphorylated. Images

Gaynor, R B; Tsukamoto, A; Montell, C; Berk, A J

1982-01-01

106

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

PubMed

Recombinant protein technology is essential for conducting protein science and using proteins as materials in pharmaceutical or industrial applications. Although obtaining soluble proteins is still a major experimental obstacle, knowledge about protein expression/solubility under standard conditions may increase the efficiency and reduce the cost of proteomics studies. In this study, we present a computational approach to estimate the probability of protein expression and solubility for two different protein expression systems: in vivo Escherichia coli and wheat germ cell-free, from only the sequence information. It implements two kinds of methods: a sequence/predicted structural property-based method that uses both the sequence and predicted structural features, and a sequence pattern-based method that utilizes the occurrence frequencies of sequence patterns. In the benchmark test, the proposed methods obtained F-scores of around 70%, and outperformed publicly available servers. Applying the proposed methods to genomic data revealed that proteins associated with translation or transcription have a strong tendency to be expressed as soluble proteins by the in vivo E. coli expression system. The sequence pattern-based method also has the potential to indicate a candidate region for modification, to increase protein solubility. All methods are available for free at the ESPRESSO server (http://mbs.cbrc.jp/ESPRESSO). PMID:23436767

Hirose, Shuichi; Noguchi, Tamotsu

2013-05-01

107

Expression Pattern of Id Proteins in Medulloblastoma  

PubMed Central

Inhibitor of DNA binding or inhibitor of differentiation (Id) proteins are up regulated in a variety of neoplasms, particularly in association with high-grade, poorly differentiated tumors, while differentiated tissues show little or no Id expression. The four Id genes are members of the helix-loop-helix (HLH) family of transcription factors and act as negative regulators of transcription by binding to and sequestering HLH complexes. We tested the hypothesis that Id proteins are overexpressed in medulloblastoma by performing immunohistochemistry using a medulloblastoma tissue microarray with 45 unique medulloblastoma and 11 normal control cerebella, and antibodies specific for Id1, Id2, Id3, and Id4. A semi-quantitative staining score that took staining intensity and the proportion of immunoreactive cells into account was used. Id1 was not detected in normal cerebella or in medulloblastoma cells, but 78 % of tumors showed strong Id1 expression in endothelial nuclei of tumor vessels. Id2 expression was scant in normal cerebella and increased in medulloblastoma (median staining score: 4). Id3 expression was noted in some neurons of the developing cerebellar cortex, but it was markedly up regulated in medulloblastoma (median staining score: 12) and in tumor endothelial cells. Id4 was not expressed in normal cerebella or in tumor cells. Id2 or Id3 overexpression drove proliferation in medulloblastoma cell lines by altering the expression of critical cell cycle regulatory proteins in favor of cell proliferation. This study shows that Id1 expression in endothelial cells may contribute to angiogenic processes and that increased expression of Id2 and Id3 in medulloblastoma is potentially involved in tumor cell proliferation and survival.

Snyder, Andrew D.; Dulin-Smith, Ashley N.; Houston, Ronald H.; Durban, Ashley N.; Brisbin, Bethany J.; Oostra, Tyler D.; Marshall, Jordan T.; Kahwash, Basil M.

2013-01-01

108

Control of expression of silk protein genes  

Microsoft Academic Search

At least three silk genes are specifically expressed in the posterior, and five other genes in middle, silk glands. The products of genes active in PSG include fibroin, L-chain fibroin and P25 protein. PSG genes as well as the Ser-1 gene, differing in structure, exhibit a striking degree of homology of their 5? flanking sequences. This suggests the presence of

Krystyna Grzelak

1995-01-01

109

Cerebellar synaptic protein expression in schizophrenia  

Microsoft Academic Search

A cortico-subcortico-cerebellar neural circuit has been postulated to be important in the pathophysiology of schizophrenia. This study investigated whether there are synaptic changes in the cerebellum to accompany its putative involvement in the disorder. We measured the expression of three synaptic proteins (synaptophysin, complexin I and complexin II) in the cerebellar cortex of 16 subjects with schizophrenia and 16 controls

S. L Eastwood; D Cotter; P. J Harrison

2001-01-01

110

Differential Modulation of P-Glycoprotein Expression and Activity by Non-Nucleoside HIV1 Reverse Transcriptase Inhibitors in Cell Culture  

Microsoft Academic Search

Purpose. This study investigated the effects of the non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTI) nevirapine (NVR), efavirenz (EFV), and delavirdine (DLV) on P-glycoprotein (P-gp) activity and expression to anticipate P-gp related drug-drug interactions associated with combination therapy.

Elke Störmer; Lisa L. von Moltke; Michael D. Perloff; David J. Greenblatt

2002-01-01

111

High expression of transgene protein in Spirodela.  

PubMed

The monocot family Lemnaceae (duckweed) is composed of small, edible, aquatic plants. Spirodela oligorrhiza SP is a duckweed with a biomass doubling time of about 2 days under controlled, axenic conditions. Stably transformed Spirodela plants were obtained following co-cultivation of regenerative calli with Agrobacterium tumefaciens. GFP activity was successfully monitored in different subcellular compartments of the plant and correlated with different targeting sequences. Transgenic lines were followed for a period of at least 18 months and more than 180 vegetative doublings (generations). The lines are stable in morphology, growth rate, transgene expression, and activity as measured by DNA-DNA and immunoblot hybridizations, fluorescence activity measurements, and antibiotic resistance. The level of transgene expression is a function of leader sequences rather than transgene copy number. A stable, transgenic, GFP expression level >25% of total soluble protein is demonstrated for the S. oligorrhiza system, making it among the higher expressing systems for nuclear transformation in a higher plant. PMID:17492286

Vunsh, Ron; Li, Jihong; Hanania, Uri; Edelman, Marvin; Flaishman, Moshe; Perl, Avihai; Wisniewski, Jean-Pierre; Freyssinet, Georges

2007-09-01

112

Engineering Genes for Predictable Protein Expression  

PubMed Central

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

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

2013-01-01

113

Engineering genes for predictable protein expression.  

PubMed

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

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

2012-05-01

114

PARP-1 protein expression in glioblastoma multiforme.  

PubMed

One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8,000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker. PMID:22472897

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

2012-01-01

115

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

116

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.

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

2004-01-01

117

Process for improved protein expression by strain engineering  

US Patent & Trademark Office Database

This invention is a process for improving the production levels of recombinant proteins or peptides or improving the level of active recombinant proteins or peptides expressed in host cells. The invention is a process of comparing two genetic profiles of a cell that expresses a recombinant protein and modifying the cell to change the expression of a gene product that is upregulated in response to the recombinant protein expression. The process can improve protein production or can improve protein quality, for example, by increasing solubility of a recombinant protein.

2013-12-10

118

Cerebellar synaptic protein expression in schizophrenia.  

PubMed

A cortico-subcortico-cerebellar neural circuit has been postulated to be important in the pathophysiology of schizophrenia. This study investigated whether there are synaptic changes in the cerebellum to accompany its putative involvement in the disorder. We measured the expression of three synaptic proteins (synaptophysin, complexin I and complexin II) in the cerebellar cortex of 16 subjects with schizophrenia and 16 controls using in situ hybridisation histochemistry and immunoautoradiography. Complexin I and II are expressed predominantly by inhibitory and excitatory neurones respectively. In schizophrenia, synaptophysin mRNA was decreased, as was complexin II and its mRNA. Complexin I mRNA and protein levels were unaltered. Expression of the mRNAs in the rat cerebellum was unaffected by 2 weeks administration of antipsychotic drugs (haloperidol, chlorpromazine, risperidone, olanzapine or clozapine). We conclude that there is synaptic pathology in the cerebellum in schizophrenia. By disrupting neural circuits, the alterations may contribute to the cerebellar dysfunction thought to occur in the disorder. PMID:11483314

Eastwood, S L; Cotter, D; Harrison, P J

2001-01-01

119

Global signatures of protein and mRNA expression levels†  

PubMed Central

Cellular states are determined by differential expression of the cell’s proteins. The relationship between protein and mRNA expression levels informs about the combined outcomes of translation and protein degradation which are, in addition to transcription and mRNA stability, essential contributors to gene expression regulation. This review summarizes the state of knowledge about large-scale measurements of absolute protein and mRNA expression levels, and the degree of correlation between the two parameters. We summarize the information that can be derived from comparison of protein and mRNA expression levels and discuss how corresponding sequence characteristics suggest modes of regulation.

Abreu, Raquel de Sousa; Penalva, Luiz O.; Marcotte, Edward M.

2013-01-01

120

Trypanosoma cruzi expresses diverse repetitive protein antigens.  

PubMed Central

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

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

1989-01-01

121

Expressed protein ligation-mediated template protein extension  

PubMed Central

Expressed protein ligation (EPL) was performed to investigate sequence requirements for a variant human apolipoprotein A-I (apoA-I) to adopt a folded structure. A C-terminal truncated apoA-I, corresponding to residues 1–172, was expressed and isolated from E. coli. Compared to full length apoA-I (243 amino acids), apoA-I(1-172) displayed less ?-helix secondary structure and lower stability in solution. To determine if extension of this polypeptide would confer secondary structure content and/or stability, 20 residues were added to the C-terminus of apoA-I(1-172) by EPL, creating apoA-IMilano(1-192). The EPL product displayed biophysical properties similar to full-length apoA-IMilano. The results provide a general protein engineering strategy to modify the length of a recombinant template polypeptide using synthetic peptides as well as a convenient, cost effective way to investigate the structure / function relations in apolipoprotein fragments or domains of different size.

Kamei, Ayako; Hauser, Paul S.; Beckstead, Jennifer A.; Weers, Paul M.M.; Ryan, Robert O.

2012-01-01

122

Bioluminescence Assay for Detecting Cell Surface Membrane Protein Expression  

PubMed Central

Abstract We have developed a method to measure the amounts of cell surface-expressed membrane proteins with bioluminescence. Dinoflagellate luciferase was expressed on the surface of a mammalian cell as a chimeric fusion protein with a membrane protein of interest. Using a membrane-impermeable substrate to quantify the membrane-displayed luciferase, the expression of the membrane protein on the cell surface was determined. By inclusion of a quenching step for the luminescent activity of luciferase on the cell surface, we were able to monitor the membrane protein expression kinetics by measuring the luminescence recovery from the cell surface after quenching. The reported methods provide a convenient way to monitor the kinetics of expression and transport of membrane proteins to the cell surface. It is applicable to the high-throughput analysis of drugs or drug candidates concerning their effects on membrane protein expression.

Kato, Mieko; Chiba, Tomoki; Li, Min

2011-01-01

123

High-efficiency protein expression in plants from agroinfection-compatible Tobacco mosaic virus expression vectors  

Microsoft Academic Search

BACKGROUND: Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that

John A Lindbo

2007-01-01

124

MDR Gene Expression Analysis of Six Drug-Resistant Ovarian Cancer Cell Lines  

PubMed Central

Ovarian cancer is the leading cause of death among gynaecological malignancies. Multiple drug resistance makes cancer cells insensitive to chemotherapy. In this study, we developed six primary ovarian cancer cell lines (W1MR, W1CR, W1DR, W1VR, W1TR, and W1PR) resistant to drugs such as methotrexate, cisplatin, doxorubicin, vincristine, topotecan, and paclitaxel. A chemosensitivity assay MTT test was performed to assess drug cross-resistance. Quantitative real-time polymerase chain reaction and Western blot were also performed to determine mRNA and protein expression of genes involved in chemoresistance. We observed high cross-resistance to doxorubicin, vincristine, and paclitaxel in the cell lines resistant to these agents. We also found a significant correlation between resistance to these drugs and increased expression of P-gp. Two different mechanisms of topotecan resistance were observed in the W1TR and W1PR cell lines. We did not observe any correlation between MRP2 transcript and protein levels. Cell lines resistant to agents used in ovarian cancer treatment remained sensitive to methotrexate. The main mechanisms of drug resistance were due to P-gp expression in the doxorubicin, vincristine, and paclitaxel resistant cell lines and BCRP expression in the topotecan resistant cell line.

Januchowski, Radoslaw; Wojtowicz, Karolina; Sujka-Kordowska, Patrycja; Andrzejewska, Malgorzata; Zabel, Maciej

2013-01-01

125

MDR gene expression analysis of six drug-resistant ovarian cancer cell lines.  

PubMed

Ovarian cancer is the leading cause of death among gynaecological malignancies. Multiple drug resistance makes cancer cells insensitive to chemotherapy. In this study, we developed six primary ovarian cancer cell lines (W1MR, W1CR, W1DR, W1VR, W1TR, and W1PR) resistant to drugs such as methotrexate, cisplatin, doxorubicin, vincristine, topotecan, and paclitaxel. A chemosensitivity assay MTT test was performed to assess drug cross-resistance. Quantitative real-time polymerase chain reaction and Western blot were also performed to determine mRNA and protein expression of genes involved in chemoresistance. We observed high cross-resistance to doxorubicin, vincristine, and paclitaxel in the cell lines resistant to these agents. We also found a significant correlation between resistance to these drugs and increased expression of P-gp. Two different mechanisms of topotecan resistance were observed in the W1TR and W1PR cell lines. We did not observe any correlation between MRP2 transcript and protein levels. Cell lines resistant to agents used in ovarian cancer treatment remained sensitive to methotrexate. The main mechanisms of drug resistance were due to P-gp expression in the doxorubicin, vincristine, and paclitaxel resistant cell lines and BCRP expression in the topotecan resistant cell line. PMID:23484165

Januchowski, Rados?aw; Wojtowicz, Karolina; Sujka-Kordowska, Patrycja; Andrzejewska, Ma?gorzata; Zabel, Maciej

2013-01-01

126

Global Expression of Cell Surface Proteins in Embryonic Stem Cells  

PubMed Central

Background Recent studies have shown that embryonic stem (ES) cells globally express most genes in the genome at the mRNA level; however, it is unclear whether this global expression is propagated to the protein level. Cell surface proteins could perform critical functions in ES cells, so determining whether ES cells globally express cell surface proteins would have significant implications for ES cell biology. Methods and Principal Findings The surface proteins of mouse ES cells were purified by biotin labeling and subjected to proteomics analysis. About 1000 transmembrane or secreted cell surface proteins were identified. These proteins covered a large variety if functional categories including signal transduction, adhesion and transporting. More over, mES cells promiscuously expressed a wide variety of tissue specific surface proteins. And many surface proteins were expressed heterogeneously on mES cells. We also find that human ES cells express a wide variety of tissue specific surface proteins. Conclusions/Significance Our results indicate that global gene expression is not simply a result of leaky gene expression, which could be attributed to the loose chromatin structure of ES cells; it is also propagated to the functional level. ES cells may use diverse surface proteins to receive signals from the diverse extracellular stimuli that initiate differentiation. Moreover, the promiscuous expression of tissue specific surface proteins illuminate new insights into the strategies of cell surface marker screening.

Gu, Bin; Zhang, Jiarong; Wang, Wei; Mo, Lijuan; Zhou, Yang; Chen, Liangbiao; Liu, Yusen; Zhang, Ming

2010-01-01

127

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

128

Cocaine increases endoplasmic reticulum stress protein expression in striatal neurons  

Microsoft Academic Search

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), Ire1? and perk via glutamate and

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

2007-01-01

129

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

PubMed Central

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

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

2011-01-01

130

Induction of expression and functional activity of P-glycoprotein efflux transporter by bioactive plant natural products.  

PubMed

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

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

2011-11-01

131

Trps1 is associated with the multidrug resistance of osteosarcoma by regulating MDR1 gene expression.  

PubMed

Multidrug resistance (MDR) is a significant clinical problem in the chemotherapy of osteosarcoma and has been linked to the cellular expression of several multidrug-efflux transporters such as MDR1/P-gp. Our inhibition of the transcription factor Trps1 led to repression of MDR1/P-gp while its overexpression resulted in upregulation of MDR1/P-gp. Flow cytometric analysis suggested Trps1 increased the release of several anti-cancer drugs, thus decreasing their accumulation. Immunohistochemical analysis of clinical samples indicated that the expression of Trps1 directly correlated with MDR1/P-gp. Trps1 inhibited TGFbeta-1 and directly bound to the MDR1 promoter. Our data demonstrate a role for Trps1 in the regulation of MDR1 expression in osteosarcoma. PMID:24491996

Jia, Ming; Hu, Jing; Li, Weiwei; Su, Peng; Zhang, Hui; Zhang, Xiaofang; Zhou, Gengyin

2014-03-01

132

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.

Dalton, Annamarie C; Barton, William A

2014-01-01

133

Proteasome inhibitors suppress expression of NPM and ARF proteins.  

PubMed

Proteasome inhibitors stabilize numerous proteins by inhibiting their degradation. Previously we have demonstrated that proteasome inhibitors thiostrepton, MG132 and bortezomib paradoxically inhibit transcriptional activity and mRNA/protein expression of FOXM1. Here we demonstrate that, in addition to FOXM1, the same proteasome inhibitors also decrease mRNA and protein expression of NPM and ARF genes. These data suggest that proteasome inhibitors may suppress gene expression by stabilizing their transcriptional inhibitors. PMID:22071628

Pandit, Bulbul; Gartel, Andrei L

2011-11-15

134

Metal-ion-coordinating properties of the dinucleotide 2'-deoxyguanylyl(5'-->3')-2'-deoxy-5'-guanylate (d(pGpG)3-): isomeric equilibria including macrochelated complexes relevant for nucleic acids.  

PubMed

The interaction between divalent metal ions and nucleic acids is well known, yet knowledge about the strength of binding of labile metal ions at the various sites is very scarce. We have therefore studied the stabilities of complexes formed between the nucleic acid model d(pGpG) and the essential metal ions Mg2+ and Zn2+ as well as with the generally toxic ions Cd2+ and Pb2+ by potentiometric pH titrations; all four ions are of relevance in ribozyme chemistry. A comparison of the present results with earlier data obtained for M(pUpU)- complexes allows the conclusion that phosphate-bound Mg2+ and Cd2+ form macrochelates by interaction with N7, whereas the also phosphate-coordinated Pb2+ forms a 10-membered chelate with the neighboring phosphate diester bridge. Zn2+ forms both types of chelates with formation degrees of about 91% and 2.4% for Zn[d(pGpG)]cl/N7 and Zn[d(pGpG)]-cl/PO, respectively; the open form with Zn2+ bound only to the terminal phosphate group, Zn[d(pGpG)]-op, amounts to about 6.8 %. The various intramolecular equilibria have also been quantified for the other metal ions. Zn2+, Cu2+, and Cd2+ also form macrochelates in the monoprotonated M[H;d(pGpG)] species (the proton being at the terminal phosphate group), that is, the metal ion at N7 interacts to some extent with the P(O)2(OH)- group. Thus, this study demonstrates that the coordinating properties of the various metal ions toward a pGpG unit in a nucleic acid differ: Mg2+, Zn2+, and Cd2+ have a significant tendency to bridge the distance between N7 and the phosphate group of a (d)GMP unit, although to various extents, whereas Pb2+ (and possibly Ca2+) prefer a pure phosphate coordination. PMID:17121397

Knobloch, Bernd; Sigel, Helmut; Okruszek, Andrzej; Sigel, Roland K O

2007-01-01

135

Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants  

Microsoft Academic Search

A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions

A. G. von Arnim; X.-W. Deng; M. G. Stacey

1998-01-01

136

Expression and Clinical Significance of Livin Protein in Hepatocellular Carcinoma  

PubMed Central

In this study, the two-step PV method of immunohistochemistry was used to determine livin protein expression in HCC tissues, pericarcinoma tissues, hepatitis/hepatic cirrhosis tissues, and normal hepatic tissues, and livin protein expression was detected in the blood plasma of patients with HCC before and after surgery, subjects with hepatic cirrhosis and hepatitis, and healthy blood donors using ELISA. Livin protein expression was significantly higher in HCC tissues than that in normal hepatic tissues and hepatitis/hepatic cirrhosis tissues, with no significant difference between HCC tissues and pericarcinoma tissues. The HCC patients with positive livin protein expression had a significantly higher survival rate than those with negative livin protein expression. Livin protein expression was significantly higher in the blood plasma of patients with HCC before and after surgery and in patients with hepatic cirrhosis and hepatitis than that in healthy blood donors, whereas livin protein expression in the blood plasma of patients with HCC was not significantly different from that of patients with hepatic cirrhosis and hepatitis. Livin protein expression in HCC tissues did not correlate with that in the blood plasma of the same HCC patients. Livin protein expression may be a potential, effective indicator for assessing prognosis in patients with HCC.

Gao, Ying-tang; Zhang, Qin; Jing, Li; Liu, Tong; Shi, Wen-xia; Zhai, Dao-kuan; Jing, Xiang; Du, Zhi

2013-01-01

137

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

PubMed

The purpose of this study was to identify conditions that will increase the sensitivity of resistant cancer cells to anti-mitotic drugs. Currently, atovaquine (ATO), chloroquine (CHL), primaquine (PRI), mefloquine (MEF), artesunate (ART), and doxycycline (DOY) are the most commonly used anti-malarial drugs. Herein, we tested whether anti-malarial drugs can sensitize drug-resistant KBV20C cancer cells. None of the six tested anti-malarial drugs was found to better sensitize the drug-resistant cells compared to the sensitive KB cells. With an exception of DOY, all other anti-malarial drugs tested could sensitize both KB and KBV20C cells to a similar extent, suggesting that anti-malarial drugs could be used for sensitive as well as resistant cancer cells. Furthermore, we examined the effects of anti-malarial drugs in combination with an antimitotic drug, vinblastine (VIN) on the sensitisation of resistant KBV20C cells. Using viability assay, microscopic observation, assessment of cleaved PARP, and Hoechst staining, we identified that two anti-malarial drugs, PRI and MEF, highly sensitized KBV20C-resistant cells to VIN treatment. Moreover, PRI- or MEF-induced sensitisation was not observed in VIN-treated sensitive KB parent cells, suggesting that the observed effect is specific to resistant cancer cells. We demonstrated that the PRI and MEF sensitisation mechanism mainly depends on the inhibition of p-glycoprotein (P-gp). Our findings may contribute to the development of anti-malarial drug-based combination therapies for patients resistant to anti-mitotic drugs. PMID:24284282

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

2013-11-22

138

Ontogeny of renal P-glycoprotein expression in mice: correlation with digoxin renal clearance.  

PubMed

Digoxin is eliminated mainly by the kidney through glomerular filtration and P-glycoprotein (P-gp) mediated tubular secretion. Toddlers and young children require higher doses of digoxin per kilogram of bodyweight than adults, although the reasons for this have not been elucidated. We hypothesized there is an age-dependant increase in P-gp expression in young children. The objectives of this study were to elucidate age-dependant expression of renal P-gp and its correlation with changes in the clearance rate of digoxin. FVB mice were killed at different ages to prepare total RNA for P-gp expression studies. Semi-quantitative RT-PCR was conducted to analyze mdr1a and mdr1b ontogeny in the kidney at: birth, 7, 14, 21, 28 and 45-d old adults. The pharmacokinetics of digoxin (7 microg/kg) was studied in mice of the same age groups. Newborn and Day 7 levels of both mdr1a and mdr1b were marginal. Day 21 mdr1b levels were significantly higher than both Day 14 and Day 28 levels. Digoxin clearance rates were the highest at Day 21, with significant correlation between P-gp expression and clearance values. Increases in digoxin clearance rates after weaning may be attributed, at least in part, to similar increases in P-gp expression. PMID:16306209

Pinto, Natasha; Halachmi, Naomi; Verjee, Zulfikarali; Woodland, Cindy; Klein, Julia; Koren, Gideon

2005-12-01

139

Saturable Active Efflux by P-Glycoprotein and Breast Cancer Resistance Protein at the Blood-Brain Barrier Leads to Nonlinear Distribution of Elacridar to the Central Nervous System  

PubMed Central

The study objective was to investigate factors that affect the central nervous system (CNS) distribution of elacridar. Elacridar inhibits transport mediated by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and has been used to study the influence of transporters on brain distribution of chemotherapeutics. Adequate distribution of elacridar across the blood-brain barrier (BBB) and into the brain parenchyma is necessary to target tumor cells in the brain that overexpress transporters and reside behind an intact BBB. We examined the role of P-gp and Bcrp on brain penetration of elacridar using Friend leukemia virus strain B wild-type, Mdr1a/b(?/?), Bcrp1(?/?), and Mdr1a/b(?/?)Bcrp1(?/?) mice. Initially, the mice were administered 2.5 mg/kg of elacridar intravenously, and the plasma and brain concentrations were determined. The brain-to-plasma partition coefficient of elacridar in the wild-type mice was 0.82, as compared with 3.5 in Mdr1a/b(?/?) mice, 6.6 in Bcrp1(?/?) mice, and 15 in Mdr1a/b(?/?)Bcrp1(?/?) mice, indicating that both P-gp and Bcrp limit the brain distribution of elacridar. The four genotypes were then administered increasing doses of elacridar, and the CNS distribution of elacridar was determined. The observed and model predicted maximum brain-to-plasma ratios (Emax) at the highest dose were not significantly different in all genotypes. However, the ED50 was lower for Mdr1a/b(?/?) mice compared with Bcrp1(?/?) mice. These findings correlate with the relative expression of P-gp and Bcrp at the BBB in these mice and demonstrate the quantitative enhancement in elacridar CNS distribution as a function of its dose. Overall, this study provides useful concepts for future applications of elacridar as an adjuvant therapy to improve targeting of chemotherapeutic agents to tumor cells in the brain parenchyma.

Sane, Ramola; Agarwal, Sagar; Mittapalli, Rajendar K.

2013-01-01

140

Mechanism of the cis-[Pt(1R,2R-DACH)(H2O)2]2+ intrastrand binding to the double-stranded (pGpG)·(CpC) dinucleotide in aqueous solution: a computational DFT study.  

PubMed

A mechanism of the intrastrand 1,2-cross-link formation between the double-stranded pGpG·CpC dinucleotide (ds(pGpG)) and fully aquated oxaliplatin cis-[Pt(DACH)(H2O)2](2+) (DACH = cyclohexane-1R,2R-diamine) is presented. All structures of the reaction pathways including the transition states (TSs) were fully optimized in water solvent using DFT methodology with dispersion corrections. Both 5' ? 3' and 3' ? 5' binding directions were considered. In the first step there is a slight kinetic preference for 5'-guanine (5'G) monoadduct formation with an activation Gibbs free energy of 18.7 kcal/mol since the N7 center of the 5'G base is fully exposed to the solvent. On the other hand, the N7 atom of 3'-guanine (3'G) is sterically shielded by 5'G. The lowest energy path for formation of the 3'G monoadduct with an activation barrier of 19.3 kcal/mol is connected with a disruption of the 'DNA-like' structure of ds(pGpG). Monoadduct formation is the rate-determining process. The second step, chelate formation, is kinetically preferred in the 3' ? 5' direction. The whole process of the platination is exergonic by up to -18.8 kcal/mol. Structural changes of ds(pGpG), charge transfer effects, and the influence of platination on the G·C base pair interaction strengths are also discussed in detail. PMID:23656523

Chval, Zden?k; Kabelá?, Martin; Burda, Jaroslav V

2013-05-20

141

Hypoxic-induced stress protein expression in rat cardiac myocytes  

Microsoft Academic Search

Mammalian stress proteins can be induced in cells and tissues exposed to a variety of conditions including hyperthermia and diminished Oâ supply. The authors have previously shown that the expression of three stress proteins (71, 85, and 95 kDa) was induced in cardiac tissue from mice exposed to hypoxic conditions. The expression of mRNAs coding for the 85 and 95

G. Howard; T. E. Geoghegan

1986-01-01

142

Sendai Viruses with Altered P, V, and W Protein Expression  

Microsoft Academic Search

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 Zn2+-binding domain (ca. 25% of the total), and a W protein representing the N-terminal half alone (ca. 5% of

Christophe Delenda; Geraldine Taylor; Stéphane Hausmann; Dominique Garcin; Daniel Kolakofsky

1998-01-01

143

A new Trichoplusia ni cell line for membrane protein expression using a baculovirus expression vector system.  

PubMed

A new cell line, MSU-TnT4 (TnT4), was established from Trichoplusia ni embryos for use with baculovirus expression vectors and evaluated for its potential for membrane protein production. To evaluate membrane protein synthesis, recombinant baculoviruses were constructed to express the human neurotensin receptor 1 as an enhanced green fluorescent protein (GFP) fusion. TnT4 cells had a doubling time of 21 h and expressed the membrane-GFP fusion protein at approximately twice the level as Sf21 cells from the p10 promoter, as evaluated by GFP intensity. Expression of secreted alkaline phosphatase (SEAP) was similar to that of Sf21 cells. Expression of membrane-GFP fusion proteins in recombinant baculoviruses provides a rapid method for evaluating the potential of new cell lines for the production of membrane proteins using a baculovirus expression vector system (BEVS). PMID:18622664

Zhang, Fengrui; Manzan, Maria Alejandra; Peplinski, Heather M; Thiem, Suzanne M

2008-01-01

144

Measurement of P-Glycoprotein expression in human neuroblastoma xenografts using in vitro quantitative autoradiography  

Microsoft Academic Search

P-glycoprotein (P-gp) has a role in multidrug resistance (MDR) encountered in human cancers. In this study, we used the colchicine-resistant cell line BE(2)-C\\/CHCb(0.2), a strain of neuroblastoma cell line BE(2)-C, as a model to measure variations of P-gp expression in cells grown in vitro and in vivo. Cells were cultured in the medium supplemented with colchicine. At the beginning of

Rosa Fonti; Andrey Levchenko; Bipin M Mehta; Jiaju Zhang; Takashi Tsuruo; Steven M Larson

1999-01-01

145

In vitro modulation of ABCB1\\/P-glycoprotein expression by polyphenols from Mangifera indica  

Microsoft Academic Search

Many plant compounds are able to modulate the activity and\\/or the expression of the major multidrug transporter ABCB1\\/P-glycoprotein (P-gp). In this study, mango (Mangifera indica L.) stem bark extract (MSBE), its main polyphenol mangiferin and the mangiferin aglycone derivative norathyriol, as well as catechin, gallic acid and quercetin, were investigated for their potential ability to influence ABCB1 gene and P-gp

Elisabetta Chieli; Nadia Romiti; Idania Rodeiro; Gabino Garrido

2010-01-01

146

Combinatorial Protein Library Screening by Periplasmic Expression.  

National Technical Information Service (NTIS)

The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating binding proteins capable of binding small molecules and peptides. In the technique, libraries of candidate binding proteins, such as antibody sequences, ...

B. L. Iverson G. Georglou J. Jeong

2005-01-01

147

Induction of a multixenobiotic resistance protein (MXR) in the Asiatic clam Corbicula fluminea after heavy metals exposure.  

PubMed

Multixenobiotic resistance mechanisms (MXR) related to the mammalian P-glycoprotein multidrug transporter protein (P-gp) are known to occur in several marine invertebrates. In the present work, we report on the induction of an MXR protein by various heavy metals in the gills of the freshwater clam Corbicula fluminea. The evaluation of the MXR protein level was assessed by Western blot using a specific monoclonal antibody raised against the human P-gp (C219). A field transplantation experiment, where clams were caged in a gradient relative to an industrial site, demonstrated a positive relationship between MXR levels and (a) metal pollution (Cd and Zn) in the environment and (b) metal bioaccumulation in the gills. To establish this correlative relationship, clams were exposed to different levels of cadmium (15-60 microg l(-1)) for up to 15 days in a controlled laboratory experiment. MXR protein levels increased in time for all treatments (including the control). However, the highest levels of MXR protein titer were expressed in clams that had been exposed to the lowest dose of cadmium. The causes for this observed inverse relationship between the exposure dose and the MXR induction is discussed. MXR protein titer was also shown to be induced by other heavy metals (zinc, inorganic mercury, and copper). PMID:15084411

Achard, M; Baudrimont, M; Boudou, A; Bourdineaud, J P

2004-05-12

148

Stress protein expression in the Alzheimer-diseased choroid plexus  

Microsoft Academic Search

Abstract. Abnormal patterns of stress protein expression are found in the cerebral cortex and hippocampus,of Alzheimer (AD) subjects. In this study, expression of various stress proteins in the Alzheimer-diseased choroid plexus (CP) was assessed immunohistochemically. We observed decreased HO-1 immunoreactivity in the AD CP, commensurate with our earlier report of suppressed HO-1 protein levels in AD cerebrospinal fluid (Schipper et

Shawn G. Anthony; Hyman M. Schipper; Rosemarie Tavares; Virginia Hovanesian; Selina C. Cortez; Edward G. Stopa; Conrad E. Johanson

149

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production.  

PubMed

Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins. PMID:24743983

Ahmad, Mudassar; Hirz, Melanie; Pichler, Harald; Schwab, Helmut

2014-06-01

150

Tools for Co-expressing Multiple Proteins in Mammalian Cells  

PubMed Central

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

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

2013-01-01

151

Strategies for the cell-free expression of membrane proteins.  

PubMed

Cell-free expression offers an interesting alternative method to produce membrane proteins in high amounts. Elimination of toxicity problems, reduced proteolytic degradation and a nearly unrestricted option to supply potentially beneficial compounds like cofactors, ligands or chaperones into the reaction are general advantages of cell-free expression systems. Furthermore, the membrane proteins may be translated directly into appropriate hydrophobic and membrane-mimetic surrogates, which might offer significant benefits for the functional folding of the synthesized proteins. Cell-free expression is a rapidly developing and highly versatile technique and several systems of both, prokaryotic and eukaryotic origins, have been established. We provide protocols for the cell-free expression of membrane proteins in different modes including their expression as precipitate as well as their direct synthesis into detergent micelles or lipid bilayers. PMID:20204858

Reckel, Sina; Sobhanifar, Solmaz; Durst, Florian; Löhr, Frank; Shirokov, Vladimir A; Dötsch, Volker; Bernhard, Frank

2010-01-01

152

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.

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

2013-01-01

153

Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins  

PubMed Central

In the last few years, the use of labeled proteins has significantly expanded in the life sciences. Now, labeled proteins are indispensable tools for a wide spectrum of biophysical and chemical biology applications. In particular, the quest for more sophisticated experimental setups requires the development of new synthetic methodology, especially for multiple site-specific labeling. In this paper, we describe a synthetic strategy based on expressed protein ligation to prepare proteins in high purity and homogeneity, in which two different molecular probes are incorporated specifically at any desired position. Proteins are sequentially labeled in solution, with the advantage that a large excess of probes is not required and the labeled fragments are not restricted to peptide synthesis length limitations. This strategy was applied to selectively label a repeat protein with a fluorophores pair in different positions along the protein sequence. The doubly labeled proteins were prepared at high purity and homogeneity, as required for single molecule FRET studies. Remarkably, this approach can be adapted to the introduction of more than two molecular probes.

De Rosa, Lucia; Cortajarena, Aitziber L.; Romanelli, Alessandra; Regan, Lynne

2012-01-01

154

Astrocytes and oligodendrocytes express different STOP protein isoforms.  

PubMed

Many cell types contain subpopulations of microtubules that resist depolymerizing conditions, such as exposure to cold or to the drug nocodazole. This stabilization is due mainly to polymer association with STOP proteins. In mouse, neurons express two major variants of these proteins, N-STOP and E-STOP (120 kDa and 79 kDa, respectively), whereas fibroblasts express F-STOP (42 kDa) and two minor variants of 48 and 89 kDa. N- and E-STOP induce microtubule resistance to both cold and nocodazole exposure, whereas F-STOP confers microtubule stability only to the cold. Here, we investigated the expression of STOP proteins in oligodendrocytes and astrocytes in culture. We found that STOP proteins were expressed in precursor cells, in immature and mature oligodendrocytes, and in astrocytes. We found that oligodendrocytes express a major STOP variant of 89 kDa, which we called O-STOP, and two minor variants of 42 and 48 kDa. The STOP variants expressed by oligodendrocytes induce microtubule resistance to the cold and to nocodazole. For astrocytes, we found the expression of two STOP variants of 42 and 48 kDa and a new STOP isoform of 60 kDa, which we called A-STOP. The STOP variants expressed by astrocytes induce microtubule resistance to the cold but not to nocodazole, as fibroblast variants. In conclusion, astrocytes and oligodendrocytes express different isoforms of STOP protein, which show different microtubule-stabilizing capacities. PMID:15389836

Galiano, M R; Bosc, C; Schweitzer, A; Andrieux, A; Job, D; Hallak, M E

2004-11-01

155

Deregulation of protein phosphatase expression in acute myeloid leukemia.  

PubMed

Acute myeloid leukemia (AML) is a highly malignant disease of myeloid cell line. AML is the most frequent adult leukemia with inadequate treatment possibility. The protein phosphatases are critical regulators of cell signaling, and deregulation of protein phosphatases always contribute to cell transformation. Although many studies established a relationship between protein phosphatases and leukemia, little is known about the role of this group of proteins in AML. To address this issue, we initially identified the complete catalog of human protein phosphatase genes and used this catalog to study deregulation of protein phosphatases in AML. Using mRNA expression data of AML patients, we show that 11 protein phosphatases are deregulated in AML within 174 protein phosphatases. The GO enrichment study suggests that these genes are involved in multiple biological processes other than protein de-phosphorylation. Expression of DUSP10, PTPRC, and PTPRE was significantly higher than average expression in AML, and a linear combination of DUSP10, MTMR11, PTPN4, and PTPRE expressions provides important information about disease subtypes. Our results provide an overview of protein phosphatase deregulation in AML. PMID:23440723

Kabir, Nuzhat N; Rönnstrand, Lars; Kazi, Julhash U

2013-06-01

156

Mobile phone radiation might alter protein expression in human skin  

PubMed Central

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

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

2008-01-01

157

Generation of multidrug resistant lymphoma cell lines stably expressing P-glycoprotein.  

PubMed

The objective of this study was to generate new P-glycoprotein (P-gp)-expressing multidrug resistant (MDR) cell lines by drug selection. Since our previous studies have been carried out with cells infected with a P-gp-containing vector, it was important to confirm our findings in cells generated by drug selection. In this report, we describe three B-lymphoma cell lines which became drug-resistant by stepwise exposure to vincristine (VCR): Raji cells resistant to 18 nM VCR (R18V), Namalwa cells resistant to 21 nM VCR (N21V) and DHL-4 cells resistant to 12 nM VCR (DHL-4/12V). Cells overexpressed P-gp and continued to express CD19, CD20 and CD22, all of which are targets for monoclonal antibody (MAb) therapy. The P-gp pump in these new cells was functional as determined by the efflux of Rhodamine 123 and DIOC2, and the three cell lines were resistant to several chemotherapeutic drugs. We further determined that their P-gp phenotype was stable in xenografted SCID mice and that the tumors were also resistant to chemotherapy. We will now use these new MDR cells to determine whether monoclonal antibodies against CD19 and -20 can reverse P-gp, as we previously demonstrated using Namalwa cells infected with a human mdr1 gene-containing retrovirus. PMID:18357372

Pop, Iliodora; Pop, Laurentiu; Vitetta, Ellen S; Ghetie, Maria-Ana

2008-04-01

158

Differential Expression of Potato Tuber Protein Genes 1  

PubMed Central

Patatin and the 22-kilodalton protein complex make up more than 50% of the soluble protein present in potato (Solanum tuberosum) tubers and these two proteins are coordinately regulated during tuber development. Although genomic sequences related to these tuber genes exist in the genome of potato species that do not bear tubers, they cannot be induced into expression under the tested conditions. These genes are not expressed during substantial starch accumulation in petioles from a model petiole-leaf cutting system in nontuber-bearing plants, indicating that starch accumulation and synthesis of the major tuber proteins occur independently. Tuber protein gene expression also has been examined in hybrid potato plants that contain genomes from both tuberizing and nontuberizing species. One such triploid hybrid produced only stolons, whereas a pentaploid hybrid with an increased number of tuber genomes produced tubers. It was shown, using immunoblotting and Northern blot hybridization, that these two hybrids actively expressed both patatin and the 22-kilodalton tuber protein in induced petioles from the leaf-cutting system. The induced accumulation of patatin transcripts was consistent in all genotypes containing some tuberizing genome. The induced accumulation of the 22-kilodalton protein transcripts, however, was lower in genotypes containing some nontuberizing genome. Sucrose induction of these genes in leaves corroborates the induction patterns in petioles. A correlation exists between 22-kilodalton protein gene expression and a potato plant's ability to produce stolons or tubers. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6

Hannapel, David J.

1990-01-01

159

Inhibition of tetramethylpyrazine on P-gp, MRP2, MRP3 and MRP5 in multidrug resistant human hepatocellular carcinoma cells.  

PubMed

Some membrane transporters in liver, such as P-glycoprotein, multidrug resistance-associated protein 2 (MRP2), MRP3, and MRP5 can lead to a complex multidrug resistance (MDR) to antineoplastic agents. How to inhibit these proteins is still an issue. Tetramethylpyrazine is a bioactive constituent isolated from the root of Ligusticum chuanxiong Hort, a Chinese herb. Recent studies showed that it can enhance the chemosensitivity effects of a drug on human hepatocellular carcinoma cells, acting as a multidrug resistance modulator. In this study, the reversal effect of TMP on MDR was evaluated and its activity mechanism in vitro was explored. The IC50 value shows that TMP reversed the multidrug resistance of BEL-7402/ADM cells 9.23-fold (P<0.01) at the concentration of 600 microM. The mean fluorescence intensity of ADM in BEL-7402/ADM cells with TMP was found to be 163.78+/-39.5% (P<0.01) versus in BEL-7402/ADM cells without TMP by flow cytometry and 126.73+/-28.72% in BEL-7402/ADM cells with TMP versus in BEL-7402/ADM cells without TMP (P<0.01) by high performance liquid chromatography, respectively. It was also found that the mRNA level of multidrug resistant gene MDR1, MRP2, MRP3 and MRP5 and the level of the proteins they encode were decreased after treatment with TMP, indicating that TMP can effectively reverse the MDR in BEL-7402/ADM cells, and its activity mechanism may be correlated with the down-regulation of expression in these transporters. PMID:19956884

Wang, Xuan-Bin; Wang, Shan-Shan; Zhang, Qiu-Fang; Liu, Ming; Li, Hong-Liang; Liu, Ying; Wang, Jia-Ning; Zheng, Fei; Guo, Ling-Yun; Xiang, Ji-Zhou

2010-01-01

160

Attenuated Influenza Virus Construct with Enhanced Hemagglutinin Protein Expression  

PubMed Central

Influenza A viruses encoding an altered viral NS1 protein have emerged as promising live attenuated vaccine platforms. A carboxy-terminal truncation in the NS1 protein compromises its interferon antagonism activity, making these viruses attenuated in the host yet still able to induce protection from challenge with wild-type viruses. However, specific viral protein expression by NS1-truncated viruses is known to be decreased in infected cells. In this report, we show that recombinant H5N1 and H1N1 influenza viruses encoding a truncated NS1 protein expressed lower levels of hemagglutinin (HA) protein in infected cells than did wild-type viruses. This reduction in HA protein expression correlated with a reduction in HA mRNA levels in infected cells. NS1 truncation affected the expression of HA protein but not that of the nucleoprotein (NP). This segment specificity was mapped to the terminal sequences of their specific viral RNAs. Since the HA protein is the major immunogenic component in influenza virus vaccines, we sought to restore its expression levels in NS1-truncated viruses in order to improve their vaccine efficacy. For this purpose, we generated an NS1-truncated recombinant influenza A/Puerto Rico/8/34 (rPR8) virus carrying the G3A C8U “superpromoter” mutations in the HA genomic RNA segment. This strategy retained the attenuation properties of the recombinant virus but enhanced the expression level of HA protein in infected cells. Finally, mice immunized with rPR8 viruses encoding a truncated NS1 protein and carrying the G3A C8U mutations in the HA segment demonstrated enhanced protection from wild-type virus challenge over that for mice vaccinated with an rPR8 virus encoding the truncated NS1 protein alone.

Maamary, Jad; Pica, Natalie; Belicha-Villanueva, Alan; Chou, Yi-ying; Krammer, Florian; Gao, Qinshan; Garcia-Sastre, Adolfo

2012-01-01

161

Alpha-1-syntrophin protein is differentially expressed in human cancers.  

PubMed

We studied the expression of ?1-syntrophin (SNTA1) protein in histologically confirmed esophageal, stomach, lung, colon, rectal and breast cancerous tissue samples. Our results suggest a significant decrease in the expression level of SNTA1 protein in both esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) compared with their respective controls while a significant increase in expression of SNTA1 protein compared with the normal tissue was observed in breast carcinoma samples. No significant difference in expression of SNTA1 protein was observed in stomach, lung, colon and rectal cancers. Our results suggest that SNTA1 has a role in carcinogenesis and could possibly be used as a novel diagnostic or prognostic marker in esophageal and breast cancers. PMID:21091386

Bhat, Hina F; Baba, Rafia A; Bashir, Muneesa; Saeed, Safder; Kirmani, Deeba; Wani, Mudassir M; Wani, Nisar A; Wani, Khursheed A; Khanday, Firdous A

2011-02-01

162

Detection of Protein Expression in Vivo Using Fluorescent Puromycin Conjugates.  

National Technical Information Service (NTIS)

Disclosed is a class of reagents for examining protein expression in vivo that does not require transfection, radiolabeling, or the prior choice of a candidate gene. Further, a series of puromycin conjugates was constructed bearing various labeling moieti...

E. Schuman H. M. Green J. Alberola-ila R. W. Roberts S. R. Starck-Green

2005-01-01

163

Telethonin protein expression in neuromuscular disorders  

Microsoft Academic Search

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, a-actinin-2, and myotilin and observed the typical cross striation pattern, suggesting

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

2002-01-01

164

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).

2013-01-01

165

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

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

Uchida, Yasuo; Tachikawa, Masanori; Obuchi, Wataru; Hoshi, Yutaro; Tomioka, Yusuke; Ohtsuki, Sumio; Terasaki, Tetsuya

2013-01-01

166

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.

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

2014-01-01

167

Membrane protein expression triggers chromosomal locus repositioning in bacteria  

PubMed Central

It has long been hypothesized that subcellular positioning of chromosomal loci in bacteria may be influenced by gene function and expression state. Here we provide direct evidence that membrane protein expression affects the position of chromosomal loci in Escherichia coli. For two different membrane proteins, we observed a dramatic shift of their genetic loci toward the membrane upon induction. In related systems in which a cytoplasmic protein was produced, or translation was eliminated by mutating the start codon, a shift was not observed. Antibiotics that block transcription and translation similarly prevented locus repositioning toward the membrane. We also found that repositioning is relatively rapid and can be detected at positions that are a considerable distance on the chromosome from the gene encoding the membrane protein (>90 kb). Given that membrane protein-encoding genes are distributed throughout the chromosome, their expression may be an important mechanism for maintaining the bacterial chromosome in an expanded and dynamic state.

Libby, Elizabeth A.; Roggiani, Manuela; Goulian, Mark

2012-01-01

168

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

169

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

PubMed Central

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 IV (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 ASIV 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.

WANG, PEI-PEI; XU, DU-JUAN; HUANG, CAN; WANG, WEI-PING; XU, WEN-KE

2014-01-01

170

[Prokaryotic expression and identification of HPV16 E7 protein].  

PubMed

HPV16 E7 fusion protein was expressed in E. coli BL21, and its applied value for HPV was evaluated. HPV16 E7 gene was amplified by PCR, and cloned into prokaryotic expression vector pGEX6p-1. The recombinant plasmid was transformed into E. coli BL21, and HPV16 E7 fusion was expressed through IPTG induction. The expressed product was analyzed by SDS-PAGE and Western blot, subsequently purified according to Glutathione Sepharose 4B purification procedure. An indirect ELISA with the purified fusion protein as the coating antigen was then established to detect E7 serum antibodies from mice immunized with recombinant Listeria monocytogenes delivering HPV16 E7. The results demonstrated that the soluble fusion protein was highly expressed at 25 degrees C after induction with 0.5 mM IPTG. Furthermore, the result of Western blot analysis showed that the fusion protein had good specific reaction with an anti-E7 monoclonal antibody. Indirect ELISA result confirmed that the fusion protein could detect the serum antibodies against E7 with a titer of 1:200. The expressed GST-E7 fusion protein was immunocompetent, which was useful in the research of E7 biological function and therapeutic vaccine. PMID:22416350

Jia, Yan-Yan; Yin, Yue-Lan; Bai, Chun-Guang; Fu, Hong; Gao, Yun-Fei; Pan, Zhi-Ming; Jiao, Xin-An

2012-01-01

171

Proteins and an Inflammatory Network Expressed in Colon Tumors  

PubMed Central

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

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

2011-01-01

172

Small-scale expression of proteins in E. coli.  

PubMed

Proteins participate in virtually every cellular activity, and a knowledge of protein function is essential for an understanding of biological systems. However, protein diversity necessitates the application of an array of in vivo and in vitro approaches for characterization of the functional and biochemical properties of proteins. Methods that enable production of proteins for in vitro studies are critical for determination of the molecular, kinetic, and thermodynamic properties of these molecules. Ideally, proteins could be purified from the original source; however, the native host is often unsuitable for a number of reasons. Consequently, systems for heterologous protein production are commonly used to produce large amounts of protein. Heterologous expression hosts are chosen using a number of criteria, including genetic tractability, advantageous production or processing characteristics (secretion or posttranslational modifications), or economy of time and growth requirements. The subcloning process also provides an opportunity to introduce purification tags, epitope tags, fusions, truncations, and mutations into the coding sequence that may be useful in downstream purification or characterization applications. Bacterial systems for heterologous protein expression have advantages in ease of use, cost, short generation times, and scalability. These expression systems have been widely used by high-throughput protein production projects and often represent an initial experiment for any expression target. Escherichia coli has been studied for many years as a model bacterial organism and is one of the most popular hosts for heterologous protein expression (Terpe, 2006). Its protein production capabilities have been intensively studied, and the ease of genetic manipulation in this organism has led to the development of strains engineered exclusively for use in protein expression. These resources are widely available from commercial sources and public repositories. Despite these advantages, many targets are unsuitable for expression in E. coli, and attempts will not yield protein that can be utilized in downstream applications. A thorough understanding of the protein target, the requirements of the final application, and available tools are all essential for planning a successful expression experiment. This protocol is designed to optimize expression and solubility using an E. coli host and expression vector with an IPTG-inducible T7 promoter. The general features of the method are easily extended to other organisms and expression systems. Small-scale expression cultures are used to identify the optimum expression parameters for a given target. Thorough analysis of the total cell content and soluble fraction is used to screen out failed targets and those unlikely to succeed in large-scale purification cultures. The protocol listed here can be used in individual tubes for a small number of targets or adapted for use in 48-well plates for high throughput applications (Abdullah et al., 2009). Using the same culture for initial expression analysis and solubility analysis reduces variability between expression trials and saves the time required to produce separate cultures. PMID:24423272

Zerbs, Sarah; Giuliani, Sarah; Collart, Frank

2014-01-01

173

Microfluidic Techniques for Single-Cell Protein Expression Analysis  

Microsoft Academic Search

Background: The analysis of single cells obtained from needle aspirates of tumors is constrained by the need for processing. To this end, we investigated two microflu- idic approaches to measure the expression of surface proteins in single cancer cells or in small populations (<50 cells). Methods: One approach involved indirect fluorescence labeling of cell-surface proteins and channeling of cells in

Ethan Fitzpatrick; Sterling McBride; Jonathan Yavelow; Saltanat Najmi; Peter Zanzucchi; Robert Wieder

2006-01-01

174

Regulation of Ped gene expression by TAP protein  

Microsoft Academic Search

The Ped (Preimplantation embryo development) gene regulates fast or slow cleavage of preimplantation mouse embryos and their subsequent survival. The protein product of the Ped gene is the major histocompatibility complex (MHC) class Ib protein Qa-2. MHC class I expression on the cell surface requires the assembly within the endoplasmic reticulum (ER) of an ? heavy chain, a ?2 microglobulin

Xiaoling Ke; Carol M. Warner

2000-01-01

175

Sperm protein 17 is expressed in human nervous system tumours  

Microsoft Academic Search

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

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

2006-01-01

176

Patterns of fluorescent protein expression in Scleractinian corals.  

PubMed

Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light. PMID:18840775

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

2008-10-01

177

Enhanced membrane protein expression by engineering increased intracellular membrane production  

PubMed Central

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

2013-01-01

178

Proteomic Analysis of Proteins Differentially Expressed in Preeclamptic Trophoblasts  

Microsoft Academic Search

Aims: To identify differential trophoblastic proteins associated with preeclampsia (PE) by proteomic analysis. Methods: We isolated and purified placental trophoblasts from normotensive pregnant women and patients with PE by a continuous Percoll gradient. The expression of proteins was determined by sliver staining after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins of interest were identified using matrix-assisted laser desorption ionization time of

Li-zhou Sun; Na-na Yang; Wei De; Yun-shan Xiao

2007-01-01

179

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

PubMed Central

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.

Unadkat, Jashvant D.

2014-01-01

180

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

181

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

PubMed Central

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

2012-01-01

182

Expression of SKP2 protein in lung carcinoma  

Microsoft Academic Search

Objective  To study the expressive characteristics of SKP2 protein in lung carcinoma and its implication for prognosis.\\u000a \\u000a \\u000a \\u000a Methods  The expression of SKP2 protein was detected in 89 non small cell lung carcinoma, 13 small cell lung carcinoma, 10 lung benign\\u000a lesion tissues by Tissue Chip and Immunohistochemistry technology.\\u000a \\u000a \\u000a \\u000a Results  The positive rate of SKP2 protein staining was (23.52±13.57)% in non small cell lung

Jun Zhao; Chun-lu Yang; Huan Zhang; Wei-zhong Ding; Zhi-ping Liu; Jing-yi Liu

2008-01-01

183

Recombinant protein expression in Escherichia coli: advances and challenges  

PubMed Central

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

Rosano, German L.; Ceccarelli, Eduardo A.

2014-01-01

184

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

Microsoft Academic Search

The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production\\u000a of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified\\u000a cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was

Kokoro Hayashi; Chojiro Kojima

2010-01-01

185

Expression of GAGE family proteins in malignant melanoma.  

PubMed

Cancer/testis antigens are considered as promising targets for immunotherapy against tumors including malignant melanoma. One group of these antigens is the GAGE antigen family. In this study, we obtained recombinant GAGE-7b protein against which a rabbit antiserum was generated. The polyclonal, monospecific antibodies were used to analyze the expression of GAGE family proteins in human melanoma tissues and cell lines. GAGE expression in melanoma cell lines ranged from 41% to 58% and in melanoma tissues from 22% to 53%. Immunohistochemical analysis of melanoma tumors revealed a rather heterogeneous expression of GAGE resulting in individual positive cells or foci of stained cells. Furthermore, we could show that autoantibodies against GAGE family proteins are detectable in 6% of melanoma patients. Besides, we first demonstrated that the expression of GAGE family proteins can be stimulated with 5'-aza-2'-deoxycytidine and trichostatin A. Through upregulation of protein expression GAGE family proteins might develop into promising targets for immunotherapy of melanoma and other tumors. PMID:17194529

Bazhin, Alexandr V; Wiedemann, Nicole; Schnölzer, Martina; Schadendorf, Dirk; Eichmüller, Stefan B

2007-06-28

186

Expression of a proline-enriched protein in Escherichia coli  

SciTech Connect

The feasibility of expressing repeated synthetic codons in bacterial cells was demonstrated by showing that repeated codons for proline were expressed in Escherichia coli. Recombinant DNA technology was used to clone synthetic polydeoxyguanylate: polydeoxycytidylate into the PstI site of plasmid pBR322. Recombinant plasmid pGC139 was shown by means of HaeIII restriction digestion to contain approximately 41 cloned base pairs; the cloned sequence was expressed as a fusion to an ampicillinase protein. The resulting protein, enriched in proline, was expressed from plasmid in pGC139 in E. coli maxicells. Extension of this technology could lead to improvement in the production of amino acids and to nutritional enrichment of single-cell protein. (Refs. 12).

Kangas, T.T.; Cooney, C.L.; Gomez, R.F.

1982-03-01

187

Detecting protein complexes from active protein interaction networks constructed with dynamic gene expression profiles  

PubMed Central

Background Protein interaction networks (PINs) are known to be useful to detect protein complexes. However, most available PINs are static, which cannot reflect the dynamic changes in real networks. At present, some researchers have tried to construct dynamic networks by incorporating time-course (dynamic) gene expression data with PINs. However, the inevitable background noise exists in the gene expression array, which could degrade the quality of dynamic networkds. Therefore, it is needed to filter out contaminated gene expression data before further data integration and analysis. Results Firstly, we adopt a dynamic model-based method to filter noisy data from dynamic expression profiles. Then a new method is proposed for identifying active proteins from dynamic gene expression profiles. An active protein at a time point is defined as the protein the expression level of whose corresponding gene at that time point is higher than a threshold determined by a standard variance involved threshold function. Furthermore, a noise-filtered active protein interaction network (NF-APIN) is constructed. To demonstrate the efficiency of our method, we detect protein complexes from the NF-APIN, compared with those from other dynamic PINs. Conclusion A dynamic model based method can effectively filter out noises in dynamic gene expression data. Our method to compute a threshold for determining the active time points of noise-filtered genes can make the dynamic construction more accuracy and provide a high quality framework for network analysis, such as protein complex prediction.

2013-01-01

188

Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins  

Microsoft Academic Search

Since its introduction into heterologous organisms as a marker of gene expression, the green fluorescent protein (GFP) has led a dramatic revolution in cell, developmental and neurobiology. By allowing breathtaking visualization of fluorescent fusion proteins as they move within and between cells, GFP has fundamentally transformed the spatial analysis of protein function. Now, new GFP technologies allow far more than

Peter van Roessel; Andrea H. Brand

2002-01-01

189

Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast.  

PubMed

We have engineered the chloroplast of eukaryotic algae to produce a number of recombinant proteins, including human monoclonal antibodies, but, to date, have achieved expression to only 0.5% of total protein. Here, we show that, by engineering the mammalian coding region of bovine mammary-associated serum amyloid (M-SAA) as a direct replacement for the chloroplast psbA coding region, we can achieve expression of recombinant protein above 5% of total protein. Chloroplast-expressed M-SAA accumulates predominantly as a soluble protein, contains the correct amino terminal sequence and has little or no post-translational modification. M-SAA is found in mammalian colostrum and stimulates the production of mucin in the gut, acting in the prophylaxis of bacterial and viral infections. Chloroplast-expressed and purified M-SAA is able to stimulate mucin production in human gut epithelial cell lines. As Chlamydomonas reinhardtii is an edible alga, production of therapeutic proteins in this organism offers the potential for oral delivery of gut-active proteins, such as M-SAA. PMID:17359495

Manuell, Andrea L; Beligni, Maria Verónica; Elder, John H; Siefker, David T; Tran, Miller; Weber, Annika; McDonald, Thomas L; Mayfield, Stephen P

2007-05-01

190

Analysis of Bcl-2 protein expression in choroidal melanomas  

PubMed Central

Background: Bcl-2 protooncogene alterations are involved in tumorigenesis and may have prognostic ramifications. Aims: To investigate normal ocular structures and choroidal melanoma for: (1) Bcl-2 protein expression (semiquantitative staining values: SI, staining intensity; PP, percentage of positive cells; and IRS, immunoreactivity score) and (2) any associations between the staining values and clinicopathological features in these lesions. Materials/Methods: Bcl-2 protein expression was analysed in 24 choroidal melanomas using immunoperoxidase staining methods. Results: Bcl-2 protein expression was seen in corneal epithelium, lens epithelium, the ciliary body, and retinal cells. In these structures, the mean (SEM) values were: 1.1 (0.1), 1.6 (0.3), 1.1 (0.1), and 2.3 (0.3), respectively, for SI; 1.6 (0.2), 1.7 (0.1), 1.7 (0.2), and 1.7 (0.2) for PP, respectively; and 1.9 (0.4), 2.7 (0.5), 1.9 (0.1), and 4.0 (0.8), respectively, for IRS. Based on Bcl-2 immunoreactivity, the lesions were divided into two groups. The first group comprised 12 tumours with Bcl-2 expression. Bcl-2 expression was significantly higher in this group compared with normal ocular structures (1.5 (0.1) v 2.8 (0.2), 1.7 (0.1) v 3.5 (0.1), and 2.6 (0.3) v 9.3 (0.9) for mean (SEM) SI, PP, and IRS scores, respectively; p ?=? 0.00). The second group comprised 12 tumours lacking Bcl-2 protein expression. There was no significant correlation between Bcl-2 protein expression and most of the clinicopathological features of these lesions. Conclusions: Bcl-2 protein expression is altered in choroidal melanomas.

Hussein, M R

2005-01-01

191

Multifactorial determinants of protein expression in prokaryotic open reading frames  

PubMed Central

A quantitative description of the relationship between protein expression levels and open reading frame nucleotide sequences (ORFs) is important for understanding natural systems, designing synthetic systems, and optimizing heterologous expression. Codon identity, mRNA secondary structure, and nucleotide composition within ORFs markedly influence expression levels. Bioinformatic analysis of ORF sequences in 816 bacterial genomes revealed that these features show distinct regional trends. To investigate their effects on protein expression, we designed 285 synthetic genes and determined corresponding expression levels in vitro using E. coli extracts. We developed a mathematical function, parameterized using this synthetic gene dataset, which enables computation of protein expression levels from ORF nucleotide sequences. In addition to its practical application in the design of heterologous expression systems, this equation provides mechanistic insight into the factors that control translation efficiency. We found that expression is strongly dependent on the presence of high AU content and low secondary structure in the ORF 5? region. Choice of high-frequency codons contributes to a lesser extent. The 3? terminal AU content makes modest, but detectable contributions. We present a model for the effect of these factors on the three phases of ribosomal function: initiation, elongation, and termination.

Allert, Malin; Cox, J. Colin; Hellinga, Homme W.

2010-01-01

192

Identification of differentially expressed serum proteins in infectious purpura fulminans.  

PubMed

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

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

2014-01-01

193

Selenomethionine incorporation in proteins expressed in Lactococcus lactis  

PubMed Central

Lactococcus lactis is a promising host for (membrane) protein overproduction. Here, we describe a protocol for incorporation of selenomethionine (SeMet) into proteins expressed in L. lactis. Incorporation efficiencies of SeMet in the membrane protein complex OpuA (an ABC transporter) and the soluble protein OppA, both from L. lactis, were monitored by mass spectrometry. Both proteins incorporated SeMet with high efficiencies (>90%), which greatly extends the usefulness of the expression host L. lactis for X-ray crystallography purposes. The crystal structure of ligand-free OppA was determined at 2.4 Å resolution by a semiautomatic approach using selenium single-wavelength anomalous diffraction phasing.

Berntsson, Ronnie P-A; Alia Oktaviani, Nur; Fusetti, Fabrizia; Thunnissen, Andy-Mark W H; Poolman, Bert; Slotboom, Dirk-Jan

2009-01-01

194

Functional specificity among ribosomal proteins regulates gene expression  

PubMed Central

Duplicated genes escape gene loss by conferring a dosage benefit or evolving diverged functions. The yeast Saccharomyces cerevisiae contains many duplicated genes encoding ribosomal proteins. Prior studies have suggested that these duplicated proteins are functionally redundant and affect cellular processes in proportion to their expression. In contrast, through studies of ASH1 mRNA in yeast, we demonstrate paralog-specific requirements for the translation of localized mRNAs. Intriguingly, these paralog-specific effects are limited to a distinct subset of duplicated ribosomal proteins. Moreover, transcriptional and phenotypic profiling of cells lacking specific ribosomal proteins reveals differences between the functional roles of ribosomal protein paralogs that extend beyond effects on mRNA localization. Finally, we show that ribosomal protein paralogs exhibit differential requirements for assembly and localization. Together, our data indicate complex specialization of ribosomal proteins for specific cellular processes, and support the existence of a ribosomal code.

Komili, Suzanne; Farny, Natalie G.; Roth, Frederick P.; Silver, Pamela A.

2008-01-01

195

Papaya fruit softening, endoxylanase gene expression, protein and activity.  

PubMed

Papaya (Carica papaya L.) cell wall matrix polysaccharides are modified as the fruit starts to soften during ripening and an endoxylanase is expressed that may play a role in the softening process. Endoxylanase gene expression, protein amount and activity were determined in papaya cultivars that differ in softening pattern and in one cultivar where softening was modified by the ethylene receptor inhibitor 1-methylcyclopropene (1-MCP). Antibodies to the endoxylanase catalytic domain were used to determine protein accumulation. The three papaya varieties used in the study, 'Line 8', 'Sunset', and 'Line 4-16', differed in softening pattern, respiration rate, ethylene production and showed similar parallel relationships during ripening and softening in endoxylanase expression, protein level and activity. When fruit of the three papaya varieties showed the respiratory climacteric and started to soften, the level of endoxylanase gene expression increased and this increase was related to the amount of endoxylanase protein at 32 kDa and its activity. Fruit when treated at less than 10% skin yellow stage with 1-MCP showed a significant delay in the respiratory climacteric and softening, and reduced ethylene production, and when ripe was firmer and had a 'rubbery' texture. The 1-MCP-treated fruit that had the 'rubbery' texture showed suppressed endoxylanase gene expression, protein and enzymatic activity. Little or no delay occurred between endoxylanase gene expression and the appearance of activity during posttranslational processing from 65 to 32 kDa. The close relationship between endoxylanase gene expression, protein accumulation and activity in different varieties and the failure of the 1-MCP-treated fruit to fully soften, supported de novo synthesis of endoxylanase, rapid posttranslation processing and a role in papaya fruit softening. PMID:18251885

Manenoi, Ashariya; Paull, Robert E

2007-11-01

196

Expression of P-glycoprotein and metallothionein in gastrointestinal stromal tumor and leiomyosarcomas. Clinical implications.  

PubMed

We investigated the expression of P-glycoprotein (P-GP) and metallothionein (MT) in a series of 92 GIST and 14 gastrointestinal leiomyosarcomas (GILMS) with the purpose to expand our knowledge on the biological bases of GIST chemo-resistance and to ascertain their significance in patients' prognosis. P-GP expression was more frequent in GIST than in GI-LMS (83.7% vs. 21.4%, p<0.001), with no difference between low- and high-risk GIST (p=1.000) or low- and high-grade GI-LMS (p=0.538). P-GP expression was unrelated to anatomic location (gastric vs. intestinal) in GIST (39/45 vs. 35/43, p=0.770) and in GI-LMS (0/2 vs. 2/6, p=1.000). MT expression was non-significantly higher in GI-LMS than in GIST (35.7% vs. 14.1%, p=0.060), with no difference between low- and high-risk GIST (p=1.000) or low- and high-grade GI-LMS (p=1.000). MT expression was unrelated to the anatomic location (gastric vs. intestinal) in GIST (7/45 vs. 6/43) and GI-LMS (0/2 vs. 1/6) (p=1.000 and p=0.1000, respectively). Overall tumor-specific survival (p< 0.001) and disease-free survival (p<0.001) were different in GIST as compared with GI-LMS, and the number of events was higher in GI-LMS. When the survival analysis took into consideration P-GP or MT expression, the overall survival in GIST was influenced by the expression of MT (p=0.021) but not by that of P-GP (p=0.638). However, in GI-LMS, P-GP expression influenced disease-free survival (p=0.050); in addition, it is important to recognize the limited value of these results because of the low number of cases involved in the study. Differential expression of P-GP and MT might explain the known variability in response to systemic chemotherapy in these tumors. Detection of P-GP and MT seems to add certain prognostic value in GIST (MT) or GI-LMS (P-GP). PMID:17922049

Pérez-Gutiérrez, Sofia; González-Cámpora, Ricardo; Amérigo-Navarro, Joaquín; Beato-Moreno, Antonio; Sánchez-León, María; Pareja Megía, Jesús María; Virizuela-Echaburu, Juan Antonio; López-Beltrán, Antonio

2007-01-01

197

Expression and purification of GST-FHL2 fusion protein.  

PubMed

Escherichia coli is the most widely used host for the production of recombinant proteins. However, most eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. The interactions between human FHL2 protein and many types of proteins, including structural proteins, kinases, and several classes of transcription factor, have been found to have important roles in a variety of fundamental processes, including arrhythmia, hypertrophy, atherosclerosis, and angiogenesis. To achieve high-level expression of soluble recombinant human FHL2 protein in E. coli, we have constructed a recombinant expression plasmid, pGEX-4T-1-FHL2, in which we merged FHL2 cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac inducible promoter. Using this plasmid, we have achieved high expression of soluble FHL2 as a GST fusion protein in E. coli BL21. We have used the engineered plasmid (pGEX-4T-1-FHL2) and the modified E. coli strain to overcome the problem of removing the GST moiety while expressing soluble FHL2. Our results show that: 1) the recombinant plasmid was successfully constructed. Sequencing results showed that FHL2 and GST are in the same reading frame; 2) at 23°C, soluble GST-FHL2 fusion protein was highly expressed after induction with 0.1 mM IPTG; and 3) GST-FHL2 can be detected by Western blotting using mouse monoclonal anti-GST antibody. Our data are the first to show that high yields of soluble FHL2 tagged with GST can be achieved in E.coli. PMID:24390986

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

2013-01-01

198

Prediction of Combinatorial Protein-Protein Interaction Networks from Expression Data Using Statistics on Conditional Probability  

Microsoft Academic Search

\\u000a In this paper we propose a method to retrieve combinatorial protein-protein interaction to predict the interaction networks\\u000a from protein expression data based on statistics on conditional probability. Our method retrieves the combinations of three\\u000a proteins A, B and C which include combinatorial effects among them. The combinatorial effect considered in this paper does\\u000a not include the ”sole effect” between two

Takatoshi Fujiki; Etsuko Inoue; Takuya Yoshihiro; Masaru Nakagawa

2010-01-01

199

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

PubMed Central

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

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

2013-01-01

200

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

PubMed

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

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

2013-01-01

201

Influence of combinations of digitonin with selected phenolics, terpenoids, and alkaloids on the expression and activity of P-glycoprotein in leukaemia and colon cancer cells.  

PubMed

P-glycoprotein (P-gp or MDR1) is an ATP-binding cassette (ABC) transporter. It is involved in the efflux of several anticancer drugs, which leads to chemotherapy failure and multidrug resistance (MDR) in cancer cells. Representative secondary metabolites (SM) including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, ?-sitosterol-O-glucoside, and ?-carotene), and alkaloids (glaucine, harmine, and sanguinarine) were evaluated as potential P-gp inhibitors (transporter activity and expression level) in P-gp expressing Caco-2 and CEM/ADR5000 cancer cell lines. Selected SM increased the accumulation of the rhodamine 123 (Rho123) and calcein-AM (CAM) in a dose dependent manner in Caco-2 cells, indicating that they act as competitive inhibitors of P-gp. Non-toxic concentrations of ?-carotene (40?M) and sanguinarine (1?M) significantly inhibited Rho123 and CAM efflux in CEM/ADR5000 cells by 222.42% and 259.25% and by 244.02% and 290.16%, respectively relative to verapamil (100%). Combination of the saponin digitonin (5?M), which also inhibits P-gp, with SM significantly enhanced the inhibition of P-gp activity. The results were correlated with the data obtained from a quantitative analysis of MDR1 expression. Both compounds significantly decreased mRNA levels of the MDR1 gene to 48% (p<0.01) and 46% (p<0.01) in Caco-2, and to 61% (p<0.05) and 1% (p<0.001) in CEM/ADR5000 cells, respectively as compared to the untreated control (100%). Combinations of digitonin with SM resulted in a significant down-regulation of MDR1. Our findings provide evidence that the selected SM interfere directly and/or indirectly with P-gp function. Combinations of different P-gp substrates, such as digitonin alone and together with the set of SM, can mediate MDR reversal in cancer cells. PMID:23999162

Eid, Safaa Yehia; El-Readi, Mahmoud Zaki; Eldin, Essam Eldin Mohamed Nour; Fatani, Sameer Hassan; Wink, Michael

2013-12-15

202

PROTEIN EXPRESSION LABORATORY Prices 2013 & 2014  

Cancer.gov

PL05051 EBV pol Taqman QPCR (DNA) (per 1 sample) $212.74 $209.96 PL05014 ELISA - Commerical Kits per 6 plates $551.18 $532.20 PL03030 Express Monoclonal Antibody, 100 - 200 Mg $2,064.58 $2,037.28 PL03012 Grow E.Coli Cells, Shake Flasks (1L), First Flask $368.70 $362.28 PL03017 Grow E.Coli In Fermentor, 30 L Auto-Induction $2,229.87 $2,153.92 PL03021 Grow E.Coli In Minifermentor , 2L Auto-Induction $702.40 $684.56 PL03026 Grow Yeast K Lactis, 1L $1,110.21 $1,069.28 PL03023 Grow Yeast P.

203

Protein expression during the embryonic development of a gastropod.  

PubMed

Despite the potential use of gastropod embryos in basic and applied research, little is known about their protein expression. We examined, for the first time, changes in proteomic profile during embryonic development of Pomacea canaliculata from an embryo without a shell (stage II) to an embryo with a fully formed shell (stage III) to understand the roles that proteins play in critical developmental events, such as the formation of shell, operculum and heart, and the differentiation of head and foot. To analyze protein expression during development, we used 2-DE to detect, MS to analyze, and de novo peptide sequencing followed by MS-BLAST to identify the proteins. The de novo cross-species protein identification method was adopted because of a lack of genomic and proteomic data in the whole class of Gastropoda. 2-DE detected approximately 700 protein spots. Among the 125 spots that were abundant, 52% were identified, a marked improvement over the conventional direct MS-BLAST method. These proteins function in perivitelline fluid utilization, shell formation, protein synthesis and folding, and cell cycle and cell fate determination, providing evidence to support that this embryonic period is a period of dynamic protein synthesis and metabolism. The data shall provide a basis for further studies of how gastropod embryos respond to natural and human-induced changes in the environment. PMID:20455212

Sun, Jin; Zhang, Yu; Thiyagarajan, Vengatesen; Qian, Pei-Yuan; Qiu, Jian-Wen

2010-07-01

204

Protein Co-Expression Network Analysis (ProCoNA)  

SciTech Connect

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

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

2013-06-01

205

Over-expression of the yeast multifunctional arom protein.  

PubMed

The pentafunctional arom protein of Saccharomyces cerevisiae is encoded by the ARO1 gene. Substantial elevation of the levels of the arom protein (25-fold) was achieved in yeast using a vector that exploited the ubiquitin-fusion cleavage system of yeast. However, attempts to express the N-terminal 3-dehydroquinate synthase domain (E1) or the internal 3-dehydroquinase domain (E2) using the same system did not succeed. The yeast arom protein was successfully purified from the over-expressing transformant, and was found to possess all five enzymatic activities in a ratio similar to that observed in crude cell extracts. The purified material consisted mainly of a polypeptide that co-migrated in SDS-PAGE with intact arom proteins from other species. PMID:8268222

Graham, L D; Gillies, F M; Coggins, J R

1993-12-14

206

Eukaryotic integral membrane protein expression utilizing the Escherichia coli glycerol-conducting channel protein (GlpF)  

Microsoft Academic Search

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous\\u000a membrane-protein expression is demonstrated by the

Irene Neophytou; Richard Harvey; Jayne Lawrence; Phil Marsh; Barry Panaretou; David Barlow

2007-01-01

207

Expression sites of two byssal protein genes of Mytilus galloprovincialis.  

PubMed

Mussels form byssal threads that can attach tenaciously to wet and irregular surfaces. The byssus consists of a fibrous collagenous core, and at least two types of polyphenolic proteins surround it. One of these proteins, designated Mgfp-1, coats the collagenous core; the other, designated Mgfp-2, is the major component of the terminal adhesive plaque of byssal threads. Both proteins contain 3,4-dihydroxyphenylalanine (DOPA) in their primary sequences. In this study, the sites of expression of the genes encoding the polyphenolic proteins were investigated in Mytilus galloprovincialis. By northern blot analysis, we found that the expression of both genes is foot-specific. Northern blot analysis of RNA isolated from the distal end and the remaining proximal portion of the foot indicated that the Mgfp-2 gene is expressed primarily in the distal part, whereas Mgfp-1 expression occurs in both parts. In situ hybridization indicated that the Mgfp-1 gene transcript is localized in the accessory gland along the ventral groove of the foot, and the Mgfp-2 gene transcript is localized in the phenol gland near the foot apex. Thus, it was shown that tissues expressing Mgfp-1 and Mgfp-2 are located around the ventral groove in an arrangement appropriate for byssus formation. PMID:8652732

Miki, D; Takeuchi, Y; Inoue, K; Odo, S

1996-04-01

208

hTERT protein expression is independent of clinicopathological parameters and c-Myc protein expression in human breast cancer  

PubMed Central

Background Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. The hTERT (human telomerase reverse transcriptase) subunit seems to be the rate-limiting determinant of telomerase and knowledge of factors controlling hTERT transcription may be useful in therapeutic strategies. The hTERT promoter contains binding sites for c-Myc and there is some experimental and in vitro evidence that c-Myc may increase hTERT expression. We previously reported no correlation between c-Myc mRNA expression and hTERT mRNA or telomerase activity in human breast cancer. This study aims to examine the correlation between hTERT expression as determined by immunohistochemistry and c-Myc expression, lymph node status, and tumour size and grade in human breast cancer. Materials and methods The immunohistochemical expression of hTERT and c-Myc was investigated in 38 malignant breast tumours. The expression of hTERT was then correlated with the lymph node status, c-Myc expression and other clinicopathological parameters of the tumours. Results hTERT expression was positive in 27 (71%) of the 38 tumours. 15 (79%) of 19 node positive tumours were hTERT positive compared with 11 (63%) of 19 node negative tumours. The expression was higher in node positive tumours but this failed to reach statistical significance (p = 0.388). There was no significant association with tumour size, tumour grade or c-Myc expression. However, hTERT expression correlated positively with patients' age (correlation coefficient = 0.415, p = 0.0097). Conclusion hTERT protein expression is independent of lymph node status, tumour size and grade and c-Myc protein expression in human breast cancer

Elkak, AE; Meligonis, G; Salhab, M; Mitchell, B; Blake, JRS; Newbold, RF; Mokbel, K

2005-01-01

209

Human C-reactive protein: expression, structure, and function  

Microsoft Academic Search

C-reactive protein (CRP) is an acute-phase protein featuring a homopentameric structure and Ca-binding specificity for phosphocholine (PCh). Expression of CRP is regulated mainly at the transcriptional level with interleukin-6 being the principal inducer of the gene during the acute phase. The crystal structure of CRP has been determined and the topology and chemical composition of its ligand-binding site determined. The

John E Volanakis

2001-01-01

210

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

PubMed

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 cultures of human cytotrophoblasts or BeWo cells were conducted with calcein-AM and vinblastine (P-gp markers) or fluorescein (MRP marker) in the presence of specific P-gp or MRP inhibitors. Results showed that the accumulation of P-gp substrates calcein-AM and vinblastine by BeWo cells or primary cultures of human cytotrophoblasts was significantly enhanced in the presence of a typical P-gp inhibitor, cyclosporin-A, or other inhibitors such as quinidine, verapamil, and dipyridamole. MRP inhibitors had no effect on the accumulation of calcein or fluorescein by BeWo cells. Western blots confirmed the presence of multidrug resistant gene product 1 (MDR1) in both primary cultures of human cytotrophoblasts and BeWo cells. This study demonstrates functional P-gp in term human trophoblasts and further supports the use of primary cultures of human cytotrophoblasts and BeWo cells as in vitro models of the trophoblast to investigate mechanisms regulating drug distribution across the placenta. PMID:10838122

Utoguchi, N; Chandorkar, G A; Avery, M; Audus, K L

2000-01-01

211

Quantitative methods to analyze subnuclear protein organization in cell populations with varying degrees of protein expression  

PubMed Central

The control of gene transcription is dependent on DNA-binding and coregulatory proteins that assemble in distinct regions of the cell nucleus. We use multispectral wide-field microscopy of cells expressing transcriptional coregulators labeled with fluorescent proteins (FP) to study the subnuclear localization and function of these factors in living cells. In coexpression studies, the glucocorticoid receptor interacting protein (GRIP) coactivator protein and the silencing mediator of retinoid and thyroid (SMRT) corepressor protein form spherical subnuclear focal bodies that are spatially distinct, suggesting that specific protein interactions concentrate these divergent proteins in separate subnuclear regions. However, the variability of these subnuclear bodies between cells within the population makes analysis based on “representative images” difficult, if not impossible. To address this issue, we develop a protocol for unbiased selection of cells from the population, followed by the automated quantification of the subnuclear organization of the labeled proteins. Statistical methods identify a significant linear correlation between the FP-coregulator expression level and subnuclear focal body formation for both FP-GRIP and FP-SMRT. Importantly, we confirm that these changes in subnuclear organization could be statistically normalized for differences in coregulator expression level. This integrated quantitative image analysis method will allow the rigorous comparison of different experimental cell populations that express variable levels of FP fusion proteins.

Voss, Ty C.; Demarco, Ignacio A.; Booker, Cynthia F.; Day, Richard N.

2005-01-01

212

Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts  

SciTech Connect

We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

Carmona-Rodriguez, Bruno [Laboratorio de Biologia Celular y Molecular, Facultad de Odontologia, UNAM, Cd. Universitaria, Coyoacan, Mexico, D.F. 04510 (Mexico); Alvarez-Perez, Marco Antonio [Laboratorio de Biologia Celular y Molecular, Facultad de Odontologia, UNAM, Cd. Universitaria, Coyoacan, Mexico, D.F. 04510 (Mexico); Narayanan, A. Sampath [Department of Pathology, School of Medicine, UW, Seattle (United States); Zeichner-David, Margarita [Center for Craniofacial Molecular Biology, School of Dentistry, USC, Los Angeles (United States); Reyes-Gasga, Jose [Instituto de Fisica, UNAM (Mexico); Molina-Guarneros, Juan [Facultad de Medicina, UNAM (Mexico); Garcia-Hernandez, Ana Lilia [Laboratorio de Biologia Celular y Molecular, Facultad de Odontologia, UNAM, Cd. Universitaria, Coyoacan, Mexico, D.F. 04510 (Mexico); Suarez-Franco, Jose Luis [Laboratorio de Biologia Celular y Molecular, Facultad de Odontologia, UNAM, Cd. Universitaria, Coyoacan, Mexico, D.F. 04510 (Mexico); Chavarria, Ivet Gil [Laboratorio de Biologia Celular y Molecular, Facultad de Odontologia, UNAM, Cd. Universitaria, Coyoacan, Mexico, D.F. 04510 (Mexico); Villarreal-Ramirez, Eduardo [Laboratorio de Biologia Celular y Molecular, Facultad de Odontologia, UNAM, Cd. Universitaria, Coyoacan, Mexico, D.F. 04510 (Mexico); Arzate, Higinio [Laboratorio de Biologia Celular y Molecular, Facultad de Odontologia, UNAM, Cd. Universitaria, Coyoacan, Mexico, D.F. 04510 (Mexico)]. E-mail: harzate@servidor.unam.mx

2007-07-06

213

Protein stickiness, rather than number of functional protein-protein interactions, predicts expression noise and plasticity in yeast  

PubMed Central

Background A hub protein is one that interacts with many functional partners. The annotation of hub proteins, or more generally the protein-protein interaction “degree” of each gene, requires quality genome-wide data. Data obtained using yeast two-hybrid methods contain many false positive interactions between proteins that rarely encounter each other in living cells, and such data have fallen out of favor. Results We find that protein “stickiness”, measured as network degree in ostensibly low quality yeast two-hybrid data, is a more predictive genomic metric than the number of functional protein-protein interactions, as assessed by supposedly higher quality high throughput affinity capture mass spectrometry data. In the yeast Saccharomyces cerevisiae, a protein’s high stickiness, but not its high number of functional interactions, predicts low stochastic noise in gene expression, low plasticity of gene expression across different environments, and high probability of forming a homo-oligomer. Our results are robust to a multiple regression analysis correcting for other known predictors including protein abundance, presence of a TATA box and whether a gene is essential. Once the higher stickiness of homo-oligomers is controlled for, we find that homo-oligomers have noisier and more plastic gene expression than other proteins, consistent with a role for homo-oligomerization in mediating robustness. Conclusions Our work validates use of the number of yeast two-hybrid interactions as a metric for protein stickiness. Sticky proteins exhibit low stochastic noise in gene expression, and low plasticity in expression across different environments.

2012-01-01

214

Controlling for gene expression changes in transcription factor protein networks.  

PubMed

The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein-protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NF?B1, NF?B2, Rel, RelB, I?B?, I?B?, and I?B?). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NF?B family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions. PMID:24722732

Banks, Charles A S; Lee, Zachary T; Boanca, Gina; Lakshminarasimhan, Mahadevan; Groppe, Brad D; Wen, Zhihui; Hattem, Gaye L; Seidel, Chris W; Florens, Laurence; Washburn, Michael P

2014-06-01

215

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

216

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

217

Heterochromatin protein 1 expression is reduced in human thyroid malignancy.  

PubMed

Owing to the loss of heterochromatin integrity that occurs during thyroid tumorigenesis, the expression of Heterochromatin Protein 1 isoforms HP1? and HP1? was assessed by immunohistochemistry in 189 thyroid tumors and non-neoplastic tissues. Expression of HP1? was significantly decreased in all thyroid lesions, except in follicular adenomas, when compared with matched adjacent normal tissue. This loss of HP1? expression may in part be caused by microRNA dysregulation. An example is miR-205, a microRNA that is abundantly upregulated in thyroid carcinomas and shown to reduce the expression of HP1?. In contrast to HP1?, HP1? expression was only reduced in metastatic carcinomas and poorly differentiated lesions. These results suggest the reduction of HP1? followed by a decrease in HP1? contributes to the pathogenesis of thyroid carcinomas, and their loss is a potential marker of thyroid malignancy and metastatic potential, respectively. PMID:24840329

Tretiakova, Maria S; Bond, Sarah D; Wheeler, David; Contreras, Alejandro; Kocherginsky, Masha; Kroll, Todd G; Hale, Tracy K

2014-07-01

218

Regenerating proteins and their expression, regulation and signaling  

PubMed Central

The regenerating (Reg) protein family comprises C-type lectin-like proteins discovered independently during pancreatitis and pancreatic islet regeneration. However, an increasing number of studies provide evidence of participation of Reg proteins in the proliferation and differentiation of diverse cell types. Moreover, Reg family members are associated with various pathologies, including diabetes and forms of gastrointestinal cancer. These findings have led to the emergence of key roles for Reg proteins as anti-inflammatory, antiapoptotic and mitogenic agents in multiple physiologic and disease contexts. Yet, there are significant gaps in our knowledge regarding the regulation of expression of different Reg genes. In addition, the pathways relaying Reg-triggered signals, their targets and potential cross-talk with other cascades are still largely unknown. In this review, the expression patterns of different Reg members in the pancreas and extrapancreatic tissues are described. Moreover, factors known to modulate Reg levels in different cell types are discussed. Several signaling pathways, which have been implicated in conferring the effects of Reg ligands to date, are also delineated. Further efforts are necessary for elucidating the biological processes underlying the action of Reg proteins and their involvement in various maladies. Better understanding of the function of Reg genes and proteins will be beneficial in the design and development of therapies utilizing or targeting this protein group.

Parikh, Abhirath; Stephan, Anne-Fleur; Tzanakakis, Emmanuel S.

2012-01-01

219

Expression and Function of Bcl-2 Proteins in Melanoma  

PubMed Central

Bcl-2 proteins are critical regulators of mitochondrial membrane permeability and the proapoptotic mitochondrial pathway. The family encloses pro- and antiapoptotic factors encoded by over 15 genes, which frequently give rise to alternative splice products. Antiapoptotic, proapoptotic multidomain, and proapoptotic BH3-only proteins are characterized by the presence of at least one of four Bcl-2 homology domains (BH 1-4). Their expression and activities are controlled by survival pathways as MAP kinases and protein kinase B/Akt, which are in touch with a number of transcription factors. In melanoma, the mitochondrial apoptosis pathways and Bcl-2 proteins appear of particular importance for apoptosis resistance, which has been addressed in clinical trials applying antisense-Bcl-2. Overexpression or induction of proapoptotic Bcl-2 proteins as well as the use of small molecule mimetics for the proapoptotic BH3 domain are further promising strategies.

Eberle, Jurgen; Hossini, Amir M

2008-01-01

220

The expression of metabolism-related proteins in phyllodes tumors.  

PubMed

The purpose of this study was to investigate the association between the expression of hypoxia-inducible factor (HIF)-1?, insulin-like growth factor (IGF)-1, glucose transporter 1 (Glut-1), carbonic anhydrase IX (CAIX), and monocarboxylate transporter (MCT)4, which are metabolism-related proteins in phyllodes tumors (PTs), and clinicopathologic factors and its implication. We used tissue microarrays to analyze 207 PTs and performed immunohistochemical staining against the glycolysis-related molecules HIF-1?, IGF-1, Glut-1, CAIX, and MCT4. We then compared the immunohistochemical results and clinicopathologic parameters. The expressions of HIF-1?, Glut-1, CAIX, and MCT4 in the stromal component of PTs increased (P?=?0.019, P?expression (P?=?0.001) and MCT4 expression (P?expression (P?=?0.012), Glut-1 expression (P?expression (P?=?0.039), and MCT4 expression (P?expression of the metabolism-related proteins HIF-1?, IGF-1, Glut-1, CAIX, and MCT4 revealed that, as the PT grade increased, the stromal expression of HIF-1?, Glut-1, CAIX, and MCT4 significantly increased. This result suggested that increasing PT grade is associated with increased glycolysis in the stromal component. PMID:22986897

Kwon, Ji Eun; Jung, Woo-Hee; Koo, Ja Seung

2013-02-01

221

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

222

Effects of Ketoconazole on the Biodistribution and Metabolism of [11C]Loperamide and [11C]N-Desmethyl-loperamide in Wild-type and P-gp Knockout Mice  

PubMed Central

Introduction [11C]Loperamide and [11C]N-desmethyl-loperamide ([11C]dLop) have been proposed as radiotracers for imaging brain P-glycoprotein (P-gp) function. A major route of [11C]loperamide metabolism is N-demethylation to [11C]dLop. We aimed to test whether inhibition of CYP3A4 with ketoconazole might reduce the metabolism of [11C]loperamide and [11C]dLop in mice, and thereby improve the quality of these radiotracers. Methods Studies were performed in wild-type and P-gp knockout (mdr–1a/b ?/?) mice. During each of seven study sessions, one pair of mice, comprising one wild-type and one knockout mouse, waspretreated with ketoconazole (50 mg/kg, i.p.) while another such pair was left untreated. Mice were sacrificed at 30 min after injection of [11C]loperamide or [11C]dLop. Whole brain and plasma samples were measured for radioactivity and analyzed with radio-HPLC. Results Ketoconazole increased the plasma concentrations of [11C]loperamide and its main radiometabolite, [11C]dLop, by about two-fold in both wild-type and knockout mice, whereas the most polar radiometabolite was decreased three-fold. Furthermore, ketoconazole increased the brain concentrations of [11C]loperamide and the radiometabolite [11C]dLop by about two-fold in knockout mice, and decreased the brain concentrations of the major and most polar radiometabolite in wild-type and knockout mice by 82 and 49%, respectively. In contrast, ketoconazole had no effect on plasma and brain distribution of administered [11C]dLop and its radiometabolites in either wild-type or knockout mice, except to increase the low plasma [11C]dLop concentration. The least polar radiometabolite of [11C]dLop was identified with LC-MSn as the N-hydroxymethyl analog of [11C]dLop and this also behaved as a P-gp substrate. Conclusion In this study, ketoconazole (50 mg/kg, i.p.) proved partiallyeffective for inhibiting the N-demethylation of [11C]loperamide in mouse in vivo but had relatively smaller or no effect on [11C]dLop.

Seneca, Nicholas; Zoghbi, Sami S.; Shetty, H. Umesha; Tuan, Edward; Kannan, Pavitra; Taku, Andrew; Innis, Robert B.; Pike, Victor W.

2010-01-01

223

Expression of myelin basic protein isoforms in nonglial cells  

Microsoft Academic Search

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, dis- play distinct developmental profiles. We expressed these major MBPs individually in HeLa cells and mapped their

Susan M. Staugaitis; P. R. Smith; David R. Colman

1990-01-01

224

Lipid Transfer Proteins from Fruit: Cloning, Expression and Quantification  

Microsoft Academic Search

Background: Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for measuring fruit LTPs. Methods: cDNA for Mal d 3 and

Laurian Zuidmeer; W. Astrid van Leeuwen; Ilona Kleine Budde; Jessica Cornelissen; Ingrid Bulder; Ilona Rafalska; Noèlia Telléz Besolí; Jaap H. Akkerdaas; Riccardo Asero; Montserrat Fernandez Rivas; Eloina Gonzalez Mancebo; Ronald van Ree

2005-01-01

225

Computational codon optimization of synthetic gene for protein expression  

PubMed Central

Background The construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression. Results In this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU. Conclusions The proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology.

2012-01-01

226

Expression Trend of Selected Ribosomal Protein Genes in Nasopharyngeal Carcinoma  

PubMed Central

Background: Ribosomal proteins are traditionally associated with protein biosynthesis until recent studies that implicated their extraribosomal functions in human diseases and cancers. Our previous studies using GeneFishing™ DEG method and microarray revealed underexpression of three ribosomal protein genes, RPS26, RPS27, and RPL32 in cancer of the nasopharynx. Herein, we investigated the expression pattern and nucleotide sequence integrity of these genes in nasopharyngeal carcinoma to further delineate their involvement in tumourigenesis. The relationship of expression level with clinicopathologic factors was also statistically studied. Methods: Quantitative Polymerase Chain Reaction was performed on nasopharyngeal carcinoma and their paired normal tissues. Expression and sequence of these three genes were analysed. Results: All three ribosomal protein genes showed no significant difference in transcript expressions and no association could be established with clinicopathologic factors studied. No nucleotide aberrancy was detected in the coding regions of these genes. Conclusion: There is no early evidence to substantiate possible involvement of RPS26, RPS27, and RPL32 genes in NPC tumourigenesis.

Ma, Xiang-Ru; Sim, Edmund Ui-Hang; Ling, Teck-Yee; Tiong, Thung-Sing; Subramaniam, Selva Kumar; Khoo, Alan Soo-Beng

2012-01-01

227

Mobile phone radiation might alter protein expression in human skin  

Microsoft Academic Search

BACKGROUND: Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin

Anu Karinen; Sirpa Heinävaara; Reetta Nylund; Dariusz Leszczynski

2008-01-01

228

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.

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

2014-01-01

229

Post-transcriptional regulator (rex) of HTLV-1 initiates expression of viral structural proteins but suppresses expression of regulatory proteins.  

PubMed Central

Gene expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by two trans-acting factors encoded by the pX region, p40tax and p27tax.p40tax is a transcriptional activator and p27rex is a post-transcriptional regulator. Using full-length viral DNA, we studied the regulatory effects of rex on HTLV-1 gene expression. p27rex is required for expression of both gag and env proteins, increasing the level of their mRNAs. The effect was dependent on the dose of p27rex expression plasmid. In parallel, increased doses of p27rex suppressed the expression of fully spliced pX mRNA, which encodes the regulatory proteins. These two effects of p27rex operated at the post-transcriptional level and were independent of transcriptional regulation. Lowering the level of pX mRNA down-regulates transcription of the proviral genome. These observations demonstrate that rex is a positive post-transcriptional regulator for gag, pol and env protein expression, and acts at the same time as an indirect negative regulator of viral transcription. Images

Hidaka, M; Inoue, J; Yoshida, M; Seiki, M

1988-01-01

230

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host  

PubMed Central

Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.

Pochon, Nathalie; Dementin, Sebastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphne; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

2011-01-01

231

Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.  

PubMed

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. PMID:8286197

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

1994-01-01

232

Mammalian cell factories for efficient and stable protein expression.  

PubMed

As the commercial market for therapeutic protein production from mammalian cells has expanded, so has the requirement for improved efficiency and stability of production. Rapid developments have been made in understanding the molecular environment of transgenes in chromatin, including elucidation of the contribution of epigenetic modifications to expression, and this understanding is being used to enhance expression from host cells. Technical advances surrounding the 'omics' revolution are enabling the rational identification of complex control factors that define the flow of information from transgene to desired protein. Using information from 'omics' interrogations, directed cell engineering has been employed to enhance the translational and secretory capacity of host cells. Taken together, these recent advances are likely to lead to improved routes for protein production in the future. PMID:16806893

Barnes, Louise M; Dickson, Alan J

2006-08-01

233

Expression of heat shock proteins in Alzheimer's disease.  

PubMed

In an investigation of heat shock proteins (HSPs) in the brains of Alzheimer's disease (AD) patients and cognitively intact control subjects, we found that 2 HSPs, termed "HSP72" and "GRP78," underwent major changes in expression in AD. HSP72, which was present at very low levels in control brains, increased dramatically in AD patients, and was localized exclusively in neuritic plaques and neurofibrillary tangles. We hypothesize that HSP72 is induced as an early response to the formation of abnormal proteins, perhaps targeting them for proteolysis. In contrast, GRP78 increased in AD only in neurons that remained cytologically normal, especially in the CA3 subfield of the hippocampus and the deep layers of the entorhinal cortex. The increased expression of GRP78 within successfully surviving neurons suggests that this protein may protect such cells from AD-specific damage. PMID:2005999

Hamos, J E; Oblas, B; Pulaski-Salo, D; Welch, W J; Bole, D G; Drachman, D A

1991-03-01

234

Expression and purification of Z protein from Junín virus.  

PubMed

Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z). The arenavirus Junín (JUNV) is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one). One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function. PMID:20652066

Goñi, S E; Borio, C S; Romano, F B; Rota, R P; Pilloff, M G; Iserte, J A; Tortorici, M A; Stephan, B I; Bilen, M F; Ghiringhelli, P D; Lozano, M E

2010-01-01

235

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

PubMed Central

Background 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. Results 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?), 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. Conclusion 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

2009-01-01

236

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.

Tuli, Leepika; Ressom, Habtom W.

2010-01-01

237

Expression and localization of surfactant proteins in human nasal epithelium.  

PubMed

Surfactant proteins (SPs), designated SP-A, SP-B, SP-C, and SP-D, play an important role in surfactant metabolism and host defense mechanisms in the lung. This study investigates expression of the different SP types in human nasal mucosa and cultured normal human nasal epithelial (NHNE) cells and whether the expression of SP mRNA is influenced by the degree of mucociliary differentiation. RT-PCR was performed with mRNA from cultured NHNE cells and nasal mucosa. Immunohistochemical staining for SPs was performed on nasal mucosa specimens. Western blot analysis was performed on cell lysates from cultured NHNE cells. SP-A2, SP-B, and SP-D mRNAs were expressed in normal NHNE cells and human nasal mucosa. SPs were localized in ciliated cells of the surface epithelium and serous acini of the submucosal glands. SP-A, SP-B, and SP-D proteins were expressed in cultured NHNE cells. The degree of mucociliary differentiation influenced expression of the SP gene. We demonstrate that SP-A, SP-B, and SP-D are expressed in human nasal mucosa and cultured NHNE cells. Further study of the functional role of SPs in the upper airway is required. PMID:17209137

Kim, Jin Kook; Kim, Sung-Shik; Rha, Keung Won; Kim, Chang-Hoon; Cho, Jae Hoon; Lee, Chang-Hoon; Lee, Jeung-Gweon; Yoon, Joo-Heon

2007-04-01

238

DNA binding proteins that amplify surfactant protein B gene expression: isolation and characterization.  

PubMed

We identified and characterized two proteins that bind the promoter of surfactant protein B (SP-B) and affect its expression. Proteins A2 and B were identified and their cDNAs cloned and sequenced. Both were novel. They bound a 212-bp functional promoter region at an NF1 site, located between -184 and -198. Effects of these DNAbp on SP-B promoter activity were studied by contransfecting a reporter construct of this 212-bp sequence + luciferase, together with expression constructs for A2 and B into H441 cells. Alone, A2 and B expression elicited modest but statistically significant increases in SP-B promoter activity. When dexamethasone was added, B further increased SP-B promoter activity. For SP-B, basal expression and glucocorticoid responsiveness may involve a number of hitherto unknown gene activators. PMID:7887923

Luzi, P; Strayer, D S

1995-03-01

239

Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii  

PubMed Central

Summary Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear if this is due to few attempts or to limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, greater than 50% expressed at levels sufficient for commercial production. Three expressed at 2% to 3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivty achieved demonstrate the utility of C. reinhardtii as a robust platform for human therapeutic protein production.

Rasala, Beth A; Muto, Machiko; Lee, Philip A; Jager, Michal; Cardoso, Rosa MF; Behnke, Craig A; Kirk, Peter; Hokanson, Craig A; Crea, Roberto; Mendez, Michael; Mayfield, Stephen P

2010-01-01

240

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.

Chan, Queenie WT; Foster, Leonard J

2008-01-01

241

Transcriptional regulation of salivary proline-rich protein gene expression.  

PubMed

Mechanisms governing gene expression and regulation in eukaryotes are remarkably complex. The results from in vivo transgenic and in vitro transfection studies designed to identify cis-element(s) and trans-factor(s) associated with the salivary proline-rich proteins (PRPs) gene expression are utilized as a paradigm to discuss the regulation of salivary-specific gene expression. Particular attention is given to the molecular mechanism(s) underlying the salivary PRP R15 gene regulation. In rodents, the PRPs are selectively expressed in the acinar cells of salivary glands, and are inducible by the beta-agonist isoproterenol as well as by dietary tannins. The results from a series of experiments using chimeric reporter constructs containing different lengths of the R15 distal enhancer region, their mutations, and various expressing constructs are analyzed and discussed. These data suggest that the inducible nuclear orphan receptor NGFI-B may participate in the regulation of salivary acinar cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites. Taken together, a model for the induction of R15 gene expression by isoproterenol is proposed. However, the exact molecular basis of this NGFI-B-mediated transactivation of cAMP-regulated R15 expression remains to be established. PMID:9599300

Ann, D K; Lin, H H

1998-04-15

242

Expression of Exocytosis Proteins in Rat Supraoptic Nucleus Neurones  

PubMed Central

In magnocellular neurones of the supraoptic nucleus (SON), the neuropeptides vasopressin and oxytocin are synthesised and packaged into large dense-cored vesicles (LDCVs). These vesicles undergo regulated exocytosis from nerve terminals in the posterior pituitary gland and from somata/dendrites in the SON. Regulated exocytosis of LDCVs is considered to involve the soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) complex [comprising vesicle associated membrane protein 2 (VAMP-2), syntaxin-1 and soluble N-ethylmaleimide attachment protein-25 (SNAP-25)] and regulatory proteins [such as synaptotagmin-1, munc-18 and Ca2+-dependent activator protein for secretion (CAPS-1)]. Using fluorescent immunocytochemistry and confocal microscopy, in both oxytocin and vasopressin neurones, we observed VAMP-2, SNAP-25 and syntaxin-1-immunoreactivity in axon terminals. The somata and dendrites contained syntaxin-1 and other regulatory exocytosis proteins, including munc-18 and CAPS-1. However, the distribution of VAMP-2 and synaptotagmin-1 in the SON was limited to putative pre-synaptic contacts because they co-localised with synaptophysin (synaptic vesicle marker) and had no co-localisation with either oxytocin or vasopressin. SNAP-25 immunoreactivity in the SON was limited to glial cell processes and was not detected in oxytocin or vasopressin somata/dendrites. The present results indicate differences in the expression and localisation of exocytosis proteins between the axon terminals and somata/dendritic compartment. The absence of VAMP-2 and SNAP-25 immunoreactivity from the somata/dendrites suggests that there might be different SNARE protein isoforms expressed in these compartments. Alternatively, exocytosis of LDCVs from somata/dendrites may use a different mechanism from that described by the SNARE complex theory.

Tobin, V.; Schwab, Y.; Lelos, N.; Onaka, T.; Pittman, Q. J.; Ludwig, M.

2012-01-01

243

Selective Replication of Coronavirus Genomes That Express Nucleocapsid Protein  

PubMed Central

The coronavirus nucleocapsid (N) protein is a structural protein that forms a ribonucleoprotein complex with genomic RNA. In addition to its structural role, it has been described as an RNA-binding protein that might be involved in coronavirus RNA synthesis. Here, we report a reverse genetic approach to elucidate the role of N in coronavirus replication and transcription. We found that human coronavirus 229E (HCoV-229E) vector RNAs that lack the N gene were greatly impaired in their ability to replicate, whereas the transcription of subgenomic mRNA from these vectors was easily detectable. In contrast, vector RNAs encoding a functional N protein were able to carry out both replication and transcription. Furthermore, modification of the transcription signal required for the synthesis of N protein mRNAs in the HCoV-229E genome resulted in the selective replication of genomes that are able to express the N protein. This genetic evidence leads us to conclude that at least one coronavirus structural protein, the N protein, is involved in coronavirus replication.

Schelle, Barbara; Karl, Nadja; Ludewig, Burkhard; Siddell, Stuart G.; Thiel, Volker

2005-01-01

244

Expression of nervous tissue nuclear protein and glial fibrillary acidic protein during morphogenesis of the neocortex.  

PubMed

We studied the distribution of glial fibrillary acidic protein (GFAP) and neuron-specific nuclear histone protein NeuN in sulci of the brain cortex during the pre- and postnatal ontogeny. The expression of GFAP during morphogenetic development of the sulci and gyri is cyclic. After functional reorientation of GFAP in human fetuses at weeks 28-30 it ceases to be a marker of morphogenetically active glia and becomes a marker of glial cells exclusively. Redistribution of NeuN expression in different layers of sulci during their formation was found: enhanced expression of NeuN in the cortical layer 6 of sulci and its reduced expression in the upper layers were noted, whereas outside the cortical sulci NeuN expression was similar in all layers. At weeks 24-25 of gestation, NeuN serves as a marker of ingrowth of secondary visual fibers from the dorsal thalamus. PMID:21165411

Godovalova, O S

2010-10-01

245

Expression of ENT1 Protein in Elderly Patients with Schizophrenia  

PubMed Central

Alterations in glutamatergic neurotransmission are thought to be involved in several psychiatric disorders, including schizophrenia. Equlibrative nucleoside transporter 1 (ENT1) regulates glutamate levels by regulating excitatory amino acid transporter expression and activity in the brain. In this study, we investigated whether ENT1 is abnormally expressed in the brain of elderly patients with schizophrenia. We measured protein expression of ENT1 in the superior temporal gyrus (STG) and anterior cingulate cortex (ACC) in patients with schizophrenia (STG, n = 22; ACC, n = 34) and a comparison group (STG, n = 24; ACC, n = 29). We found decreased ENT1 expression in the superior temporal gyrus in patients with schizophrenia, supporting the hypothesis of altered glutamate transport in this illness.

Shan, Dan; Haroutunian, Vahram; Meador-Woodruff, James H.; McCullumsmith, Robert E.

2012-01-01

246

Identification of differential expressed proteins and characterization their mRNA expression in thermally stressed Apostichopus japonicus.  

PubMed

In this study, we present a comparative proteomic analysis of the global protein expression changes in sea cucumber after 7 days exposure at 25°C. Using two-dimensional electrophoresis followed by MALDI-TOF MS/MS, 27 protein spots with significant differences in abundance were identified and characterized. The identified proteins belonged primarily to the following four functional categories: cytoskeletal, material and energy metabolism, calcium homeostasis and extracellular matrix. The mRNA expression levels of 7 differentially expressed proteins were further assessed by qRT-PCR. The expression levels of 6 genes, including collagen, ATP synthase, major yolk protein, ferritin, nectin and protein disulfide isomerase showed significant differences under thermal stress, and among them, only two genes-ATP synthase and major yolk protein-showed consistent levels of protein and mRNA expression. Our results offer insight into the complex changes in protein turnover during higher temperature exposure in sea cucumber. PMID:23727926

Zhang, Peng; Lu, Yali; Li, Chenghua; Su, Xiurong; Wang, Zhonghua; Jin, Chunhua; Li, Ye; Li, Taiwu

2013-09-01

247

Effect of pregnancy on cytochrome P450 3a and P-glycoprotein expression and activity in the mouse: mechanisms, tissue specificity, and time course  

PubMed Central

The plasma concentrations of orally administered anti-HIV protease inhibitors are significantly reduced during human and mouse pregnancy. We have shown that in the mouse, at gestational day 19, this reduction is due to increased hepatic cytochrome P450 3a (Cyp3a) protein expression and activity. In the current study, we investigated the mechanisms by which Cyp3a activity is increased by pregnancy and the time course of change in expression of Cyp3a and P-gp in various tissues. We found hepatic transcripts of Cyp3a16, Cyp3a41 and Cyp3a44 were significantly increased during pregnancy, while those of Cyp3a11 and Cyp3a25 were significantly decreased. This resulted in a net increase in Cyp3a protein expression and activity in the liver during pregnancy. The increase in Cyp3a41 and Cyp3a44 transcripts was positively correlated (p<0.05) with HNF6 and ER? transcripts. The pregnancy-related factors that transcriptionally activated mouse Cyp3a isoforms also activated the human CYP3A4 promoter in pregnant CYP3A4-promoter-luciferase transgenic (CYP3A4-tg) mice. In contrast, intestinal Cyp3a protein expressions were not significantly affected by pregnancy. No change in P-gp protein expression was observed in the liver or kidney during pregnancy, though a significant decrease was observed in the placenta. Since hepatic CYP3A activity also appears to be induced during human pregnancy, the mouse (including CYP3A4-tg mouse) appears to be an excellent animal model to determine the molecular mechanisms for such induction.

Zhang, Huixia; Wu, Xiaohui; Wang, Honggang; Mikheev, Andrei M.; Mao, Qingcheng; Unadkat, Jashvant D.

2008-01-01

248

The role of bromodomain proteins in regulating gene expression.  

PubMed

Histone modifications are important in regulating gene expression in eukaryotes. Of the numerous histone modifications which have been identified, acetylation is one of the best characterised and is generally associated with active genes. Histone acetylation can directly affect chromatin structure by neutralising charges on the histone tail, and can also function as a binding site for proteins which can directly or indirectly regulate transcription. Bromodomains specifically bind to acetylated lysine residues on histone tails, and bromodomain proteins play an important role in anchoring the complexes of which they are a part to acetylated chromatin. Bromodomain proteins are involved in a diverse range of functions, such as acetylating histones, remodeling chromatin, and recruiting other factors necessary for transcription. These proteins thus play a critical role in the regulation of transcription. PMID:24704920

Josling, Gabrielle A; Selvarajah, Shamista A; Petter, Michaela; Duffy, Michael F

2012-01-01

249

Ca(2+)-binding protein expression in primary human thyrocytes.  

PubMed

We recently identified several Ca(2+)-binding proteins (CaBP) from the S100 and annexin family to be regulated by TSH in FRTL-5 cells. Here, we study the regulation of S100A4, S100A6 and ANXA2 in primary human thyrocytes (PHT) derived from surrounding tissues (ST), cold benign thyroid nodules (CTN) and autonomously functioning thyroid nodules (AFTN). We investigated the expression and regulation of CaBP and the effect of their expression on Ca(2+) and TSHR signaling. We used an approach that accounts for the potential of an individual PHT culture to proliferate or to express thyroid differentiation features by assessing the expression of FOS and TPO. We found a strong correlation between the regulation of CaBP and the proliferation-associated transcription factor gene FOS. PKA and MEK1/2 were regulators of ANXA2 expression, while PI3-K and triiodothyronine were additionally involved in S100 regulation. The modulated expression of CaBP was reflected by changes in ATP-elicited Ca(2+) signaling in PHT. S100A4 increased the ratio of subsequent Ca(2+) responses and showed a Ca(2+) buffering effect, while ANXA2 affected the first Ca(2+) response to ATP. Overexpression of S100A4 led to a reduced activation of NFAT by TSH. Using S100A4 E33Q, D63N, F72Q and Y75K mutants we found that the effects of S100A4 expression on Ca(2+) signaling are mediated by protein interaction. We present evidence that TSH has the ability to fine-tune Ca(2+) signals through the regulation of CaBP expression. This represents a novel putative cross-regulating mechanism in thyrocytes that could affect thyrocyte signaling and physiology. PMID:23886630

Lorenz, S; Aust, G; Krohn, K

2013-12-01

250

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.

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

2014-01-01

251

Heat shock protein expression during gametogenesis and embryogenesis.  

PubMed Central

When cells are subjected to various stress factors, they increase the production of a group of proteins called heat shock proteins (hsp). Heat shock proteins are highly conserved proteins present in organisms ranging from bacteria to man. Heat shock proteins enable cells to survive adverse environmental conditions by preventing protein denaturation. Thus the physiological and pathological potential of hsps is enormous and has been studied widely over the past two decades. The presence or absence of hsps influences almost every aspect of reproduction. They are among the first proteins produced during mammalian embryo development. In this report, the production of hsps in gametogenesis and early embryo development is described. It has been suggested that prolonged and asymptomatic infections trigger immunity to microbial hsp epitopes that are also expressed in man. This may be relevant for human reproduction, since many couples with fertility problems have had a previous genital tract infection. Antibodies to bacterial and human hsps are present at high titers in sera of many patients undergoing in vitro fertilization. In a mouse embryo culture model, these antibodies impaired the mouse embryo development at unique developmental stages. The gross morphology of these embryos resembled cells undergoing apoptosis. The TUNEL (terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling) staining pattern, which is a common marker of apoptosis, revealed that embryos cultured in the presence of hsp antibodies stained TUNEL-positive more often than unexposed embryos. These data extend preexisting findings showing the detrimental effect of immune sensitization to hsps on embryo development.

Neuer, A; Spandorfer, S D; Giraldo, P; Jeremias, J; Dieterle, S; Korneeva, I; Liu, H C; Rosenwaks, Z; Witkin, S S

1999-01-01

252

Expression and purification of a truncated recombinant streptococcal protein G.  

PubMed

The gene for Protein G from Streptococcus strain G148 was cloned and expressed in Escherichia coli. The regions on the gene corresponding to the albumin-binding domains and the Fab-binding region were then deleted by site-directed mutagenesis. The translation of regions corresponding to the cell-wall- and membrane-binding domains was prevented by introduction of stop codons upstream of these domains. This recombinant DNA sequence codes for a protein (G') that contains repetitive regions and that binds only the Fc portion of IgG, analogously to Protein A. Translation of the sequence produces a protein with an Mr of about 20,000. The nucleotide sequence differs from those published previously [Guss, Eliasson, Olsson, Uhlén, Frej, Jornvall, Flock & Lindberg (1986) EMBO J. 5, 1567-1575; Olsson, Eliasson, Guss, Nilsson, Hellman, Lindberg & Uhlén (1987) Eur. J. Biochem. 168, 319-324]. The protein can be substantially purified on a large scale by chromatography on IgG-Sepharose 4B. Homogeneous Protein G' can be prepared by anion-exchange f.p.l.c. on Mono Q HR. This Protein G' has a pI of 4.19 and SDS/PAGE gives an apparent anomalous Mr of 35,000. PMID:2183792

Goward, C R; Murphy, J P; Atkinson, T; Barstow, D A

1990-04-01

253

Differential protein expression in perfusates from metastasized rat livers  

PubMed Central

Background Liver perfusates exhibit theoretical advantages regarding the discovery of disease biomarkers because they contain proteins that readily enter the blood-stream, and perfusion preserves the disease state in its natural context. The purpose of the study is to explore the value of liver perfusate proteome in the biomarker discovery of liver diseases. Results In this study, 86 differentially expressed proteins were identified in perfusates from isolated rat livers metastasized by Walker-256 tumor cells. Among these proteins, 27 were predicted to be secreted, and 59 were intracellular or membrane proteins. Most of the secretory proteins (70.4%) were decreased in metastasized liver perfusates. The main canonical ingenuity pathway to which these secretory proteins belonged was acute phase response, which indicated that the liver-associated immune reaction was damaged by the metastasis. In contrast, most of the intracellular or membrane proteins (86.4%) exhibited higher relative abundances in the metastasized liver perfusates. Some of these proteins, including Rpl21, Atic, Eif3s2, Echs1, Eps15 and Ywhab, have previously been reported to be involved in cancer genesis and progression. As a member of the 14-3-3 protein family, Ywhab plays a key role in cellular proliferation and oncogenic transformation and has been reported to be involved in the development of breast cancer. Its abundance was elevated by 3.5-fold in the metastasized perfusates. Validation by Western blotting revealed a 3.7-fold increase in the abundance of this protein in metastasized plasma. Conclusions These results show that perfusate proteome can be used as an alternative initial resource for biomarker identification, which ultimately requires validation in serum.

2013-01-01

254

Proteins in cancer multidrug resistance.  

PubMed

Multidrug Resistance (MDR) is defined as insensitivity to administered medicines that are structurally unrelated and have different molecular targets. Cancers possess numerous mechanisms of drug resistance, involving various aspects of cell biology. A pivotal role in this phenomenon is played by proteins - enzymatic or structural parts of the cell. Membrane transporters, including the main members of ABC protein family - P-gp, MRP1 and BCRP, as well as LRP, which builds structure of vaults, determine the multidrug-resistant phenotype by decreasing drug concentration within the cell or modifying its distribution to intracellular compartments. The ? isoform of protein enzyme - glutathione S-transferase (GSTP-1), is responsible for excessive intensity of detoxification of cytostatics. A common example of altered drug target site that does not respond to chemotherapy is topoisomerase II ? (TopoIIa). Alterations of programmed cell death result from expression of metallothionein (MT) - inhibitor of the process, and cytokeratin 18 (CK18), which, if in high concentration, also prevents apoptosis of cells. Several methods of decreasing activity of these proteins have been developed, aiming to overcome MDR in cancer cells. However, for a variety of reasons, their clinical suitability is still very low, leading to continuous increase in death rate among patients. This paper presents current state of knowledge on the most important examples of proteins responsible for MDR of cancer cells and molecular mechanisms of their action. PMID:24864112

Pop?da, Marta; P?uciennik, El?bieta; Bednarek, Andrzej K

2014-01-01

255

Expression cloning screen for modifiers of amyloid precursor protein shedding.  

PubMed

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the amyloid beta peptide (Abeta), which is thought to provoke the pathogenesis of Alzheimer's disease. To better understand the cellular processes that regulate ectodomain shedding of APP we used human embryonic kidney 293 cells and applied a sib-selection expression cloning approach. In addition to a known activator of APP shedding -- protein kinase A -- the following cDNAs were identified: the endocytic proteins endophilin A1 and A3, the metabotropic glutamate receptor 3 (mGluR3), palmitoyl-protein thioesterase 1 (PPT1), Numb-like and the kinase MEKK2. Endophilins A1 and A3, as well as mGluR3 activated APP shedding relatively specifically. They had little or no effect on the shedding of the unrelated membrane proteins TNF receptor 2 and P-selectin glycoprotein ligand-1. In contrast, MEKK2 and PKA also increased shedding of TNF receptor 2, suggesting that these kinases contribute to a general program regulating ectodomain shedding. The strongest activator of APP shedding, endophilin A3, reduced the rate of APP endocytosis and specifically increased APP shedding by the protease alpha-secretase, as measured in an antibody uptake assay and by immunoblot analysis. This suggests that endophilin A3 is a novel modulator of APP trafficking affecting access of APP to alpha-secretase. In summary, this study shows that expression cloning is a suitable way to identify proteins controlling ectodomain shedding of membrane proteins. PMID:16446073

Schöbel, Susanne; Neumann, Stephanie; Seed, Brian; Lichtenthaler, Stefan F

2006-01-01

256

Bone morphogenetic protein expression in human atherosclerotic lesions.  

PubMed

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. PMID:8473518

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

1993-04-01

257

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

258

Expression and immunogenicity of six putative variable surface proteins in Mycoplasma mycoides subsp. mycoides SC  

Microsoft Academic Search

Variable surface protein Vmm and five Vmm-type proteins from Mycoplasma mycoides subsp. mycoides SC were analysed to determine whether these proteins are expressed in vivo in animals affected by contagious bovine pleuropneumonia (CBPP) and in vitro. Recombinant versions of these proteins were constructed and expressed in Escherichia coli after mutation of the TGA Trp codons to TGG. These proteins were

Carl Hamsten; Joakim Westberg; Goran Bolske; Roger Ayling; Mathias Uhlen; Anja Persson

2008-01-01

259

Transcriptional regulation of neurofilament expression by protein kinase A.  

PubMed

RN46A cells, a conditionally immortalized neuronal cell line derived from E12 rat medullary raphe nucleus, upregulate low M(r) (68 kDa, neurofilament [NF]-L) and medium M(r) (160 kDa, NF-M) neurofilament protein expression upon activation of protein kinase A (PKA). To examine possible transcriptional regulation of neurofilament protein expression by PKA, two cell lines were used; RN46A cells and C alpha EV6 cells, a cell line derived from RN46A cells that stably expresses the catalytic subunit of PKA under the control of the metallothionein promoter. Treatment of RN46A cells with dbcAMP resulted in an increase in the steady-state levels of both NF-L and NF-M, but not high M(r) (200 kDa, NF-H) neurofilament mRNA. These increases were both time and dose dependent and were sensitive to treatment with the protein synthesis inhibitor cycloheximide. In C alpha EV6 cells, activation of PKA by 80 microM ZnSO4 upregulated the expression of C alpha mRNA with maximal levels reached 8 hr post-treatment and maintained at 24 hr. Reporter gene assays in C alpha EV6 cells following transfection with increasing lengths of the NF-L promoter demonstrated that both a putative Sp1-like and a cAMP response (CRE), but not a NGFI-A, element were likely involved in PKA-dependent activation of the NF-L promoter. Electrophoretic mobility shift assays confirmed these results but showed that the nuclear proteins induced by PKA which bound to the NF-L promoter Sp1-like sequence were not Sp1. Collectively, these data suggest that constitutively expressed Sp1 may be involved in basal NF-L promoter activity, and newly synthesized, PKA-dependent nuclear proteins may synergistically activate the rat NF-L promoter. PMID:9039646

White, L A; Reeben, M; Saarma, M; Whittemore, S R

1997-02-01

260

Recombinant EPO therapy increases erythrocyte expression of complement regulatory proteins  

Microsoft Academic Search

Background: One of the complications of hemodialysis (HD) therapy is anemia caused by erythropoietin (EPO) deficiency. Recombinant EPO (rEPO) has been used routinely as a supplemental treatment. Erythrocyte expression of the complement regulatory proteins decay accelerating factor (DAF) and CD59 restricts complement activation and inhibits hemolysis. We hypothesized that the efficacy of rEPO treatment may be caused in part by

Hiroyuki Ohi; Mariko Tamano; Sukemasa Sudo; Noriko Okada

2003-01-01

261

Expression of Water Channel Proteins in Mesembryanthemum crystallinum  

Microsoft Academic Search

We have characterized transcripts for nine major intrinsic proteins (MIPs), some of which function as water channels (aquaporins), from the ice plant Mesembryanthemum crystallinum. To determine the cellular distribution and expression of these MIPs, oligopeptide-based antibodies were generated against MIP-A, MIP-B, MIP-C, or MIP-F, which, according to sequence and functional characteristics, are located in the plasma membrane (PM) and tonoplast,

Hans-Hubert Kirch; Rosario Vera-Estrella; Dortje Golldack; Francoise Quigley; Christine B. Michalowski; Bronwyn J. Barkla; Hans J. Bohnert

2000-01-01

262

MicroRNA regulation of Alzheimer's Amyloid precursor protein expression  

Microsoft Academic Search

Gene dosage effects of Amyloid precursor protein (APP) can cause familial AD. Recent evidence suggest that microRNA (miRNA) pathways, implicated in gene transcriptional control, could be involved in the development of sporadic Alzheimer's disease (AD). We therefore investigated whether miRNAs could participate in the regulation of APP gene expression. We show that miRNAs belonging to the miR-20a family (that is,

Sébastien S. Hébert; Katrien Horré; Laura Nicolaï; Bruno Bergmans; Aikaterini S. Papadopoulou; André Delacourte; Bart De Strooper

2009-01-01

263

Effects of recombinant protein expression on green fluorescent protein diffusion in Escherichia coli.  

PubMed

Fluorescence recovery after photobleaching was used to measure the diffusion coefficient of green fluorescent protein (GFP, 27 kDa) in Escherichia coli in the presence or absence of four coexpressed proteins: cytoplasmic maltose binding protein (42 kDa), tau-40 (45 kDa), alpha-synuclein (14 kDa), or calmodulin (17 kDa). The GFP diffusion coefficient remains constant regardless of the type of coexpresseed protein and whether or not the coexpressed protein was induced. We conclude that expression of these soluble proteins has little to no effect on the diffusion of GFP. These results have implications for the utility of in-cell nuclear magnetic resonance spectroscopy. PMID:19413350

Slade, Kristin M; Baker, Rachael; Chua, Michael; Thompson, Nancy L; Pielak, Gary J

2009-06-16

264

Expression of dengue virus structural proteins and nonstructural protein NS1 by a recombinant vaccinia virus.  

PubMed

A recombinant vaccinia virus containing cloned DNA sequences coding for the three structural proteins and nonstructural proteins NS1 and NS2a of dengue type 4 virus was constructed. Infection of CV-1 cells with this recombinant virus produced dengue virus structural proteins as well as the nonstructural protein NS1. These proteins were precipitated by specific antisera and exhibited the same molecular size and glycosylation patterns as authentic dengue virus proteins. Infection of cotton rats with the recombinant virus induced NS1 antibodies in 1 of 11 animals. However, an immune response to the PreM and E glycoproteins was not detected. A reduced level of gene expression was probably the reason for the limited serologic response to these dengue virus antigens. PMID:3316711

Zhao, B T; Prince, G; Horswood, R; Eckels, K; Summers, P; Chanock, R; Lai, C J

1987-12-01

265

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

Lohr, M.; Trautmann, B.; Gottler, M.; Peters, S.; Zauner, I.; Maillet, B.; Kloppel, G.

1994-01-01

266

Expression of XPG protein in human normal and tumor tissues.  

PubMed

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

267

Expression of mutant p53 protein and CD44 variant proteins in colorectal tumorigenesis.  

PubMed Central

Colorectal tumorigenesis evolves through a series of molecular genetic changes, providing putative markers for tumour progression. This study investigated the relation between expression of the tumour suppressor gene p53 and splice variants v5 and v6 of the cell adhesion molecule CD44 by immunohistochemistry on tissue samples of early adenomas (n = 12), late adneomas (n = 12), Dukes's A and B carcinomas (n = 21), and Dukes's C and D carcinomas (n = 22) and compared these results with expression of these proteins in normal colonic mucosa (n = 17). A statistically significant trend of increasing expression was seen for both p53 (p < 0.005) and CD44 variant exon v6 (p < 0.0005) in subsequent stages of this tumour progression model. High expression of CD44 v5 was seen in most colorectal neoplasms (83%-96%), independent of stage. A statistically significant correlation was present between p53 expression and expression of variant v6 of CD44 (p < 0.01). Both p53 expression and CD44 v6 expression in adenomas increased with the degree of dysplasia (p < 0.05). The results of this study show that mutant p53 protein and variant v6 of the CD44 glycoprotein are markers of tumour progression in colorectal cancer. Images Figure 1

Mulder, J W; Wielenga, V J; Polak, M M; van den Berg, F M; Adolf, G R; Herrlich, P; Pals, S T; Offerhaus, G J

1995-01-01

268

Expression of SFRP Family Proteins in Human Keratoconus Corneas  

PubMed Central

We investigated the expression of the secreted frizzled-related proteins (SFRPs) in keratoconus (KC) and control corneas. KC buttons (?8 mm diameter) (n?=?15) and whole control corneas (n?=?7) were fixed in 10% formalin or 2% paraformaldehyde and subsequently paraffin embedded and sectioned. Sections for histopathology were stained with hematoxylin and eosin, or Periodic Acid Schiff’s reagent. A series of sections was also immunolabelled with SFRP 1 to 5 antibodies, visualised using immunofluorescence, and examined with a Zeiss LSM700 scanning laser confocal microscope. Semi-quantitative grading was used to compare SFRP immunostaining in KC and control corneas. Overall, KC corneas showed increased immunostaining for SFRP1 to 5, compared to controls. Corneal epithelium in all KC corneas displayed heterogeneous moderate to strong immunoreactivity for SFRP1 to 4, particularly in the basal epithelium adjacent to cone area. SFRP3 and 5 were localised to epithelial cell membranes in KC and control corneas, with increased SFRP3 cytoplasmic expression observed in KC. Strong stromal expression of SFRP5, including extracellular matrix, was seen in both KC and control corneas. In control corneas we observed differential expression of SFRP family proteins in the limbus compared to more central cornea. Taken together, our results support a role for SFRPs in maintaining a healthy cornea and in the pathogenesis of epithelial and anterior stromal disruption observed in KC.

You, Jingjing; Wen, Li; Roufas, Athena; Madigan, Michele C.; Sutton, Gerard

2013-01-01

269

Molecular cloning and expression of starfish protein kinase C isoforms.  

PubMed

To investigate the roles of protein kinase C (PKC) isoforms in Echinoderms, we cloned starfish cDNAs for novel, atypical, and conventional PKCs. They showed highest homology with PKCdelta, iota, and alpha isoforms respectively. It was predicted from the whole genome sequence and by RT-PCR that sea urchin has only one isoform of each PKC subgroups. It is thus likely that these isoforms are the prototypes or ancestors of the PKC subgroups. The phylogenetic tree suggests that atypical PKC was first formed by evolution from the common prototype of AGC protein kinase family, and novel and conventional PKCs next. RT-PCR analysis indicated that novel and atypical PKC mRNAs are expressed ubiquitously in all tissues of adult starfish, whereas conventional PKC mRNA is expressed mainly in the ovary and oocytes, and only slightly in the tube foot and stomach. Upon heterologous expression, only atypical PKC was expressed in the functional form in insect cells. PMID:19584538

Miyake, Yasuo; Yasui, Masaru; Ikeda, Kaori; Kondo, Tsunenori; Tsukamoto, Shinpei; Hori, Chinami; Okemoto, Naomi; Mashou, Keiko; Bando, Reiko; Nakamura, Naoko; Toraya, Tetsuo

2009-07-01

270

In vivo Expression of Signaling Proteins in Reconstituted NK Cells  

PubMed Central

Natural Killer cells are cells of the innate immune system that are important for the recognition and clearance of virally infected cells or tumors. Examination of the development and signaling of these cells has been severely hampered due to an inability to over-express proteins in these cells. We developed a novel technique to generate NK cells in vivo, all of which express a gene of interest. IL2R?c-/-/Rag2-/- mice do not develop NK cells due to the lack of IL15 signaling. We infected bone marrow from IL2R?c-/-/Rag2-/- mice with a retroviral construct encoding EGFP and IL2R?c connected by an IRES. NK cells selectively developed through expression of IL2R?c and 100% of these NK cells were found to be EGFP+. In order to test the utilization of this method to examine the function of biologically relevant proteins, constitutively active PI3K p110? and p110? isoforms were over-expressed in this system. Constitutively active p110? revealed profound effects on NK cell development and function in vivo while p110? had little effect.

Orr, Selinda J.; Quigley, Laura; McVicar, Daniel W.

2009-01-01

271

A Novel Protein Is Lower Expressed in Renal Cell Carcinoma  

PubMed Central

Engrailed-2 (EN2) has been identified as a candidate oncogene in breast cancer and prostate cancer. It is usually recognized as a mainly nuclear staining in the cells. However, recent studies showed a cytoplasmic staining occurred in prostate cancer, bladder cancer and clear cell renal cell carcinoma. The inconsistency makes us confused. To clarify the localization and expression of EN2 in renal cell carcinoma, anti-EN2 antibody (ab28731) and anti-EN2 antibody (MAB2600) were used for immunohistochemistry (IHC) respectively. Interestingly, we found that EN2 detected by ab28731 was mainly presented in cytoplasm while EN2 detected by MAB2600 was mainly presented in nucleus. To further investigate the different patterns observed above, lysates from full-length EN2 over expression in HEK293T cells were used to identify which antibody the EN2 molecule bound by western blot. Results showed ab28731 did not react with the lysates. For this reason, the novel specific protein detected by ab28731 was not the EN2 molecule and was named nonEN2. Then using the renal carcinoma tissue microarray and renal tissues, we found that the protein expression levels of nonEN2 in kidney tumor tissues was significantly lower than that in kidney normal tissues (p < 0.05), so was in renal cell lines. Taken together, nonEN2 is lower expressed and may play an important role in renal cell carcinoma.

Guan, Ruili; Xu, Yongde; Lei, Hongen; Gao, Zhezhu; Xin, Zhongcheng; Guo, Yinglu

2014-01-01

272

Amplified expression and large-scale purification of protein G'.  

PubMed

PCR was used to isolate the gene fragment coding for Protein G' (SpG'), a truncated bacterial cell surface protein from Streptococcus G148 which binds to the Fc region of IgG and expressed in E. coli [Goward et al. (1990) Biochem. J. 267: 171-177]. The PCR primer was designed to change the TTG initiation triplet to ATG and to incorporate it into an NdeI restriction site (CATATG), allowing the gene to be cloned in frame into an NdeI restriction site immediately downstream of a trp promoter. Expression of SpG' was estimated as about 30% total soluble cell protein which compares very favourably to the less than 1% total soluble cell protein obtained from the original system [Goward, et al. (1990) Biochem. J. 267: 171-177]. Homogeneous SpG' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in a process which avoided use of an affinity adsorbent. Even though SpG' consists of almost identical repetitive domains from amino acid sequence analysis, different proteolytic sensitivity of each domain was observed indicating their structural dissimilarity. PMID:1369218

Murphy, J P; Atkinson, T; Hartwell, R; Stevens, G B; Hinton, R J; Goward, C R

1992-01-01

273

The expression and induction of heat shock proteins in molluscs.  

PubMed

Living cells respond to stress stimuli by triggering rapid changes in the protein profiles, and the induction of heat shock proteins (HSPs) plays an important part in this process. HSPs, mainly acting as molecular chaperones, are constitutively expressed in cells and involved in protein folding, assembly, degradation, and intracellular localization. The overexpression of HSPs represents a ubiquitous molecular mechanism to cope with stress. Compared to vertebrates, molluscs have a biphasic life cycle where pelagic larvae go through settlement and metamorphosis. HSPs may play an important role in the survival strategy of molluscs during the biphasic life stages. Since aquatic environments are highly dynamic, molluscs may be subject to a variety of sources of stress and HSPs might play a more important role in the adaptation of these animals. Moreover, the mechanisms of stress tolerance in molluscs can offer fundamental insights into the adaptation of organisms for a wide range of environmental challenges. The cDNA of HSPs has been cloned from some molluscs, and HSPs can be induced by heat stress, hypoxia, heavy metal contamination, and aestivation, etc. The expression of HSPs was detected in the neuroendocrine system, mollusc development, and reproductive process. Furthermore, the induction of HSPs is related with the phosphorylation of stress-activated p38 mitogen-activated protein kinase (p38 MAPK) and cJun-N-terminal kinases (JNKs) in molluscs. PMID:23092135

Liu, Dongwu; Chen, Zhiwei

2013-05-01

274

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.  

PubMed

The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes. PMID:11404393

Yousefi, S; Cooper, P R; Potter, S L; Mueck, B; Jarai, G

2001-06-01

275

Amyloid precursor protein expression modulates intestine immune phenotype.  

PubMed

Amyloid precursor protein (APP) is widely expressed across many tissue and cell types. Proteolytic processing of the protein gives rise to a plethora of protein fragments with varied biological activities. Although a large amount of data has been generated describing the metabolism of the protein in neurons, its role in regulating the phenotype of other cells remains unclear. Based upon prior work demonstrating that APP regulates the activation phenotype of monocytic lineage cells, we hypothesized that APP can regulate macrophage activation phenotype in tissues other than brain. Ileums of the small intestines from C57BL6/J wild type and APP(-/-) mice were compared as a representative tissue normally associated with abundant macrophage infiltration. APP(-/-) intestines demonstrated diminished CD68 immunoreactivity compared to wild type mice. This correlated with significantly less cyclooxygenase-2 (cox-2), CD68, CD40, CD11c, and ?III-tubulin protein levels. Peritoneal macrophages from APP(-/-) mice demonstrated decreased in vitro migratory ability compared to wild type cells and diminished basal KC cytokine secretion. Whereas, APP(-/-) intestinal macrophages had an increase in basal KC cytokine secretion compared to wild type cells. Conversely, there was a significant decrease in multiple cytokine levels in APP(-/-) compared to wild type ileums. Finally, APP(-/-) mice demonstrated impaired absorption and increased motility compared to wild type mice. These data demonstrate the APP expression regulates immune cell secretions and phenotype and intestinal function. This data set describes a novel function for this protein or its metabolites that may be relevant not only for Alzheimer's disease but a range of immune-related disorders. PMID:22124967

Puig, Kendra L; Swigost, Adam J; Zhou, Xudong; Sens, Mary Ann; Combs, Colin K

2012-03-01

276

Accessory proteins are vital for the functional expression of certain G protein-coupled receptors.  

PubMed

Certain G protein-coupled receptors (GPCRs) fail to be expressed in a functional form at the cell surface. This may be due to the improper folding and maturation of GPCRs which are highly intricate events that need to take place before these integral membrane proteins can be transported from the endoplasmic reticulum (ER), where they are synthesised, to the plasma membrane which is their site of action. Once at the plasma membrane they act as the recognition elements for a vast range of endogenous ligands including biogenic amines, peptides, glycoproteins, lipids, nucleotides, ions and proteases. The assistance of molecular chaperones has been widely implicated in the trafficking and function of these proteins. Characterisation of certain GPCRs has identified a novel group of membrane proteins collectively named 'accessory proteins' as being important for the expression and function of GPCRs. In this review we will summarise the importance of these accessory proteins for the function of their respective GPCRs. Understanding their roles in GPCR expression would not only give us an insight into these receptors from a cell biological point of view but may also potentially lead to the development of novel therapeutics. PMID:19000738

Cooray, Sadani N; Chan, Li; Webb, Tom R; Metherell, Louise; Clark, Adrian J L

2009-03-01

277

Paeonol reverses paclitaxel resistance in human breast cancer cells by regulating the expression of transgelin 2.  

PubMed

Paclitaxel (PTX) is a first-line antineoplastic drug that is commonly used in clinical chemotherapy for breast cancer treatment. However, the occurrence of drug resistance in chemotherapeutic treatment has greatly restricted its use. There is thus an urgent need to find ways of reversing paclitaxel chemotherapy resistance in breast cancer. Plant-derived agents have great potential in preventing the onset of the carcinogenic process and enhancing the efficacy of mainstream antitumor drugs. Paeonol, a main compound derived from the root bark of Paeonia suffruticosa, has various biological activities, and is reported to have reversal drug resistance effects. This study established a paclitaxel-resistant human breast cancer cell line (MCF-7/PTX) and applied the dual-luciferase reporter gene assay, MTT assay, flow cytometry, transfection assay, Western blotting and the quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the reversing effects of paeonol and its underlying mechanisms. It was found that transgelin 2 may mediate the resistance of MCF-7/PTX cells to paclitaxel by up-regulating the expressions of the adenosine-triphosphate binding cassette transporter proteins, including P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1), and breast cancer resistance protein (BCRP). Furthermore, the ability of paeonol to reverse paclitaxel resistance in breast cancer was confirmed, with a superior 8.2-fold reversal index. In addition, this study found that paeonol down-regulated the transgelin 2-mediated paclitaxel resistance by reducing the expressions of P-gp, MRP1, and BCRP in MCF-7/PTX cells. These results not only provide insight into the potential application of paeonol to the reversal of paclitaxel resistance, thus facilitating the sensitivity of breast cancer chemotherapy, but also highlight a potential role of transgelin 2 in the development of paclitaxel resistance in breast cancer. PMID:24680370

Cai, Jiangxia; Chen, Siying; Zhang, Weipeng; Hu, Sasa; Lu, Jun; Xing, Jianfeng; Dong, Yalin

2014-06-15

278

Expression of a novel nuclear protein in activated and in tat-I expressing T cells.  

PubMed

The intracellular events that occur in T lymphoid cells after activation or after infection with HIV-1 are not well defined. In the case of HIV-1 infection, it is unknown whether the tat-I gene, an essential gene for viral replication, affects host cell nuclear factors. Using two-dimensional PAGE, we have identified a novel nuclear protein, designated nuclear protein-28,000 (NP-28), which is induced in Jurkat T cells by stimulation with PMA and/or PHA or ionomycin. This nuclear protein has an apparent molecular mass of 28,000 Da and an isoelectric point of 4.6. Interestingly, Jurkat cells transfected with tat-I express higher levels of NP-28 constitutively, without added stimulation. Incubation of Jurkat cells expressing tat-I with PMA and/or PHA or ionomycin causes superinduction of NP-28. We have therefore identified a novel lymphoid nuclear protein induced by T cell activation that occurs in tat-I expressing cells in the absence of activating agents. PMID:1988491

Bielinska, A; Baier, L; Hailat, N; Strahler, J R; Nabel, G J; Hanash, S

1991-02-01

279

Bacterial expression and photoaffinity labeling of a pheromone binding protein.  

PubMed Central

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.

Prestwich, G. D.

1993-01-01

280

Preliminary identification of differentially expressed tear proteins in keratoconus  

PubMed Central

Purpose To examine the proteins differentially expressed in the tear film of people with keratoconus and normal subjects. Methods Unstimulated tears from people with keratoconus (KC) and controls (C) were collected using a capillary tube. Tear proteins from people with KC and controls were partitioned using a novel in-solution electrophoresis, Microflow 10 (ProteomeSep), and analyzed using linear ion trap quadrupole fourier transform mass spectrometry. Spectral counting was used to quantify the individual tear proteins. Results Elevated levels of cathepsin B (threefold) were evident in the tears of people with KC. Polymeric immunoglobulin receptor (ninefold), fibrinogen alpha chain (eightfold), cystatin S (twofold), and cystatin SN (twofold) were reduced in tears from people with KC. Keratin type-1 cytoskeletal-14 and keratin type-2 cytoskeletal-5 were present only in the tears of people with KC. Conclusions The protein changes in tears, that is, the decrease in protease inhibitors and increase in proteases, found in the present and other previously published studies reflect the pathological events involved in KC corneas. Further investigations into tear proteins may help elucidate the underlying molecular mechanisms of KC, which could result in better treatment options.

Wasinger, Valerie C.; Pye, David C.; Willcox, Mark D. P.

2013-01-01

281

Expression and purification of recombinant NFI proteins for functional analysis.  

PubMed

Nuclear factor I (NFI) is a transcription factor playing wide role in signal transduction pathways and developmental processes in higher eukaryotes. In order to produce recombinant NFI proteins for functional and structural studies, full length cDNAs of individual isoforms were subcloned into pETM30 vector and expressed in Escherichia coli. Although the fusion proteins containing both glutathione S-transferase (GST) and His6 tags at the N-terminus could be overexpressed in detectable amounts, they were found mainly, if not exclusively, in insoluble form. Purification yield was improved by modification of cell disruption procedure and by the use of detergent Tween 20. The final purification strategy represents a triple-helix affinity chromatography consisting of prepurification of bacterial lysate on Heparin-Sepharose with subsequent immobilized metal affinity and glutathione affinity chromatography. Heparin chromatography was crucial for obtaining active NFI proteins, whereas the other steps significantly improved the purity of isolated proteins. As demonstrated by EMSA and DNase I protection assay, the recombinant proteins were able to recognize their cognate DNA sequences. PMID:20097955

Kollárovic, Gabriel; Majera, Dusana; Luciaková, Katarína; Baráth, Peter

2009-12-01

282

Expression of P53 protein after exposure to ionizing radiation  

NASA Astrophysics Data System (ADS)

One of the most important tumor suppressor genes is p53 gene, which is involved in apoptotic cell death, cell differentiation and cell cycle arrest. The expression of p53 gene can be evaluated by determining the presence of P53 protein in cells using Western Blot assay with a chemiluminescent method. This technique has shown variabilities that are due to biological factors. Film developing process can influence the quality of the p53 bands obtained. We irradiated tumor cell lines and human peripheral lymphocytes with 137Cs and 60Co gamma rays to standardize irradiation conditions, to compare ionizing radiation with actinomycin D and to reduce the observed variability of P53 protein induction levels. We found that increasing radiation doses increase P53 protein induction while it decreases viability. We also conclude that ionizing radiation could serve as a positive control for Western Blot analysis of protein P53. In addition, our results show that the developing process may play an important role in the quality of P53 protein bands and data interpretation. .

Salazar, A. M.; Salvador, C.; Ruiz-Trejo, C.; Ostrosky, P.; Brandan, M. E.

2001-10-01

283

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

PubMed Central

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 func-tions of WT1 by clinically available Hsp90 inhibitors.

Bansal, Hima; 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.

2010-01-01

284

Protein expression in salivary glands of rats with streptozotocin diabetes.  

PubMed

Diabetes mellitus (DM) is a widespread disease with high morbidity and health care costs. An experimental animal model was employed, using morphological and biochemical methods, to investigate the effects of DM on the expression and compartmentation of salivary gland proteins. The distribution of proline-rich proteins (PRP), submandibular mucin (Muc10) and the regulatory (RI and RII) subunits of cyclic AMP-dependent protein kinase type I and type II was determined in the parotid and submandibular (SMG) glands of rats treated with streptozotocin. Quantitative immunocytochemistry of secretory granules in diabetic glands revealed decreases of 30% for PRP in both the parotid and SMG, and a 40% decrease in Muc10 in the SMG. Immunogold labelling showed that RII decreased in nuclei and the cytoplasm in diabetic acinar cells while labelling of secretory granules was similar in control and diabetic parotid. Electrophoresis and Western blotting of tissue extracts of two secretory proteins showed that the response to DM and insulin treatment was gland specific: PRP showed little change in the SMG, but decreased in the parotid in DM and was partially restored after insulin treatment. Photoaffinity labelling showed only RI present in the SMG and mainly RII in the parotid. The results of this and previous studies demonstrating highly specific changes in salivary protein expression indicate that the oral environment is significantly altered by DM, and that oral tissues and their function can be compromised. These findings may provide a basis for future studies to develop tests using saliva for diabetic status or progression in humans. PMID:19659899

Mednieks, Maija I; Szczepanski, Andrew; Clark, Brett; Hand, Arthur R

2009-08-01

285

A gene family encoding RING finger proteins in rice: their expansion, expression diversity, and co-expressed genes  

Microsoft Academic Search

The proteins harboring RING finger motif(s) have been shown to mediate protein–protein interactions that are relevant to a\\u000a variety of cellular processes. In an effort to elucidate the evolutionary dynamics of the rice RING finger protein family,\\u000a we have attempted to determine their genomic locations, expression diversity, and co-expressed genes via in silico analysis\\u000a and semi-quantitative RT–PCR. A total of

Sung Don Lim; Won Cheol Yim; Jun-Cheol Moon; Dong Sub Kim; Byung-Moo Lee; Cheol Seong Jang

2010-01-01

286

Development of novel self-assembled DS-PLGA hybrid nanoparticles for improving oral bioavailability of vincristine sulfate by P-gp inhibition.  

PubMed

To improve the encapsulation efficiency and oral bioavailability of vincristine sulfate (VCR), novel self-assembled dextran sulphate-PLGA hybrid nanoparticles (DPNs) were successfully developed using self-assembly and nanoprecipitation method. By introducing the negative polymer of dextran sulphate sodium (DS), VCR was highly encapsulated (encapsulation efficiency up to 93.6%) into DPNs by forming electrostatic complex. In vitro release of VCR solution (VCR-Sol) and VCR-loaded DPNs (VCR-DPNs) in pH 7.4 PBS showed that about 80.4% of VCR released from VCR-DPNs after 96h and burst release was effectively reduced, indicating pronounced sustained-release characteristics. In vivo pharmacokinetics in rats after oral administration of VCR-Sol and VCR-DPNs indicated that the apparent bioavailability of VCR-DPNs was increased to approximate 3.3-fold compared to that of VCR-Sol. The cellular uptake experiments were conducted by quantitative assay of VCR cellular accumulation and fluorescence microscopy imaging of fluorescent labeled DPNs in two human breast cancer cells including MCF-7 and P-glycoprotein over-expressing MCF-7/Adr cells. The relative cellular uptake of VCR-DPNs was 12.4-fold higher than that of VCR-Sol in MCF-7/Adr cells implying that P-glycoprotein-mediated drug efflux was diminished by the introduction of DPNs. The new DPNs might provide an effective strategy for oral delivery of VCR with improved encapsulation efficiency and oral bioavailability. PMID:20727928

Ling, Guixia; Zhang, Peng; Zhang, Wenping; Sun, Jin; Meng, Xiaoxue; Qin, Yimeng; Deng, Yihui; He, Zhonggui

2010-12-01

287

Expression of green fluorescent protein fused to magnetosome proteins in microaerophilic magnetotactic bacteria.  

PubMed

The magnetosomes of magnetotactic bacteria are prokaryotic organelles consisting of a magnetite crystal bounded by a phospholipid bilayer that contains a distinct set of proteins with various functions. Because of their unique magnetic and crystalline properties, magnetosome particles are potentially useful as magnetic nanoparticles in a number of applications, which in many cases requires the coupling of functional moieties to the magnetosome membrane. In this work, we studied the use of green fluorescent protein (GFP) as a reporter for the magnetosomal localization and expression of fusion proteins in the microaerophilic Magnetospirillum gryphiswaldense by flow cytometry, fluorescence microscopy, and biochemical analysis. Although optimum conditions for high fluorescence and magnetite synthesis were mutually exclusive, we established oxygen-limited growth conditions, which supported growth, magnetite biomineralization, and GFP fluorophore formation at reasonable rates. Under these optimized conditions, we studied the subcellular localization and expression of the GFP-tagged magnetosome proteins MamC, MamF, and MamG by fluorescence microscopy and immunoblotting. While all fusions specifically localized at the magnetosome membrane, MamC-GFP displayed the strongest expression and fluorescence. MamC-GFP-tagged magnetosomes purified from cells displayed strong fluorescence, which was sensitive to detergents but stable under a wide range of temperature and salt concentrations. In summary, our data demonstrate the use of GFP as a reporter for protein localization under magnetite-forming conditions and the utility of MamC as an anchor for magnetosome-specific display of heterologous gene fusions. PMID:18539817

Lang, Claus; Schüler, Dirk

2008-08-01

288

Expression of Green Fluorescent Protein Fused to Magnetosome Proteins in Microaerophilic Magnetotactic Bacteria? †  

PubMed Central

The magnetosomes of magnetotactic bacteria are prokaryotic organelles consisting of a magnetite crystal bounded by a phospholipid bilayer that contains a distinct set of proteins with various functions. Because of their unique magnetic and crystalline properties, magnetosome particles are potentially useful as magnetic nanoparticles in a number of applications, which in many cases requires the coupling of functional moieties to the magnetosome membrane. In this work, we studied the use of green fluorescent protein (GFP) as a reporter for the magnetosomal localization and expression of fusion proteins in the microaerophilic Magnetospirillum gryphiswaldense by flow cytometry, fluorescence microscopy, and biochemical analysis. Although optimum conditions for high fluorescence and magnetite synthesis were mutually exclusive, we established oxygen-limited growth conditions, which supported growth, magnetite biomineralization, and GFP fluorophore formation at reasonable rates. Under these optimized conditions, we studied the subcellular localization and expression of the GFP-tagged magnetosome proteins MamC, MamF, and MamG by fluorescence microscopy and immunoblotting. While all fusions specifically localized at the magnetosome membrane, MamC-GFP displayed the strongest expression and fluorescence. MamC-GFP-tagged magnetosomes purified from cells displayed strong fluorescence, which was sensitive to detergents but stable under a wide range of temperature and salt concentrations. In summary, our data demonstrate the use of GFP as a reporter for protein localization under magnetite-forming conditions and the utility of MamC as an anchor for magnetosome-specific display of heterologous gene fusions.

Lang, Claus; Schuler, Dirk

2008-01-01

289

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

290

Simvastatin enhances bone morphogenetic protein receptor type II expression  

SciTech Connect

Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function.

Hu Hong [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Sung, Arthur [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Zhao, Guohua [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Shi, Lingfang [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Qiu Daoming [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Nishimura, Toshihiko [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Kao, Peter N. [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States)]. E-mail: peterkao@stanford.edu

2006-01-06

291

Expression of Smad proteins in human colorectal cancer.  

PubMed

Escape from transforming growth factor-beta (TGF-beta)-induced inhibition of proliferation has been observed in many tumor cells and may contribute to loss of growth control. Smad proteins have been identified as major components in the intracellular signaling of TGF-beta family members. In this study, we examined the expression of receptor-activated, common-mediator and inhibitory Smads by immunohistochemistry in human colorectal cancers. We found increased expression of receptor-activated Smads in a fraction of the tumor cells, while no immunostaining for Smad2, Smad3 or Smad5 and only occasional staining for Smad1/8 was found in epithelial mucosa of normal colon. No or only weak staining for receptor-activated Smads, common-mediator Smad4 and inhibitory Smads was observed in the tumor stroma. Common-mediator Smad4 and inhibitory Smads were detected in cells of both tumor and normal tissues. We observed a distinct pattern of Smad4 immunostaining of epithelial cells along colon crypts, with high expression in zones of terminal differentiation. Our data show selective up-regulation of receptor-activated Smad proteins in human colorectal cancers and suggest involvement of Smad4 in differentiation and apoptosis of surface epithelial cells of normal crypts. PMID:10389752

Korchynskyi, O; Landström, M; Stoika, R; Funa, K; Heldin, C H; ten Dijke, P; Souchelnytskyi, S

1999-07-19

292

A novel chalcone derivative which acts as a microtubule depolymerising agent and an inhibitor of P-gp and BCRP in in-vitro and in-vivo glioblastoma models  

PubMed Central

Background Over the past decades, in spite of intensive search, no significant increase in the survival of patients with glioblastoma has been obtained. The role of the blood-brain barrier (BBB) and especially the activity of efflux pumps belonging to the ATP Binding Cassette (ABC) family may, in part, explain this defect. Methods The in-vitro activities of JAI-51 on cell proliferation were assessed by various experimental approaches in four human and a murine glioblastoma cell lines. Using drug exclusion assays and flow-cytometry, potential inhibitory effects of JAI-51 on P-gp and BCRP were evaluated in sensitive or resistant cell lines. JAI-51 activity on in-vitro microtubule polymerization was assessed by tubulin polymerization assay and direct binding measurements by analytical ultracentrifugation. Finally, a model of C57BL/6 mice bearing subcutaneous GL26 glioblastoma xenografts was used to assess the activity of the title compound in vivo. An HPLC method was designed to detect JAI-51 in the brain and other target organs of the treated animals, as well as in the tumours. Results In the four human and the murine glioblastoma cell lines tested, 10 ?M JAI-51 inhibited proliferation and blocked cells in the M phase of the cell cycle, via its activity as a microtubule depolymerising agent. This ligand binds to tubulin with an association constant of 2 × 105 M-1, overlapping the colchicine binding site. JAI-51 also inhibited the activity of P-gp and BCRP, without being a substrate of these efflux pumps. These in vitro studies were reinforced by our in vivo investigations of C57BL/6 mice bearing GL26 glioblastoma xenografts, in which JAI-51 induced a delay in tumour onset and a tumour growth inhibition, following intraperitoneal administration of 96 mg/kg once a week. In accordance with these results, JAI-51 was detected by HPLC in the tumours of the treated animals. Moreover, JAI-51 was detected in the brain, showing that the molecule is also able to cross the BBB. Conclusion These in vitro and in vivo data suggest that JAI-51 could be a good candidate for a new treatment of tumours of the CNS. Further investigations are in progress to associate the title compound chemotherapy to radiotherapy in a rat model.

2009-01-01

293

Proteome analysis of abundantly expressed proteins from unfed larvae of the cattle tick, Boophilus microplus  

Microsoft Academic Search

Protein expression in unfed larvae of the cattle tick, Boophilus microplus, was characterized using gel electrophoresis and mass spectrometry in an effort to assemble a database of proteins produced at this stage of development. Soluble and insoluble proteins were extracted and resolved by two-dimensional (2D) gel electrophoresis. Twenty abundantly expressed larval proteins were selected for peptide mass mapping and for

P. M. Untalan; F. D. Guerrero; L. R. Haines; T. W. Pearson

2005-01-01

294

Combinatorial expression vector engineering for tuning of recombinant protein production in Escherichia coli  

Microsoft Academic Search

The complex and integrated nature of both genetic and protein level factors influencing recombinant protein production in Escherichia coli makes it difficult to predict the optimal expression strategy for a given protein. Here, two combinatorial library strategies were evaluated for their capability of tuning recombinant protein production in the cyto- plasm of E. coli. Large expression vector libraries were constructed

Nina Bandmann; Per-Ake Nygren

2007-01-01

295

[Effects of gene design on recombinant protein expression: a review].  

PubMed

It has become a hotspot and keystone in gene engineering and bioengineering to produce recombinant proteins through heterologous expression systems. Unfortunately, not all the genes could be successfully and effectively expressed in heterologous hosts. The role of gene itself in regulating translation process through its intrinsic sequence characteristics such as codon bias, codon pair bias, GC content, mRNA secondary structure and mRNA stability, has been gradually elucidated. Here we review these factors that influence the translation processes and their corresponding optimization methods in the process of gene design. We emphatically discussed codon bias and codon pair bias and their optimization methods. In particular, the latest theories of codon harmonization and codon pair harmonization were discussed and compared with the traditional codon and codon pair optimization strategies in gene design. PMID:24409684

Cai, Haiying; Li, Yang; Zhang, Hui; Feng, Fengqin

2013-09-01

296

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.

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

2014-01-01

297

TRANSFECTED MDCK CELL LINE WITH ENHANCED EXPRESSION OF CYP3A4 AND P-GLYCOPROTEIN AS A MODEL TO STUDY THEIR ROLE IN DRUG TRANSPORT AND METABOLISM  

PubMed Central

The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and P-glycoprotein as an in vitro model for evaluating drug-herb and drug-drugs of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 ?M cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Similar studies were also carried out in the presence of 50 ?M naringin and 3 ?M morphine. Samples were analyzed by HPLC for drug and its CYP3A4 metabolite. PCR, qPCR and western blot studies confirmed the enhanced expression of the proteins in the transfected cells. The vivid CYP3A4 assay and ketoconazole inhibition studies further confirmed the presence of active protein. Apical to basal transport of cortisol was found to be ten and three fold lower in MMC as compared to WT and MDCKMDR1 respectively. Higher amount of metabolite was formed in MMC than in MDCK-WT indicating enhanced expression of CYP3A4. Highest cortisol metabolite formation was observed in MMC cell line due to the combined metabolic activities of CYP3A4 and P-gp. Transport of cortisol increased fivefold in presence of naringin in MMC and doubled in MDCKMDR1. Cortisol transport in MMC was significantly lower than that in WT in presence of naringin. The permeability increased three fold in presence of morphine which is a weaker inhibitor of CYP3A4. Formation of 6?-hydroxy cortisol was found to decrease in presence of morphine and naringin. This new model cell line with its enhanced CYP3A4 and P-gp levels in addition to short culture time can serve as an invaluable model to study drug-drug interactions. This cell line can also be used to study the combined contribution of efflux transporter and metabolizing enzymes towards drug-drug interactions.

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

2012-01-01

298

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

299

Identification of differentially expressed proteins and phosphorylated proteins in rice seedlings in response to strigolactone treatment.  

PubMed

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

300

Decreased Dicer Expression Enhances SRP-Mediated Protein Targeting  

PubMed Central

We have shown that Dicer processes 7SL RNA into different fragments ranging from ?20 to more than 200 nucleotides. Here we addressed the molecular functions of these 7SL RNA fragments and found that some of them functioned as dominant-negative regulators of the full-length 7SL RNA, interfering with signal recognition particle (SRP) complex formation. Transfection of these 7SL RNA fragments inhibited the expression of cell surface glycoproteins, the targeting of a reporter protein to the endoplasmic reticulum, and the secretion of secreted alkaline phosphatase. These results suggest that some Dicer-processed 7SL RNA fragments interfered with SRP-mediated protein targeting. Moreover, we showed that Dicer knockdown enhanced SRP-mediated protein targeting and that transfection of a mixture of the 7SL RNA fragments partially restored this effect. Our data indicate that Dicer can fine-tune the efficiency of SRP-mediated protein targeting via processing a proportion of 7SL RNA into fragments of different lengths.

Zhang, Xue-Jiao; Song, Yi-Jiang; Lv, Lu; Wu, Jianmin; Fang, Yu-Xiao; Wang, Yu-Qun; Shi, Ke-Qing; Chen, Yong-ping; Tang, Kai-Fu

2013-01-01

301

The tetraspan protein EMP2 regulates expression of caveolin-1.  

PubMed

Caveolin-1 is the primary component of caveolae and functions in a variety of intracellular activities, including membrane trafficking and signal transduction. EMP2 (epithelial membrane protein 2) is a tetraspan protein recently identified as a novel regulator of caveolin-1 expression. In this study, we analyzed the mechanism of EMP2-mediated caveolin-1 regulation. In NIH 3T3 cells and in the human retinal pigment epithelium cell line (ARPE-19), EMP2 regulates caveolin-1 transcription and more substantially its protein levels. EMP2-mediated down-regulation of caveolin-1 does not affect caveolin-1 translational efficiency, phosphorylation, or proteasome-mediated degradation. Analysis of caveolin-1 protein half-life indicates the EMP2-mediated loss of caveolin-1 occurs rapidly. Protease inhibition and laser confocal microscopy associates this fate with specific intracellular compartmentalization, including early lysosomal delivery. These findings elucidate a new mechanism of caveolin-1 regulation and define an additional role for EMP2 as a key regulator of cell membrane composition. PMID:17609206

Forbes, Ashley; Wadehra, Madhuri; Mareninov, Sergei; Morales, Shawn; Shimazaki, Kaori; Gordon, Lynn K; Braun, Jonathan

2007-09-01

302

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.

2010-01-01

303

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.

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

2005-01-01

304

Globally predicting protein functions based on co-expressed protein–protein interaction networks and ontology taxonomy similarities  

Microsoft Academic Search

Determining protein functions is an important task in the post-genomic era. Most of the current methods work on some large-sized functional classes selected from functional categorization systems prior to the prediction processes. GESTs, a prediction approach previously proposed by us, is based on gene expression similarity and taxonomy similarity of the functional classes. Unlike many conventional methods, it does not

Mingzhu Zhu; Lei Gao; Zheng Guo; Yanhui Li; Dong Wang; Jing Wang; Chenguang Wang

2007-01-01

305

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.  

PubMed

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity. PMID:20072815

Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

2010-10-01

306

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

Moi, P; Kan, Y W

1990-01-01

307

A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila  

Microsoft Academic Search

In Drosophila, enhancer trap strategies allow rapid access to expression patterns, molecular data, and mutations in trapped genes. However, they do not give any information at the protein level, e.g., about the protein subcellular localization. Using the green fluorescent protein (GFP) as a mobile artificial exon carried by a transposable P-element, we have developed a protein trap system. We screened

Xavier Morin; Richard Daneman; Michael Zavortink; William Chia

2001-01-01

308

Altered expression of carbonic anhydrase-related protein XI in neuronal cells expressing mutant ataxin-3.  

PubMed

Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a late-onset neurodegenerative disorder caused by the expansion of a polyglutamine tract within the gene product, ataxin-3. Microarray analysis revealed a dramatic differential expression of carbonic anhydrase-related protein XI (CA-RPXI/CA11) in the presence or absence of mutant ataxin-3. Therefore, we examined the expression and distribution of all three CA-RPs (CA8, 10, and 11) in human neuronal cells that stably express mutant ataxin-3. Compared with the cells containing normal ataxin-3, protein expression of CA8 and CA11 is significantly increased in human neuroblastoma cells harboring mutant ataxin-3. Semi-quantitative RT-PCR demonstrated that all three CA-RPs exhibited significantly higher transcript levels in neuronal cells expressing mutant ataxin-3. Interestingly, CA11 is distributed not only in the cytoplasm but also within the nuclei of the stably transfected mutant cells when compared with the sole cytoplasmic distribution in cells containing normal ataxin-3. In addition, results from transient transfection assays in SK-N-SH and Neuro2a (N2a) cells also confirmed the nuclear localization of CA11 in the presence of truncated ataxin-3. Most importantly, immunohistochemical staining of the MJD transgenic mouse and post-mortem MJD human brain also revealed that CA11 is highly expressed in both cytoplasm and nuclei of the brain cells. Recruitment of CA11 into nuclear inclusions containing mutant ataxin-3 revealed a possible correlation between CA11 and disease progression. Although the exact function of CA-RPs is still undefined in the central nervous system, our findings suggest that CA-RPs, especially CA11, may play specific roles in the pathogenesis of Machado-Joseph disease. PMID:23184527

Hsieh, Mingli; Chang, Wei-Hsiu; Hsu, Chi-Fu; Nishimori, Isao; Kuo, Cheng-Liang; Minakuchi, Tomoko

2013-06-01

309

Localisation of breast cancer resistance protein in microvessel endothelium of human brain.  

PubMed

Movement of substrates between blood and brain is known to be influenced by P-glycoprotein (P-gp) at the luminal surface of the endothelium lining brain microvessels and by multidrug resistance associated protein 1 (MRP1) at the basolateral surface of the choroid plexus epithelium. Here, using RT-PCR and Western blotting, we investigate other ABC transporters in both normal and tumour human brain tissue and demonstrate the presence of breast cancer resistance protein (BCRP). Immunofluorescence confocal microscopy demonstrates that BCRP is located at the blood-brain barrier, mainly at the luminal surface of microvessel endothelium. This localization closely resembles that of P-gp. BCRP has several substrates in common with P-gp and may pose an additional barrier to drug access to the brain. PMID:12438926

Cooray, Hiran C; Blackmore, Colin G; Maskell, Lynn; Barrand, Margery A

2002-11-15

310

Induction of a multixenobiotic resistance protein (MXR) in the Asiatic clam Corbicula fluminea after heavy metals exposure  

Microsoft Academic Search

Multixenobiotic resistance mechanisms (MXR) related to the mammalian P-glycoprotein multidrug transporter protein (P-gp) are known to occur in several marine invertebrates. In the present work, we report on the induction of an MXR protein by various heavy metals in the gills of the freshwater clam Corbicula fluminea. The evaluation of the MXR protein level was assessed by Western blot using

M Achard; M Baudrimont; A Boudou; J. P Bourdineaud

2004-01-01

311

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

312

Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes  

PubMed Central

Arsenic trioxide (arsenite, AsIII) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of AsIII on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of AsIII on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent AsIII-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of AsIII were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to AsIII than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by AsIII in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to AsIII-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to AsIII cytotoxicity between these cells.

YOSHINO, Yuta; YUAN, Bo; KAISE, Toshikazu; TAKEICHI, Makoto; TANAKA, Sachiko; HIRANO, Toshihiko; KROETZ, Deanna L.; TOYODA, Hiroo

2012-01-01

313

SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm  

PubMed Central

Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains.

2012-01-01

314

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

315

Ozone inhalation stimulates expression of a neutrophil chemotactic protein, macrophage inflammatory protein 2  

Microsoft Academic Search

Short-term exposure of humans and animals to ozone results in increased lung neutrophils; however, the mechanisms underlying this response are not completely understood. We examined the potential involvement of the neutrophil chemotactic factor, macrophage inflammatory protein 2 (MIP-2), in ozone-induced inflammation. Exposure-response relationships for ozone and MIP-2 expression were characterized by exposing C57B1\\/6 mice to 0.1-2 ppm ozone for 3

K. E. Driscoll; L. Simpson; J. Carter; D. Hassenbein; G. D. Leikauf

1993-01-01

316

Breast Cancer Resistance Protein and P-glycoprotein in Brain Cancer: Two Gatekeepers Team Up  

PubMed Central

Brain cancer is a devastating disease. Despite extensive research, treatment of brain tumors has been largely ineffective and the diagnosis of brain cancer remains uniformly fatal. Failure of brain cancer treatment may be in part due to limitations in drug delivery, influenced by the ABC drug efflux transporters P-gp and BCRP at the blood-brain and blood-tumor barriers, in brain tumor cells, as well as in brain tumor stem-like cells. P-gp and BCRP limit various anti-cancer drugs from entering the brain and tumor tissues, thus rendering chemotherapy ineffective. To overcome this obstacle, two strategies – targeting transporter regulation and direct transporter inhibition – have been proposed. In this review, we focus on these strategies. We first introduce the latest findings on signaling pathways that could potentially be targeted to down-regulate P-gp and BCRP expression and/or transport activity. We then highlight in detail the new paradigm of P-gp and BCRP working as a “cooperative team of gatekeepers” at the blood-brain barrier, discuss its ramifications for brain cancer therapy, and summarize the latest findings on dual P-gp/BCRP inhibitors. Finally, we provide a brief summary with conclusions and outline the perspectives for future research endeavors in this field.

Agarwal, Sagar; Hartz, Anika M.S.; Elmquist, William F.; Bauer, Bjorn

2012-01-01

317

Small envelope protein E of SARS: cloning, expression, purification, CD determination, and bioinformatics analysis1  

Microsoft Academic Search

AIM: To obtain the pure sample of SARS small envelope E protein (SARS E protein), study its properties and analyze its possible functions. METHODS: The plasmid of SARS E protein was constructed by the polymerase chain reaction (PCR), and the protein was expressed in the E coli strain. The secondary structure feature of the protein was determined by circular dichroism

SHEN Xu; XUE Jian-Hua; YU Chang-Ying; QIN Lei; YU Xiao-Jing; CHEN Jing; CHEN Li-Li; XIONG Bin; YUE Li-Duo; CAI Jian-Hua; SHEN Jian-Hua; LUO Xiao-Min; CHEN Kai-Xian; SHI Tie-Liu; LI Yi-Xue; HU Geng-Xi; JIANG Hua-Liang

318

Engineering Cowpea Mosaic Virus RNA2 into a Vector to Express Heterologous Proteins in Plants  

Microsoft Academic Search

A series of new cowpea mosaic virus (CPMV) RNA-2-based expression vectors were designed. The jellyfish green fluorescent protein (GFP) was introduced between the movement protein (MP) and the large (L) coat protein or downstream of the small (S) coat protein. Release of the GFP inserted between the MP and L proteins was achieved by creating artificial processing sites each side

Kodetham Gopinath; Joan Wellink; Claudine Porta; Kathryn M. Taylor; George P. Lomonossoff; Ab van Kammen

2000-01-01

319

Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.  

PubMed

To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and ?-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5-6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean. PMID:24435987

Zhang, Yunfang; Schernthaner, Johann; Labbé, Natalie; Hefford, Mary A; Zhao, Jiping; Simmonds, Daina H

2014-06-01

320

Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression  

PubMed Central

Background In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. Results Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. Conclusions By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli.

Dyson, Michael R; Shadbolt, S Paul; Vincent, Karen J; Perera, Rajika L; McCafferty, John

2004-01-01

321

Excystment-Dependent Alteration of Protein Expression in Terrestrial Ciliate Colpoda cucullus  

PubMed Central

Protein expression during the excystment of Colpoda cucullus was studied by SDS-PAGE. The expression levels of 60-, 50- and 49-kDa proteins were markedly changed from the early to later stage of excystment. The 60-kDa protein (p60) was temporarily expressed first, and its expression was inhibited by actinomycin D. LC-MS/MS analysis showed that the amino acid sequences of p60 partially coincided with those of the Paramecium tetraurelia unnamed protein homologous to DEAD-box RNA helicase. These results suggest that p60 expression is enhanced by transcriptional regulation and may be involved in initiating the molecular events leading to cellular morphogenesis.

Sogame, Yoichiro; Kojima, Katsuhiko; Takeshita, Toshikazu; Kinoshita, Eiji; Funadani, Ryoji; Matsuoka, Tatsuomi

2013-01-01

322

Changes related to the oestrous cycle in the expression of endometrial and oviductal proteins of mice.  

PubMed

Soluble proteins extracted from the endometria and oviducts of normal sexually mature cycling Swiss Webster mice were analysed by two-dimensional high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Thirty endometrial and 25 oviductal proteins showed differential expression related to the oestrous cycle. In the endometrium, 19 proteins were maximally expressed in the oestrous phase, and significantly decreased or could not be detected in dioestrus. Eleven additional proteins were more prominent in dioestrus. Most of these endometrial cyclic proteins were acidic. In the oviduct, almost two-thirds of cycle-related, differentially expressed proteins were more strongly expressed in dioestrus and were significantly less prominent or could not be detected in the oestrous phase. In contrast to the endometrial proteins, most of the oviductal cyclic proteins were basic. Fourteen proteins appeared to be identical in both organs, and five of these showed the same cyclic pattern of expression. The remaining cyclic proteins were organ specific and showed uterus- or oviduct-specific changes during the oestrous cycle. Among the cyclic proteins, four endometrial and two oviductal proteins were restricted to oestrus, whereas two endometrial and seven oviductal proteins were restricted to dioestrus. These proteins could serve as markers for specific phases of the oestrous cycle. Our data show that the mouse oestrous cycle is associated with consistent and predictable changes in protein expression in both the endometrium and oviduct. PMID:1625236

Horvat, B; Vrci?, H; Damjanov, I

1992-05-01

323

Highly efficient protein expression and purification using bacterial hemoglobin fusion vector  

Microsoft Academic Search

Recently developed bacterial hemoglobin (VHb) fusion expression vector has been widely used for the production of many target proteins due to its distinctive properties of expressing fusion protein with red color which facilitates visualization of the steps in purification, and increasing solubility of the target proteins. However, after intensive use of the vector, several defects have been found. In this

Soo-Young Kwon; Yoon-Joo Choi; Tae-Hong Kang; Kwang-Hoon Lee; Sun-Shin Cha; Gyung-Hwa Kim; Heung-Soo Lee; Kyong-Tai Kim; Kyung-Jin Kim

2005-01-01

324

C-Reactive Protein Inhibits Survivin Expression via Akt/mTOR Pathway Downregulation by PTEN Expression in Cardiac Myocytes  

PubMed Central

C-reactive protein (CRP) is one of the most important biomarkers for arteriosclerosis and cardiovascular disease. Recent studies have shown that CRP affects cell cycle and inflammatory process in cardiac myocytes. Survivin is also involved in cardiac myocytes replication and apoptosis. Reduction of survivin expression is associated with less favorable cardiac remodeling in animal models. However, the effect of CRP on survivin expression and its cellular mechanism has not yet been studied. We demonstrated that treatment of CRP resulted in a significant decrease of survivin protein expression in a concentration-dependent manner in cardiac myocytes. The upstream signaling proteins of survivin, such as Akt, mTOR and p70S6K, were also downregulated by CRP treatment. In addition, CRP increased the protein and mRNA levels of PTEN. The siRNA transfection or specific inhibitor treatment for PTEN restored the CRP-induced downregulation of Akt/mTOR/p70S6K pathway and survivin protein expression. Moreover, pretreatment with a specific p53 inhibitor decreased the CRP-induced PTEN expression. ERK-specific inhibitor also blocked the p53 phosphorylation and PTEN expression induced by CRP. Our study provides a novel insight into CRP-induced downregulation of survivin protein expression in cardiac myocytes through mechanisms that involved in downregulation of Akt/mTOR/p70S6K pathway by expression of PTEN.

Lee, Beom Seob; Kim, Soo Hyuk; Oh, Jaewon; Jin, Taewon; Choi, Eun Young; Park, Sungha; Lee, Sang-Hak; Chung, Ji Hyung; Kang, Seok-Min

2014-01-01

325

Anatomical profiling of G protein-coupled receptor expression  

PubMed Central

Summary G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane signaling molecules and regulate a host of physiological and disease processes. To better understand the functions of GPCRs in vivo, we quantified transcript levels of 353 non-odorant GPCRs in 41 adult mouse tissues. Cluster analysis placed many GPCRs into anticipated anatomical and functional groups and predicted novel roles for less studied receptors. From one such prediction, we showed that the Gpr91 ligand succinate can regulate lipolysis in white adipose tissue suggesting that signaling by this citric acid cycle intermediate may regulate energy homeostasis. We also showed that pairwise analysis of GPCR expression across tissues may help predict drug side effects. This resource will aid studies to understand GPCR function in vivo and may assist in the identification of therapeutic targets.

Regard, Jean B.; Sato, Isaac T.; Coughlin, Shaun R.

2008-01-01

326

Expression of ATP-binding cassette membrane transporters in a HIV-1 transgenic rat model.  

PubMed

P-glycoprotein (P-gp, product of Mdr1a and Mdr1b genes), multidrug resistance associated proteins (Mrps), and breast cancer resistance protein (Bcrp), all members of the ATP-binding cassette (ABC) membrane-associated drug transporters superfamily, can significantly restrict the entry of antiretroviral drugs (ARVs) into organs which exhibit a barrier function such as the central nervous system (CNS) and the male genital tract (MGT). In vitro, HIV-1 viral proteins such as glycoprotein-120 (gp120) and transcriptional transactivator (tat) have been shown to alter the expression of these transporters and ARVs permeability. The objective of this study was to compare mRNA expression of these transporters, in vivo, in several tissues obtained from HIV-1 transgenic rats (Tg-rat) (8 and 24 weeks) with those of age-matched wild-type rats. At 24 weeks, significant changes in several drug transporter mRNA expressions were observed, in particular, in brain, kidney, liver and testes. These findings suggest that HIV-1 viral proteins can alter the expression of ABC drug transporters, in vivo, in the context of HIV-1 and further regulate ARVs permeability in several organs including the CNS and MGT, two sites which have been reported to display very low ARVs permeability in the clinic. PMID:24472536

Robillard, Kevin R; Hoque, Md Tozammel; Bendayan, Reina

2014-02-21

327

Expression, purification, and crystallization of the adipocyte lipid binding protein.  

PubMed

The murine adipocyte lipid binding protein (ALBP/aP2) has been cloned and expressed in Escherichia coli, purified to homogeneity, biochemically characterized, and crystallized for x-ray diffraction study. In the cloning, the ALBP coding region was placed under control of the recA promoter and downstream of the phage T7 g-10 translation enhancer sequence. Nalidixic acid (50 micrograms/ml) induced the expression of ALBP 20-fold over that attained using the pT7 system previously reported (Chinander, L. L., and Bernlohr, D. A. (1989) J. Biol. Chem. 264, 19564-19572). Recombinant ALBP was purified to homogeneity using a combination of pH fractionation, gel filtration, and immobilized metal affinity chromatography. The fluorescent affinity ligand 12-(9-anthroyloxy)oleic acid bound to homogeneous ALBP with an apparent Kd of 0.5 microM. rALBP was devoid of endogenous fatty acid, and oleic acid inhibited cysteine 117 modification by 5,5' -dithiobis-(2-nitrobenzoic acid) indicating integrity of the binding domain. Recombinant ALBP was phosphorylated by the soluble kinase domain of the insulin receptor with a Vmax of 11 nmol.min.mg of kinase and an apparent Km of 270 microM. Purified protein was crystallized using the hanging drop method with seeding. Crystalline ALBP was orthorhombic with cell dimensions of a = 34.4 A, b = 54.8 A, and c = 76.3 A. The space group was P212121, and there was one molecule per asymmetric unit. PMID:1650358

Xu, Z H; Buelt, M K; Banaszak, L J; Bernlohr, D A

1991-08-01

328

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.

2014-01-01

329

Expression of a Y-box protein, YB2\\/RYB-a, precedes protamine 2 expression during spermatogenesis in rodents  

Microsoft Academic Search

Y-box binding proteins, a large family of proteins, are involved in a variety of functions. The present study describes the expression of YB2, a rat Y-box binding protein, and\\/or RYB-a, an alternatively spliced product of the YB2 gene during spermatogenesis. YB2\\/RYB-a is thought to be the rat orthologue of mouse Y-box protein 3 (MSY3). An antibody which recognizes YB2\\/RYB-a was

Yoshihito Iuchi; Takashi Kobayashi; Tomoko Kaneko; Masatoshi Takahara; Toshihiko Ogino; Junichi Fujii

2001-01-01

330

Human Articular Chondrocytes Express Multiple Gap Junction Proteins  

PubMed Central

Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 ?m of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas.

Mayan, Maria D.; Carpintero-Fernandez, Paula; Gago-Fuentes, Raquel; Martinez-de-Ilarduya, Oskar; Wang, Hong-Zhang; Valiunas, Virginijus; Brink, Peter; Blanco, Francisco J.

2014-01-01

331

Phorbol esters alter the expression of lymphocyte membrane proteins  

SciTech Connect

T cell activation via the T cell receptor (T3-Ti complex) by OKT3 results in modulation of the T3-Ti complex, but does not affect T4, T8, or T11 antigen expression. To study the effect of other T cell activators on these T cell membrane antigens, the authors incubated mononuclear cells for 0-3 days with lectins or pharmacologic agents and stained with monoclonal antibodies to their antigens. The median fluorescence intensity (MFI) was measured with a fluorescence activated cell sorter. Activation of PBL with Con A, PHA, calcium ionophore A23187, or with dbcAMP, isoproterenol, or theophyllin had minimal effects on the MFI of T3, T4, T8, or T11. Phorbol myristate acetate (PMA), a protein kinase C activator which stimulates PBL though an alternate pathway, caused a 90-100% reduction of T3 and T4 MFI, a 25% reduction in T8 MFI, and a 400% increase in T11 MFI after 2 days. Addition of A23187 slightly increased these effects. PMA induced a 2-3-fold increase in cell diameter concomitant with the alterations in membrane antigens. These data suggest that T cell activation through pathways not directly linked to the T cell antigen receptor can result in surface antigen expression different from that which follows activation via the T cell receptor.

Reder, A.T.; Antel, J.P.

1986-03-01

332

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

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

1995-01-01

333

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.

Fenneteau, Frederique; Turgeon, Jacques; Couture, Lucie; Michaud, Veronique; Li, Jun; Nekka, Fahima

2009-01-01

334

Prion Protein Expression and Functional Importance in Skeletal Muscle  

PubMed Central

Abstract Skeletal muscle expresses prion protein (PrP) that buffers oxidant activity in neurons. Aims We hypothesize that PrP deficiency would increase oxidant activity in skeletal muscle and alter redox-sensitive functions, including contraction and glucose uptake. We used real-time polymerase chain reaction and Western blot analysis to measure PrP mRNA and protein in human diaphragm, five murine muscles, and muscle-derived C2C12 cells. Effects of PrP deficiency were tested by comparing PrP-deficient mice versus wild-type mice and morpholino-knockdown versus vehicle-treated myotubes. Oxidant activity (dichlorofluorescin oxidation) and specific force were measured in murine diaphragm fiber bundles. Results PrP content differs among mouse muscles (gastrocnemius>extensor digitorum longus, EDL>tibialis anterior, TA; soleus>diaphragm) as does glycosylation (di-, mono-, nonglycosylated; gastrocnemius, EDL, TA=60%, 30%, 10%; soleus, 30%, 40%, 30%; diaphragm, 30%, 30%, 40%). PrP is predominantly di-glycosylated in human diaphragm. PrP deficiency decreases body weight (15%) and EDL mass (9%); increases cytosolic oxidant activity (fiber bundles, 36%; C2C12 myotubes, 7%); and depresses specific force (12%) in adult (8–12?mos) but not adolescent (2?mos) mice. Innovation This study is the first to directly assess a role of prion protein in skeletal muscle function. Conclusions PrP content varies among murine skeletal muscles and is essential for maintaining normal redox homeostasis, muscle size, and contractile function in adult animals. Antioxid. Redox Signal. 15, 2465—2475.

Smith, Jeffrey D.; Moylan, Jennifer S.; Hardin, Brian J.; Chambers, Melissa A.; Estus, Steven; Telling, Glenn C.

2011-01-01

335

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.

2013-01-01

336

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.

Mbikay, M; Seidah, N G; Chretien, M

2001-01-01

337

Endogenous gene and protein expression of drug-transporting proteins in cell lines routinely used in drug discovery programs.  

PubMed

The aim of this study was to investigate the gene and protein expression profiles of important drug-transporting proteins in human cell lines commonly used for studies of drug transport mechanisms. Human cell lines used to transiently or stably express single transporters [HeLa, human embryonic kidney (HEK) 293] and leukemia cell lines used to study drug resistance by ATP-binding cassette transporters (HL-60, K562) were investigated and compared with organotypic cell lines (HepG2, Saos-2, Caco-2, and Caco-2 TC7). For gene expression studies, real-time polymerase chain reaction was used, whereas monospecific polyclonal antibodies were generated and used to investigate protein expression by immunohistochemistry. Thirty-six transporters were studied for gene expression, and nine were studied for protein expression. The antibodies were validated using expression patterns in human tissues. Finally, the function of one ubiquitously expressed transporter, MCT1/SLC16A1, was investigated using [(14)C]lactic acid as a substrate. In general, the adherent cell lines (HeLa, HEK293) displayed low transporter expression, and the expression patterns were barely affected by transfection. The leukemia cell lines (K562, HL-60) and Saos-2 also had low endogenous transporter expression, whereas the organotypic cell lines (HepG2 and Caco-2) showed higher expression of some transporters. Comparison of gene and protein expression profiles gave poor correlations, but better agreement was obtained for antibodies with a good validation score, indicating that antibody quality was a significant variable. It is noteworthy that the monocarboxylic acid-transporting protein MCT1 was significantly expressed in all and was functional in most of the cell lines, indicating that MCT1 may be a confounding factor when the transport of small anionic drugs is investigated. PMID:19741037

Ahlin, Gustav; Hilgendorf, Constanze; Karlsson, Johan; Szigyarto, Cristina Al-Khalili; Uhlén, Mathias; Artursson, Per

2009-12-01

338

Proteomic identification of abnormally expressed proteins in early-stage placenta derived from cloned cat embryos.  

PubMed

It is unknown whether gene expression in cloned placenta during pre- and postimplantation is associated with early pregnancy failure in the cat. In this study, protein expression patterns were examined in early-stage (21-day-old) domestic cat placentas of fetuses derived from AI (CP; N = 4) and cloned embryo transfer (CEP; N = 2). Differentially expressed proteins were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry (MS). A total of 21 proteins were aberrantly expressed (P < 0.05) by >1.5-fold in CEP compared with CP. Compared with CP, 12 proteins were upregulated in CEP (peptidyl-prolyl cis-trans isomerase A, annexin A2, protein DJ-1, adenylate kinase isoenzyme 1, protein disulfide-isomerase A3, actin cytoplasmic 1, serum albumin, protein disulfide-isomerase A6, and triosephosphate isomerase), and nine proteins were downregulated (triosephosphate isomerase; heterogeneous nuclear ribonucleoprotein H; tropomyosin alpha-4; triosephosphate isomerase 1; 60 kDa heat shock protein, mitochondrial; serum albumin; calumenin; keratin type 1; and prohibitin). The identities of the differentially expressed proteins were validated by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-TOF/TOF MS/MS. The abnormally expressed proteins identified in this study might be associated with impaired development and dysfunction of CEP during early pregnancy. Abnormal protein expression might also induce fetal loss and contribute to failure to maintain pregnancy to term. PMID:23146403

Bang, Jae-Il; Lee, Hyo-Sang; Deb, Gautam Kumar; Ha, A-Na; Kwon, Young-Sang; Cho, Seong-Keun; Kim, Byeong-Woo; Cho, Kyu-Woan; Kong, Il-Keun

2013-01-15

339

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

340

Protective Efficacy of Baculovirus Dual Expression System Vaccine Expressing Plasmodium falciparum Circumsporozoite Protein  

PubMed Central

We have previously developed a new malaria vaccine delivery system based on the baculovirus dual expression system (BDES). In this system, expression of malaria antigens is driven by a dual promoter consisting of the baculovirus-derived polyhedrin and mammal-derived cytomegalovirus promoters. To test this system for its potential as a vaccine against human malaria parasites, we investigated immune responses against the newly developed BDES-based Plasmodium falciparum circumsporozoite protein vaccines (BDES-PfCSP) in mice and Rhesus monkeys. Immunization of mice with BDES-PfCSP induced Th1/Th2-mixed type immune responses with high PfCSP-specific antibody (Ab) titers, and provided significant protection against challenge from the bites of mosquitoes infected with a transgenic P. berghei line expressing PfCSP. Next, we evaluated the immunogenicity of the BDES-PfCSP vaccine in a rhesus monkey model. Immunization of BDES-PfCSP elicited high levels of anti-PfCSP Ab responses in individual monkeys. Moreover, the sera from the immunized monkeys remarkably blocked sporozoite invasion of HepG2 cells. Taken together with two animal models, our results indicate that this novel vaccine platform (BDES) has potential clinical application as a vaccine against malaria.

Iyori, Mitsuhiro; Nakaya, Hiroki; Inagaki, Katsuya; Pichyangkul, Sathit; Yamamoto, Daisuke S.; Kawasaki, Masanori; Kwak, Kyungtak; Mizukoshi, Masami; Goto, Yoshihiro; Matsuoka, Hiroyuki; Matsumoto, Makoto; Yoshida, Shigeto

2013-01-01

341

Efficient expression, processing and secretion of a biologically active mammalian protein by Vibrio cholerae.  

PubMed

The use of Vibrio cholerae as a secretory expression system for the expression of a mammalian protein, namely human growth hormone, under the control of the heat labile enterotoxin chain B signal sequence is reported. The protein is efficiently expressed and processed. The mature protein is exported to the periplasm after which it is secreted to the extracellular milieu. The expressed and secreted hGH actively binds to its receptor as established by its receptor binding activity. The biological activity of the protein is demonstrated in vitro in a Nb2 proliferation assay. PMID:8674542

Ghorpade, A; Garg, L C

1996-06-01

342

Cellular lipid binding proteins: expression, function, and nutritional regulation  

Microsoft Academic Search

The membrane transport and cytosolic solubiization of hydrophobic ligands, including sterols, fatty acids, reti- noids, and certain hydrophobic carcinogens, are facili- tated by a group of similar low molecular weight pro- teins: plasma membrane transport protein, fatty acid binding proteins, sterol carrier protein, and retinoid binding proteins. The cellular content of these proteins, which establishes the capacity of a cell

STEVEN D. CLARKE; MICHAEL K. ARMSTRONG

1989-01-01

343

G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions.  

PubMed

G protein-coupled receptor 84 (GPR84) is a recently discovered member of the seven transmembrane receptor superfamily whose function and regulation are unknown. Here, we report that in mice suffering from endotoxemia, microglia express GPR84 in a strong and sustained manner. This property is shared by subpopulations of peripheral macrophages and, to a much lesser extent, monocytes. The induction of GPR84 expression by endotoxin is mediated, at least in part, by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1), because mice lacking either one or both of these molecules have fewer GPR84-expressing cells in their cerebral cortex than wild-type mice during the early phase of endotoxemia. Moreover, when injected intracerebrally or added to microglial cultures, recombinant TNF stimulates GPR84 expression through a dexamethasone-insensitive mechanism. Finally, we show that microglia produce GPR84 not only during endotoxemia, but also during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In conclusion, this study reports the identification of a new sensitive marker of microglial activation, which may play an important regulatory role in neuroimmunological processes, acting downstream to the effects of proinflammatory mediators. PMID:17390309

Bouchard, Caroline; Pagé, Julie; Bédard, Andréanne; Tremblay, Pierrot; Vallières, Luc

2007-06-01

344

GA-binding protein is involved in altered expression of ribosomal protein L32 gene.  

PubMed

Differentiation of BC3H1 myoblasts to myocytes is accompanied by a 67% drop in the rate of rpL32 gene transcription. Addition of high concentrations of serum to resting myocyte populations stimulates cell growth and subsequent dedifferentiation to proliferating myoblasts with a return to the normal rate of rpL32 gene transcription. During these growth rate changes the binding activities of previously identified factors (beta, gamma, delta) which interact with the rpL32 gene promoter were examined by mobility shift assays. Binding of the beta factor (an Ets related protein) to an oligonucleotide containing the beta element was reduced significantly in myocyte nuclear extracts, but subsequent dedifferentiation increased binding within 30 min in either the presence or absence of the cycloheximide. Binding of the gamma and delta factors to their respective elements changed only slightly during these processes. Dephosphorylation of either myoblast or myocyte extracts resulted in increased binding of the beta factor suggesting that binding activity of the beta factor is modulated by phosphorylation during the changes in BC3H1 myoblasts growth rate. In addition, mobility shift assays with recombinant GABP alpha and beta proteins and their specific antibodies revealed that GABP proteins bind to the rpL32 gene promoter in a sequence dependent manner, and that similar proteins are present in BC3H1 myoblast/myocyte extracts. These results support the premise that the GABP heterodimer is the rpL32 beta factor. Furthermore, during BC3H1 myoblast differentiation and dedifferentiation neither the levels of the GABP alpha and beta proteins nor their respective mRNAs change. These results suggest that GABP is a constitutively expressed protein and is involved in regulating rpL32 gene by post-transcriptional modifications. PMID:9138087

Curci?, D; Glibeti?, M; Larson, D E; Sells, B H

1997-06-01

345

Identification of proteins with altered expression in colorectal cancer by means of 2D-proteomics  

Microsoft Academic Search

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumors as compared to normal\\u000a tissues. Although proteomics is currently widely used, identification of proteins differentially expressed in particular types\\u000a of cancer remains a challenging task. The goal of our study was to detect novel protein markers of colorectal cancer using\\u000a comparative proteomics of protein extracts

G. S. Krasnov; N. Yu. Oparina; S. L. Hankin; T. D. Mashkova; A. N. Ershov; O. G. Zatsepina; V. L. Karpov; S. F. Beresten

2009-01-01

346

Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1?  

PubMed Central

Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.

Yoshino, Tomoko; Shimojo, Akiko; Maeda, Yoshiaki; Matsunaga, Tadashi

2010-01-01

347

Inducible expression of transmembrane proteins on bacterial magnetic particles in Magnetospirillum magneticum AMB-1.  

PubMed

Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs. PMID:20038711

Yoshino, Tomoko; Shimojo, Akiko; Maeda, Yoshiaki; Matsunaga, Tadashi

2010-02-01

348

Proteome analysis of abundantly expressed proteins from unfed larvae of the cattle tick, Boophilus microplus.  

PubMed

Protein expression in unfed larvae of the cattle tick, Boophilus microplus, was characterized using gel electrophoresis and mass spectrometry in an effort to assemble a database of proteins produced at this stage of development. Soluble and insoluble proteins were extracted and resolved by two-dimensional (2D) gel electrophoresis. Twenty abundantly expressed larval proteins were selected for peptide mass mapping and for peptide sequencing by matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) and quadrupole time-of-flight (Q-ToF) tandem mass spectrometry (MS), respectively. Only one protein, tropomyosin, was unequivocally identified from its peptide mass map. Ten proteins were assigned putative identities based on BLAST searching of heterologous databases with peptide sequences. These included a cytoskeletal protein (troponin I), multiple cuticular proteins, a glycine-rich salivary gland-associated protein and proteins with a presumed housekeeping role (arginine kinase, a high-mobility group protein and a small heat shock protein). Eight additional proteins were identified by searching translated open reading frames of a B. microplus EST database (unpublished): putative fatty-acid binding protein, thioredoxin, glycine-rich salivary gland protein and additional cuticular proteins. One remaining protein was not identifiable, suggesting it may be a novel molecule. The ongoing assembly of this database contributes to our understanding of proteins expressed by the tick and provides a resource that can be mined for molecules that play a role in tick-host interactions. PMID:15681224

Untalan, P M; Guerrero, F D; Haines, L R; Pearson, T W

2005-02-01

349

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

PubMed Central

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.

Kim, Youngmin; Ganesan, Prabhakar; Ihee, Hyotcherl

2013-01-01

350

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

351

Identities of Sequestered Proteins in Aggregates from Cells with Induced Polyglutamine Expression  

PubMed Central

Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle–regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

Suhr, Steven T.; Senut, Marie-Claude; Whitelegge, Julian P.; Faull, Kym F.; Cuizon, Denise B.; Gage, Fred H.

2001-01-01

352

Expression of selected proteins in breast cancer brain metastases.  

PubMed

The aim of the study was to assess the immunohistochemical (IHC) profiles of SRC3, Pax2, ER, PgR, Her2, EGFR, CK5/6, and Ki67 proteins in breast-cancer brain metastasis. The study utilized tumor samples from 30 metastatic patients and calculated correlations between all IHC variables. In fourteen cases, primary breast cancers paired with secondary deposits were analyzed. We evaluated the association between IHC status in the primary and secondary deposits, grade, and histotype of the tumors. The examination of the metastatic deposits in all 30 patients resulted in positive detection in the following cases: SRC3 in 20 cases (66.6%), Pax2 in 22 (73.3%), ER in 22 (73.3%), PgR in 25 (83.3%), Her2 in 10 (33.3%), EGFR in 12 (40%), CK5/6 in 7 (23.3%), and Ki67 in 23 (76.6%). Grade 2 was found in 13.3% of all patients, and grade 3 in 86.7%. SRC3 and Pax2 were positive in both G2 and G3. Invasive lobular carcinoma and invasive ductal carcinoma were diagnosed in 23.3% and 76.7% of cases, respectively. There were no differences between the IHC expression of the studied proteins in either grading or histotype of the tumors. In the IHC profiles, which included SRC3, Pax2, ER, PgR, Her2, CK5/6, Ki67, and EGFR, we found no statistically significant differences between the primary cancer and the brain metastasis. In our study of metastatic breast carcinoma deposits, there was no correlation between SRC3, Pax2 status and histotype, and tumor grade. The IHC status of the paired primary and metastatic deposits did not differ in a statistically significant manner. PMID:24203627

Gojis, Ondrej; Kubecova, Martina; Rosina, Jozef; Vranova, Jana; Celko, Martin; Frajerova, Denisa; Zmrhal, Jan; Zahumensky, Jozef; Bacova, Tereza; Baca, Vaclav; Mandys, Vaclav; Kucera, Eduard

2013-01-01

353

Role of untranslated regions in regulation of gene expression, replication, and pathogenicity of Newcastle disease virus expressing green fluorescent protein.  

PubMed

To gain insight into the role of untranslated regions (UTRs) in regulation of foreign gene expression, replication, and pathogenicity of Newcastle disease virus (NDV), a green fluorescent protein (GFP) gene flanked by 5' and 3' UTRs of each NDV gene was individually expressed by recombinant NDVs. UTRs of each gene modulated GFP expression positively or negatively. In particular, UTRs of the M and F genes enhanced levels of GFP expression at the junction of the P and M genes without altering replication of NDV, suggesting that UTRs could be used for enhanced expression of a foreign gene by NDV. PMID:20015997

Kim, Shin-Hee; Samal, Siba K

2010-03-01

354

Glucose enhances collectrin protein expression in insulin-producing MIN6 {beta} cells  

SciTech Connect

Collectrin is a novel target gene of hepatocyte nuclear factor-1{alpha} in pancreatic {beta}-cells and controls insulin exocytosis. Although glucose is known to stimulate the expression of genes of the insulin secretory pathway, there is no information on how glucose regulates collectrin expression. We investigated the effects of glucose on the expression of collectrin in MIN6 {beta}-cell line. Glucose, in a dose-dependent manner, increased collectrin protein levels without changing collectrin mRNA levels and protein stability, indicating that glucose stimulation of collectrin protein expression is primarily mediated at a translational level. Although mannose and pyruvate also increased collectrin protein expression level, neither 2-deoxyglucose, mitochondrial fuels leucine and glutamate, sulphonylurea nor Ca{sup 2+} channel blockers, mimicked the effects of glucose. These data indicate the involvement of mitochondrial TCA cycle intermediates, distal to pyruvate, in the regulation of collectrin protein expression in {beta}-cells.

Saisho, Kenji; Fukuhara, Atsunori [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan)] [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan); Yasuda, Tomoko [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan)] [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan); Sato, Yoshifumi; Fukui, Kenji; Iwahashi, Hiromi; Imagawa, Akihisa [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan)] [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan); Hatta, Mitsutoki [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan)] [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan); Shimomura, Iichiro [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan)] [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan); Yamagata, Kazuya, E-mail: k-yamaga@kumamoto-u.ac.jp [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan)] [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan)

2009-11-06

355

Proteomic analysis of protein expression profiles during hyperthermia-induced apoptosis in Tca8113 cells  

PubMed Central

The aim of the present study was to explore protein expression profiles during cancer cell apoptosis induced by hyperthermia. A hyperthermia-induced apoptosis model was established using a Tca8113 cell line derived from a human tongue squamous cell carcinoma, which underwent fluorescent differential display two-dimensional (2D) gel electrophoresis at 2, 6, 8, 12 and 24 h following the induction of hyperthermia. Proteins were identified by mass spectrometry analysis. Expression changes in the proteins were detected by western blot analysis. A total of 107 proteins were detected that exhibited different expression levels in the hyperthermia-treated cells compared with the controls, and 57 of these proteins were identified. Expression changes in the representative proteins were further verified by western blot analysis. These 57 proteins were identified according to the following functional groups: energy metabolism-related enzymes, cytoskeleton-related proteins, chaperones, transcription factors, protein synthesis-related proteins and cell division- and proliferation-related proteins. These groups included 44 upregulated and 13 downregulated proteins. Among the 44 upregulated proteins, 27 were upregulated continuously, eight were upregulated at an early time-point and nine were upregulated at a middle to late time-point. Among the 13 downregulated proteins, five were downregulated continuously, six were downregulated at an early time-point and two were downregulated at a middle to late time-point. These results indicate that hyperthermia-induced Tca8113 cell apoptosis is controlled by multiple factors, which include time and regulatory proteins.

JIANG, WEN; BIAN, LI; WANG, NING; HE, YONGWEN

2013-01-01

356

The Predicted Candidates of Arabidopsis Plastid Inner Envelope Membrane Proteins and Their Expression Profiles1[w  

PubMed Central

Plastid envelope proteins from the Arabidopsis nuclear genome were predicted using computational methods. Selection criteria were: first, to find proteins with NH2-terminal plastid-targeting peptides from all annotated open reading frames from Arabidopsis; second, to search for proteins with membrane-spanning domains among the predicted plastidial-targeted proteins; and third, to subtract known thylakoid membrane proteins. Five hundred forty-one proteins were selected as potential candidates of the Arabidopsis plastid inner envelope membrane proteins (AtPEM candidates). Only 34% (183) of the AtPEM candidates could be assigned to putative functions based on sequence similarity to proteins of known function (compared with the 69% function assignment of the total predicted proteins in the genome). Of the 183 candidates with assigned functions, 40% were classified in the category of “transport facilitation,” indicating that this collection is highly enriched in membrane transporters. Information on the predicted proteins, tissue expression data from expressed sequence tags and microarrays, and publicly available T-DNA insertion lines were collected. The data set complements proteomic-based efforts in the increased detection of integral membrane proteins, low-abundance proteins, or those not expressed in tissues selected for proteomic analysis. Digital northern analysis of expressed sequence tags suggested that the transcript levels of most AtPEM candidates were relatively constant among different tissues in contrast to stroma and the thylakoid proteins. However, both digital northern and microarray analyses identified a number of AtPEM candidates with tissue-specific expression patterns.

Koo, Abraham J.K.; Ohlrogge, John B.

2002-01-01

357

Polyplex Exposure Inhibits Cell Cycle, Increases Inflammatory Response, and Can Cause Protein Expression Without Cell Division  

PubMed Central

We sought to evaluate the relationship between cell division and protein expression when using commercial poly(ethylenimine) (PEI)-based polyplexes. The membrane dye PKH26 was used to assess cell division, and cyan fluorescent protein (CFP) was used to monitor protein expression. When analyzed at the whole population level, a greater number of cells divided than expressed protein, regardless of the level of protein expression observed, giving apparent consistency with the hypothesis that protein expression requires cells to pass through mitosis in order for the transgene to overcome the nuclear membrane. However, when the polyplex-exposed population was evaluated for the amount of division in the protein-expressing subpopulation, it was observed that substantial amounts of expression had occurred in the absence of division. Indeed, in HeLa S3 cells, this represented the majority of expressing cells. Of interest, the doubling time for both cell lines was slowed by ~2-fold upon exposure to polyplexes. This change was not altered by the origin of the plasmid DNA (pDNA) transgene promoter (cytomegalovirus (CMV) or elongation factor-1 alpha (EF1?)). Gene expression arrays in polyplex-exposed HeLa S3 cells showed upregulation of cell cycle arrest genes and downregulation of genes related to mitosis. Chemokine, interleukin, and toll-like receptor genes were also upregulated, suggesting activation of proinflammatory pathways. In summary, we find evidence that a cell division-independent expression pathway exists, and that polyplex exposure slows cell division and increases inflammatory response.

Matz, Rebecca L.; Erickson, Blake; Vaidyanathan, Sriram; Kukowska-Latallo, Jolanta F.; Baker, James R.; Orr, Bradford G.; Banaszak Holl, Mark M.

2013-01-01

358

Loss of HLA class I and mismatch repair protein expression in sporadic endometrioid endometrial carcinomas.  

PubMed

Tumor cells can escape from cytotoxic T-cell responses by downregulation of human leukocyte antigen (HLA) class I molecules expressed at the cell surface which has been associated with a deficient mismatch repair (MMR) system in colorectal carcinomas. Our study investigated the association between expression of MMR proteins and HLA class I in sporadic endometrioid endometrial carcinomas (EC). In a consecutively selected cohort of 486 EC patients, MMR proteins (MLH1, MSH2 and MSH6) and HLA class I (HLA-A, -B, -C or ?(2) m) were investigated by immunohistochemistry. Expression levels of MMR proteins and HLA class I were compared between low-grade and high-grade ECs. HLA class I expression was compared between tumors with loss (negative immunostaining of ?1 MMR protein) and expression of MMR proteins. Associations between previously determined numbers of intratumoral CD8(+) T-lymphocytes and expression of MMR proteins and HLA class I and the influence on survival was determined. ECs with loss of MMR protein expression (33.5%) more frequently have loss of HLA-B/C (37.3%), compared to ECs with MMR protein expression (25.5%, p = 0.007). Patients with loss of MMR proteins have a worse disease-specific survival compared to patients with expression (p = 0.039). CD8(+) T-lymphocytes have a positive influence on disease-free and disease-specific survival in the total EC cohort but not in patients with loss of MMR protein expression. In conclusion, our results indicate that loss of MMR protein expression is related to selective downregulation of HLA class I which contributes to immune escape in EC with an abnormal MMR system. PMID:22287095

de Jong, Renske A; Boerma, Annemarie; Boezen, H Marike; Mourits, Marian J E; Hollema, Harry; Nijman, Hans W

2012-10-15

359

Differential expression of proteins and phosphoproteins during larval metamorphosis of the polychaete Capitella sp. I  

PubMed Central

Background The spontaneous metamorphosis of the polychaete Capitella sp. I larvae into juveniles requires minor morphological changes, including segment formation, body elongation, and loss of cilia. In this study, we investigated changes in the expression patterns of both proteins and phosphoproteins during the transition from larvae to juveniles in this species. We used two-dimensional gel electrophoresis (2-DE) followed by multiplex fluorescent staining and MALDI-TOF mass spectrometry analysis to identify the differentially expressed proteins as well as the protein and phosphoprotein profiles of both competent larvae and juveniles. Results Twenty-three differentially expressed proteins were identified in the two developmental stages. Expression patterns of two of those proteins were examined at the protein level by Western blot analysis while seven were further studied at the mRNA level by real-time PCR. Results showed that proteins related to cell division, cell migration, energy storage and oxidative stress were plentifully expressed in the competent larvae; in contrast, proteins involved in oxidative metabolism and transcriptional regulation were abundantly expressed in the juveniles. Conclusion It is likely that these differentially expressed proteins are involved in regulating the larval metamorphosis process and can be used as protein markers for studying molecular mechanisms associated with larval metamorphosis in polychaetes.

2011-01-01