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1

Activation of ERM-family proteins via RhoA-ROCK signaling increases intestinal P-gp expression and leads to attenuation of oral morphine analgesia.  

PubMed

Previously, we reported that repeated oral treatment with etoposide (ETP) causes attenuation of oral morphine analgesia through upregulation of ileal P-glycoprotein (P-gp) mediated by Ras homolog gene family, member A (RhoA) activation. However, the detailed mechanism of the increase in ileal P-gp via RhoA activation remains unknown. Recently, it has been reported that ezrin-radixin-moesin (ERM) proteins, linking several plasma-membrane proteins to the actin cytoskeleton, are involved in the membrane localization and functional activity of P-gp. Moreover, the cross-linking activities of ERM are known to be regulated by RhoA and Rho-associated coiled-coil containing kinase (ROCK). Here, we examined the involvement of ERM in the changes in expression of P-gp via RhoA and ROCK in ileal membrane induced by ETP. Repeated oral treatment with ETP significantly increased the ileal membrane localization of ERM and phosphorylated ERM (p-ERM) in association with upregulation of P-gp and activation of RhoA and ROCK. Interestingly, coadministration of rosuvastatin (inhibitor of RhoA activation) and fasudil (ROCK inhibitor) prevented increments in the activation and phosphorylation of ERM, respectively. In conclusion, upregulation of ileal membrane localization of ERM and p-ERM via activation of RhoA/ROCK induced by ETP treatment may be involved in the regulation of ileal membrane localization of P-gp. PMID:23303573

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

2013-01-09

2

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

PubMed

Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD), a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) expression, and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp) for comparison). Study design. Following the establishment of the basal expression of these transporters in the membrane fraction of all three cell lines, P-gp and BCRP protein and mRNA levels were determined following chronic (24-72 h) exposure to CBD, by Western Blot and qPCR. CBD impact on P-gp efflux function was examined by uptake of specific P-gp fluorescent substrates (calcein-AM, DiOC2(3) and rhodamine123(rh123)). Cyclosporine A (CsA) served as a positive control. Results. Chronic exposure to CBD resulted in significant changes in the protein and mRNA levels of both transporters. While P-gp was down-regulated, BCRP levels were up-regulated in the choriocarcinoma cell lines. CBD had a remarkably different influence on P-gp and BCRP expression in MCF7/P-gp cells, demonstrating that these are cell type specific effects. P-gp dependent efflux (of calcein, DiOC2(3) and rh123) was inhibited upon short-term exposure to CBD. Conclusions. Our study shows that CBD might alter P-gp and BCRP expression in the human placenta, and inhibit P-gp efflux function. We conclude that marijuana use during pregnancy may reduce placental protective functions and change its morphological and physiological characteristics. PMID:24058883

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

2013-09-12

3

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

4

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

PubMed

Abstract Context: Previous studies have demonstrated that both 3'-azido-3'-deoxythymidine (AZT) and emodin, a traditional chemotherapy agent, can inhibit the growth of many types of cancer cells. Objective: This study aimed to evaluate the effect of AZT and emodin on adriamycin-resistant human chronic myelogenous leukemia (K562/ADM) cells, determine the expression of multidrug resistance 1 (MDR1) mRNA/p-glycoprotein (p-gp) protein, a protein known to induce resistance to anticancer agents, and to elucidate the underlying molecular mechanisms. Materials and methods: K562/ADM cells were treated with AZT (10-160??M) or emodin (5-80??M) for 24,?48 and 72?h and cell viability was measured using the MTT assay. The effect of AZT (16.5, 33 and 66??M) and emodin (6.1,?17.6 and 33.2??M) on K562/ADM cell cycle distribution was determined by flow cytometry, and MDR1 mRNA/p-gp protein expression was determined by real time RT-PCR and western blotting. Results: The growth suppression of emodin was dramatically enhanced by AZT in K562/ADM cells. The IC50 of AZT and emodin was lower than that of emodin alone. All examined combinations of AZT and emodin yielded a synergetic effect (CI?expression of MDR1 mRNA/p-gp protein was markedly decreased. Discussion and conclusion: These results show a synergistic growth-inhibitory effect of AZT and emodin in K562/ADM cells, which is achieved through S phase arrest. MDR1 might ultimately be responsible for these phenomena. PMID:24004004

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

2013-09-05

5

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

PubMed

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

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

2008-03-18

6

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

7

Study of expression and functional activity of P-GP membrane glycoprotein in children with acute leukemia.  

PubMed

We studied expression and functional activity of tumor cell P-gp in children with various leukemia variants and analyzed its prognostic role in acute lymphoblastic leukemia. Functional activity of P-gp increased in acute myeloid leukemia, relapses of acute lymphoblastic leukemia (in comparison with primary disease), and in a group of patients in whom no remission was attained. The survival of patients with acute lymphoblastic leukemia was lower in cases with increased expression and function of P-gp. PMID:17364061

Shman, T V; Savitskii, V P; Potapnev, M P; Aleinikova, O V

2006-06-01

8

Effects of capsaicin on P-gp function and expression in Caco-2 cells  

Microsoft Academic Search

Capsaicin is the pungent component of hot chilli, a popular spice in many populations. The aim of the present study was to evaluate the chronicity and reversibility of the modulating effect of capsaicin on both the P-gp expression and activity in the Caco-2 cell monolayers. Capsaicin at concentrations ranging from 10 to 100?M, which were found to be non-cytotoxic towards

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

2006-01-01

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

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. PMID:23976943

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

2013-08-16

10

Arsenic trioxide induces apoptosis equally in T lymphoblastoid leukemia MOLT4 cells and P-gp-expressing daunorubicin-resistant MOLT4 cells  

Microsoft Academic Search

Purpose. To investigate the effects of arsenic trioxide (As2O3) on human T-lymphoblastoid leukemia MOLT-4 cells and P-gp-expressing daunorubicin-resistant MOLT-4 (MOLT-4\\/DNR) cells. Methods. Cell growth was measured by an MTT assay. Cell viability was determined by a dye exclusion test. The level of P-gp expression was estimated using phycoerythrin-conjugated anti-P-gp monoclonal antibody 17F9. The function of P-gp was evaluated in terms

Xiao-Mei Hu; Toshihiko Hirano; Kitaro Oka

2003-01-01

11

Protein kinase C-mediated down-regulation of MDR3 mRNA expression in Chang liver cells 1 1 Abbreviations: MDR, multidrug resistance; P-gp, P-glycoprotein; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; 4?-PDD, 4?-phorbol 12,13-didecanoate; and PKC, protein kinase C  

Microsoft Academic Search

MDR3 is a phospholipid translocator homologous to MDR1 P-glycoprotein. MDR3 localizes to the canalicular membrane and contributes to the secretion of bile. To elucidate the role of protein kinase C in the regulation of MDR3 gene expression, we investigated the effect of phorbol 12-myristate 13-acetate (PMA) on the level of MDR3 mRNA in human Chang liver cells by a reverse

Ritsuko Ikeda; Yuhta Shiono; Hisao Hayashi

2001-01-01

12

The two enantiomers of tetrahydropalmatine are inhibitors of P-gp, but not inhibitors of MRP1 or BCRP.  

PubMed

Tetrahydropalmatine (THP), with one chiral centre, is one of the major constituents of Rhizoma corydalis. THP is considered to possess analgesic, sedative, hypnotic actions and cardiac protection. The aim of this study was to elucidate the stereoselective interaction between THP and ABC transporters. The present study investigated three most important ABC transporters, including P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). The intracellular accumulation and bidirectional transport suggested THP enantiomers were inhibitors of P-gp, but not of MRP1 or BCRP. The IC(50) values of (-)-THP and (+)-THP on rhodamine 123 (P-gp substrate) efflux were 48.6 and 20.0 µM, respectively, which showed obvious stereoselective difference. In the bidirectional transport, THP enantiomers showed high passive permeability and the contribution of P-gp could not be testified. The western blot and real-time RT-PCR assays showed that THP enantiomers reduced the protein expression of P-gp, but did not affect its mRNA expression. In in vitro cytotoxicity test, THP enantiomers showed the potential of increasing the cytotoxicity of doxorubicin in P-gp-mediated multidrug resistant tumour cells. The present study showed the stereoselective interaction between THP enantiomers and P-gp, which should be considered in clinical practice. PMID:22900779

Sun, Siyuan; Chen, Zhongjian; Li, Liping; Sun, Dongli; Tian, Ye; Pan, Hao; Bi, Huichang; Huang, Min; Zeng, Su; Jiang, Huidi

2012-08-19

13

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

14

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

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

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

2012-05-23

15

The co-expression of p53 protein and P-glycoprotein is correlated to a poor prognosis in osteosarcoma  

Microsoft Academic Search

Multidrug resistance (MDR), mediated by P-glycoprotein (P-gp), is an in vitro phenomenon observed within tumour cells, suggesting cross-resistance to unrelated drugs, and expression of P-gp may therefore affect the prognosis and incidence of recurrence after treatment. The mutant p53 protein causes reduced tumour suppression. Co-expression of p53 and P-gp is related to short survival, increased tumour activity and drug resistance.

Y. B. Park; H. S. Kim; J. H. Oh; S. H. Lee

2001-01-01

16

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

17

Involvement of P-gp in the process of apoptosis in peripheral blood mononuclear cells  

Microsoft Academic Search

Multidrug resistance mediated by the drug-efflux protein P (P-gp) is one of mechanisms that cells use to escape death induced by drugs and other agents. The aim of the study was to evaluate the effect of P-gp inhibition on apoptosis of PHA-activated peripheral blood mononuclear cells (MNC) as well as apoptosis induced by methotrexate (MTX), dexamethasone (DEX), methylprednisolone (MP) and

A. Pawlik; M. Baskiewicz-Masiuk; B. Machalinski; B. Gawronska-Szklarz

2005-01-01

18

FBXO15 regulates P-glycoprotein/ABCB1 expression through the ubiquitin--proteasome pathway in cancer cells.  

PubMed

Expression of P-glycoprotein (P-gp)/ABCB1 on cancer cell surfaces is a critical determinant of anticancer drug resistance. Regulators of P-gp expression and function are key molecules controlling drug resistance. Here we report the mechanism underlying the ubiquitin-proteasome pathway-mediated degradation of P-gp. The proteasome inhibitor MG132 increased the P-gp level, enhanced its ubiquitination, and delayed the disappearance of the ubiquitinated P-gp. To search for regulators of P-gp ubiquitination, MALDI-time of flight mass spectrometry analyses were carried out, and 22 candidates were identified as P-gp binding partners. Among them, FBXO15/Fbx15 is known as an F-box protein in the ubiquitin E3 ligase complex, Skp1-Cullin1-FBXO15 (SCF(Fbx15) ); therefore, we further studied the involvement of FBXO15 on P-gp degradation. Coprecipitation assays revealed that FBXO15 bound to P-gp. We screened ubiquitin-conjugating enzyme E2s that bind to FBXO15 and P-gp; Ube2r1/Cdc34/Ubc3 was found to be a binding partner. Exogenous FBXO15 expression enhanced P-gp ubiquitination, but FBXO15 knockdown suppressed it. FBXO15 knockdown increased P-gp expression without affecting its mRNA level. Ube2r1 knockdown decreased P-gp ubiquitination, and simultaneous knockdown of Ube2r1 with FBXO15 further suppressed the ubiquitination. Ube2r1 knockdown increased P-gp expression, suggesting that Ube2r1 is a partner of FBXO15 in P-gp ubiquitination. FBXO15 knockdown enhanced vincristine resistance and lowered intracellular levels of rhodamine 123. These data suggest that FBXO15 and Ube2r1 regulate P-gp expression through the ubiquitin-proteasome pathway. PMID:23465077

Katayama, Kazuhiro; Noguchi, Kohji; Sugimoto, Yoshikazu

2013-04-15

19

Localization of P-gp (Abcb1) and Mrp2 (Abcc2) in Freshly Isolated Rat Hepatocytes  

PubMed Central

Freshly isolated hepatocytes are widely accepted as the “gold standard” for providing reliable data on drug uptake across the sinusoidal (basolateral) membrane. However, the suitability of freshly isolated hepatocytes in suspension to assess efflux by canalicular (apical) proteins or predict biliary excretion in the intact organ is unclear. After collagenase digestion, hepatocytes rapidly lose polarity, but localization of canalicular transport proteins in the first few hours after isolation has not been well characterized. In this study, immunostaining and confocal microscopy have provided, for the first time, a detailed examination of canalicular transport protein localization in freshly isolated rat hepatocytes fixed within 1 h of isolation and in cells cultured for 1 h. Organic anion transporting polypeptide 1a1 (Oatp1a1) was expressed in all hepatocytes and distributed evenly across the basolateral membrane; there was no evidence for colocalization of Oatp1a1 with P-glycoprotein (P-gp) or multidrug resistance-associated protein 2 (Mrp2). In contrast, P-gp and Mrp2 expression was lower than Oatp1a1 and confined to junctions between adjacent cells, intracellular compartments, and “legacy” network structures at or near the cell surface. P-gp and Mrp2 staining was more predominant in regions adjacent to former canalicular spaces, identified by zonula occludens-1 staining. Functional analysis of rat hepatocytes cultured for 1 h demonstrated that the fluorescent anion and Mrp2 substrate, 5-(and-6)-carboxy-2?,7?-dichlorofluorescein (CDF), accumulated in cellular compartments; compartmental accumulation of CDF was sensitive to (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571, Mrp inhibitor) and was not observed in hepatocytes isolated from Mrp2-deficient rats. Drug efflux from freshly isolated hepatocytes as an estimate of apical efflux/biliary excretion would give an inaccurate assessment of true apical elimination and, as such, should not be used to make in vivo extrapolations.

Bow, Daniel A. J.; Perry, Jennifer L.; Miller, David S.; Pritchard, John B.; Brouwer, Kim L. R.

2009-01-01

20

Lobeline, a piperidine alkaloid from Lobelia can reverse P-gp dependent multidrug resistance in tumor cells.  

PubMed

Multidrug resistance (MDR) can limit efficacy of chemotherapy. The best studied mechanism involves P-gp (P-glycoprotein) mediated drug efflux. This study focuses on MDR reversal agents from medicinal plants, which can interfere with P-gp. Rhodamine 123 accumulation assay and flow cytometry analysis were employed to screen for P-gp dependant efflux inhibitors. Lobeline, a piperidine alkaloid from Lobelia inflata and several other Lobelia species, inhibited P-gp activity. MDR reversal potential of lobeline could be demonstrated in cells treated with doxorubicin in that lobeline can sensitize resistant tumor cells at non-toxic concentrations. However, lobeline cannot block BCRP (Breast Cancer Resistance Protein) dependent mitoxantrone efflux. Lobeline could be a good candidate for the development of new MDR reversal agents. PMID:18222670

Ma, Yonggang; Wink, Michael

2008-01-28

21

The distribution of drug-efflux pumps, P-gp, BCRP, MRP1 and MRP2, in the normal blood–testis barrier and in primary testicular tumours  

Microsoft Academic Search

The drug-efflux pumps P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) are present in the blood–testis barrier (BTB) and may hamper the delivery of cytotoxic drugs to the testis. The precise localisation of P-gp and MRP1 in testicular tissue and the presence of the efflux pumps MRP2 and breast cancer resistance protein (BCRP) in the BTB are unknown. We therefore

J. Bart; H. Hollema; H. J. M. Groen; E. G. E. de Vries; N. H. Hendrikse; D. T. Sleijfer; T. D. Wegman; W. Vaalburg

2004-01-01

22

Abamectin resistance in Drosophila is related to increased expression of P-glycoprotein via the dEGFR and dAkt pathways.  

PubMed

Many insects have evolved resistance to abamectin but the mechanisms involved in this resistance have not been well characterized. P-glycoprotein (P-gp), an ATP-dependent drug-efflux pump transmembrane protein, may be involved in abamectin resistance. We investigated the role of P-gp in abamectin (ABM) resistance in Drosophila using an ABM-resistant strain developed in the laboratory. A toxicity assay, Western blotting analysis and a vanadate-sensitive ATPase activity assay all demonstrated the existence of a direct relationship between P-gp expression and ABM resistance in these flies. Our observations indicate that P-gp levels in flies' heads were higher than in their thorax and abdomen, and that both P-gp levels and LC(50) values were higher in resistant than in susceptible and P-gp-deficient strains. In addition, P-gp levels in the blood-brain barrier (BBB) of resistant flies were higher than in susceptible and P-gp-deficient flies, which is further evidence that a high level of P-gp in the BBB is related to ABM resistance. Furthermore, we found greater expression of Drosophila EGFR (dEGFR) in the resistant strain than in the susceptible strain, and that the level of Drosophila Akt (dAkt) was much higher in resistant than in susceptible flies, whereas that in P-gp-deficient flies was very low. Compared to susceptible flies, P-gp levels in the resistant strain were markedly suppressed by the dEGFR and dAkt inhibitors lapatinib and wortmannin. These results suggest that the increased P-gp in resistant flies was regulated by the dEGFR and dAkt pathways and that increased expression of P-gp is an important component of ABM resistance in insects. PMID:23648830

Luo, Liang; Sun, Ying-Jian; Wu, Yi-Jun

2013-05-03

23

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

Microsoft Academic Search

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

S Withoff; EGE de Vries; WN Keith; EF Nienhuis; WTA van der Graaf; DRA Uges; NH Mulder

1996-01-01

24

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

2012-06-09

25

In Silico Quantitative StructureActivity Relationship Studies on P-gp Modulators of Tetrahydroisoquinoline-Ethyl-Phenylamine Series  

Microsoft Academic Search

BACKGROUND: Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The drug efflux by a transport protein is the main reason for MDR. In humans, MDR mainly occurs when the ATP-binding cassette (ABC) family of proteins is overexpressed simultaneously. P-glycoprotein (P-gp) is most commonly associated with human MDR; it utilizes energy from adenosine triphosphate (ATP) to transport a number

Changdev G Gadhe; Thirumurthy Madhavan; Gugan Kothandan; Seung Joo Cho

2011-01-01

26

Small P-gp modulating molecules: SAR studies on tetrahydroisoquinoline derivatives  

Microsoft Academic Search

The development of small molecules as P-gp modulating agents and SAR studies on these ligands represented the aim of the present work. A series of 6,7-dimethoxytetrahydroisoquinoline derivatives was prepared and their ability to inhibit P-gp activity has been evaluated. The basic nucleus of these compounds, common to the best P-gp inhibitors such as Tariquidar and Elacridar, has been functionalized with

Nicola Antonio Colabufo; Francesco Berardi; Mariangela Cantore; Maria Grazia Perrone; Marialessandra Contino; Carmela Inglese; Mauro Niso; Roberto Perrone; Amalia Azzariti; Grazia Maria Simone; Letizia Porcelli; Angelo Paradiso

2008-01-01

27

[11C]sorafenib: radiosynthesis and preliminary PET study of brain uptake in P-gp/Bcrp knockout mice.  

PubMed

Sorafenib (Nexavar, BAY43-9006, 1) is a second-generation, orally active multikinase inhibitor that is approved for the treatment of some cancers in patients. In this Letter, we developed [(11)C]1 as a novel positron emission tomography (PET) probe, and evaluated the influence of ABC transporters-mediated efflux on brain uptake using PET with [(11)C]1 in P-glycoprotein (P-gp)/breast cancer resistance protein (Bcrp) knockout mice versus wild-type mice. [(11)C]1 was synthesized by the reaction of hydrochloride of aniline 2 with [(11)C]phosgene ([(11)C]COCl(2)) to give isocyanate [(11)C]6, followed by reaction with another aniline 3. Small-animal PET study with [(11)C]1 indicated that the radioactivity level (AUC(0-60 min), SUV×min) in the brains of P-gp/Bcrp knockout mice was about three times higher than in wild-type mice. PMID:21419625

Asakawa, Chiharu; Ogawa, Masanao; Kumata, Katsushi; Fujinaga, Masayuki; Kato, Koichi; Yamasaki, Tomoteru; Yui, Joji; Kawamura, Kazunori; Hatori, Akiko; Fukumura, Toshimitsu; Zhang, Ming-Rong

2011-03-06

28

MDR1-P-glycoprotein behaves as an oncofetal protein that promotes cell survival in gastric cancer cells.  

PubMed

P-glycoprotein (P-gp), traditionally linked to cancer poor prognosis and multidrug resistance, is undetectable in normal gastric mucosa and overexpressed in gastric cancer (GC). We propose that P-gp may be involved in Helicobacter pylori (Hp)-related gastric carcinogenesis by inhibiting apoptosis. Aim of the study was to evaluate the expression of P-gp in fetal stomach and in Hp-related gastric carcinogenesis, the epigenetic control of the multi-drug resistance-1 (MDR1) gene, the localization and interaction between P-gp and Bcl-x(L) and the effect of the selective silencing of P-gp on cell survival. P-gp and Bcl-xl expression was evaluated by immunohistochemistry on 28 spontaneously abortive human fetuses, 66 Hp-negative subjects, 138 Hp-positive chronic gastritis (CG) of whom 28 with intestinal metaplasia (IM) and 45 intestinal type GCs. P-gp/Bcl-x(L) colocalization was investigated by confocal immunofluorescence microscopy and protein-protein interaction by co-immunoprecipitation, in basal conditions and after stress-induced apoptosis, in GC cell lines AGS and MKN-28 and hepatocellular carcinoma cell line Hep-G2. The role of P-gp in controlling apoptosis was evaluated by knocking down its expression with a specific small interfering RNAs in stressed AGS and MKN-28 cell lines. P-gp is expressed in the gastric mucosa of all human fetuses while, it is undetectable in adult normal mucosa and re-expressed in 30/110 Hp-positive non-IM-CG, 28/28 IM-CG and 40/45 GCs. P-gp expression directly correlates with that of Bcl-x(L) and with the promoter hypomethylation of the MDR1 gene. In GC cell lines, P-gp is localized on the plasma membrane and mitochondria where it colocalizes with Bcl-x(L). Co-immunoprecipitation confirms the physical interaction between P-gp and Bcl-x(L) in AGS, MKN-28 and Hep-G2, at both basal level and after stress-induced apoptosis. The selective silencing of P-gp sensitizes GC cells to stress-induced apoptosis. P-gp behaves as an oncofetal protein that, by cross-talking with Bcl-x(L), acts as an anti-apoptotic agent in Hp-related gastric carcinogenesis. PMID:22751348

Rocco, Alba; Compare, Debora; Liguori, Eleonora; Cianflone, Alessandra; Pirozzi, Giuseppe; Tirino, Virginia; Bertoni, Alessandra; Santoriello, Margherita; Garbi, Corrado; D'Armiento, Maria; Staibano, Stefania; Nardone, Gerardo

2012-07-02

29

The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis  

PubMed Central

Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.

Smyth, Mark J.; Krasovskis, Erika; Sutton, Vivien R.; Johnstone, Ricky W.

1998-01-01

30

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

2013-08-25

31

Small P-gp modulating molecules: SAR studies on tetrahydroisoquinoline derivatives.  

PubMed

The development of small molecules as P-gp modulating agents and SAR studies on these ligands represented the aim of the present work. A series of 6,7-dimethoxytetrahydroisoquinoline derivatives was prepared and their ability to inhibit P-gp activity has been evaluated. The basic nucleus of these compounds, common to the best P-gp inhibitors such as Tariquidar and Elacridar, has been functionalized with no-basic moiety from our studied sigma receptor ligands displaying potent P-gp inhibition. The best results were obtained for compounds 3c and 3a (EC(50)=1.64 and 4.86 microM, respectively) and these results were remarkable because Elacridar showed in the same biological evaluation similar inhibitory activity (EC(50)=2 microM). SAR studies displayed that the removal of double bond on the spacer or its shifting into tetraline ring decreased the P-gp inhibiting activity. Moreover, the P-gp inhibition mechanism of tested compounds was investigated by three selected biological experiments. The results displayed that only compound 3c was P-gp inhibitor as Elacridar, while compound 3a and reference compounds Cyclosporin A and Verapamil modulated P-gp activity saturating the efflux pump as substrates. Flow cytometry studies carried out in Doxorubicin resistant breast cancer cell line (MCF7/Adr) confirmed that compound 3c increased Doxorubicin cell accumulation 5.7-fold. In addition, in MCF7/Adr, antiproliferative effect of 5 microM Doxorubicin shifted from 5% to 95% when co-administered with compound 3c (20 microM). The present study suggested a new class of small molecules displaying P-gp inhibitor activity differing from reference compounds Elacridar and Tariquidar for a simplified, and in the meantime, efficacious no-basic moiety. PMID:17936633

Colabufo, Nicola Antonio; Berardi, Francesco; Cantore, Mariangela; Perrone, Maria Grazia; Contino, Marialessandra; Inglese, Carmela; Niso, Mauro; Perrone, Roberto; Azzariti, Amalia; Simone, Grazia Maria; Porcelli, Letizia; Paradiso, Angelo

2007-09-25

32

Regulation of P-glycoprotein 1 and 2 gene expression and protein activity in two MCF-7/Dox cell line subclones.  

PubMed Central

The MCF-7 doxorubicin-resistant cell line MCF-7/Dox has been used extensively for studies of the multidrug resistance phenomenon. Using fluorescence-activated cell sorting (FACS), these cells were separated into two populations on the basis of rhodamine 123 (R123) accumulation. We designated these as low P-glycoprotein (LP-gp) and high P-gp (HP-gp) cells on the basis of their P-gp content. Using the reverse transcriptase polymerase chain reaction technique controlled by homologous internal standards, we analysed levels of MDR1 and MDR2 mRNA in each cell type. LP-gp and HP-gp cells had MDR1 mRNA levels of 2.17 +/- 0.17 and 6.65 +/- 2.29 amol ng-1 total RNA respectively, compared with 0.00088 +/- 0.00005 amol ng-1 in wild-type MCF-7 cells (MCF-7/WT). MCF-7/WT cells additionally contained 0.023 +/- 0.016 amol ng-1 of MDR2 mRNA, which was unchanged in LP-gp cells, but lower than in HP-gp cells, which contained 0.42 +/- 0.08 amol ng-1. Both LP-gp and HP-gp cells contained increased copies of the MDR1 gene. However, the degree of gene amplification did not correlate with the changes in MDR1 mRNA levels, indicating further regulatory levels of gene expression. The level of P-gp detected by MRK 16 correlated with R123 accumulation. HP-gp cells expressed a 10-fold higher level of P-gp1 than LP-gp cells. However, there was only a 3-fold increase in MDR1 mRNA level in HP-gp cells compared with LP-gp cells. These data suggest that some regulation of P-gp1 expression also occurred at the post-translational level. Phosphorylation of P-gp by protein kinase C (PKC)-alpha is necessary for its activity. Our analysis of PKC-alpha, 0 and epsilon isozyme levels, and subcellular distribution, shows a co-regulation of expression with P-gp, suggesting a necessary role for PKC in P-gp regulation. Images Figure 6

Davies, R.; Budworth, J.; Riley, J.; Snowden, R.; Gescher, A.; Gant, T. W.

1996-01-01

33

Vitamin E formulation affects digoxin absorption by inhibiting P-glycoprotein (P-GP) in humans  

Microsoft Academic Search

Water-soluble vitamin E(D-?-tocopheryl polyethylene glycol 1000 succinate (TPGS) may increase oral drug absorption. In vitro and animal data suggest that TPGS inhibits P-gp. It is unknown whether ?-tocopherol acetate(TA), the more common formulation of vitamin E, has similar drug interaction potential. This study aimed to determine whether TPGS and TA cause comparable P-gp inhibition in humans. Ten healthy subjects received

L. Chan; L. M. Humma; C. A. Schriever; L. A. Fahsingbauer; C. P. Dominguez; C. L. Baum

2004-01-01

34

Enhanced complement resistance in drug-selected P-glycoprotein expressing multi-drug-resistant ovarian carcinoma cells  

PubMed Central

Multi-drug resistance (MDR) is a major obstacle in cancer chemotherapy. There are contrasting data on a possible correlation between the level of expression of the drug transporter P-glycoprotein (P-gp) and susceptibility to complement-dependent cytotoxicity (CDC). We therefore investigated the sensitivity of human ovarian carcinoma cells and their P-gp expressing MDR variants to complement. Chemoselected P-gp expressing MDR cells showed increased resistance to CDC associated with overexpression of membrane-bound complement regulatory proteins (mCRP) and increased release of the soluble inhibitors C1 inhibitor and factor I. MDR1 gene transfection alone did not alter the susceptibility of P-gp expressing A2780-MDR and SKOV3-MDR cells to CDC. However, subsequent vincristine treatment conferred an even higher resistance to complement to these cells, again associated with increased expression of mCRP. Blocking the function of P-gp with verapamil, cyclosporine A or the anti-P-gp-antibody MRK16 had no impact on their complement resistance, whereas blocking of mCRP enhanced their susceptibility to complement. These results suggest that enhanced resistance of chemoselected MDR ovarian carcinoma cells to CDC is not conferred by P-gp, but is due at least partly to overexpression of mCRP, probably induced by treatment with the chemotherapeutic agents.

Odening, K E; Li, W; Rutz, R; Laufs, S; Fruehauf, S; Fishelson, Z; Kirschfink, M

2009-01-01

35

Acetaminophen modulates p-glycoprotein functional expression at the blood-brain barrier by a constitutive androstane receptor-dependent mechanism.  

PubMed

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

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

2013-09-09

36

P-glycoprotein expression does not change the apoptotic pathway induced by curcumin in HL60 cells  

Microsoft Academic Search

Purpose One of the mechanisms responsible for the multidrug resistance (MDR) phenotype of cancer cells is overexpression of so-called ATP-dependent drug efflux proteins: the 170-kDa P-glycoprotein (P-gp) encoded by the MDR1 gene and the 190-kDa multidrug resistance-associated protein 1 encoded by the MRP1 gene. The purpose of the present study was to verify the hypothesis postulating that P-gp expression, apart

Anna Bielak-?mijewska; Katarzyna Piwocka; Adriana Magalska; Ewa Sikora

2004-01-01

37

1?,25-Dihydroxyvitamin D3-liganded vitamin D receptor increases expression and transport activity of P-glycoprotein in isolated rat brain capillaries and human and rat brain microvessel endothelial cells.  

PubMed

Induction of the multidrug resistance protein 1 (MDR1)/P-glycoprotein (P-gp) by the vitamin D receptor (VDR) was investigated in isolated rat brain capillaries and rat (RBE4) and human (hCMEC/D3) brain microvessel endothelial cell lines. Incubation of isolated rat brain capillaries with 10 nM of the VDR ligand, 1?,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] for 4 h increased P-gp protein expression fourfold. Incubation with 1,25(OH)(2)D(3) for 4 or 24 h increased P-gp transport activity (specific luminal accumulation of NBD-CSA, the fluorescent P-gp substrate) by 25-30%. In RBE4 cells, Mdr1b mRNA was induced in a concentration-dependent manner by exposure to 1,25(OH)(2)D(3). Concomitantly, P-gp protein expression increased 2.5-fold and was accompanied by a 20-35% reduction in cellular accumulation of the P-gp substrates, rhodamine 6G (R6G), and HiLyte Fluor 488-labeled human amyloid beta 1-42 (hA?(42)). In hCMEC/D3 cells, a 3 day exposure to 100 nM 1,25(OH)(2)D(3) increased MDR1 mRNA expression (40%) and P-gp protein (threefold); cellular accumulation of R6G and hA?(42) was reduced by 30%. Thus, VDR activation up-regulates Mdr1/MDR1 and P-gp protein in isolated rat brain capillaries and rodent and human brain microvascular endothelia, implicating a role for VDR in increasing the brain clearance of P-gp substrates, including hA?(42), a plaque-forming precursor in Alzheimer's disease. PMID:23035695

Durk, Matthew R; Chan, Gary N Y; Campos, Christopher R; Peart, John C; Chow, Edwin C Y; Lee, Eason; Cannon, Ronald E; Bendayan, Reina; Miller, David S; Pang, K Sandy

2012-11-01

38

The terminal phosphate in d(pGpG) reduces self-association.  

PubMed

An investigation of the self-association behavior of 2'-deoxy[5'-phosphate-guanylyl-(3'-5')-guanosine] (d(pGpG)) in the presence of Na+ and K+ ions has been carried out by 1H and 31P NMR and FTIR spectroscopy. A comparison has been made of the self- association behavior of d(pGpG) with that of the related dinucleotide d(GpG), which has been shown to form extended structures based on stacked G-tetrads. Chemically, d(pGpG) monomer differs from d(GpG) only by the addition of a phosphate at the 5'-OH of the sugar residue. It was found that the addition of the second phosphate interferes with self-association. A suitable counterion is all that is required by d(GpG) to induce the formation of large super structures, but for d(pGpG) a large excess of salt is needed to produce the same effect. However, once self-association occurs, d(pGpG) forms similar structures to d(GpG) and has nearly the same properties. For both compounds, the K+ ion induces a more stable structure than the Na+ ion. The 31P NMR chemical shift ranges of d(pGpG) were consistent with the reported data for a phosphodiester and terminal phosphate. The small change in the chemical shift of the terminal phosphate with increasing temperature suggests that no major change in the terminal phosphate conformation occurred upon self-association. It was concluded that the terminal phosphate did not result in steric hindrance to self-association, but that interference to self-association was due to electrostatic repulsion effects. PMID:10636090

Kawasaki, M M; Walmsley, J A

1999-12-01

39

Tanshinone IIA potentiates the efficacy of 5-FU in Colo205 colon cancer cells in vivo through downregulation of P-gp and LC3-II  

PubMed Central

Traditional Chinese herbal medicines are widely accepted as an option for the treatment of colorectal cancers. Danshen (Salviae miltiorrhizae Radix) is widely prescribed in traditional Chinese medicine for cardiovascular diseases. Tanshinone IIA (Tan-IIA) is extracted from Danshen. Our previous studies have shown that Tan-IIA induces apoptosis in Colo205 human colon cancer cells in vitro and in vivo. In the present study, we investigated the efficacy of Tan-IIA and 5-fluorouracil (5-FU) in a Colo205 cell xenograft model. For in vivo studies, SCID mice were engrafted with Colo205 cells and from day 10 onwards were randomly divided into 3 groups and treated with 5-FU plus Tan-IIA, 5-FU plus corn oil, and the vehicle alone. At the end of a 4-week dosing schedule, the SCID mice were sacrificed and xenograft tumors were dissected for protein western blot analysis. Our results showed that the Colo205 xenograft model co-treated with Tan-IIA plus 5-FU caused a reduction in the xenograft tumor volumes and decreased P-glycoprotein (P-gp) and microtubule-associated protein light chain 3 (LC3)-II expression compared to 5-FU alone. Based on these observations, it may be possible to develop Tan-IIA plus 5-FU as therapeutic agents for human colon cancer.

SU, CHIN-CHENG

2012-01-01

40

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

PubMed

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

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

2012-02-02

41

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

PubMed Central

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

2012-01-01

42

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

Microsoft Academic Search

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

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

2012-01-01

43

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

44

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

PubMed

Venlafaxine, and to a lesser extent desvenlafaxine, has previously been shown to induce the expression of the drug efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in whole cells and alter the cellular permeability of a known drug efflux probe (rhodamine 123). To validate these in vitro findings, wild-type mice were treated for 4 days with 10 mg/kg venlafaxine or desvenlafaxine, and drug efflux transporter expression was examined in the brain, liver, and intestine. P-gp and BCRP expression was significantly upregulated in the intestine, following a treatment with venlafaxine (2.6- and 6.7-fold, respectively) or desvenlafaxine (2.3- and 4.8-fold, respectively). In addition, venlafaxine increased the BCRP expression in the brain (40%) and liver (60%), whereas desvenlafaxine had no effect on drug efflux transporter levels in these tissues. Using the same treatment paradigm, we observed a minimal impact of either drug on the brain disposition of the known drug efflux probe, topotecan. However, in the periphery, venlafaxine treatment significantly reduced the topotecan oral bioavailability by nearly 40%, whereas the impact of desvenlafaxine on topotecan plasma levels was more modest (23%). These studies demonstrate an effect of venlafaxine on the drug efflux transport activity and the potential for clinical drug-drug interactions. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3838-3843, 2013. PMID:23897419

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

2013-07-29

45

Beta-Amyloid Downregulates MDR1-P-Glycoprotein (Abcb1) Expression at the Blood-Brain Barrier in Mice  

PubMed Central

Neurovascular dysfunction is an important component of Alzheimer's disease, leading to reduced clearance across the blood-brain barrier and accumulation of neurotoxic ?-amyloid (A?) peptides in the brain. It has been shown that the ABC transport protein P-glycoprotein (P-gp, ABCB1) is involved in the export of A? from the brain into the blood. To determine whether A? influences the expression of key A? transporters, we studied the effects of 1-day subcutaneous A?1-40 and A?1-42 administration via Alzet mini-osmotic pumps on P-gp, BCRP, LRP1, and RAGE expression in the brain of 90-day-old male FVB mice. Our results demonstrate significantly reduced P-gp, LRP1, and RAGE mRNA expression in mice treated with A?1-42 compared to controls, while BCRP expression was not affected. The expression of the four proteins was unchanged in mice treated with A?1-40 or reverse-sequence peptides. These findings indicate that, in addition to the age-related decrease of P-gp expression, A?1-42 itself downregulates the expression of P-gp and other A?-transporters, which could exacerbate the intracerebral accumulation of A? and thereby accelerate neurodegeneration in Alzheimer's disease and cerebral ?-amyloid angiopathy.

Brenn, Anja; Grube, Markus; Peters, Michele; Fischer, Andrea; Jedlitschky, Gabriele; Kroemer, Heyo K.; Warzok, Rolf W.; Vogelgesang, Silke

2011-01-01

46

In vivo evaluation of an oral delivery system for P-gp substrates based on thiolated chitosan  

Microsoft Academic Search

Recently, thiolated polymers, so called thiomers, have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a proof-of-principle for a delivery system based on thiolated chitosan in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. In vitro, the permeation enhancing effect of unmodified chitosan, chitosan-4 thiobutylamidine

Florian Föger; Thierry Schmitz; Andreas Bernkop-Schnürch

2006-01-01

47

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

PubMed

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

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

2011-10-01

48

Identification of Novel Specific and General Inhibitors of the Three Major Human ATP-Binding Cassette Transporters P-gp, BCRP and MRP2 Among Registered Drugs  

Microsoft Academic Search

Purpose  To study the inhibition patterns of the three major human ABC transporters P-gp (ABCB1), BCRP (ABCG2) and MRP2 (ABCC2), using\\u000a a dataset of 122 structurally diverse drugs.\\u000a \\u000a \\u000a \\u000a Methods  Inhibition was investigated in cellular and vesicular systems over-expressing single transporters. Computational models discriminating\\u000a either single or general inhibitors from non-inhibitors were developed using multivariate statistics.\\u000a \\u000a \\u000a \\u000a Results  Specific (n?=?23) and overlapping (n?=?19) inhibitors of

Pär Matsson; Jenny M. Pedersen; Ulf Norinder; Christel A. S. Bergström; Per Artursson

2009-01-01

49

Histone deacetylase inhibitor induction of P-glycoprotein transcription requires both histone deacetylase 1 dissociation and recruitment of CAAT/enhancer binding protein beta and pCAF to the promoter region.  

PubMed

Although histone deacetylase (HDAC) inhibitors are appreciated as a promising class of anticancer drugs, recent reports show that P-glycoprotein (P-gp) is induced by HDAC inhibitor treatment in cancer cells, resulting in multidrug resistance of cancer cells to other chemotherapeutic agents. In this study, we investigated the molecular mechanism of HDAC inhibitor induction of P-gp expression. HDAC inhibitor treatment causes cell type-specific induction of P-gp expression without changes in the CpG methylation status of the promoter region. In addition, our data show that HDAC inhibitor does not alter the DNA binding activity of Sp1 but facilitates both the recruitment of a coactivator complex that includes CAAT/enhancer binding protein beta and pCAF and the dissociation of the repressive complex, HDAC1, to the Sp1 binding region. Subsequently, the hyperacetylated histone H3 becomes enriched in the promoter region, leading to RNA polymerase II recruitment to activate P-gp gene transcription. Furthermore, specific down-regulation of HDAC1, but not HDAC2, by RNA silencing was enough to induce P-gp expression in HeLa cells, strongly supporting the essential role of HDAC1 in HDAC inhibitor induction of P-gp. Concomitantly, cell type-specific induction of P-gp expression seems to be dependent on phosphatidylinositol 3-kinase activity. Taken together, our findings show that HDAC inhibitor treatment leads to an increase in P-gp expression through dynamic changes in chromatin structure and transcription factor association within the promoter region. PMID:19435809

Kim, Su-Nam; Kim, Nam Hyun; Lee, Woojung; Seo, Dong-Wan; Kim, Yong Kee

2009-05-12

50

Expression of P-glycoprotein in southeastern oysters, Crassostrea virginica  

Microsoft Academic Search

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,

C. J. Keppler; A. H. Ringwood

2001-01-01

51

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

2013-03-29

52

mdm2 gene mediates the expression of mdr1 gene and P-glycoprotein in a human glioblastoma cell line.  

PubMed Central

The overexpression of the multidrug resistance (mdr1) gene and its product, P-glycoprotein (P-gp), is thought to limit the successful chemotherapy of human tumours. The mechanism by which mdr1 gene and P-gp are overexpressed in human tumours, however, is not yet clear. In this report, we show that the mdm2 (murine double minute 2) gene induced the expression of the mdr1 gene and P-gp in human glioblastoma U87-MG cells, which did not express the MDM2 protein or P-gp. The mdm2 gene, in addition, conferred the resistance of U87-MG cells to the apoptotic cell death induced by etoposide (VP-16) or doxorubicin. Furthermore, treatment with mdm2 antisense oligonucleotides inhibited the expression of P-gp in MDM2-expressing U87-MG cells. These findings suggest that the mdm2 gene may play an important role in the development of MDR phenotype in human tumours. Images Figure 1 Figure 3 Figure 5

Kondo, S.; Kondo, Y.; Hara, H.; Kaakaji, R.; Peterson, J. W.; Morimura, T.; Takeuchi, J.; Barnett, G. H.

1996-01-01

53

4-Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoline derivatives as potent P-gp modulators.  

PubMed

Starting from lead compound 1 (EC(50)=1.64 microM), its non-basic nucleus has been conformationally restricted by 4-biphenyl and 2-naphthyl moieties. In each series we investigated if the presence of H-bond donor or acceptor substituents, the basicity and the lipophilicity (clogP) were correlated with the P-gp inhibiting activity of tested compounds. In the biphenyl series, derivative 4d displayed the best results (EC(50)=0.05 microM). The corresponding amide 3d was found less active (EC(50)=3.5 microM) ascertaining the importance of basicity in this series whilst the presence of hydroxy or methoxy substituents seems to be negligible. In the naphthyl series, both the basicity and the presence of H-bond donor or acceptor groups seem to be negligible. Moreover, the lipophilicity did not influence the P-gp inhibition activity of each series. Specific biological assays have been carried out to establish the P-gp interacting mechanism of tested compounds discriminating between substrates and inhibitors. Moreover, compound 4d displayed a potent P-gp inhibition activity with good selectivity towards BCRP pump. PMID:18276145

Colabufo, Nicola Antonio; Berardi, Francesco; Cantore, Mariangela; Perrone, Maria Grazia; Contino, Marialessandra; Inglese, Carmela; Niso, Mauro; Perrone, Roberto; Azzariti, Amalia; Simone, Grazia Maria; Paradiso, Angelo

2008-02-02

54

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

PubMed

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

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

2012-11-29

55

[Pharmaceutical evaluation of hydroxycamptothecin nanosuspensions with the action of inhibiting P-gp].  

PubMed

Oral hydroxycamptothecin nanosuspension (HCPT-Nano) with high supersaturated dissolution level, high permeation and well physical stability, was manufactured by microprecipitation-high press homogenization method. Its pharmaceutical properties were investigated, such as size and distribution, zeta potential, particle shape, physical existence condition, supersaturated dissolution level and so on. Particle size was measured by laser diffraction, and the mean diameters before and after lyophilization were 138 +/- 11.72 nm and 175 +/- 12.74 nm, respectively, for HCPT-Nano. Zeta potentials of HCPT-Nano was over -20 mV. The nanoparticles, being observed by transmission electron microscopy (TEM), were claviform or column in shape. DSC and X-ray diffraction revealed that HCPT existed in the form of crystal for HCPT-Nano. And HCPT-Nano could maintain higher supersaturated dissolution level for long time. So it supplied the possibility of improving oral bioavailability of HCPT when combining together admoveatur of P-gp inhibitor, CsA. PMID:22010354

Pu, Xiao-Hui; Sun, Jin; Qin, Yi-Meng; Zhang, Xiao; Zhang, Peng; He, Zhong-Gui

2011-07-01

56

In Silico Quantitative Structure-Activity Relationship Studies on P-gp Modulators of Tetrahydroisoquinoline-Ethyl-Phenylamine Series  

PubMed Central

Background Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The drug efflux by a transport protein is the main reason for MDR. In humans, MDR mainly occurs when the ATP-binding cassette (ABC) family of proteins is overexpressed simultaneously. P-glycoprotein (P-gp) is most commonly associated with human MDR; it utilizes energy from adenosine triphosphate (ATP) to transport a number of substrates out of cells against concentration gradients. By the active transport of substrates against concentration gradients, intracellular concentrations of substrates are decreased. This leads to the cause of failure in cancer chemotherapy. Results Herein, we report Topomer CoMFA (Comparative Molecular Field Analysis) and HQSAR (Hologram Quantitative Structure Activity Relationship) models for third generation MDR modulators. The Topomer CoMFA model showed good correlation between the actual and predicted values for training set molecules. The developed model showed cross validated correlation coefficient (q2) = 0.536 and non-cross validated correlation coefficient (r2) = 0.975 with eight components. The best HQSAR model (q2 = 0.777, r2 = 0.956) with 5-8 atom counts was used to predict the activity of test set compounds. Both models were validated using test set compounds, and gave a good predictive values of 0.604 and 0.730. Conclusions The contour map near R1 indicates that substitution of a bulkier and polar group to the ortho position of the benzene ring enhances the inhibitory effect. This explains why compounds with a nitro group have good inhibitory potency. Molecular fragment analyses shed light on some essential structural and topological features of third generation MDR modulators. Fragments analysis showed that the presence of tertiary nitrogen, a central phenyl ring and an aromatic dimethoxy group contributed to the inhibitory effect. Based on contour map information and fragment information, five new molecules with variable R1 substituents were designed. The activity of these designed molecules was predicted by the Topomer CoMFA and HQSAR models. The novel compounds showed higher potency than existing compounds.

2011-01-01

57

4Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoline derivatives as potent P-gp modulators  

Microsoft Academic Search

Starting from lead compound 1 (EC50=1.64?M), its non-basic nucleus has been conformationally restricted by 4-biphenyl and 2-naphthyl moieties. In each series we investigated if the presence of H-bond donor or acceptor substituents, the basicity and the lipophilicity (clogP) were correlated with the P-gp inhibiting activity of tested compounds. In the biphenyl series, derivative 4d displayed the best results (EC50=0.05?M). The

Nicola Antonio Colabufo; Francesco Berardi; Mariangela Cantore; Maria Grazia Perrone; Marialessandra Contino; Carmela Inglese; Mauro Niso; Roberto Perrone; Amalia Azzariti; Grazia Maria Simone; Angelo Paradiso

2008-01-01

58

Gateway cloning for protein expression.  

PubMed

The rate-limiting step in protein production is usually the generation of an expression clone that is capable of producing the protein of interest in soluble form at high levels. Although cloning of genes for protein expression has been possible for some time, efficient generation of functional expression clones, particularly for human proteins, remains a serious bottleneck. Often, such proteins are hard to produce in heterologous systems because they fail to express, are expressed as insoluble aggregates, or cannot be purified by standard methods. In many cases, researchers are forced to return to the cloning stages to make a new construct with a different purification tag, or perhaps to express the protein in a different host altogether. This usually requires identifying new cloning schemes to move a gene from one vector to another, and frequently requires multistep, inefficient cloning processes, as well as lengthy verification and sequence analysis. Thus, most researchers view this as a linear pathway - make an expression clone, try it out, and if it fails, go back to the beginning and start over. Because of this, protein expression pipelines can be extremely expensive and time consuming.The advent of recombinational cloning has dramatically changed the way protein expression can be handled. Rapid production of parallel expression clones is now possible at relatively low cost, opening up many possibilities for both low- and high-throughput protein expression, and increasing the flexibility of expression systems that researchers have available to them. While many different recombinational cloning systems are available, the one with the highest level of flexibility remains the Gateway system. Gateway cloning is rapid, robust, and highly amenable to high-throughput parallel generation of expression clones for protein production. PMID:18988017

Esposito, Dominic; Garvey, Leslie A; Chakiath, Chacko S

2009-01-01

59

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

PubMed

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. PMID:19794400

Zhong, Yu; Hennig, Bernhard; Toborek, Michal

2009-09-30

60

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

61

Restricted brain penetration of the tyrosine kinase inhibitor erlotinib due to the drug transporters P-gp and BCRP  

Microsoft Academic Search

Summary  \\u000a Purpose Erlotinib (Tarceva®, OSI-774) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase.\\u000a As high-grade gliomas frequently show amplification, overexpression and\\/or mutation of EGFR, this drug has been tested in\\u000a several clinical trials with glioblastoma patients, but unfortunately, with little success. As erlotinib is a known substrate\\u000a of P-glycoprotein (P-gp) and Breast Cancer Resistance

Nienke A. de Vries; Tessa Buckle; Jin Zhao; Jos H. Beijnen; Jan H. M. Schellens; Olaf van Tellingen

62

Effect of methotrexate treatment on expression levels of organic anion transporter polypeptide 2, P-glycoprotein and bile salt export pump in rats.  

PubMed

High-dose methotrexate (HDMTX) chemotherapy with leucovorin (LV) rescue has been used as a therapeutic strategy in oncology since the 1970s. Adverse reactions following extension of methotrexate (MTX) elimination are a crucial problem in HDMTX chemotherapy. MTX is a substrate for drug transporters, which are multidrug resistance protein 2 (Mrp2), organic anion transporter polypeptide 2 (Oatp2) and other transporters. We previously reported that MTX treatment downregulated the expression level of Mrp2 in rats. Here we examined the effect of MTX treatment on the expression of Oatp2, P-glycoprotein (P-gp) and bile salt export pump (Bsep) in rats. MTX was single-injected intraperitoneally at a dose of 150 mg/kg, and Western blot analysis was performed. The levels of Oatp2, P-gp and Bsep in the liver on day 4 after treatment were downregulated to 36.3 +/- 6.9%, 51.5 +/- 5.2% and 61.8 +/- 5.5% (mean +/- S.E.M.) of controls, respectively. Expression levels of P-gp in the kidney and ileum were also downregulated to 38.5 +/- 1.6% and 16.2 +/- 1.6% of controls, respectively. These effects of MTX were partially recovered by LV, which rescues normal cells from MTX toxicity. In conclusion, the result indicates that MTX treatment downregulates expression levels of Oatp2, P-gp and Bsep. PMID:19252302

Shibayama, Yoshihiko; Takeda, Yasuo; Yamada, Katsushi

2009-03-01

63

Factors affecting the expression and function of P-glycoprotein in rats: drug treatments and diseased states.  

PubMed

The expression and function of P-glycoprotein (P-gp), an ATP-dependent efflux pump, were examined in rats pretreated with dexamethasone (DEX), an inducer of P-gp, and in rats with glycerol-induced acute renal failure (ARF) and with CCl4-induced acute hepatic failure (AHF). DEX pretreatment increased the P-gp level and its functional activity in the intestine. In contrast, in ARF and AHF rats, the in vivo P-gp function was systemically suppressed, even though the level of P-gp remained unchanged or rather increased. In Caco-2 cells, the plasma collected from diseased rats exhibited a greater inhibitory effect on P-gp function than did plasma from control rats. A higher-plasma level of corticosterone, an endogenous P-gp substrate/inhibitor, was observed in the disease rats. These findings indicate that the actual in vivo function of P-gp cannot be predicted merely from the expression level of P-gp, and suggest that some endogenous P-gp-related compounds such as corticosterone participate in the regulation of in vivo P-gp function in diseased states. PMID:11878184

Murakami, T; Yumoto, R; Nagai, J; Takano, M

2002-02-01

64

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

2011-06-09

65

P-glycoprotein expression as a predictor of response to neoadjuvant chemotherapy in breast cancer.  

PubMed

Background: Chemoresistance is an important factor determining the response of tumor to neoadjuvant chemotherapy (NACT). P-glycoprotein (P-gp) expression-mediated drug efflux is one of the mechanisms responsible for multi-drug resistance. Our study was aimed to determine the role of P-gp expression as a predictor of response to NACT in locally advanced breast cancer (LABC) patients. Materials and Methods: P-gp expression was performed by real-time quantitative polymerase chain reaction [qRT-PCR] in 76 patients with LABC. Response to adriamycin-based regimen was assessed both clinically and with contrast enhanced computed tomography (CECT) scan before and after NACT. The significance of correlation between tumor and P-gp levels was determined with Chi-square test. Results: Twenty-one had high and 55 had low P-gp expression. On analyzing P-gp expression with response by World Health Organization (WHO) criteria, statistical significance was obtained (P = 0.038). Similarly, assessment of P-gp expression with response by Response Evaluation in Solid Tumors (RECIST) criteria in 48 patients showed statistical significance (P = 0.0005). Conclusion: This study proves that P-gp expression is a determinant factor in predicting response to NACT. Finally, detection of P-gp expression status before initiation of chemotherapy can be used as a predictive marker for NACT response and will also aid in avoiding the toxic side effects of NACT in non-responders. PMID:24061458

Vishnukumar, S; Umamaheswaran, G; Anichavezhi, D; Indumathy, S; Adithan, C; Srinivasan, K; Kadambari, D

66

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

67

Modulation and absorption of xenobiotics: the synergistic role of CYP450 and P-gp activities in cancer and neurodegenerative disorders.  

PubMed

P-glycoprotein (P-gp) is involved in MDR and in neurodegenerative disorders such as Parkinson disease (PD), Alzheimer disease (AD) and epilepsy. Cytochrome P450 enzymes (CYP450s) catalyze the metabolism of a wide variety of endogenous and exogenous compounds including xenobiotics, drugs, environmental toxins, steroids, and fatty acids. P-gp substrates, inhibitors and inducers should be designed and developed studying interacting mechanism with both P-gp an CYP450 enzymes before they could be employed in MDR and/or in neurodegenerative disorders. Here, the ex vivo rat everted gut sac assay has been proposed as an immediate approach to simultaneously study metabolism and transport of drugs. Elacridar, verapamil and cyclosporine A (CsA), P-gp inhibitor, substrate and modulator respectively, have been tested to validate this ex vivo approach. The new model have been used yet to develop our ligands MC18, MC266 and MC80, both as potential drugs for MDR and radiotracers for diagnosis of neurodegenerative disorders. Herein, a comparative evaluation of transport and metabolic results, by using in vitro, ex vivo and in vivo assays, is reported. PMID:21434860

Inglese, Carmela; Perrone, Maria Grazia; Berardi, Francesco; Perrone, Roberto; Colabufo, Nicola Antonio

2011-10-01

68

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

PubMed

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. PMID:24066157

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

2013-09-16

69

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

70

Conformational Characteristics of Mixed Sugar Puckered Deoxydinucleoside Triphosphate Units d-pCpGp and d-pGpCp from Energy Minimization Studies  

Microsoft Academic Search

The deoxydinucleoside triphosphate units d-pCpGp and d-pGpCp were subjected to a rigorous theoretical investigation with a view to describing their distinctive conformational characteristics. For each unit 216 probable three-dimensional forms defined by the backbone-base dihedral angles and sugar pucker modes were considered for conformational energy minimization process and scrutinized with reference to properties, such as base-stacking, hydrogen-bonding, internal flexibility and

P. K. Ponnuswamy; A. Anukanth

1989-01-01

71

Methylated ?-cyclodextrin as P-gp modulators for deliverance of doxorubicin across an in vitro model of blood–brain barrier  

Microsoft Academic Search

Co-incubations of various ?-cyclodextrins and doxorubicin have been evaluated on an in vitro model of blood–brain barrier in order to increase the delivery of this P-gp substrate to the brain. Among these cyclodextrins used, the Rame-?-cyclodextrin and Crysme-?-cyclodextrin increased the transport by a factor of 2 and 3.7, respectively. This increase was attributed to the cholesterol extraction property of these

Sébastien Tilloy; Véronique Monnaert; Laurence Fenart; Hervé Bricout; Roméo Cecchelli; Eric Monflier

2006-01-01

72

Upregulation of drug transporter expression by osteopontin in prostate cancer cells.  

PubMed

Multidrug resistance is a major cause of chemotherapy failure. Recent studies indicate that drug resistance can be rapidly induced by some soluble factors, such as cytokines, chemokines, growth factors, and cell adhesion factors in the tumor microenvironment. Osteopontin (OPN), an extracellular matrix protein, has a functional arginine-glycine-aspartic acid (RGD) domain for binding to integrin. Here we found OPN expression to be upregulated by hypoxic condition in PC-3 prostate tumor cells. OPN increased the mRNA and protein expression of p-glycoprotein (P-gp), a subfamily of ATP-binding cassette transporter in a concentration- and time-dependent manner. The increase in P-gp transporter by OPN was mediated by binding to ?v?3 integrin. Daunomycin (DUN), a chemotherapeutic agent with autofluorescence, was used to evaluate the pump activity, and OPN increased the drug pumping-out activity. OPN inhibited DUN-induced cell death, which was antagonized by ?v?3 monoclonal antibody. Long-term treatment with DUN further enhanced the expression of OPN. Knockdown of endogenous OPN potentiated the DUN-induced apoptosis of PC-3 cells. Furthermore, knockdown of OPN enhanced cell death caused by other drugs, including paclitaxel, doxorubicin, actinomycin-D, and rapamycin, which are also P-gp substrates. The animal studies also showed that OPN knockdown enhanced the cytotoxic action of DUN. These results indicate that OPN is a potential therapeutic target for cancer therapy to reduce drug resistance in sensitive tumors. PMID:23434829

Hsieh, I-Shan; Huang, Wei-Hsun; Liou, Houng-Chi; Chuang, Woei-Jer; Yang, Rong-Sen; Fu, Wen-Mei

2013-02-22

73

Effect of MDR modulators verapamil and promethazine on gene expression levels of MDR1 and MRP1 in doxorubicin-resistant MCF7 cells  

Microsoft Academic Search

Purpose  One of the major problems of cancer chemotherapy is the development of multidrug resistance (MDR) phenotype. Among the numerous\\u000a mechanisms of MDR, a prominent one is the increased expression of membrane transporter proteins, the action of which leads\\u000a to decreased intracellular drug concentration and cytotoxicity of drugs. Among them, P-gp and MRP1, encoded by MDR1 and MRP1 genes, respectively, have

Yaprak Dönmez; Laila Akhmetova; Özlem Darcansoy ??eri; Meltem Demirel Kars; Ufuk Gündüz

2011-01-01

74

Reversal of different drug-resistant phenotypes by an autocatalytic multitarget multiribozyme directed against the transcripts of the ABC transporters MDR1\\/P-gp, MRP2, and BCRP  

Microsoft Academic Search

A “multitarget multiribozyme” (MTMR) was constructed. It consists of three trans-acting hammerhead ribozymes directed against the transcripts of the ABC transporters MDR1\\/P-gp, BCRP, and MRP2; three cis-acting MDR1\\/P-gp-specific ribozymes; and three MDR1\\/P-gp-homologous spacer sequences. The trans-acting hammerhead ribozymes are liberated from the MTMR through autocatalytic self-cleavage by the cis-acting ribozymes. The MTMR was characterized with regard to its kinetic parameters.

Petra Kowalski; Pawel Surowiak; Hermann Lage

2005-01-01

75

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

76

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

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter

1998-07-01

77

Desipramine treatment has minimal effects on the brain accumulation of glucocorticoids in P-gp-deficient and wild-type mice  

PubMed Central

Summary Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis in patients with depression can be reduced by antidepressants, which are thought to improve endogenous glucocorticoid-mediated negative feedback. A proportion of peripherally released glucocorticoids need to enter brain tissue, protected by the blood–brain barrier (BBB), in order to achieve this negative feedback effect at the level of the central nervous systems (CNS). The multidrug resistance transporter P-glycoprotein (P-gp) has been shown to actively transport glucocorticoid hormones and has been implicated in the regulation of glucocorticoid access to the CNS. Using an in situ brain/choroid plexus perfusion method, we tested the hypothesis that the antidepressant desipramine increases glucocorticoid accumulation in the mouse brain by inhibiting P-gp, following either chronic treatment (8 days, 20 mg/kg/day, IP) or acute administration (20 min brain perfusion in the presence of either 0.9 ?M or 10 ?M desipramine). Contrary to our hypothesis, chronic treatment with desipramine did not affect the accumulation of [3H]dexamethasone in any sample compared to saline-treated mice. Acute desipramine had limited and variable effects on glucocorticoid accumulation in the CNS, with accumulation of [3H]dexamethasone increased in the cerebellum, accumulation of [3H]cortisol reduced in the frontal cortex, hypothalamus, and cerebellum, and accumulation of [3H]corticosterone (the endogenous glucocorticoid in rodents) not affected. Overall, under the conditions tested, these results do not support the hypothesis that treatment with desipramine can inhibit P-gp at the BBB and subsequently increase the accumulation of glucocorticoids in the brain.

Mason, Brittany L.; Thomas, Sarah A.; Lightman, Stafford L.; Pariante, Carmine M.

2011-01-01

78

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

PubMed

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

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

2013-05-23

79

p53 and P-glycoprotein are often co-expressed and are associated with poor prognosis in breast cancer.  

PubMed Central

Expression of both P-glycoprotein (P-gp) and mutant p53 have recently been reported to be associated with poor prognosis of breast cancer. The expression of P-gp is associated in vitro and in vivo with cross-resistance to several anti-cancer drugs. p53 plays a regulatory role in apoptosis, and mutant p53 has been suggested to be involved in drug resistance. Interestingly, in vitro experiments have shown that mutant p53 can activate the promoter of the MDR1 gene, which encodes P-gp. We investigated whether p53 and P-gp are simultaneously expressed in primary breast cancer cells and analysed the impact of the co-expression on patients prognosis. Immunohistochemistry was used to investigate P-gp expression (JSB-1, C219) and nuclear p53 accumulation (DO-7) in 20 operable chemotherapy untreated and 30 locally advanced breast cancers undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. Double immunostaining showed that P-gp expression and nuclear p53 accumulation often occur concomitantly in the same tumour cells. A correlation between p53 and P-gp expression was found in all 50 breast cancers (P = 0.003; Fisher's exact test). P-gp expression, nuclear p53 accumulation, and co-expression of p53 and P-gp were more frequently observed in locally advanced breast cancers than in operable breast cancers (P = 0.0004, P = 0.048; P = 0.002 respectively. Fisher's exact test). Co-expression of p53 and P-gp was the strongest prognostic factor for shorter survival by multivariate analysis (P = 0.004) in the group of locally advanced breast cancers (univariate analysis: P = 0.0007). Only 3 out of 13 samples sequentially taken before and after chemotherapy displayed a change in P-gp or p53 staining. In conclusion, nuclear p53 accumulation is often associated with P-gp expression in primary breast cancer, and simultaneous expression of p53 and P-gp is associated with shorter survival in locally advanced breast cancer patients. Co-expression of P-gp and mutant p53 belong to a series of molecular events resulting in a more aggressive phenotype, drug resistance and poor prognosis. Images Figure 1

Linn, S. C.; Honkoop, A. H.; Hoekman, K.; van der Valk, P.; Pinedo, H. M.; Giaccone, G.

1996-01-01

80

Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies  

Microsoft Academic Search

Tumor cells may display a multidrug resistance phenotype by overex- pression of ATP binding cassette transporter genes such as multidrug resistance (MDR) 1 P-glycoprotein (P-gp) or the multidrug resistance pro- tein 1 (MRP1). MDR3 P-gp is a close homologue of MDR1 P-gp, but its role in MDR is probably minor and remains to be established. The MRP1 protein belongs to

George L. Scheffer; Marcel Kool; Marc Heijn; Haas de M; A. C. L. M. Pijnenborg; J. Wijnholds; Helvoort van A; M. C. Jong; J. H. Hooijberg; C. A. A. M. Mol; Linden van der M; Vree de J. M. L; Valk van der P; R. P. J. Oude Elferink; P. Borst; R. J. Scheper

2000-01-01

81

In vivo induction of P-glycoprotein expression at the mouse blood-brain barrier: an intracerebral microdialysis study.  

PubMed

Intracerebral microdialysis was utilized to investigate the effect of P-glycoprotein (a drug efflux transporter) induction at the mouse blood-brain barrier (BBB) on brain extracellular fluid concentrations of quinidine, an established substrate of P-glycoprotein. Induction was achieved by treating male CD-1 mice for 3 days with 5 mg/kg/day dexamethasone (DEX), a ligand of the nuclear receptor, pregnane X receptor, and a P-glycoprotein inducer. Tandem liquid chromatography mass spectrometric method was used to quantify analytes in dialysate, blood and plasma. P-glycoprotein, pregnane X receptor and Cyp3a11 (metabolizing enzyme for quinidine) protein expression in capillaries and brain homogenates was measured by immunoblot analysis. Following quinidine i.v. administration, the average ratio of unbound quinidine concentrations in brain extracellular fluid (determined from dialysate samples) to plasma at steady state (375-495 min) or Kp, uu, ECF /Plasma in the DEX-treated animals was 2.5-fold lower compared with vehicle-treated animals. In DEX-treated animals, P-glycoprotein expression in brain capillaries was 1.5-fold higher compared with vehicle-treated animals while Cyp3a11 expression in brain capillaries was not significantly different between the two groups. These data demonstrate that P-gp induction mediated by DEX at the BBB can significantly reduce quinidine brain extracellular fluid concentrations by decreasing its brain permeability and further suggest that drug-drug interactions as a result of P-gp induction at the BBB are possible. Applying microdialysis, distribution of quinidine, a P-gp substrate, in mouse brain extracellular fluid (ECF) was investigated following ligand-mediated P-glycoprotein (P-gp) induction at the blood-brain barrier (BBB). We demonstrated that a PXR agonist (dexamethasone) significantly up-regulated P-gp in brain capillaries and reduced quinidine brain ECF concentrations. Our data suggest that drug-drug interactions as a result of P-gp induction at the BBB are possible. PMID:23777437

Chan, Gary N Y; Saldivia, Victor; Yang, Yingbo; Pang, Henrianna; de Lannoy, Inés; Bendayan, Reina

2013-07-15

82

Membrane protein expression: no cells required.  

PubMed

Structural and functional studies of membrane proteins have been severely hampered by difficulties in producing sufficient quantities of properly folded protein products. It is well established that cell-based expression of membrane proteins is generally problematic and frequently results in low yield, cell toxicity, protein aggregation and misfolding. Owing to its inherent open nature, cell-free protein expression has become a highly promising tool for the fast and efficient production of these difficult-to-express proteins. Here we review the most recent advances in this field, underscoring the potentials and weaknesses of the newly developed approaches and place specific emphasis on the use of nanolipoprotein particles (NLPs or nanodiscs). PMID:19616329

Katzen, Federico; Peterson, Todd C; Kudlicki, Wieslaw

2009-07-16

83

Cultured CD4T cells and primary human lymphocytes express hOATPs: intracellular accumulation of saquinavir and lopinavir  

PubMed Central

Background and purpose: Drug efflux tranporters (P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP)) limit the cellular uptake of human immunodeficiency virus protease inhibitors but the contribution of influx transporters in cells that (over)express P-gp or MRP is less clear. Here, we studied the expression of one influx transporter system, human organic anion-transporting polypeptide (hOATP), in some T-cell lines (CEM, CEMVBL, CEME1000) and in peripheral blood mononuclear cells (PBMCs) and examined the effects of manipulation of influx/efflux transporters on the uptake of saquinavir and lopinavir. Experimental approach: The expression of hOATPs was studied by PCR. We used hOATP substrate or inhibitor (estrone-3-sulphate (E-3-S) or montelukast, respectively) and inhibitors of P-gp (XR9576) and MRP (MK571 and frusemide) to study functional interactions between influx and efflux transporters in the uptake of saquinavir and lopinavir. Lipophilicity of the drugs was measured by octanol/saline partition coefficient. Key results: CEM cells, their variants and PBMCs express various hOATP isoforms, with OATP3A1 detected in all of the cells. MK571, XR9576 and frusemide increased the uptake of saquinavir and lopinavir. E-3-S and montelukast reduced the uptake of saquinavir and lopinavir in some, but not all, of the cells. Pretreatment of the cells with MK571, XR9576 or frusemide, followed by E-3-S co-incubation reduced the cellular accumulation of saquinavir and lopinavir. Lopinavir is much more lipophilic than saquinavir. Conclusions and implications: Human OATPs, MRP, P-gp and lipophilicity determine the cellular uptake and retention of saquinavir and lopinavir. These data may have important implications for drug–drug interactions, drug safety and efficacy.

Janneh, O; Hartkoorn, R C; Jones, E; Owen, A; Ward, S A; Davey, R; Back, D J; Khoo, S H

2008-01-01

84

A new bacterial co-expression system for over-expressing soluble protein and validating protein-protein interaction.  

PubMed

Toxic, membrane, and hydrophobic proteins are usually difficult to individually over-express in Escherichia coli because they require a binding-partner protein for folding and stability. To obtain these types of soluble proteins or protein complexes, protein co-expression is used. Such co-expression systems are extremely suitable for the high-throughput validation of protein-protein interactions. In a previous study, we developed a novel co-expression vector, pHEX, which is compatible, and thus can be partnered, with many commercially available E. coli vectors, such as pGEX and pMAL. Either of the vectors allows proteins to be expressed individually as a tagged fusion protein and can be used directly for protein co-purification. This protocol presents the experimental procedure for the co-expression method. PMID:22160902

Zeng, Jumei; He, Zheng-Guo

2012-01-01

85

Insight into the Cooperation of P-glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) at the Blood-Brain Barrier: A Case Study Examining Sorafenib Efflux Clearance  

PubMed Central

The ATP-binding cassette transporters p-glycoprotein and breast cancer resistance protein have been shown to be critical determinants limiting drug transport across the BBB into the brain. Several therapeutic agents have been shown to be substrates for these two transporters, and as a result they have limited distribution to the brain. Recently, it has been shown that these two drug transporters cooperate at the BBB and brain penetration of dual substrates increase significantly only when both are absent, e.g., in the Mdr1a/1b-/-Bcrp1-/- mice. The present study uses the brain penetration of sorafenib to investigate these findings and attempts to explain the mechanistic basis of this cooperation with a simple theory based on affinity and capacity dependent carrier-mediated transport. The brain efflux index method, combined with the organotypic brain slices, were used to determine the net contribution of P-gp and BCRP to the total clearance of sorafenib out of the brain and show that its efflux at the BBB is mediated primarily by BCRP. Sorafenib clearance out of the brain decreased 2-fold in the Bcrp1-/- mice and 2.5-fold in the Mdr1a/1b-/-Bcrp1-/- mice. Clearance out of brain when P-gp was absent did not change significantly compared to wild-type. We also investigated the expression of P-gp and BCRP in the genetic knockout animals and saw no differences in either P-gp or BCRP in the transporter deficient mice compared to the wild-type mice. In conclusion, this study explains the cooperation of P-gp and BCRP by analysis of the efflux clearance of sorafenib and correlating it to the ‘mechanisms’ that determine the clearance, i.e., affinity and capacity.

Agarwal, Sagar; Elmquist, William F.

2012-01-01

86

Heterologous Protein Expression in Psychrophilic Hosts  

Microsoft Academic Search

The vast number of candidate proteins generated from genome projects are creating enormous opportunities for biologists. However,\\u000a efficient expression of genes in homologous\\/heterologous expression systems and rapid purification steps are actually major\\u000a bottlenecks. In fact, although many recombinant proteins have been successfully produced from common prokaryotic (Escherichia coli) and eukaryotic (yeast and CHO cells) hosts, these conventional systems have often

Ermenegilda Parrilli; Angela Duilio; Maria Luisa Tutino

87

Expression and Evaluation of RING Finger Proteins  

Microsoft Academic Search

RING finger proteins represent the largest class of potential ubiquitin ligases. This chapter describes methods used to express and assess the activity of proteins containing RING fingers based on our experience with a number of different family members. In addition to general protocols for assessing activity, specific protocols are provided for evaluating the ubiquitylation of p53 by the RING finger

Yili Yang; Kevin L. Lorick; Jane P. Jensen; Allan M. Weissman

2005-01-01

88

Connectivity and expression in protein networks: Proteins in a complex are uniformly expressed  

NASA Astrophysics Data System (ADS)

We explore the interplay between the protein-protein interactions network and the expression of the interacting proteins. It is shown that interacting proteins are expressed in significantly more similar cellular concentrations. This is largely due to interacting pairs which are part of protein complexes. We solve a generic model of complex formation and show explicitly that complexes form most efficiently when their members have roughly the same concentrations. Therefore, the observed similarity in interacting protein concentrations could be attributed to optimization for efficiency of complex formation.

Carmi, Shai; Levanon, Erez Y.; Havlin, Shlomo; Eisenberg, Eli

2006-03-01

89

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

PubMed

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 the study the drug was withdrawn and, after 22 days, added back to the culture medium. Cells were harvested at various time points and xenografted in nude mice. P-gp content in cells was measured by self-competitive binding assay and in tumors, by quantitative autoradiography (QAR). Both assays were carried out using 125I-labeled monoclonal antibody MRK16, reactive with P-gp. Concentration of P-gp in cells varied from a maximum of 1,361 pmol/g in the presence of colchicine to a minimum of 374 pmol/g in the absence of colchicine in the culture medium. P-gp concentration in the tumors ranged from 929 to 188 pmol/g, which correlated with P-gp content in the cells at the time of their injection in the mice. QAR is an accurate and reliable method to quantify P-gp expression in tumors. Changes in colchicine concentration in the ambient medium of BE(2)-C/CHCb(0.2) cells growing in vitro resulted in a change in phenotype of P-gp expression, which was stable under conditions of in vivo growth over approximately 9 cell divisions in nude mice xenografts. Therefore, P-gp content in xenografts depends only on the level of resistance of the cells at the time of their injection in the mice. PMID:10096499

Fonti, R; Levchenko, A; Mehta, B M; Zhang, J; Tsuruo, T; Larson, S M

1999-01-01

90

Thioredoxin-interacting Protein (Txnip) Gene Expression  

PubMed Central

Thioredoxin-interacting protein (Txnip) has important functions in regulating cellular metabolism including glucose utilization; the expression of the Txnip gene is sensitive to the availability of glucose and other fuels. Here, we show that Txnip expression is down-regulated at the transcriptional level by diverse inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). The effect of these OXPHOS inhibitors is mediated by earlier identified carbohydrate-response elements (ChoREs) on the Txnip promoter and the ChoRE-associated transcription factors Max-like protein X (MLX) and MondoA (or carbohydrate-response element-binding protein (ChREBP)) involved in glucose-induced Txnip expression, suggesting that inhibited oxidative phosphorylation compromises glucose-induced effects on Txnip expression. We also show that the OXPHOS inhibitors repress the Txnip transcription most likely by inducing the glycolytic rate, and increased glycolytic flux decreases the levels of glycolytic intermediates important for the function of MLX and MondoA (or ChREBP). Our findings suggest that the Txnip expression is tightly correlated with glycolytic flux, which is regulated by oxidative phosphorylation status. The identified link between the Txnip expression and glycolytic activity implies a mechanism by which the cellular glucose uptake/homeostasis is regulated in response to various metabolic cues, oxidative phosphorylation status, and other physiological signals, and this may facilitate our efforts toward understanding metabolism in normal or cancer cells.

Yu, Fa-Xing; Chai, Tin Fan; He, Hongpeng; Hagen, Thilo; Luo, Yan

2010-01-01

91

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

92

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

93

Differential Liver Protein Expression during Schistosomiasis  

Microsoft Academic Search

The arrival of eggs in the liver during Schistosoma mansoni infection initiates a protective granulomatous response; however, as the infection progresses, this response results in chronic liver fibrosis. To better understand the impact of schistosomiasis on liver function, we used a proteomic approach to identify proteins whose expression was significantly altered in schistosome-infected mice 8 weeks postinfection. Identification of differentially

Marina Harvie; Thomas William Jordan; Anne Camille La Flamme

2007-01-01

94

Expression pattern of id proteins in medulloblastoma.  

PubMed

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. PMID:23397264

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; Pierson, Christopher R

2013-02-09

95

Detecting Selective Expression of Genes and Proteins  

PubMed Central

Selective expression of a gene product (mRNA or protein) is a pattern in which the expression is markedly high, or markedly low, in one particular tissue compared with its level in other tissues or sources. We present a computational method for the identification of such patterns. The method combines assessments of the reliability of expression quantitation with a statistical test of expression distribution patterns. The method is applicable to small studies or to data mining of abundance data from expression databases, whether mRNA or protein. Though the method was developed originally for gene-expression analyses, the computational method is, in fact, rather general. It is well suited for the identification of exceptional values in many sorts of intensity data, even noisy data, for which assessments of confidences in the sources of the intensities are available. Moreover, the method is indifferent as to whether the intensities are experimentally or computationally derived. We show details of the general method and examples of computational results on gene abundance data.

Greller, Larry D.; Tobin, Frank L.

1999-01-01

96

Effects of monoglycerides on P-glycoprotein: modulation of the activity and expression in Caco-2 cell monolayers.  

PubMed

The purpose of this study was to analyze the effects of two common monoglyceride components of lipid excipients, 1-monoolein and 1-monostearin, on the activity and expression of P-glycoprotein (P-gp) in Caco-2 cells. Non-cytotoxic concentrations of 1-monoolein and 1-monostearin were determined by assessing membrane permeability and mitochondrial activity in Caco-2 cells, a human colon adenocarcinoma cell line. Concentrations of 500 and 100 microM were used to evaluate P-gp activity through Rh123 accumulation and bifunctional transport studies. The P-gp protein expression levels were quantified through the use of immunoblots. The changes in cell membrane fluidity and nuclear membrane integrity upon the addition of monoglycerides were analyzed by fluorescence anisotropy using DPH and TMA-DPH as the fluorescent labels and by using increasing salt concentrations to release the nuclear contents, respectively. The absorptive flux (apical to basolateral) in the bifunctional transport studies was not found to be statistically significant for the non-cytotoxic concentrations of 1-monoolein and 1-monostearin. However, treatments of 500 and 100 microM of 1-monoolein or 1-monostearin displayed statistically lowered efflux (basolaterial to apical, P < 0.05) compared to the controls (7.9 +/- 0.8, 12.9 +/- 2.6 x 10 (6) cm/s for 1-monoolein or 11.1 +/- 2.0, 11.4 +/- 2.3 x 10 (6) cm/s for 1-monostearin, respectively, compared to the untreated control, 21.1 +/- 2.9 x 10 (6) cm/s, n = 5). Rh123 accumulation was also found to be enhanced upon 24 h incubation with both concentrations of the monoglycerides; however, only concentrations of 500 muM of the monoglycerides were shown to significantly reduce the P-gp protein expression. The results from this study suggest that these two monoglycerides, common components in various lipid excipients, are inhibitors of P-gp. PMID:18651749

Barta, Cheri A; Sachs-Barrable, Kristina; Feng, Florina; Wasan, Kishor M

2008-07-24

97

Streamlined Expressed Protein Ligation Using Split Inteins  

PubMed Central

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

2012-01-01

98

Polarized P-glycoprotein expression by the immortalised human brain endothelial cell line, hCMEC/D3, restricts apical-to-basolateral permeability to rhodamine 123.  

PubMed

P-glycoprotein (P-gp) expression at the blood-brain barrier prevents unwanted blood-borne toxins and signalling molecules from entering the brain. Primary and immortalised human brain endothelial cells (BECs) represent two suitable options for studying P-gp function in vitro. The limited supply of primary human BECs and their instability over passage number make this choice unattractive for medium/high throughput studies. The aim of this study was to further characterise the expression of P-gp by an immortalised human BEC line, hCMEC/D3, in order to evaluate their use as an in vitro human blood-brain barrier model. P-gp expression was stable over a high passage number (up to passage 38) and was polarised on the apical plasma membrane, consistent with human BECs in vivo. In addition, hCMEC/D3 cell P-gp expression was comparable, albeit slightly lower to that observed in primary isolated human BECs although P-gp function was similar in both cell lines. The P-gp inhibitors tariquidar and vinblastine prevented the efflux of rhodamine 123 (rh123) from hCMEC/D3 cells, indicative of functional P-gp expression. hCMEC/D3 cells also displayed polarised P-gp transport, since both tariquidar and vinblasine selectively increased the apical-to-basolateral permeability of hCMEC/D3 cells to rh123. The results presented here demonstrate that hCMEC/D3 cells are a suitable model to investigate substrate specificity of P-gp in BECs of human origin. PMID:19631619

Tai, Leon M; Reddy, P Sreekanth; Lopez-Ramirez, M Alejandro; Davies, Heather A; Male, David K; Male, A David K; Loughlin, A Jane; Romero, Ignacio A

2009-07-23

99

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

100

Expression of heat shock proteins in medulloblastoma.  

PubMed

Object Medulloblastoma (MB) is the most common malignant brain tumor in children. Heat shock proteins (HSPs) comprise a superfamily of proteins that serve as molecular chaperones and are overexpressed in a wide range of human cancers. The purpose of the present study was to investigate the expression of HSP27 (pSer(82)), HSP27 (pSer(15)), HSP40, HSP60, HSP70, HSP90-?, Akt, and phospho-Akt by multiplex bead array assay of MBs. The results of HSP and Akt expression were correlated with MB subtype; immunohistochemical expression of Ki-67 index, bcl-2, and p53; and patients' prognosis. Methods The authors retrospectively evaluated 25 children with MB who underwent surgery. Immunohistochemical analysis of Ki-67, p53, and bcl-2 expression was performed in all cases. By using multiplex bead array assay, a simultaneous detection of HSP27 (pSer(82)), HSP27 (pSer(15)), HSP40, HSP60, HSP70, HSP90-?, Akt, and phospho-Akt was performed. Results Medulloblastoma with extensive nodularity had significantly lower HSP27 (pSer(15)) expression (p = 0.039) but significantly higher HSP60 expression (p = 0.021) than classic MB. Large-cell MB had significantly higher HSP70 expression (p = 0.028) than classic MB. No significant difference was found between HSP27 (pSer(82)), HSP40, HSP90-?, Akt, or phospho-Akt expression and MB subtype. Large-cell MBs had significantly higher Ki-67 index compared with classic MBs (p = 0.033). When analyzing all MBs, there was a significant negative correlation between HSP27 (pSer(15)) and Ki-67 index (r = -0.475, p = 0.016); a significant positive correlation between HSP70 expression and Ki-67 index (r = 0.407, p = 0.043); and a significant positive correlation between HSP70 expression and bcl-2 index (r = 0.491, p = 0.023). Patients with large-cell MB had a worse survival than those with classic MB, but the difference did not reach statistical significance (p = 0.076). Conclusions A substantial expression of several HSPs in MB was observed. Given that HSPs represent an attractive strategy for anticancer therapy, further studies, involving larger series of patients, are obviously necessary to clarify the relationship of HSPs with tumor aggressiveness and prognosis. PMID:23992239

Alexiou, George A; Vartholomatos, George; Stefanaki, Kalliopi; Patereli, Amalia; Dova, Lefkothea; Karamoutsios, Achilleas; Lallas, George; Sfakianos, George; Moschovi, Maria; Prodromou, Neofytos

2013-08-30

101

Global analysis of protein expression in yeast  

Microsoft Academic Search

The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins-and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect

Sina Ghaemmaghami; Won-Ki Huh; Kiowa Bower; Russell W. Howson; Archana Belle; Noah Dephoure; Erin K. O'Shea; Jonathan S. Weissman

2003-01-01

102

The "Specific" P-Glycoprotein Inhibitor Tariquidar Is Also a Substrate and an Inhibitor for Breast Cancer Resistance Protein (BCRP/ABCG2)  

PubMed Central

Tariquidar was developed as a specific inhibitor of the efflux transporter ABCB1. Recent positron emission tomographic brain imaging studies using [11C]tariquidar to measure ABCB1 (P-gp, P-glycoprotein) density in mice indicate that the inhibitor may not be as specific as previously thought. We examined its selectivity as an inhibitor and a substrate for the human transporters P-gp, breast cancer resistance protein (BCRP, ABCG2), and multidrug resistance protein 1 (MRP1, ABCC1). Our results show that at low concentrations, tariquidar acts selectively as an inhibitor of P-gp and also as a substrate of BCRP. At much higher concentrations (?100 nM), tariquidar acts as an inhibitor of both P-gp and BCRP. Thus, the in vivo specificity of tariquidar depends on concentration and the relative density and capacity of P-gp vs BCRP.

2010-01-01

103

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

PubMed Central

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

2012-01-01

104

Conversion of Bacterially Expressed Recombinant Prion Protein  

PubMed Central

The infectivity associated with prion disease sets it apart from a large group of late-onset neurodegenerative disorders that shares the characteristics of protein aggregation and neurodegeneration. The unconventional infectious agent, PrPSc, is an aberrantly folded form of the normal prion protein (PrPC) and the PrPC-to-PrPSc conversion is a critical pathogenic step in prion disease. Using the Protein Misfolding Cyclic Amplification technique, we converted folded bacterially expressed recombinant PrP into a Proteinase K-resistant and aggregated conformation (rPrP-res) in the presence of anionic lipid and RNA molecules. Moreover, high prion infectivity was demonstrated by intracerebral inoculation of rPrP-res into wild-type mice, which caused prion disease with a short incubation period. The establishment of the in vitro recombinant PrP conversion assay makes it feasible for us to explore the molecular basis behind the intriguing properties associated with prion infectivity.

Wang, Fei; Wang, Xinhe; Ma, Jiyan

2011-01-01

105

Characterization of P-glycoprotein Expression as a Multixenobiotic Resistance Mechanism in Fish.  

National Technical Information Service (NTIS)

P-glycoproteins, responsible for multidrug resistance, function as energy dependent efflux flippases that prevent the cellular accumulation of moderately hydrophobic chemicals. We characterized P-gp expression in populations of several fish species expose...

S. M. Bard

2001-01-01

106

Effect of fractionated irradiation on the expression of multidrug resistance genes in the CNE1 human nasopharyngeal carcinoma cell line.  

PubMed

Nasopharyngeal carcinoma (NPC) often develops drug resistance following radiotherapy. The molecular basis of radiotherapy-related multidrug resistance (MDR) remains unclear. In the present study, we investigated the effect of fractionated irradiation on the expression of the MDR-1 gene and the MDR-associated protein P-glycoprotein (P-gp) in CNE1 human NPC cells. CNE1 cells were treated with fractionated X-rays. Drug resistance was determined by MTT assay. The expression levels of MDR-1 and P-gp were analyzed by RT-PCR and western blot analysis, respectively. Differential expression was analyzed by gene chips. The results revealed that low levels of mRNA expression of MDR1 were present in non-irradiated CNE1 cells. Compared with the control, the expression of MDR1 mRNA was gradually increased following fractionated irradiation. On day 21, the expression of MDR1 mRNA was increased 1.59- and 2.19-fold, compared with the control, by treatment with 10 and 20 Gy, respectively. We observed decreased MDR1 expression following treatment with 10 and 20 Gy irradiation on days 28 and 35, compared with day 21. On days 21, 28 and 35, expression was increased 1.37-, 1.40- and 1.15-fold by treatment with 20 Gy compared with 10 Gy. Expression of MDR1 was significantly upregulated by treatment with 50 Gy irradiation compared with the control on days 78 and 106. P-gp expression was consistent with that of MDR1 mRNA expression. The sensitivity of CNE1 cells to cisplatin was reduced following irradiation compared with the control. A total of 26 genes were significantly upregulated and 8 genes were significantly downregulated compared with the control. Results of the present study have shown that MDR1 and P-gp are upregulated in CNE1 cells following irradiation. Multiple genes were involved in the mechanism of radiation-induced drug resistance. PMID:23128850

Ji, Xue-Ning; Yang, Fang; Sui, Xiao-Mei; Wang, Fu-Guang; Ge, Ri-Guang; Quan, Xiu-Lian; Zhao, Tong; Gao, Bin Wen; Wang, Ruo-Yu

2012-10-24

107

Quercetin as a potential modulator of P-glycoprotein expression and function in cells of human pancreatic carcinoma line resistant to daunorubicin.  

PubMed

P-glycoprotein (P-gp) is one of the ABC transporters responsible for the resistance of several tumours to successful chemotherapy. Numerous agents are capable of interfering with the P-gp-mediated export of drugs but unfortunately most of them produce serious side effects. Some plant polyphenols, including the flavonol quercetin (Q), manifest anti-neoplastic activity mainly due to their influence on cell cycle control and apoptosis. Reports are also available which show that Q may intensify action of cytostatic drugs and suppress the multidrug resistance (MDR) phenomenon. The study aimed at determination if Q sensitizes cells resistant to daunorubicin (DB) through its effect on P-gp expression and action. The experiments were conducted on two cell lines of human pancreatic carcinoma, resistant to DB EPP85-181RDB and sensitive EPP85-181P as a comparison. Cells of both lines were exposed to selected concentrations of Q and DB, and then membranous expression of P-gp and its transport function were examined. The influence on expression of gene for P-gp (ABCB1) was also investigated. Results of the studies confirmed that Q affects expression and function of P-gp in a concentration-dependent manner. Moreover it decreased expression of ABCB1. Thus, Q may be considered as a potential modulator of P-gp. PMID:20335952

Borska, Sylwia; Sopel, Miroslaw; Chmielewska, Magdalena; Zabel, Maciej; Dziegiel, Piotr

2010-02-09

108

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

Microsoft Academic Search

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

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

2001-01-01

109

Influence of the pro-inflammatory cytokines on P-glycoprotein expression and functionality  

Microsoft Academic Search

Purpose: P-glycoprotein (P-gp) is involved in the transport of many drugs at different barriers with consequence in terms of drug distribution and elimina- tion. The expression and activity of P-gp can be modu- lated by different factors and pathologies. The present article reviews the knowledge regarding the effect of pro-inflammatory cytokines (TNF?, IL-1?, IL-6, IL-2, IFN? ) on the expression

Christine Fernandez; Marion Buyse; Michèle German-Fattal; François Gimenez

110

Altered intestinal P-glycoprotein expression levels in a monosodium glutamate-induced obese mouse model  

Microsoft Academic Search

AimsP-glycoprotein (P-gp) is an important drug efflux transporter located in many tissues such as the blood–brain barrier, intestines, liver and kidneys. We have previously reported that ileal P-gp expression levels decrease via a nitric oxide synthase (NOS)-mediated pathway in a streptozotocin (STZ)-induced type 1 diabetic mouse model. Herein, our objective was to assess whether there are differences in the expression

Ayaka Nawa; Wakako Fujita-Hamabe; Shogo Tokuyama

111

Modulation of vinca-alkaloid induced P-glycoprotein expression by indole-3-carbinol  

Microsoft Academic Search

The over-expression of mdr-1 gene transcript P-glycoprotein (P-gp), responsible for multiple drug resistance, is one of the major obstacles in cancer chemotherapy. In the present study, indole-3-carbinol (I3C), a well-known chemopreventive agent present in cruciferous vegetables, has been evaluated for its potential to modulate the over-expression of P-gp induced by vinblastine or vincristine, which are known inducers of mdr-1 gene.

Annu Arora; Yogeshwer Shukla

2003-01-01

112

Expression of P-glycoprotein in killifish (Fundulus heteroclitus) exposed to environmental xenobiotics.  

PubMed

P-glycoproteins (P-gp) are transmembrane efflux flippases that prevent the cellular accumulation of moderately hydrophobic compounds and are responsible for certain multidrug resistance phenotypes in tumor cell lines and human patients. We investigated whether P-gps could be involved in a contaminant resistant phenotype observed in a population of fish exposed over generations to high levels of planar halogenated aromatic hydrocarbons (PHAHs). Hepatic and intestinal epithelial P-gp expression was examined by immunoblot and immunohistochemistry in killifish (Fundulus heteroclitus) from New Bedford Harbor, MA (NBH), a Superfund site highly contaminated with PHAHs, and from Scorton Creek on Cape Cod, MA (SC), a relatively unpolluted site. The NBH population has developed resistance to the toxicity of PHAHs. Hepatic P-gp levels were more than 40% greater in fish freshly collected from SC than in fish freshly collected from NBH. When killifish from either site were maintained in clean water for up to 78 days to permit depuration of bioaccumulated contaminants, hepatic P-gp levels decreased approximately 50% by day 8. P-glycoprotein expression was detected in the intestinal epithelium in 55% of freshly collected NBH fish. However, depurated NBH fish and freshly caught and depurated SC fish rarely expressed P-gp in the intestine. In an effort to determine whether environmental chemicals at the two sites might contribute to altered P-gp expression, depurated fish were exposed either to sediment collected from SC or 2,3,7,8-tetrachlorodibenzofuran, a contaminant found at the NBH site and a model aryl hydrocarbon receptor agonist. Neither exposure affected hepatic P-gp levels in killifish. Elevated intestinal P-gp in NBH fish might counter the absorption of P-gp substrates/inducers and thus limit the amount of these compounds reaching the liver, which might account for the lower hepatic P-gp levels in NBH fish compared to SC fish. The differences in hepatic P-gp levels (SC>NBH) and intestinal P-gp (NBH>SC) in freshly collected fish also might reflect environmental exposure to different anthropogenic contaminants or microbial, algal, plant or other natural products via the water column, sediment, or diet at each site. PMID:12127740

Bard, Shannon Mala; Bello, Susan M; Hahn, Mark E; Stegeman, John J

2002-09-24

113

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

2011-08-10

114

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

115

Blood-brain barrier (BBB) pharmacoproteomics: reconstruction of in vivo brain distribution of 11 P-glycoprotein substrates based on the BBB transporter protein concentration, in vitro intrinsic transport activity, and unbound fraction in plasma and brain in mice.  

PubMed

The purpose of this study was to examine whether in vivo drug distribution to the brain can be reconstructed by integrating P-glycoprotein (P-gp)/mdr1a expression levels, P-gp in vitro activity, and drug unbound fractions in mouse plasma and brain. For 11 P-gp substrates, in vitro P-gp transport activities were determined by measuring transcellular transport across monolayers of mouse P-gp-transfected LLC-PK1 (L-mdr1a) and parental cells. P-gp expression amounts were determined by quantitative targeted absolute proteomics. Unbound drug fractions in plasma and brain were obtained from the literature and by measuring brain slice uptake, respectively. Brain-to-plasma concentration ratios (K(p brain)) and its ratios between wild-type and mdr1a/1b(-/-) mice (K(p brain) ratio) were obtained from the literature or determined by intravenous constant infusion. Unbound brain-to-plasma concentration ratios (K(p,uu,brain)) were estimated from K(p brain) and unbound fractions. Based on pharmacokinetic theory, K(p brain) ratios were reconstructed from in vitro P-gp transport activities and P-gp expression amounts in L-mdr1a cells and mouse brain capillaries. All reconstructed K(p brain) ratios were within a 1.6-fold range of observed values. K(p brain) then was reconstructed from the reconstructed K(p brain) ratios and unbound fractions. K(p,uu,brain) was reconstructed as the reciprocal of the reconstructed K(p brain) ratios. For quinidine, loperamide, risperidone, indinavir, dexamethasone, paclitaxel, verapamil, loratadine, and diazepam, the reconstructed K(p brain) and K(p,uu,brain) agreed with observed and estimated in vivo values within a 3-fold range, respectively. Thus, brain distributions of P-gp substrates can be reconstructed from P-gp expression levels, in vitro activity, and drug unbound fractions. PMID:21828264

Uchida, Yasuo; Ohtsuki, Sumio; Kamiie, Junichi; Terasaki, Tetsuya

2011-08-09

116

Imaging bacterial protein expression using genetically encoded RNA sensors.  

PubMed

The difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome by using fluorescent sensors based on Spinach, an RNA that activates the fluorescence of a small-molecule fluorophore. These RNAs selectively bind target proteins and exhibit fluorescence increases that enable protein expression to be imaged in living Escherichia coli. These sensors are key components of a generalizable strategy to image protein expression in a single bacterium in real time. PMID:23872791

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

2013-07-21

117

Flavonoids inhibit breast cancer resistance protein-mediated drug resistance: transporter specificity and structure–activity relationship  

Microsoft Academic Search

Purpose  ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug\\u000a resistance-related protein 1 (MRP1), confer resistance to various anticancer agents. We previously reported that some flavonoids\\u000a have BCRP-inhibitory activity. Here we show the reversal effects of an extensive panel of flavonoids upon BCRP-, P-gp-, and\\u000a MRP1-mediated drug resistance.\\u000a \\u000a \\u000a \\u000a Methods  Reversal effects of flavonoids upon BCRP-, P-gp-,

Kazuhiro Katayama; Kazuto Masuyama; Sho Yoshioka; Hitomi Hasegawa; Junko Mitsuhashi; Yoshikazu Sugimoto

2007-01-01

118

Heat shock protein expression in diabetic nephropathy.  

PubMed

Heat shock protein (HSP) HSP27, HSP60, HSP70, and HSP90 are induced by cellular stresses and play a key role in cytoprotection. Both hyperglycemia and glomerular hypertension are crucial determinants in the pathogenesis of diabetic nephropathy and impose cellular stresses on renal target cells. We studied both the expression and the phosphorylation state of HSP27, HSP60, HSP70, and HSP90 in vivo in rats made diabetic with streptozotocin and in vitro in mesangial cells and podocytes exposed to either high glucose or mechanical stretch. Diabetic and control animals were studied 4, 12, and 24 wk after the onset of diabetes. Immunohistochemical analysis revealed an overexpression of HSP25, HSP60, and HSP72 in the diabetic outer medulla, whereas no differences were seen in the glomeruli. Similarly, exposure neither to high glucose nor to stretch altered HSP expression in mesangial cells and podocytes. By contrast, the phosphorylated form of HSP27 was enhanced in the glomerular podocytes of diabetic animals, and in vitro exposure of podocytes to stretch induced HSP27 phosphorylation via a P38-dependent mechanism. In conclusion, diabetes and diabetes-related insults differentially modulate HSP27, HSP60, and HSP70 expression/phosphorylation in the glomeruli and in the medulla, and this may affect the ability of renal cells to mount an effective cytoprotective response. PMID:18922888

Barutta, Federica; Pinach, Silvia; Giunti, Sara; Vittone, Ferdinando; Forbes, Josephine M; Chiarle, Roberto; Arnstein, Maryann; Perin, Paolo Cavallo; Camussi, Giovanni; Cooper, Mark E; Gruden, Gabriella

2008-10-15

119

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

120

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

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. PMID:23397054

Sane, Ramola; Agarwal, Sagar; Mittapalli, Rajendar K; Elmquist, William F

2013-02-08

121

TFPI-2 downregulates multidrug resistance protein in 5-FU-resistant human hepatocellular carcinoma BEL-7402/5-FU cells.  

PubMed

Tissue factor pathway inhibitor-2 (TFPI-2) is known to induce apoptosis and to suppress tumor metastasis in several types of cancer cells. However, there is little known about its reversal effect on chemoresistant tumor cells. This study investigated the effect of TFPI-2 in 5-fluorouracil (5-FU)-resistant human hepatocellular cancer BEL-7402/5-FU cells in vitro. We constructed TFPI-2 overexpression BEL-7402/5-FU cell lines and explored resistance index (RI) of 5-FU, function of the P-glycoprotein (P-gp) efflux pump, and the mRNA and protein expression of drug resistance gene, including multidrug resistance gene (MDR1), lung-resistance protein (LRP), multidrug resistance-associated protein (MRP1), glutathione-S-transferase-? (GST-?), excision repair cross-complementing gene 1 (ERCC1), and p38 phosphorylation. We found that TFPI-2 improved the RI of 5-FU and inhibited P-gp function. Western blotting and real-time PCR revealed that TFPI-2 also decreased mRNA and protein expression of MDR1, LRP, MRP1, GST-?, and ERCC1, whereas p38 phosphorylation was increased. We considered that TFPI-2 reduces 5-FU resistance in BEL-7402/5-FU cells, and the mechanism appears to involve p38-mediated downregulation of drug resistance gene expression such as MDR1, LRP, MRP1, GST-?, and ERCC1. PMID:23125179

Lu, Fei; Hou, Yong-Qiang; Song, Ying; Yuan, Zheng-Jiang

2012-11-04

122

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

123

Discordant Protein and mRNA Expression in Lung Adenocarcinomas  

Microsoft Academic Search

The relationship between gene expression measured at the mRNA level and the corresponding protein level is not well characterized in human cancer. In this study, we compared mRNA and protein expression for a cohort of genes in the same lung adenocarcinomas. The abun- dance of 165 protein spots representing 98 individual genes was analyzed in 76 lung adenocarcinomas and nine

Guoan Chen; Tarek G. Gharib; Chiang-Ching Huang; Jeremy M. G. Taylor; David E. Misek; Sharon L. R. Kardia; Thomas J. Giordano; Mark D. Iannettoni; Mark B. Orringer; Samir M. Hanash; David G. Beer

2002-01-01

124

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

Microsoft Academic Search

Six protoberberine alkaloids were isolated from the chloroform layer of the rhizome ofCoptis japonica Makino (Ranunculaceae). The structures of the isolated compounds were determined to be 6-([1,3]dioxolo[4,5-g]isoquinoline-5-carbonyl)-2,3-dimethoxy-benzoic\\u000a acid methyl ester (1), oxyberberine (2), 8-oxo-epiberberine (3), 8-oxocoptisine (4), berberine (5) and palmatine (6) by physicochemical and spectroscopic methods. The compound3 (8-oxo-epiberberine) was first isolated from natural sources. The compounds were tested

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

2006-01-01

125

Strain engineering for improved expression of recombinant proteins in bacteria  

PubMed Central

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

2011-01-01

126

Lymphokine-activated killer cell susceptibility and adhesion molecule expression of multidrug resistant breast carcinoma  

Microsoft Academic Search

Reports showing susceptibility of multidrug resistant (MDR) cancer cells to immune effectors, together with P-glycoprotein (P-gp) expression in immune effector subsets, including immature natural killer (NK) cells, and some activated T cells, suggest P-gp or some changes associated with it, have implications in immune-mediated mechanisms. A series of experiments were done to determine the nature of alterations associated with susceptibility

Burhan Savas; Pauline E Kerr; Hugh F Pross

2006-01-01

127

High-throughput expression of C. elegans proteins.  

PubMed

Proteome-scale studies of protein three-dimensional structures should provide valuable information for both investigating basic biology and developing therapeutics. Critical for these endeavors is the expression of recombinant proteins. We selected Caenorhabditis elegans as our model organism in a structural proteomics initiative because of the high quality of its genome sequence and the availability of its ORFeome, protein-encoding open reading frames (ORFs), in a flexible recombinational cloning format. We developed a robotic pipeline for recombinant protein expression, applying the Gateway cloning/expression technology and utilizing a stepwise automation strategy on an integrated robotic platform. Using the pipeline, we have carried out heterologous protein expression experiments on 10,167 ORFs of C. elegans. With one expression vector and one Escherichia coli strain, protein expression was observed for 4854 ORFs, and 1536 were soluble. Bioinformatics analysis of the data indicates that protein hydrophobicity is a key determining factor for an ORF to yield a soluble expression product. This protein expression effort has investigated the largest number of genes in any organism to date. The pipeline described here is applicable to high-throughput expression of recombinant proteins for other species, both prokaryotic and eukaryotic, provided that ORFeome resources become available. PMID:15489332

Luan, Chi-Hao; Qiu, Shihong; Finley, James B; Carson, Mike; Gray, Rita J; Huang, Wenying; Johnson, David; Tsao, Jun; Reboul, Jérôme; Vaglio, Philippe; Hill, David E; Vidal, Marc; Delucas, Lawrence J; Luo, Ming

2004-10-01

128

Protein Expression Profiling in the Spectrum of Renal Cell Carcinomas  

PubMed Central

In this study, we aimed to evaluate the protein expression profile of a spectrum of renal cell carcinomas (RCC) to find potential biomarkers for disease onset and progression and therefore, prospective therapeutic targets. A 2D-gel based proteomic analysis was used to outline differences in protein levels among different subtypes of renal cell carcinomas, including clear cell carcinomas, papillary lesions, chromophobe tumors and renal oncocytomas. Spot pattern was compared to the corresponding normal kidney from the same patients and distinctive, differentially expressed proteins were characterized by mass spectrometry. Twenty-one protein spots were found differentially expressed between clear cell RCC and normal tissue and 38 spots were found expressed in chromophobe tumors. Eleven proteins were identified, with most differentially expressed -by fold change- between clear cell tumors and the corresponding normal tissue. Two of the identified proteins, Triosephosphate isomerase 1 (TPI-1) and Heat Shock protein 27 (Hsp27), were further validated in a separate set of tumors by immunohistochemistry and expression levels were correlated with clinicopathologic features of the patients. Hsp27 was highly expressed in 82% of the tumors used for validation, and all cases showed strong immunoreactivity for TPI-1. In both Hsp27 and TPI-1, protein expression positively correlated with histologic features of the disease. Our results suggest that the subjacent cytogenetic abnormalities seen in different histological types of RCC are followed by specific changes in protein expression. From these changes, Hsp27 and TPI-1 emerged as potential candidates for the differentiation and prognosis in RCC.

Valera, Vladimir A; Li-Ning-T, Elsa; Walter, Beatriz A; Roberts, David D.; Linehan, W M; Merino, Maria J

2010-01-01

129

Strain engineering for improved expression of recombinant proteins in bacteria  

Microsoft Academic Search

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

Tomohiro Makino; Georgios Skretas; George Georgiou

2011-01-01

130

Advanced genetic strategies for recombinant protein expression in Escherichia coli  

Microsoft Academic Search

Preparations enriched by a specific protein are rarely easily obtained from natural host cells. Hence, recombinant protein pro- duction is frequently the sole applicable procedure. The ribosomal machinery, located in the cytoplasm is an outstanding catalyst of recombinant protein biosynthesis. Escherichia colifacilitates protein expression by its relative simplicity, its inexpensive and fast high-density cultivation, the well-known genetics and the large

Hans Peter Sørensen; Kim Kusk Mortensen

2005-01-01

131

Expression and Localization of Plant Protein Disulfide lsomerase  

Microsoft Academic Search

A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa 1.) was expressed in Fscherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on

Basil S. Shorrosh; Jayaram Subramaniam; Karel R. Schubert; Richard A. Dixon

1993-01-01

132

Improving expression and solubility of rice proteins produced as fusion proteins in Escherichia coli.  

PubMed

For proteins of higher eukaryotes, such as plants, which have large genomes, recombinant protein expression and purification are often difficult. Expression levels tend to be low and the expressed proteins tend to misfold and aggregate. We tested seven different expression vectors in Escherichia coli for rapid subcloning of rice genes and for protein expression and solubility levels. Each expressed gene product has an N-terminal fusion protein and/or tag, and an engineered protease site upstream of the mature rice protein. Several different fusion proteins/tags and protease sites were tested. We found that the fusion proteins and the protease sites have significant and varying effects on expression and solubility levels. The expression vector with the most favorable characteristics is pDEST-trx. The vector, which is a modified version of the commercially available expression vector, pET-32a, contains an N-terminal thioredoxin fusion protein and a hexahistidine tag, and is adapted to the Gateway expression system. However, addition of an engineered protease site could drastically change the expression and solubility properties. We selected 135 genes corresponding to potentially interesting rice proteins, transferred the genes from cDNAs to expression vectors, and engineered in suitable protease sites N-terminal to the mature proteins. Of 135 genes, 131 (97.0%) could be expressed and 72 (53.3%) were soluble when the fusion proteins/tags were present. Thirty-eight mature-length rice proteins and domains (28.1%) are suitable for NMR solution structure studies and/or X-ray crystallography. Our expression systems are useful for the production of soluble plant proteins in E. coli to be used for structural genomics studies. PMID:15914031

Tsunoda, Yuki; Sakai, Nobuya; Kikuchi, Koji; Katoh, Shizue; Akagi, Kayo; Miura-Ohnuma, Jun; Tashiro, Yumiko; Murata, Katsuyoshi; Shibuya, Naoto; Katoh, Etsuko

2005-08-01

133

Large-scale protein expression for proteome research.  

PubMed

Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility, as determined at two different induction temperatures. Affinity-purified fusion proteins were confirmed by MALDI-TOF MS. PMID:16127724

Korf, Ulrike; Kohl, Thorsten; van der Zandt, Hans; Zahn, Regina; Schleeger, Simone; Ueberle, Barbara; Wandschneider, Silke; Bechtel, Stephanie; Schnölzer, Martina; Ottleben, Holger; Wiemann, Stefan; Poustka, Annemarie

2005-09-01

134

Semliki Forest virus capsid protein expressed by a baculovirus recombinant  

Microsoft Academic Search

Summary We have constructed a recombinant baculovirus which expressed the Semliki Forest virus (SFV) capsid (C) gene as a fusion protein under the control of the polyhedrin gene promoter. The sequence coding for C and part of the envelope E3 region were expressed as a polyprotein precursor.Spodoptera frugiperda (Sf9) insect cells infected with the recombinant virus produced a protein reacting

D. Favre; E. Studer; T. Nishimura; M. Weitz; M. R. Michel

1993-01-01

135

Adenovirus vector designed for expression of toxic proteins.  

PubMed

To construct recombinant adenoviruses expressing biologically active proteins may be impossible, or result in a significant reduction in virus yield, if the protein expressed has an inhibitory effect on virus replication or cellular growth. To overcome this problem, we previously designed adenovirus vectors expressing foreign proteins from inducible promoters. However, during our work with a replication-deficient virus expressing the ASF/SF2 splicing factor from a progesterone antagonist-inducible gene cassette, we discovered that ASF/SF2 was expressed at a significant level in the 293 producer cell line, even in the absence of inducer. 293 cells code for adenovirus E1A and E1B proteins and thus support the growth of E1-deficient adenoviruses. Here we show that this background ASF/SF2 expression results from a low level of E1A-mediated transactivation of the basal promoter driving transgene expression. To overcome the problem of leaky expression, we reconstructed a novel gene cassette that combines an inducible promoter and a Lac repressor protein-based block to reduce transcriptional elongation. We show that this novel vector system dramatically reduced background transgene expression and therefore should be useful for the rescue and propagation of high-titer stocks of recombinant adenoviruses expressing toxic proteins. PMID:11559789

Edholm, D; Molin, M; Bajak, E; Akusjärvi, G

2001-10-01

136

Effects of protein maturation on the noise in gene expression.  

PubMed

Fluorescent proteins are frequently used as reporters for gene expression in living cells, either by being expressed in tandem with a protein of interest or through the creation of fusion proteins. The data yielded by the fluorescence output are of considerable interest in efforts to formulate quantitative models of cellular behavior underway in fields such as systems biology and synthetic biology. An often neglected aspect of these proteins, however, is their maturation: Before a fluorescent protein can generate a fluorescent signal, it must mature through a series of steps (folding, cyclization, and oxidation) that may take from many minutes to over a day. The presence of these maturation steps creates a distinction between the observed gene expression profile and the actual profile. We examine this effect through a simplified gene expression model and conclude that fluorescent protein maturation can have significant effects on estimates of both the mean protein levels and the variability in gene expression. The model shows that in many regimes, the observed variability will be increased by the maturation process, but indicates the existence of regimes in which the observed variability will actually be less than the true variability of the target protein. The latter effect arises from a low-pass filtering effect introduced by the chain of maturation reactions. The results suggest that the maturation of fluorescent proteins should be taken into account when using such proteins as quantitative indicators of gene expression levels. PMID:18352052

Dong, Guang Qiang; McMillen, David R

2008-02-13

137

Effects of protein maturation on the noise in gene expression  

NASA Astrophysics Data System (ADS)

Fluorescent proteins are frequently used as reporters for gene expression in living cells, either by being expressed in tandem with a protein of interest or through the creation of fusion proteins. The data yielded by the fluorescence output are of considerable interest in efforts to formulate quantitative models of cellular behavior underway in fields such as systems biology and synthetic biology. An often neglected aspect of these proteins, however, is their maturation: Before a fluorescent protein can generate a fluorescent signal, it must mature through a series of steps (folding, cyclization, and oxidation) that may take from many minutes to over a day. The presence of these maturation steps creates a distinction between the observed gene expression profile and the actual profile. We examine this effect through a simplified gene expression model and conclude that fluorescent protein maturation can have significant effects on estimates of both the mean protein levels and the variability in gene expression. The model shows that in many regimes, the observed variability will be increased by the maturation process, but indicates the existence of regimes in which the observed variability will actually be less than the true variability of the target protein. The latter effect arises from a low-pass filtering effect introduced by the chain of maturation reactions. The results suggest that the maturation of fluorescent proteins should be taken into account when using such proteins as quantitative indicators of gene expression levels.

Dong, Guang Qiang; McMillen, David R.

2008-02-01

138

Relating protein adduction to gene expression changes: a systems approach  

PubMed Central

Modification of proteins by reactive electrophiles such as the 4-hydroxy-2-nonenal (HNE) plays a critical role in oxidant-associated human diseases. However, little is known about protein adduction and the mechanism by which protein damage elicits adaptive effects and toxicity. We developed a systems approach for relating protein adduction to gene expression changes through the integration of protein adduction, gene expression, protein-DNA interaction, and protein-protein interaction data. Using a random walk strategy, we expanded a list of responsive transcription factors inferred from gene expression studies to upstream signaling networks, which in turn allowed overlaying protein adduction data on the network for the prediction of stress sensors and their associated regulatory mechanisms. We demonstrated the general applicability of transcription factor-based signaling network inference using 103 known pathways. Applying our workflow on gene expression and protein adduction data from HNE-treatment not only rediscovered known mechanisms of electrophile stress but also generated novel hypotheses regarding protein damage sensors. Although developed for analyzing protein adduction data, the framework can be easily adapted for phosphoproteomics and other types of protein modification data.

Zhang, Bing; Shi, Zhiao; Duncan, Dexter T; Prodduturi, Naresh; Marnett, Lawrence J; Liebler, Daniel C

2013-01-01

139

mir-29 Regulates Mcl-1 Protein Expression and Apoptosis  

PubMed Central

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

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

2008-01-01

140

The effects of cannabinoids on P-glycoprotein transport and expression in multidrug resistant cells.  

PubMed

Cannabis is the most widely used illicit drug in the world. Cannabinoids are used therapeutically by some patients as they have analgesic, anti-emetic and appetite stimulant properties which palliate adverse symptoms. Use of these agents in an oncology setting raises the question of whether they act to modulate the effectiveness of concurrently administered anti-cancer drugs. The transporter, P-glycoprotein (P-gp) confers multiple drug resistance (MDR) by effluxing a diverse array of anti-cancer agents. This study was undertaken to examine the effect of cannabinoids on P-gp. Unlike the known P-gp inhibitor, PSC833, short 1h exposure to three plant-derived cannabinoids, cannabinol (CBN), cannabidiol (CBD) and Delta(9)-tetrahydrocannabinol (THC) and the synthetic cannabinoid receptor agonist, WIN55, 212-2 (WIN) did not inhibit the efflux of the P-gp substrate Rhodamine 123 (Rh123) in either a drug-selected human T lymphoblastoid leukaemia cell line (CEM/VLB(100)) or in a mouse fibroblast MDR1 transfected cell line (77.1). However, in CEM/VLB(100) cells, prolonged 72 h exposure to the cannabinoids, THC and CBD, decreased P-gp expression to a similar extent as the flavonoid, curcumin (turmeric). This correlated with an increase in intracellular accumulation of Rh123 and enhanced sensitivity of the cells to the cytotoxic actions of the P-gp substrate, vinblastine. Taken together, these results provide preliminary evidence that cannabinoids do not exacerbate P-gp mediated MDR. Further, plant-derived cannabinoids are moderately effective in reversing MDR in CEM/VLB(100) cells by decreasing P-gp expression. PMID:16458258

Holland, M L; Panetta, J A; Hoskins, J M; Bebawy, M; Roufogalis, B D; Allen, J D; Arnold, J C

2006-02-02

141

An expression system for screening of proteins for glycan and protein interactions.  

PubMed

Here we describe a versatile high-throughput expression system that permits genome-wide screening of type 1 membrane and secreted proteins for interactions with glycans and proteins using both cell-expressed and soluble forms of the expressed proteins. Based on Gateway cloning methodology, we have engineered a destination vector that directs expression of enhanced green fluorescent protein (EGFP)-tagged proteins at the cell surface via a glycosylphosphatidylinositol tail. The EGFP fusion proteins can then be cleaved with PreScission protease to release soluble forms of proteins that can be optionally biotinylated. We demonstrate the utility of this cloning and expression system for selected low-affinity membrane lectins from the siglec family of sialic acid-binding immunoglobulin-like lectins, for the glycosaminoglycan-binding proteins FGF-1 and BACE, and for the heterotypic adhesion molecules JAM-B and JAM-C. Cell-expressed proteins can be evaluated for glycan interactions using polyvalent soluble glycan probes and for protein interactions using either cells or soluble proteins. Following cleavage from the cell surface, proteins were complexed in solution and sufficient avidity was achieved to measure weak protein-glycan and weak protein-protein interactions using glycan arrays and surface plasmon resonance, respectively. PMID:21211507

Otto, Diana M E; Campanero-Rhodes, Maria A; Karamanska, Rositsa; Powell, Andrew K; Bovin, Nicolai; Turnbull, Jeremy E; Field, Robert A; Blackburn, Jonathan; Feizi, Ten; Crocker, Paul R

2011-01-04

142

An expression system for screening of proteins for glycan and protein interactions  

PubMed Central

Here we describe a versatile high-throughput expression system that permits genome-wide screening of type 1 membrane and secreted proteins for interactions with glycans and proteins using both cell-expressed and soluble forms of the expressed proteins. Based on Gateway cloning methodology, we have engineered a destination vector that directs expression of enhanced green fluorescent protein (EGFP)-tagged proteins at the cell surface via a glycosylphosphatidylinositol tail. The EGFP fusion proteins can then be cleaved with PreScission protease to release soluble forms of proteins that can be optionally biotinylated. We demonstrate the utility of this cloning and expression system for selected low-affinity membrane lectins from the siglec family of sialic acid-binding immunoglobulin-like lectins, for the glycosaminoglycan-binding proteins FGF-1 and BACE, and for the heterotypic adhesion molecules JAM-B and JAM-C. Cell-expressed proteins can be evaluated for glycan interactions using polyvalent soluble glycan probes and for protein interactions using either cells or soluble proteins. Following cleavage from the cell surface, proteins were complexed in solution and sufficient avidity was achieved to measure weak protein–glycan and weak protein–protein interactions using glycan arrays and surface plasmon resonance, respectively.

Otto, Diana M.E.; Campanero-Rhodes, Maria A.; Karamanska, Rositsa; Powell, Andrew K.; Bovin, Nicolai; Turnbull, Jeremy E.; Field, Robert A.; Blackburn, Jonathan; Feizi, Ten; Crocker, Paul R.

2011-01-01

143

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

144

Correlation between gene expression profiles and protein-protein interactions within and across genomes  

Microsoft Academic Search

Motivation: Function annotation of an unclassified protein on the basis of its interaction partners is well documented in the literat- ure. Reliable predictions of interactions from other data sources such as gene expression measurements would provide a useful route to function annotation. We investigate the global relationship of protein- protein interactions with gene expression. This relationship is studied in four

Nitin Bhardwaj; Hui Lu

2005-01-01

145

Expression of heat shock protein genes in insect stress responses  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

146

The S100 protein family: History, function, and expression  

Microsoft Academic Search

The S100 family of calcium binding proteins contains approximately 16 members each of which exhibits a unique pattern of tissue\\/cell type specific expression. Although the distribution of these proteins is not restricted to the nervous system, the implication of several members of this family in nervous system development, function, and disease has sparked new interest in these proteins. We now

Danna B. Zimmer; Emily H. Cornwall; Aimee Landar; Wei Song

1995-01-01

147

Expression screening of membrane proteins with cell-free protein synthesis.  

PubMed

Detailed biophysical studies of integral membrane proteins are often hampered by sample preparation difficulties. Membrane proteins are typically difficult to express in sufficient amounts to enable the use of demanding techniques such as nuclear magnetic resonance and X-ray crystallography for structural biology. Here, we show that an inexpensive batch-based cell-free expression system can be a viable alternative for production of a wide range of different membrane proteins, both of prokaryotic and eukaryotic origin. Out of 38 tested protein constructs, 37 express at levels suitable for structural biology, i.e. enough to produce several milligrams of protein routinely and without excessive costs. This success rate was not anticipated and is even more impressive considering that more than half of the expressed proteins where of mammalian origin. A detergent screen identified Brij-58 as the, in general, most successful choice for co-translational solubilization of the expressed proteins. PMID:22270086

Isaksson, Linnéa; Enberg, Johan; Neutze, Richard; Göran Karlsson, B; Pedersen, Anders

2012-01-18

148

Exact protein distributions for stochastic models of gene expression  

NASA Astrophysics Data System (ADS)

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

Kulkarni, Rahul; Pendar, Hodjat; Platini, Thierry

2013-03-01

149

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

150

Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris.  

PubMed

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

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

2013-02-21

151

Tumor imaging with multicolor fluorescent protein expression  

Microsoft Academic Search

Imaging with fluorescent proteins has been revolutionary and has led to the new field of in vivo cell biology. Many new applications\\u000a of this technology have been developed. Green fluorescent protein (GFP)-labeled or red fluorescent protein (RFP)-labeled HT-1080\\u000a human fibrosarcoma cells were used to determine clonality of metastasis by imaging of metastatic colonies after mixed implantation\\u000a of the red and

Norio Yamamoto; Hiroyuki Tsuchiya; Robert M. Hoffman

2011-01-01

152

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

PubMed

The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals. PMID:19156736

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

2009-02-01

153

Modular cloning and protein expression of long, repetitive resilin-based proteins.  

PubMed

Resilin has emerged as a promising new biomaterial possessing attractive properties for tissue engineering applications. To date, proteins with repeating resilin motifs have been expressed with molecular weights less than 30 kDa. This work describes the development of resilin-based proteins (repeating motif derived from Anopheles gambiae) 50 kDa in size. A modular cloning scheme was utilized and features a recursive cloning technique that can seamlessly and precisely tune the number of resilin repeats. Previously-established resilin expression protocols (based on the Studier auto-induction method) were employed to express the proteins in Escherichia coli BL21(DE3)pLysS. Western blot and densitometry results demonstrated that only ~50% of expressed proteins were the desired molecular weight. This finding suggested that either protein truncation or degradation occurred during protein expression. Preventing leaky expression, lowering the culture temperature, and harvesting during exponential phase resulted in up to 94% of the expressed proteins having the desired molecular weight. These expression conditions differ from previously-published resilin expression methods and are recommended when expressing proteins with a larger number of repetitive resilin sequences. PMID:22173203

Renner, Julie N; Kim, Yeji; Cherry, Kevin M; Liu, Julie C

2011-12-08

154

Relating Whole-Genome Expression Data with Protein-Protein Interactions  

Microsoft Academic Search

We investigate the relationship of protein-protein interactions with mRNA expression levels, by integrating a variety of data sources for yeast. We focus on known protein complexes that have clearly defined interactions between their subunits. We find that subunits of the same protein complex show significant coexpression, both in terms of similarities of absolute mRNA levels and expression profiles, e.g., we

Ronald Jansen; Dov Greenbaum; Mark Gerstein

2002-01-01

155

Automated and quantitative immunocytochemical assays of Bcl-2 protein in breast carcinomas.  

PubMed Central

Expression of the bcl-2 gene was investigated in 218 human breast carcinomas by immunohistochemical analysis. Immunodetections were assessed using (1) frozen sections, (2) documented commercially available monoclonal antibody (bcl-2/124, Dako), (3) automation of immunoperoxidase technique (Ventana) and (4) quantitative evaluation of results by image analysis (SAMBA) and statistical analysis of quantitative data (BMDP software). Bcl-2 protein expression was correlated with current prognostic indicators and with molecular markers detected by the same procedure as for Bcl-2. It was shown that Bcl-2 expression is not related to patients' age, tumour size and type or lymph node status, but an inverse relationship was observed between Bcl-2 and tumour grade (P < 0.0001). An inverse relationship was also observed between Bcl-2 expression and p53 (P < 0.0001), Ki67/MIB1 antigen- (P = 0.0012), and P-gp- (P = 0.002) positive immunoreactions. In contrast, anti-Bcl-2 positive reaction was significantly associated with ER-positive (P < 0.001) and with ER/PR-positive or ER/PR/pS2-positive immunoreactions (P < or = 0.005). Bcl-2 expression was independent of CD31 and cathepsin D expression. Thus, Bcl-2 protein, thought to be antiapoptotic, exhibits parodoxical expression in human breast carcinomas. It is strongly detected in low-grade tumours (well-differentiated) with low (MIB1) growth fraction, but is independent of the tumour progression (size, node status, CD31, and cathepsin D). Bcl-2 acting on apoptosis is related to p53 gene abnormalities in breast carcinomas. Bcl-2 protein expression may also be involved in response to endocrine therapy (associated to ER/PR/pS2 positive immunoreactions) and probably with chemoresistance mechanisms (inverse relationship with P-gp). Images Figure 1 Figure 2

Charpin, C.; Garcia, S.; Bouvier, C.; Devictor, B.; Andrac, L.; Lavaut, M. N.; Allasia, C.

1997-01-01

156

Expression of trisomic proteins in Down syndrome model systems.  

PubMed

Down syndrome (DS) is the most common genetic aberration leading to intellectual disability. DS results from an extra copy of the long arm of human chromosome 21 (HSA21) and the increased expression of trisomic genes due to gene dosage. While expression in DS and DS models has been studied extensively at the RNA level, much less is known about expression of trisomic genes at the protein level. We have used quantitative Western blotting with antibodies to 20 proteins encoded by HSA21 to assess trisomic protein expression in lymphoblastoid cell lines (LCLs) from patients with DS and in brains from two mouse models of DS. These antibodies have recently become available and the 20 proteins largely have not been investigated previously for their potential contributions to the phenotypic features of DS. Twelve proteins had detectable expression in LCLs and three, CCT8, MX1 and PWP2, showed elevated levels in LCLs derived from patients with DS compared with controls. Antibodies against 15 proteins detected bands of appropriate sizes in lysates from mouse brain cortex. Genes for 12 of these proteins are trisomic in the Tc1 mouse model of DS, but only SIM2 and ZNF295 showed elevated expression in Tc1 cortex when compared with controls. Genes for eight of the 15 proteins are trisomic in the Ts65Dn mouse model of DS, but only ZNF294 was over expressed in cortex. Comparison of trisomic gene expression at the protein level with previous reports at the mRNA level showed many inconsistencies. These may be caused by natural inter-individual variability, differences in the age of mice analyzed, or post-transcriptional regulation of gene dosage effects. These antibodies provide resources for further investigation of the molecular basis of intellectual disability in DS. PMID:23103828

Spellman, Claire; Ahmed, Md Mahiuddin; Dubach, Daphne; Gardiner, Katheleen J

2012-10-26

157

Expression of Hedgehog Proteins in the Human Thymus  

PubMed Central

SUMMARY The Hedgehog (Hh) family of secreted proteins includes intercellular signaling molecules that specify cell fate and patterning during the development of many tissues. In this study we show that the different components of the Hh signaling pathway are expressed in human thymus. The three mammalian Hh proteins, Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog, are produced by thymic epithelial cells. Shh-expressing epithelial cells are restricted to the thymic subcapsula and medulla, whereas Ihh- and Dhh-producing epithelial cells are distributed throughout the thymus. The requisite Hh receptors, Patched 1(Ptc1) and Smoothened (Smo), and the Gli transcription factors are expressed by thymocytes and also by epithelial cells. Ptc1 is expressed in most thymocyte subsets, whereas Smo expression is mainly associated with immature thymocytes. The isoform of the Ptc receptor, Ptc2, is expressed only by intrathymic progenitor cells and epithelial cells. Other Hh-binding proteins with modulating functions, such as Hedgehog-interacting protein (Hip) and growth arrest-specific gene-1 (Gas-1), are also expressed in human thymus. Our study shows that the intrathymic expression pattern of the Hh signaling pathway components is complex and suggests that Hh proteins may regulate human thymocyte differentiation from the earliest developmental stages, as well as thymic epithelial cell function.

Sacedon, Rosa; Varas, Alberto; Hernandez-Lopez, Carmen; Gutierrez-deFrias, Cruz; Crompton, Tessa; Zapata, Agustin G.; Vicente, Angeles

2005-01-01

158

Induction of multidrug resistance in MOLT4 cells by anticancer agents is closely related to increased expression of functional P-glycoprotein and MDR1 mRNA  

Microsoft Academic Search

Purpose: The aim of this study was to investigate the multidrug resistance (MDR) pattern, MDR gene and P-glycoprotein (P-gp) expression, and P-gp function in drug-induced human T-lymphoblastoid leukemia MOLT-4 sublines. Methods: The MDR sublines were developed by exposing the parental MOLT-4 cells to stepwise increasing concentrations of anticancer drugs daunorubicin (DNR), vinblastine (VBL) and doxorubicin (DOX). Degrees of resistance were

Zhen-Li Liu; Kenji Onda; Sachiko Tanaka; Tsugutoshi Toma; Toshihiko Hirano; Kitaro Oka

2002-01-01

159

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

160

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

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

161

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

162

Expression of human salivary protein genes  

Microsoft Academic Search

Human proline-rich proteins (PRPs) are polymorphic, homologous in sequence, and linked in a cluster called the human salivary protein complex (SPC). Recently this complex was localized to human chromosome band 12p13.2 (Mamulaet al., Cytogenet. Cell Genet. 39:279, 1985). We have isolated a PRP cDNA, EO27, from a human parotid gland library, identified it by DNA sequencing, and used it to

Paul W. Mamula; Debra J. Morley; Steven H. Larsen; Robert C. Karn

1988-01-01

163

DYSREGULATION OF PROMYELOCYTIC LEUKEMIA (PML) PROTEIN EXPRESSION IN PREECLAMPTIC PLACENTAE  

PubMed Central

Promyelocytic leukemia (PML) protein is a nucleoprotein that can regulate a variety of cellular stress responses. The aim of this study was to determine qualitative and quantitative changes in PML expression in preeclamptic placentae. Immunoblot, qRT-PCR and immunohistochemistry techniques were used to determine PML gene expression and localization in normal (n=6) and preeclamptic (n=6) placentae and primary cells. PML protein was immunolocalized within nuclei of villus mesenchyme, but largely absent in trophoblast nuclei, with a trend for increased PML reactivity in preeclamptic placenta. Immunoblot analyses of nuclear extracts confirmed relative increases (~3 fold) of PML expression in preeclamptic placentae (p<0.05). Conversely, less PML mRNA (~2 fold) was detected in preeclamptic versus normal placental samples. In vitro, PML expression could be increased by hypoxia in cultured endothelial cells but not trophoblast. Increased PML protein expression in preeclamptic villi suggests it could contribute to decreased vascularity and placental growth and/or function.

LEAVENWORTH, Jonathan D.; GROESCH, Kathleen A.; HU, Xin; MALM, Scott; TORRY, Ronald J.; ABRAMS, Robert; TORRY, Donald S.

2010-01-01

164

Heterologous gene expression in a membrane-protein-specific system.  

PubMed

We have constructed an expression system for heterologous proteins which uses the molecular machinery responsible for the high level production of bacteriorhodopsin in Halobacterium salinarum. Cloning vectors were assembled that fused sequences of the bacterio-opsin gene (bop) to coding sequences of heterologous genes and generated DNA fragments with cloning sites that permitted transfer of fused genes into H. salinarum expression vectors. Gene fusions include: (i) carboxyl-terminal-tagged bacterio-opsin; (ii) a carboxyl-terminal fusion with the catalytic subunit of the Escherichia coli aspartate transcarbamylase; (iii) the human muscarinic receptor, subtype M1; (iv) the human serotonin receptor, type 5HT2c; and (v) the yeast alpha mating factor receptor, Ste2. Characterization of the expression of these fusions revealed that the bop gene coding region contains previously undescribed molecular determinants which are critical for high level expression. For example, introduction of immunogenic and purification tag sequences into the C-terminal coding region significantly decreased bop gene mRNA and protein accumulation. The bacteriorhodopsin-aspartate transcarbamylase fusion protein was expressed at 7 mg per liter of culture, demonstrating that E. coli codon usage bias did not limit the system's potential for high level expression. The work presented describes initial efforts in the development of a novel heterologous protein expression system, which may have unique advantages for producing multiple milligram quantities of membrane-associated proteins. PMID:10545281

Turner, G J; Reusch, R; Winter-Vann, A M; Martinez, L; Betlach, M C

1999-11-01

165

Combining gene expression profiles and protein–protein interaction data to infer gene functions  

Microsoft Academic Search

The ever-increasing flow of gene expression profiles and protein–protein interactions has catalyzed many computational approaches for inference of gene functions. Despite all the efforts, there is still room for improvement, for the information enriched in each biological data source has not been exploited to its fullness. A composite method is proposed for classifying unannotated genes based on expression data and

Kang Tu; Hui Yu; Yi-Xue Li

2006-01-01

166

Heterologous Gene Expression in a Membrane-Protein-Specific System  

Microsoft Academic Search

We have constructed an expression system for heterologous proteins which uses the molecular machinery responsible for the high level production of bacteriorhodopsin in Halobacterium salinarum. Cloning vectors were assembled that fused sequences of the bacterio-opsin gene (bop) to coding sequences of heterologous genes and generated DNA fragments with cloning sites that permitted transfer of fused genes into H. salinarum expression

George J. Turner; Regina Reusch; Ann M. Winter-Vann; Lynell Martinez; Mary C. Betlach

1999-01-01

167

Production of membrane proteins using cell-free expression systems  

Microsoft Academic Search

Production of membrane proteins (MPs) is a challenging task as their hydrophobic nature and their specific requirements in cellular expression systems frequently prevent an efficient syn- thesis. Cell-free (CF) expression systems have been developed in recent times as promising tools by offering completely new approaches to synthesize MPs directly into artificial hydrophobic environments. A considerable variety of CF produced MPs

Daniel Schwarz; Volker Dötsch; Frank Bernhard

2008-01-01

168

Functional modules by relating protein interaction networks and gene expression  

Microsoft Academic Search

Genes and proteins are organized on the basis of their particular mutual relations or according to their interactions in cellular and genetic networks. These include metabolic or signaling pathways and protein interaction, regulatory or co-expression net- works. Integrating the information from the different types of networks may lead to the notion of a func- tional network and functional modules. To

Sabine Tornow; H. W. Mewes

2003-01-01

169

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

170

Protein Kinase C Alpha Expression in Breast and Ovarian Cancer  

Microsoft Academic Search

In recent years research has focused on the development of specific, targeted drugs to treat cancer. One approach has been to block intracellular signaling proteins, such as protein kinase C alpha (PKC-?). To help support the rationale for clinical studies of a PKC-?-targeted therapy in breast and ovarian cancers, we reviewed publications studying PKC-? expression in these tumors. Since these

Michael Lahn; Gabriele Köhler; Karen Sundell; Chen Su; Shuyu Li; Blake M. Paterson; Thomas F. Bumol

2004-01-01

171

Ribozymes, riboswitches and beyond: regulation of gene expression without proteins  

Microsoft Academic Search

Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and

Alexander Serganov; Dinshaw J. Patel

2007-01-01

172

Plasmids for expression of heterologous proteins in Rhizopus oryzae.  

PubMed

Rhizopus oryzae has long been used for enzyme production (e.g., glucoamylase and lipase), organic acid synthesis, and various fermented food applications. In this work, we describe a set of plasmid-based expression vectors that can be used for the production of heterologous proteins in R. oryzae. Three plasmid vectors have been created using either the glucoamylase A (amyA), pyruvate decarboxylase (pdcA), or phosphoglycerate kinase (pgk1) promoters to drive expression of heterologous proteins. All three plasmids use the pdcA terminator for transcription termination, the pyrG gene for restoration of uracil prototrophy, and an ampicillin resistance gene and origin of replication for maintenance in Escherichia coli. We have expressed green fluorescent protein (GFP) and compared transcription and protein accumulation for each of the expression vectors. Accumulation of GFP transcript and protein was directly correlated with the choice of promoter with pdcA > amyA > pgk1. Transcript level appears to parallel GFP protein accumulation. Plasmid copy number had little impact on transcription or protein accumulation. These vectors should be useful for overexpression of heterologous proteins and potentially, metabolic engineering of Rhizopus strains. PMID:16804680

Mertens, Jeffrey A; Skory, Christopher D; Ibrahim, Ashraf S

2006-06-28

173

A Novel Protein Expressed in Mammalian Cells Undergoing Apoptosis  

Microsoft Academic Search

Human and rodent cells undergoing apoptosis were observed to express high levels of a novel 45,000 Mr protein. The protein, which we have termed apoptosis specific protein (ASP), was found in Burkitt lymphoma (BL) cells and in adenovirus-transformed human and rat embryo cells induced into apoptosis by a variety of stimuli, including serum deprivation, exposure to the Ca2+ ionophore, ionomycin,

Roger J. A. Grand; Anne E. Milner; Tracey Mustoe; Gerald D. Johnson; Darerca Owen; Michael L. Grant; Christopher D. Gregory

1995-01-01

174

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

175

Expression of Recombinant Proteins with Uniform N-Termini  

PubMed Central

Heterologously expressed proteins in Escherichia coli may undergo unwanted N-terminal processing by methionine and proline aminopeptidases. To overcome this problem, we present a system where the gene of interest is cloned as a fusion to a self-splicing mini-intein. Furthermore, this fusion construct is expressed in an engineered Escherichia coli strain from which the pepP gene coding for aminopeptidase P has been deleted. We describe a protocol using human cationic trypsinogen as an example to demonstrate that recombinant proteins produced in this expression system contain homogeneous, unprocessed N-termini.

Kiraly, Orsolya; Guan, Lan; Sahin-Toth, Miklos

2011-01-01

176

Secretory expression of heterologous protein in Kluyveromyces cicerisporus  

Microsoft Academic Search

To explore the potential of heterologous protein expression in Kluyveromyces cicerisporus, three expression plasmids, pUK1-PIT, pUKD-PIT and pUKD-S-PIT, based on the vector pUK1 or pUKD were constructed and transformed, respectively, into yeast strain K. cicerisporus Y179U. Human interferon a-2a, used as an example protein, was successfully expressed and secreted by transformant Y179U\\/pUKD-PIT and Y179U\\/pUKD-S-PIT. In the flask culture, strain Y179U\\/pUKD-S-PIT

X. P. Cai; J. Zhang; H.-Y. Yuan; Z.-A. Fang; Y.-Y. Li

2005-01-01

177

Translation Levels Control Multi-Spanning Membrane Protein Expression  

PubMed Central

Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane.

Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.

2012-01-01

178

Evaluation of genetically expressed absorbing proteins using photoacoustic spectroscopy  

NASA Astrophysics Data System (ADS)

Genetically expressed contrast agents are of great interest in the life sciences as they allow the study of structure and function of living cells and organisms. However, many commonly used fluorescent proteins present disadvantages when used in mammalian organisms, such as low near-infrared absorption and photostability. In this study, a variety of genetically expressed fluorescent proteins and novel chromoproteins were evaluated using photoacoustic spectroscopy. The results showed that chromoproteins provide stronger photoacoustic signals, better spectral stability, and exhibit less photobleaching than fluorescent proteins.

Laufer, Jan; Jathoul, Amit; Pule, Martin; Beard, Paul

2013-03-01

179

Expression of Extracellular Superoxide Dismutase Protein in Diabetes  

PubMed Central

Background Diabetes is characterized by chronic hyperglycemia, which can increase reactive oxygen species (ROS) production by the mitochondrial electron transport chain. The formation of ROS induces oxidative stress and activates oxidative damage-inducing genes in cells. No research has been published on oxidative damage-related extracellular superoxide dismutase (EC-SOD) protein levels in human diabetic skin. We investigated the expression of EC-SOD in diabetic skin compared with normal skin tissue in vivo. Methods The expression of EC-SOD protein was evaluated by western blotting in 6 diabetic skin tissue samples and 6 normal skin samples. Immunohistochemical staining was also carried out to confirm the EC-SOD expression level in the 6 diabetic skin tissue samples. Results The western blotting showed significantly lower EC-SOD protein expression in the diabetic skin tissue than in the normal tissue. Immunohistochemical examination of EC-SOD protein expression supported the western blotting analysis. Conclusions Diabetic skin tissues express a relatively small amount of EC-SOD protein and may not be protected against oxidative stress. We believe that EC-SOD is related to the altered metabolic state in diabetic skin, which elevates ROS production.

2013-01-01

180

Protein expression analysis of inflammation-related colon carcinogenesis  

PubMed Central

Background: Chronic inflammation is a risk factor for colorectal cancer (CRC) development. The aim of this study was to determine the differences in protein expression between CRC and the surrounding nontumorous colonic tissues in the mice that received azoxymethane (AOM) and dextran sodium sulfate (DSS) using a proteomic analysis. Materials and Methods: Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight), followed by 2% (w/v) DSS in their drinking water for seven days, starting one week after the AOM injection. Colonic adenocarcinoma developed after 20 weeks and a proteomics analysis based on two-dimensional gel electrophoresis and ultraflex TOF/TOF mass spectrometry was conducted in the cancerous and nontumorous tissue specimens. Results: The proteomic analysis revealed 21 differentially expressed proteins in the cancerous tissues in comparison to the nontumorous tissues. There were five markedly increased proteins (beta-tropomyosin, tropomyosin 1 alpha isoform b, S100 calcium binding protein A9, and an unknown protein) and 16 markedly decreased proteins (Car1 proteins, selenium-binding protein 1, HMG-CoA synthase, thioredoxin 1, 1 Cys peroxiredoxin protein 2, Fcgbp protein, Cytochrome c oxidase, subunit Va, ETHE1 protein, and 7 unknown proteins). Conclusions: There were 21 differentially expressed proteins in the cancerous tissues of the mice that received AOM and DSS. Their functions include metabolism, the antioxidant system, oxidative stress, mucin production, and inflammation. These findings may provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies to treat carcinogenesis in the inflamed colon.

Yasui, Yumiko

2009-01-01

181

Protein expression analysis of inflammation-related colon carcinogenesis.  

PubMed

Background: Chronic inflammation is a risk factor for colorectal cancer (CRC) development. The aim of this study was to determine the differences in protein expression between CRC and the surrounding nontumorous colonic tissues in the mice that received azoxymethane (AOM) and dextran sodium sulfate (DSS) using a proteomic analysis. Materials and Methods: Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight), followed by 2% (w/v) DSS in their drinking water for seven days, starting one week after the AOM injection. Colonic adenocarcinoma developed after 20 weeks and a proteomics analysis based on two-dimensional gel electrophoresis and ultraflex TOF/TOF mass spectrometry was conducted in the cancerous and nontumorous tissue specimens. Results: The proteomic analysis revealed 21 differentially expressed proteins in the cancerous tissues in comparison to the nontumorous tissues. There were five markedly increased proteins (beta-tropomyosin, tropomyosin 1 alpha isoform b, S100 calcium binding protein A9, and an unknown protein) and 16 markedly decreased proteins (Car1 proteins, selenium-binding protein 1, HMG-CoA synthase, thioredoxin 1, 1 Cys peroxiredoxin protein 2, Fcgbp protein, Cytochrome c oxidase, subunit Va, ETHE1 protein, and 7 unknown proteins). Conclusions: There were 21 differentially expressed proteins in the cancerous tissues of the mice that received AOM and DSS. Their functions include metabolism, the antioxidant system, oxidative stress, mucin production, and inflammation. These findings may provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies to treat carcinogenesis in the inflamed colon. PMID:19491504

Yasui, Yumiko; Tanaka, Takuji

2009-01-01

182

Expression of Focal Adhesion Proteins in the Developing Rat Kidney  

PubMed Central

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

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

2011-01-01

183

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

PubMed

Cell-free protein synthesis (CFPS) has major advantages over traditional cell-based methods in the capability of high-throughput protein synthesis and special protein production. During recent decades, CFPS has become an alternative protein production platform for both fundamental and applied purposes. Using Renilla luciferase as model protein, we describe a typical process of CFPS in wheat germ extract system, including wheat germ extract preparation, expression vector construction, in vitro protein synthesis (transcription/translation), and target protein assay. PMID:22160920

Ge, Xumeng; Xu, Jianfeng

2012-01-01

184

Individual expression of influenza virus PA protein induces degradation of coexpressed proteins.  

PubMed Central

In the process of in vivo reconstitution of influenza virus transcriptase-replicase complex, an inhibitory effect was observed when the level of PA protein expression was increased. This inhibition was paralleled by a decrease in the accumulation of the other influenza virus core proteins. The sole expression of PA protein was sufficient to reduce the accumulation level of the proteins encoded by the coexpressed genes. The PA effect was observed upon influenza virus and non-influenza virus proteins and independently of the expression system chosen and the origin of cell line used. The expression of PA protein did not induce variations in the translation of the target proteins but did induce variations on their half-lives, which were clearly reduced. A functional PA subunit seems to be necessary to induce this negative effect, because an inactive point mutant was unable to decrease the steady-state levels or the half-lives of the reporter proteins. The PA effect was observed as early as 5 h after its expression, and continuous synthesis of proteins was not required for performance of its biological activity. The results presented represent the first biological activity of individually expressed PA polymerase subunit.

Sanz-Ezquerro, J J; de la Luna, S; Ortin, J; Nieto, A

1995-01-01

185

Ewing's sarcoma can express bone morphogenetic protein  

Microsoft Academic Search

Paraffin sections from 6 cases of Ewing's sarcoma were immunostained by ABC method using BMP—McAb for investigating the existence\\u000a of BMP in Ewing's sarcoma. All cases were positive. The result was coincided with human tumor transplants in athymic nude\\u000a mouse by Bauer. It is shown that Ewing's sarcoma can express BMP. So we support the hypothesis that Ewing's sarcoma originates

Zhang Suixiang; Li Defang; Liu Jie; Yang Lianjia; Jin Yan

1994-01-01

186

Neisseria meningitidis rifampicin resistant strains: analysis of protein differentially expressed  

PubMed Central

Background Several mutations have been described as responsible for rifampicin resistance in Neisseria meningitidis. However, the intriguing question on why these strains are so rare remains open. The aim of this study was to investigate the protein content and to identify differential expression in specific proteins in two rifampicin resistant and one susceptible meningococci using two-dimensional electrophoresis (2-DE) combined with mass spectrometry. Results In our experimental conditions, able to resolve soluble proteins with an isoelectric point between 4 and 7, twenty-three proteins have been found differentially expressed in the two resistant strains compared to the susceptible. Some of them, involved in the main metabolic pathways, showed an increased expression, mainly in the catabolism of pyruvate and in the tricarboxylic acid cycle. A decreased expression of proteins belonging to gene regulation and to those involved in the folding of polypeptides has also been observed. 2-DE analysis showed the presence of four proteins displaying a shift in their isoelectric point in both resistant strains, confirmed by the presence of amino acid changes in the sequence analysis, absent in the susceptible. Conclusions The analysis of differentially expressed proteins suggests that an intricate series of events occurs in N. meningitidis rifampicin resistant strains and the results here reported may be considered a starting point in understanding their decreased invasion capacity. In fact, they support the hypothesis that the presence of more than one protein differentially expressed, having a role in the metabolism of the meningococcus, influences its ability to infect and to spread in the population. Different reports have described and discussed how a drug resistant pathogen shows a high biological cost for survival and that may also explain why, for some pathogens, the rate of resistant organisms is relatively low considering the widespread use of a particular drug. This seems the case of rifampicin resistant meningococci.

2010-01-01

187

Protein Inhibitor of Activated STAT-3 Expression in Lung Cancer  

PubMed Central

Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is an endogenous inhibitor of STAT3 transcriptional activity. We have previously demonstrated the concentration-dependent negative regulatory effect of PIAS3 on STAT3 signaling and its capacity to decrease lung cancer proliferation and synergize with epidermal growth factor inhibition. We now investigate PIAS3 expression in both non-small cell lung cancer (NSCLC) cell lines and human resected NSCLC specimens. We also investigated the mechanism by which some lung cancers have significantly decreased PIAS3 expression. Expression of PIAS3 is variable in lung cancer cells lines with 2 of 3 squamous cell carcinoma (SCC) cell lines having no or little PIAS3 protein expression. Similarly, the majority of human SCCs of the lung lack PIAS3 expression by immunohistochemistry; this despite the finding that SCCs have significantly higher levels of PIAS3 mRNA compared to adenocarcinomas. High PIAS3 expression generally correlates with decreased phosphorylated STAT3 in both SCC cell lines and human specimens compatible with the negative regulatory effect of this protein on STAT3 signaling. To investigate this variable expression of PIAS3 we first performed sequencing of the PIAS3 gene that demonstrated single nucleotide polymorphisms but no mutations. Exposure of lung cancer cells to 5-azacytidine and trichostatin A results in a significant increase in PIAS3 mRNA and protein expression. However, methylation-specific PCR demonstrates a lack of CpG island methylation in the promoter region of PIAS3. Exposure of cells to an agent blocking proteosomal degradation results in a significant increase in PIAS3. Our data thus shows that SCC of the lung commonly lacks PIAS3 protein expression and that post-translational modifications may explain this finding in some cases. PIAS3 is a potential therapeutic molecule to target STAT3 pathway in lung cancer.

Kluge, Amy; Dabir, Snehal; Vlassenbroeck, Ilse; Eisenberg, Rosana; Dowlati, Afshin

2011-01-01

188

Prolonged morphine administration alters protein expression in the rat myocardium  

PubMed Central

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

2011-01-01

189

Functional specificity among ribosomal proteins regulates gene expression.  

PubMed

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. PMID:17981122

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

2007-11-01

190

KAI-1 protein expression in odontogenic cysts.  

PubMed

The KAI-1 tumor suppressor gene is widely distributed in normal tissues and its down-regulation may be correlated with the invasive phenotype and metastases in several different epithelial tumors. The aim of the present study was an evaluation of KAI-1 expression in radicular cysts (RC), follicular cysts (FC), orthokeratinized keratocysts (OOKC), and parakeratinized keratocysts (POKC). Eighty-five odontogenic cysts, 28 RC, 22 FC, and 35 OKC (16 OOKC, 19 POKC) were selected. All the POKC were negative and only four of 16 of the OOKC were positive for KAI-1. On the contrary, all RC and FC cases were positive and immunoreactivity for KAI-1 was detected throughout all the layers of the cyst epithelium. The lack of KAI-1 expression in POKC could help to explain the differences in the clinical and pathologic behavior of OKC and, according to what has been reported for epithelial tumors, could be related to the increased aggressive behavior and invasiveness of OKC. PMID:17320703

Iezzi, Giovanna; Piattelli, Adriano; Artese, Luciano; Goteri, Gaia; Fioroni, Massimiliano; Rubini, Corrado

2007-03-01

191

Expression of an active spinach acyl carrier protein-I\\/protein-A gene fusion  

Microsoft Academic Search

A synthetic gene encoding spinach acyl carrier protein I (ACP-I) was fused to a gene encoding the Fc-binding portion of staphylococcal protein A. This gene fusion, under the control of the ?PR promoter, was expressed at high levels in Escherichia coli producing a 42 kDa fusion protein. This fusion protein was phosphopantethenylated in E. coli. In vitro the ACP portion

Phillip D. Beremand; DonitaDoyle Elmore; Katarzyna Dziewanowska; Daniel J. Guerra

1989-01-01

192

Green fluorescent protein and factorial approach: An effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli  

Microsoft Academic Search

We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach (“InFFact”) made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli).In the first part of

Bruno Coutard; Mathieu Gagnaire; Aude-Agnès Guilhon; Marine Berro; Stéphane Canaan; Christophe Bignon

2008-01-01

193

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

2012-11-26

194

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

195

Express Your LOV: An Engineered Flavoprotein as a Reporter for Protein Expression and Purification  

PubMed Central

In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an “effector” protein normally produced by enterohemorrhagic E. coli strains and “injected” into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.

Gawthorne, Jayde A.; Reddick, L. Evan; Akpunarlieva, Snezhana N.; Beckham, Katherine S. H.; Christie, John M.; Alto, Neal M.; Gabrielsen, Mads; Roe, Andrew J.

2012-01-01

196

Protein co-expression network analysis (ProCoNA)  

PubMed Central

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

2013-01-01

197

Identification of prognostic biomarkers for glioblastomas using protein expression profiling.  

PubMed

A set of proteins reflecting the prognosis of patients have clinical significance since they could be utilized as predictive biomarkers and/or potential therapeutic targets. With the aim of finding novel diagnostic and prognostic markers for glioblastoma (GBM), a tissue microarray (TMA) library consisting of 62 GBMs and 28 GBM-associated normal spots was constructed. Immunohistochemistry against 78 GBM-associated proteins was performed. Expression levels of each protein for each patient were analyzed using an image analysis program and converted to H-score [summation of the intensity grade of staining (0-3) multiplied by the percentage of positive cells corresponding to each grade]. Based on H-score and hierarchical clustering methods, we divided the GBMs into two groups (n=19 and 37) that had significantly different survival lengths (p<0.05). In the two groups, expression of nine proteins (survivin, cyclin E, DCC, TGF-?, CDC25B, histone H1, p-EGFR, p-VEGFR2/3, p16) was significantly changed (q<0.05). Prognosis-predicting potential of these proteins were validated with another independent library of 82 GBM TMAs and a public GBM DNA microarray dataset. In addition, we determined 32 aberrant or mislocalized subcellular protein expression patterns in GBMs compared with relatively normal brain tissues, which could be useful for diagnostic biomarkers of GBM. We therefore suggest that these proteins can be used as predictive biomarkers and/or potential therapeutic targets for GBM. PMID:22179774

Jung, Yong; Joo, Kyeung Min; Seong, Dong Ho; Choi, Yoon-La; Kong, Doo-Sik; Kim, Yonghyun; Kim, Mi Hyun; Jin, Juyoun; Suh, Yeon-Lim; Seol, Ho Jun; Shin, Chul Soo; Lee, Jung-Il; Kim, Jong-Hyun; Song, Sang Yong; Nam, Do-Hyun

2011-12-15

198

Myofibrillar protein and gene expression in acute quadriplegic myopathy  

PubMed Central

The dramatic muscle wasting, preferential loss of myosin and impaired muscle function in intensive care unit (ICU) patients with acute quadriplegic myopathy (AQM) have traditionally been suggested to be the result of proteolysis via specific proteolytic pathways. In this study we aim to investigate the mechanisms underlying the preferential loss of thick vs. thin filament proteins and the reassembly of the sarcomere during the recovery process in muscle samples from ICU patients with AQM. Quantitative and qualitative analyses of myofibrillar protein and mRNA expression were analyzed using SDS-PAGE, confocal microscopy, histochemistry and real-time PCR. The present results demonstrate that the transcriptional regulation of myofibrillar protein synthesis plays an important role in the loss of contractile proteins, as well as the recovery of protein levels during clinical improvement, myosin in particular, presumably in concert with proteolytic pathways, but the mechanisms are specific to the different thick and thin filament proteins studied.

Norman, Holly; Zackrisson, Hakan; Hedstrom, Yvette; Andersson, Per; Nordquist, Jenny; Eriksson, Lars I.; Libelius, Rolf; Larsson, Lars

2009-01-01

199

Combinatorial peptidomics: a generic approach for protein expression profiling  

PubMed Central

Traditional approaches to protein profiling were built around the concept of investigating one protein at a time and have long since reached their limits of throughput. Here we present a completely new approach for comprehensive compositional analysis of complex protein mixtures, capable of overcoming the deficiencies of current proteomics techniques. The Combinatorial methodology utilises the peptidomics approach, in which protein samples are proteolytically digested using one or a combination of proteases prior to any assay being carried out. The second fundamental principle is the combinatorial depletion of the crude protein digest (i.e. of the peptide pool) by chemical crosslinking through amino acid side chains. Our approach relies on the chemical reactivities of the amino acids and therefore the amino acid content of the peptides (i.e. their information content) rather than their physical properties. Combinatorial peptidomics does not use affinity reagents and relies on neither chromatography nor electrophoretic separation techniques. It is the first generic methodology applicable to protein expression profiling, that is independent of the physical properties of proteins and does not require any prior knowledge of the proteins. Alternatively, a specific combinatorial strategy may be designed to analyse a particular known protein on the basis of that protein sequence alone or, in the absence of reliable protein sequence, even the predicted amino acid translation of an EST sequence. Combinatorial peptidomics is especially suitable for use with high throughput micro- and nano-fluidic platforms capable of running multiple depletion reactions in a single disposable chip.

Soloviev, Mikhail; Barry, Richard; Scrivener, Elaine; Terrett, Jonathan

2003-01-01

200

Nicotinamide N-methyltransferase protein expression in renal cell cancer*  

PubMed Central

Objective: To understand the function of nicotinamide N-methyltransferase (NNMT) protein as tumor biomarker in renal carcinoma. Methods: Recombinant NNMT protein was used to prepare monoclonal antibodies by hybridoma technique. The diagnostic and prognostic function of NNMT protein in renal carcinoma was evaluated by analyzing 74 renal cancer tissues through immunohistochemical staining for NNMT by using the prepared antibodies. Results: Two hybridomas named 2F8 and 1E7 stably secreting the monoclonal antibodies were isolated successfully, and characters such as isotypes and specificity were determined. NNMT protein was significantly up-regulated in renal cancer and significantly associated with tumor histology and ages. The univariate survival analysis demonstrated that the pT-status, high levels of NNMT, and distant metastasis were significant prognosticators. Conclusion: NNMT is over-expressed in a large proportion in renal cell cancers. High NNMT expression is significantly associated with unfavorable prognosis. However, the prognostic value of NNMT needs further verification in larger sample sizes.

Zhang, Jun; Xie, Xin-you; Yang, Su-wen; Wang, Jin; He, Chao

2010-01-01

201

Sequential expression of matrix protein genes in developing rat teeth.  

PubMed

Tooth organogenesis is dependent on reciprocal and sequential epithelial-mesenchymal interactions and is marked by the appearance of phenotypic matrix macromolecules in both dentin and enamel. The organic matrix of enamel is composed of amelogenins, ameloblastin/amelin, enamelins and tuftelin. Dentin is mainly composed of type I collagen, but its specificity arises from the nature of the non-collagenous proteins (NCPs) involved in mineralization, phosphophoryn (DPP), dentin sialoprotein (DSP), osteocalcin, bone sialoprotein and dentin matrix protein-1 (Dmp1). In this paper, we studied the pattern of expression of four mineralizing protein genes (type I collagen, amelogenin, DSPP and osteocalcin) during the development of rat teeth by in situ hybridization on serial sections. For this purpose, we used an easy and rapid procedure to prepare highly-specific labeled single-stranded DNA probes using asymmetric polymerase chain reaction (PCR). Our results show that type I collagen is primarily expressed in polarizing odontoblasts, followed by the osteocalcin gene expression in the same polarized cells. Concomitantly, polarized ameloblasts start to accumulate amelogenin mRNAs and transiently express the DSPP gene. This latter expression switches over to odontoblasts whereas mineralization occurs. At the same time, osteocalcin gene expression decreases in secretory odontoblasts. Osteocalcin may thus act as an inhibitor of mineralization whereas DSP/DPP would be involved in more advanced steps of mineralization. Amelogenin and type I collagen gene expression increases during dentin mineralization. Their expression is spatially and temporally controlled, in relation with the biological role of their cognate proteins in epithelial-mesenchymal interactions and mineralization. PMID:10372553

Bleicher, F; Couble, M L; Farges, J C; Couble, P; Magloire, H

1999-04-01

202

The protein expression landscape of the Arabidopsis root  

PubMed Central

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

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

2012-01-01

203

Primate Cytomegaloviruses Encode and Express an IL10-like Protein  

Microsoft Academic Search

An open reading frame (ORF) with homology to interleukin-10 (IL-10) has been identified in rhesus cytomegalovirus (RhCMV). The IL-10-like protein is generated from a multispliced, polyadenylated early gene transcript encompassing part of the corresponding UL111A ORF of human CMV (HCMV). Immunological analyses confirm expression of the IL-10-like protein both in tissue culture and in RhCMV-infected rhesus macaques. Conserved ORFs were

Kristen M. Lockridge; Shan-Shan Zhou; Rachel H. Kravitz; Jennifer L. Johnson; Earl T. Sawai; Earl L. Blewett; Peter A. Barry

2000-01-01

204

Protein synthesis rate is the predominant regulator of protein expression during differentiation  

PubMed Central

External perturbations, by forcing cells to adapt to a new environment, often elicit large-scale changes in gene expression resulting in an altered proteome that improves the cell's fitness in the new conditions. Steady-state levels of a proteome depend on transcription, the levels of transcripts, translation and protein degradation but system-level contribution that each of these processes make to the final protein expression change has yet to be explored. We therefore applied a systems biology approach to characterize the regulation of protein expression during cellular differentiation using quantitative proteomics. As a general rule, it seems that protein expression during cellular differentiation is largely controlled by changes in the relative synthesis rate, whereas the relative degradation rate of the majority of proteins stays constant. In these data, we also observe that the proteins in defined sub-structures of larger protein complexes tend to have highly correlated synthesis and degradation rates but that this does not necessarily extend to the holo-complex. Finally, we provide strong evidence that the generally poor correlation observed between transcript and protein levels can fully be explained once the protein synthesis and degradation rates are taken into account.

Kristensen, Anders R; Gsponer, Joerg; Foster, Leonard J

2013-01-01

205

Expression of Yes Associated Protein, YAP, Modulates Survivin Expression in Primary Liver Malignancies  

PubMed Central

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

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

2012-01-01

206

Spatiotemporal expression profiling of proteins in rat sciatic nerve regeneration using reverse phase protein arrays  

PubMed Central

Background Protein expression profiles throughout 28 days of peripheral nerve regeneration were characterized using an established rat sciatic nerve transection injury model. Reverse phase protein microarrays were used to identify the spatial and temporal expression profile of multiple proteins implicated in peripheral nerve regeneration including growth factors, extracellular matrix proteins, and proteins involved in adhesion and migration. This high-throughput approach enabled the simultaneous analysis of 3,360 samples on a nitrocellulose-coated slide. Results The extracellular matrix proteins collagen I and III, laminin gamma-1, fibronectin, nidogen and versican displayed an early increase in protein levels in the guide and proximal sections of the regenerating nerve with levels at or above the baseline expression of intact nerve by the end of the 28 day experimental course. The 28 day protein levels were also at or above baseline in the distal segment however an early increase was only noted for laminin, nidogen, and fibronectin. While the level of epidermal growth factor, ciliary neurotrophic factor and fibroblast growth factor-1 and -2 increased throughout the experimental course in the proximal and distal segments, nerve growth factor only increased in the distal segment and fibroblast growth factor-1 and -2 and nerve growth factor were the only proteins in that group to show an early increase in the guide contents. As expected, several proteins involved in cell adhesion and motility; namely focal adhesion kinase, N-cadherin and ?-catenin increased earlier in the proximal and distal segments than in the guide contents reflecting the relatively acellular matrix of the early regenerate. Conclusions In this study we identified changes in expression of multiple proteins over time linked to regeneration of the rat sciatic nerve both demonstrating the utility of reverse phase protein arrays in nerve regeneration research and revealing a detailed, composite spatiotemporal expression profile of peripheral nerve regeneration.

2012-01-01

207

Expression of surface hydrophobic proteins by Candida albicans in vivo.  

PubMed

Candida albicans modulates cell surface hydrophobicity during growth and morphogenesis in vitro. To determine if surface hydrophobicity is expressed during pathogenesis, we generated a polyclonal antiserum against yeast hydrophobic proteins. The antiserum was then used for indirect immunofluorescence analysis of tissues from mice colonized and chronically infected with C. albicans. Results demonstrated that yeast hydrophobic proteins are exposed on fungal cells present in host tissues. The polyclonal antiserum distinguished between hydrophobic and hydrophilic cell surfaces in vitro and gave similar staining patterns and intensities for C. albicans cells in vivo. Of the yeast forms present within tissue lesions, approximately half exhibited moderate to intense immunofluorescence with the antiserum. Immunoblot analysis indicated that antigens recognized by the antiserum are predominantly low-molecular-mass hydrophobic proteins that are expressed by different C. albicans isolates and are expressed regardless of growth temperature. Taken together, the immunofluorescence and immunoblot analyses of antigens indicate that C. albicans displays surface hydrophobic proteins during pathogenesis and these proteins are available for hydrophobic interactions with host tissues. The effect of hydrophobic protein exposure on the virulence of C. albicans is discussed. PMID:7890397

Glee, P M; Sundstrom, P; Hazen, K C

1995-04-01

208

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

209

Examination of Neisseria Gonorrhoeae Opacity Protein Expression During Experimental Murine Genital Tract Infection.  

National Technical Information Service (NTIS)

The opacity (Opa) proteins of Neisseria gonorrhoeae are a family of phasevariable outer membrane proteins that bind to host cells. Phase variable expression occurs via a reversible frameshift mechanism within each opa gene. Opa protein expression is selec...

A. N. Simms

2005-01-01

210

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

211

Expression of pokeweed antiviral proteins in creeping bentgrass.  

PubMed

Fungal diseases of creeping bentgrass, an important amenity grass used extensively on golf courses, are a serious problem in golf course management. Transgenic approaches to improving disease resistance to fungal diseases are being explored in many species, and in some cases ribosome-inactivating proteins have been found to be effective. We have generated transgenic creeping bentgrass plants expressing three forms of ribosome-inactivating proteins from pokeweed, which are termed pokeweed antiviral proteins (PAP). PAP-Y and PAP-C are nontoxic mutants of PAP; PAPII is the native form of another ribosome-inactivating protein from pokeweed. In creeping bentgrass, PAP-C transformants did not accumulate the protein, suggesting that it is unstable, and in a field test these plants were not protected from infection by the fungal pathogen Sclerotinia homoeocarpa, the causal agent of dollar spot disease. PAPII transformants could accumulate stable levels of the protein but had symptoms of toxicity; one low-expressing line exhibited good disease resistance. PAP-Y transformants accumulated stable levels of protein, and under greenhouse conditions they appeared to be phenotypically normal. PMID:12789454

Dai, W D; Bonos, S; Guo, Z; Meyer, W A; Day, P R; Belanger, F C

2002-12-04

212

Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.  

PubMed

Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers. PMID:24132420

Nebenführ, Andreas

2014-01-01

213

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

214

PROTEIN EXPRESSION AND SECRETION BY TRICHODERMA REESEI UNDER LOW ENDOGENOUS PROTEIN BACKGROUND  

Technology Transfer Automated Retrieval System (TEKTRAN)

Trichoderma reesei (Hypocrea jecorina) is one of the most commonly used fungi for the manufacturing of industrial enzyme products. The fungus is capable of secreting proteins in levels up to 100 grams per liter. A number of homologous and heterologous proteins have been successfully over-expressed...

215

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

216

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

2012-09-18

217

Protein kinases expressed by interstitial cells of Cajal  

Microsoft Academic Search

Interstitial cells of Cajal (ICC) are involved in the generation of electrical rhythmicity of intestinal muscle and in the transduction of neural inputs in the gut. Although the expression of receptors for neurotransmitters and hormones and some second messengers have been investigated in ICC, the protein kinases present in these cells have not been well documented. This study has demonstrated

Daniel P. Poole; Trung Van Nguyen; Mitsuhisa Kawai; John B. Furness

2004-01-01

218

Expression cloning screen for modifiers of amyloid precursor protein shedding  

Microsoft Academic Search

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the amyloid ? peptide (A?), 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

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

2006-01-01

219

Manipulating heat shock protein expression in laboratory animals  

Microsoft Academic Search

Upregulation of heat shock proteins (Hsps) has been observed to impart resistance to a wide variety of physical and chemical insults. Elucidation of the role of Hsps in cellular defense processes depends, in part, on the ability to manipulate Hsp expression in laboratory animals. Simple methods of inducing whole body hyperthermia, such as warm water immersion or heating pad application,

J. Keith Tolson; Stephen M. Roberts

2005-01-01

220

Protein tyrosine kinase expression in the porcine ovary  

Microsoft Academic Search

Various growth factor receptors contain intrinsic tyrosine kinase activity, indicating that protein tyrosine kinases (PTK) play an important role in signal transduction pathways for cell proliferation and differentiation. To identify oocyte-derived factors which control follicle cells as well as oocyte-controlling factors produced by follicle cells, we examined the expression of genes which contain the PTK domain in the porcine ovary,

Yoshinori Okamura; Akira Myoumoto; Noboru Manabe; Nobuyuki Tanaka; Hitoshi Okamura; Manabu Fukumoto

2001-01-01

221

Heterologous protein expression in the methylotrophic yeast Pichia pastoris  

Microsoft Academic Search

During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to

Joan Lin Cereghino; James M. Cregg

2000-01-01

222

The matricellular protein SPARC is expressed in human trabecular meshwork  

Microsoft Academic Search

Purpose. This investigation was undertaken to determine whether the matricellular protein SPARC is expressed in the human trabecular meshwork (TM) and cultured human trabecular meshwork cells.Methods. Human donor trabecular meshwork and cultured cells obtained from trabecular meshwork were used in this study. Total RNA was obtained from TM and cultured TM endothelial cells, and RT-PCR was done with primers specific

Douglas J. Rhee; Robert N. Fariss; Rolf Brekken; E. Helene Sage; Paul Russell

2003-01-01

223

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

224

SURFACTANT PROTEIN D EXPRESSION IN NORMAL AND PNEUMONIC OVINE LUNG  

Technology Transfer Automated Retrieval System (TEKTRAN)

Surfactant Protein D (SP-D), a hydrophilic pulmonary surfactant collagenous calcium-dependent lectin with opsonizing activity, binds to surface glycoconjugates expressed by a wide variety of microorganisms such as Gram-negative bacteria, Influenza A virus, and various fungi as well as surface carboh...

225

Developmental expression of the tuberous sclerosis proteins tuberin and hamartin  

Microsoft Academic Search

Tuberous sclerosis complex is an autosomal dominant multisystem disorder, characterized by the development of hamartomas in multiple organs, primarily the skin, heart, kidney, and brain. The tuberous sclerosis genes, TSC1 and TSC2, encode hamartin and tuberin, respectively. Employing specific antibodies for hamartin and tuberin, we analyzed the expression of these two proteins by Western blot analyses in normal developing human

Vanishree Murthy; Anat O. Stemmer-Rachamimov; Luciana A. Haddad; Jennifer E. Roy; Andrea N. Cutone; Roberta L. Beauchamp; Nicole Smith; David N. Louis; Vijaya Ramesh

2001-01-01

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

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

228

Frontiers of protein expression control with conditional degrons.  

PubMed

It is useful to artificially control the expression levels of a protein of interest (POI), not only for its characterization in vivo, but also for the modulation of biological pathways. Overexpression of a POI is relatively easy because it is possible to drive its expression from a transgene encoding the POI under the control of a strong promoter. However, it is more challenging to reduce or deplete the expression of a POI. A protein domain called "degron", which induces rapid proteolysis by the proteasome, can be used for this purpose. Degron-based technologies for the conditional depletion of POI--degron fusion proteins have been developed by exploiting various pathways leading to proteasomal degradation. Compared with other depletion technologies that control the expression levels of POIs at the DNA or mRNA levels, these protein-depletion approaches are advantageous in terms of specificity, reversibility, and the time required for depletion. Current conditional degron-based technologies are described and discussed. PMID:23271452

Kanemaki, Masato T

2012-12-28

229

Heat shock protein expression in canine malignant mammary tumours  

Microsoft Academic Search

BACKGROUND: Abnormal levels of Heat Shock Proteins (HSPs) have been observed in many human neoplasms including breast cancer and it has been demonstrated that they have both prognostic and therapeutic implications. In this study, we evaluated immunohistochemical expression of HSPs in normal and neoplastic canine mammary glands and confronted these results with overall survival (OS), in order to understand the

Mariarita Romanucci; Alessia Marinelli; Giuseppe Sarli; Leonardo Della Salda

2006-01-01

230

PDGFR? Gene Mutation and Protein Expression in Gastrointestinal Stromal Tumors  

Microsoft Academic Search

Objective: Mutation of the PDGFR? is a potential candidate in the pathogenesis of KIT wild-type gastrointestinal tumors (GISTs). In this study, we evaluated the prevalence of PDGFR? mutations and corresponding protein expression in GISTs, to determine their usefulness in obtaining a prognosis. Methods: Genomic DNA was extracted from paraffin-embedded tumor tissues from 194 GISTs. Exons 12, 14 and 18 of

Heung Moon Chang; Min-Hee Ryu; Hyoungnam Lee; Se Jin Jang; Jae-Lyun Lee; Tae Won Kim; Jeong Hwan Yook; Sung Tae Oh; Byung Sik Kim; Jung Shin Lee; Yoon-Koo Kang

2008-01-01

231

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

232

Glucocorticoid receptor protein expression in human hippocampus; stability with age.  

PubMed

The glucocorticoid receptor (GR) exerts numerous functions in the body and brain. In the brain, it has been implicated, amongst others, in feedback regulation of the hypothalamic-pituitary-adrenal axis, with potential deficits during aging and in depression. GRs are abundantly expressed in the hippocampus of rodent, except for the Ammon's horn (CA) 3 subregion. In rhesus monkey however, GR protein was largely absent from all hippocampal subregions, which prompted us to investigate its distribution in human hippocampus. After validation of antibody specificity, we investigated GR? protein distribution in the postmortem hippocampus of 26 human control subjects (1-98 years of age) and quantified changes with age and sex. In contrast to monkey, abundant GR-immunoreactivity was present in nuclei of almost all neurons of the hippocampal CA subfields and dentate gyrus (DG), although neurons of the CA3 subregion displayed lower levels of immunoreactivity. Colocalization with glial fibrillary acidic protein confirmed that GR was additionally expressed in approximately 50% of the astrocytes in the CA regions, with lower levels of colocalization (approximately 20%) in the DG. With increased age, GR expression remained stable in the CA regions in both sexes, whereas a significant negative correlation was found with age only in the DG of females. Thus, in contrast to the very low levels previously reported in monkey, GR protein is prominently expressed in human hippocampus, indicating that this region can form an important target for corticosteroid effects in human. PMID:23290588

Wang, Qian; Van Heerikhuize, Joop; Aronica, Eleonora; Kawata, Mitsuhiro; Seress, Laszlo; Joels, Marian; Swaab, Dick F; Lucassen, Paul J

2013-01-03

233

Expression of Aequorea green fluorescent protein in plant cells  

Microsoft Academic Search

The coding region of the green fluorescent protein (GFP) from Aequorea victoria has been fused to the cauliflower mosaic virus 35S promoter and introduced into maize leaf protoplasts. Transient expression of GFP was observed. In addition, the coding region of GFP was fused to an Arabidopsis heat shock promoter and co-transformed with another construct in which GFP has been replaced

Wei Hu; Chi-Lien Cheng

1995-01-01

234

CO-EXPRESSION AMONG CONSTITUENTS OF A MOTIF IN THE PROTEIN-PROTEIN INTERACTION NETWORK  

PubMed Central

Almost all cellular functions are results of well-coordinated interactions between various proteins. A more connected hub or motif in the interaction network is expected to be more important and any perturbation in this motif would be more damaging to the smooth performance of the related functions. Thus, some coherent robustness of these hubs has to be derived. Here we provide the global evidence that interaction hubs obtain their robustness against uneven protein concentrations through co-expression of the constituents and the degree of co-expression correlates strongly with the complexity of the embedded motif. We calculate the gene expression correlations between the proteins embedded in 3-, 4-, 5- and 6-node interaction motifs of increasing complexities and compared them to those between proteins from random motifs of similar complexities. We find that as the connectedness of these motifs increase, there is a higher co-expression between the constituent proteins. For example, when the expression correlation is 0.7, the kernel density of the correlation increases from 0.152 for 4-node motifs with 3 edges to 0.403 for 4-node cliques. This implies that the robustness of the interaction system emerges from a proportionate synchronicity among the constituents of the motif via co-expression. We further show that such biological coherence via co-expression of component proteins can be reinforced by integrating conservation data in the analysis. For example, on addition of evolutionary information from other genomes, the ratio of kernel density for interaction and random data in the case of 5- and 6-node cliques in yeast increases from 37.8 to 123 and 98.4 to 1300, respectively, when their expression correlation is 0.8. Our results show that genes whose products are involved in motifs have transcription and translation properties that that minimize the noise in final protein concentrations, compared to random sets of genes.

BHARDWAJ, NITIN

2009-01-01

235

Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins  

PubMed Central

Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics.

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

2012-01-01

236

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

SciTech Connect

With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

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

2009-12-01

237

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

238

Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins  

PubMed Central

Background Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. Results The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. Conclusion The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels of the recombinant protein and induce the formation of PBs regardless of the cultivar used. However, a specific level of recombinant protein accumulation needs to be reached for PBs to form.

2013-01-01

239

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

240

Expression and Localization of Plant Protein Disulfide Isomerase.  

PubMed Central

A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules.

Shorrosh, B. S.; Subramaniam, J.; Schubert, K. R.; Dixon, R. A.

1993-01-01

241

Differential Expression of Borrelia burgdorferi Proteins during Growth In Vitro  

PubMed Central

In an earlier paper we described the transcriptionally regulated differential levels of expression of two lipoproteins of Borrelia burgdorferi, P35 and P7.5, during growth of the spirochetes in culture from logarithmic phase to stationary phase (K. J. Indest, R. Ramamoorthy, M. Solé, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165–1171, 1997). Here we further assess this phenomenon by investigating whether the expression of other antigens of B. burgdorferi, including some well-characterized ones, are also regulated in a growth-phase-dependent manner in vitro. These studies revealed 13 additional antigens, including OspC, BmpD, and GroEL, that were upregulated 2- to 66-fold and a 28-kDa protein that was downregulated 2- to 10-fold, during the interval between the logarithmic- and stationary-growth phases. Unlike with these in vitro-regulated proteins, the levels of expression of OspA, OspB, P72, flagellin, and BmpA remained unchanged throughout growth of the spirochetes in culture. Furthermore, ospAB, bmpAB, groEL, and fla all exhibited similar mRNA profiles, which is consistent with the constitutive expression of these genes. By contrast, the mRNA and protein profiles of ospC and bmpD indicated regulated expression of these genes. While bmpD exhibited a spike in mRNA expression in early stationary phase, ospC maintained a relatively higher level of mRNA throughout culture. These findings demonstrate that there are additional genes besides P7.5 and P35 whose regulated expression can be investigated in vitro and which may thus serve as models to facilitate the study of regulatory mechanisms in an organism that cycles between an arthropod and a vertebrate host.

Ramamoorthy, Ramesh; Philipp, Mario T.

1998-01-01

242

A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins  

Microsoft Academic Search

We have developed and evaluated a highly parallel protein expression and purification system using ORFs derived from the pathogenic bacterium Streptococcus pneumoniae as a representative test case in conjunction with the Gateway cloning technology. Establishing high throughput protein production capability is essential for genome-wide characterization of protein function. In this study, we focused on protein expression and purification outcomes generated

Keehwan Kwon; Rembert Pieper; Shamira Shallom; Carissa Grose; Erika Kwon; Yu Do; Saeeda Latham; Hamid Alami; Shih-Ting Huang; Christine Gatlin; Leka Papazisi; Robert Fleischmann; Scott Peterson

2007-01-01

243

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

244

Expression determinants of mammalian argonaute proteins in mediating gene silencing  

PubMed Central

RNA interference occurs by two main processes: mRNA site-specific cleavage and non-cleavage-based mRNA degradation or translational repression. Site-specific cleavage is carried out by argonaute-2 (Ago2), while all four mammalian argonaute proteins (Ago1–Ago4) can carry out non-cleavage-mediated inhibition, suggesting that Ago1, Ago3 and Ago4 may have similar but potentially redundant functions. It has been observed that in mammalian tissues, expression of Ago3 and Ago4 is dramatically lower compared with Ago1; however, an optimization of the Ago3 and Ago4 coding sequences to include only the most common codon at each amino acid position was able to augment the expression of Ago3 and Ago4 to levels comparable to that of Ago1 and Ago2. Thus, we examined whether particular sequence features exist in the coding region of Ago3 and Ago4 that may prevent a high level of expression. Swapping specific sub-regions of wild-type and optimized Ago sequence identified the portion of the coding region (nucleotides 1–1163 for Ago-3 and 1–1494 for Ago-4) that is most influential for expression. This finding has implications for the evolutionary conservation of Ago proteins in the mammalian lineage and the biological role that potentially redundant Ago proteins may have.

Valdmanis, Paul N.; Gu, Shuo; Schuermann, Nina; Sethupathy, Praveen; Grimm, Dirk; Kay, Mark A.

2012-01-01

245

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

246

Expression analysis of Tudor-SN protein in mouse tissues.  

PubMed

Tudor-SN (SND1, p100) has been shown to function as a transcriptional coactivator as well as a modulator of RNA metabolism and biogenesis and a component in the RNA-induced silencing complex (RISC). Tudor-SN consists of five repeats of staphylococcus nuclease-like domains (SN1-SN5) and, a Tudor domain implicated in binding to methylated ligands. The protein is highly conserved through evolution from fission yeast to mammals and it exists as a single gene without any close homologs. Tudor-SN is found to be overexpressed in several cancers such as colon adenocarcinomas and prostate cancer. The conservation of Tudor-SN along evolution suggests it may have important functions; however, the physiological function of Tudor-SN has not yet been characterized. In this study we analyzed the expression and localization of Tudor-SN in mouse tissues and organs by immunohistochemistry, fluorescent immunostaining, Western blotting and RT-qPCR. Expression analysis indicated that Tudor-SN is widely expressed in most organs with the exception of muscle cells. Up-regulated expression was observed in rapidly dividing cells and progenitor cells such as in spermatogonial cells in testis, in the follicular cells of ovary, in the cells of crypts of Lieberkühn of ileum and basal keratinocytes of skin and hair follicle when compared to more differentiated or terminally differentiated cells in the respective organs. Moreover, Tudor-SN was robustly expressed in T-cells and Tudor-SN was co-expressed with CD3 in T-cells in the Peyer's patch, spleen and lymph node. The wide expression pattern of Tudor-SN and high expression in proliferating and self-differentiating cells suggests that the protein serves functions related to activated state of cells. PMID:23068188

Fashe, Tekele; Saarikettu, Juha; Isomäki, Pia; Yang, Jie; Silvennoinen, Olli

2012-10-12

247

Bacteria and Protozoa Differentially Modulate the Expression of Rab Proteins  

PubMed Central

Phagocytic cells represent an important line of innate defense against microorganisms. Uptake of microorganisms by these cells involves the formation of a phagosome that matures by fusing with endocytic compartments, resulting in killing of the enclosed microbe. Small GTPases of the Rab family are key regulators of vesicular trafficking in the endocytic pathway. Intracellular pathogens can interfere with the function of these proteins in order to subvert host immune responses. However, it is unknown if this subversion can be achieved through the modulation of Rab gene expression. We compared the expression level of 23 distinct Rab GTPases in mouse macrophages after infection with the protozoan Plasmodium berghei, and the bacteria Escherichia coli and Salmonella enterica. We found that P. berghei induces an increase in the expression of a different set of Rab genes than E. coli and S. enterica, which behaved similarly. Strikingly, when one of the Rab proteins whose expression was increased by P. berghei, namely Rab14, was silenced, we observed a significant increase in the phagocytosis of P. berghei, whereas Rab14 overexpression led to a decrease in phagocytosis. This suggests that the parasite might induce the increase of Rab14 expression for its own advantage. Similarly, when Rab9a, whose expression was increased by E. coli and S. enterica, was silenced, we observed an increase in the phagocytosis of both bacterial species, whereas Rab9a overexpression caused a reduction in phagocytosis. This further suggests that the modulation of Rab gene expression could represent a mechanism of immune evasion. Thus, our study analyzes the modulation of Rab gene expression induced by bacteria and protozoa and suggests that this modulation could be necessary for the success of microbial infection.

Seixas, Elsa; Ramalho, Jose S.; Mota, Luis J.; Barral, Duarte C.; Seabra, Miguel C.

2012-01-01

248

Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.  

PubMed

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization. PMID:21912076

Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

2011-09-09

249

Distribution and Expression of Protein Kinase C Interactive Protein (PKCI\\/HINT1) in Mouse Central Nervous System (CNS)  

Microsoft Academic Search

Protein kinase C interactive protein (PKCI; also known as histidine triad protein, HINT1) is a small intracellular protein\\u000a widely expressed in tissues from both the peripheral and CNS. Although the structure of this protein is well characterized,\\u000a the functional aspect and cellular distribution of the protein remain unknown, especially in CNS. To analyze the expression\\u000a pattern of PKCI\\/HINT1 we used

Qing Liu; Adam C. Puche; Jia Bei Wang

2008-01-01

250

Manipulating heat shock protein expression in laboratory animals.  

PubMed

Upregulation of heat shock proteins (Hsps) has been observed to impart resistance to a wide variety of physical and chemical insults. Elucidation of the role of Hsps in cellular defense processes depends, in part, on the ability to manipulate Hsp expression in laboratory animals. Simple methods of inducing whole body hyperthermia, such as warm water immersion or heating pad application, are effective in producing generalized expression of Hsps. Hsps can be upregulated locally with focused direct or indirect heating, such as with ultrasound or with laser or microwave radiation. Increased Hsp expression in response to toxic doses of xenobiotics has been commonly observed. Some pharmacologic agents are capable of altering Hsps more specifically by affecting processes involved in Hsp regulation. Gene manipulation offers the ability to selectively increase or decrease individual Hsps. Knockout mouse strains and Hsp-overexpressing transgenics have been used successfully to examine the role of specific Hsps in protection against hyperthermia, chemical insults, and ischemia-reperfusion injury. Gene therapy approaches also offer the possibility of selective alteration of Hsp expression. Some methods of increasing Hsp expression have application in specialized areas of research, such cold response, myocardial protection from exercise, and responses to stressful or traumatic stimuli. Each method of manipulating Hsp expression in laboratory animals has advantages and disadvantages, and selection of the best method depends upon the experimental objectives (e.g., the alteration in Hsp expression needed, its timing, and its location) and resources available. PMID:15649841

Tolson, J Keith; Roberts, Stephen M

2004-12-19

251

Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes.  

PubMed Central

We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal luciferase expression observed prior to inflammatory challenge, markedly increased expression from the C3 promoter was detected in liver in response to both lipopolysaccharide (LPS) and turpentine, and lower-level inducible expression was also found in lung. In contrast, expression from the SAA3 promoter was found only in liver and was much more responsive to LPS than to turpentine. After LPS challenge, hepatic luciferase expression increased rapidly and in proportion to the LPS dose. Use of cytokine-inducible promoters in gene transfer vectors may make it possible to produce antiinflammatory proteins in vivo in direct relationship to the intensity and duration of an individual's inflammatory response. By providing endogenously controlled production of recombinant antiinflammatory proteins, this approach might limit the severity of the inflammatory response without interfering with the beneficial components of host defense and immunity.

Varley, A W; Coulthard, M G; Meidell, R S; Gerard, R D; Munford, R S

1995-01-01

252

Viral proteins expressed on the surface of murine leukemia cells.  

PubMed Central

Leukemic cells of AKR mice contain as constituents of their membranes the murine leukemia virus envelope protein gp70 and the precursor polyprotein of the viral internal (core) structural proteins. Both gp70 and the core polyprotein are represented on the cell surface as glycoproteins, as evidenced by incorporation of [3H]glucosamine into their structure and the binding of these proteins to lectins. The glycosylated core polyprotein exists in at least two serologically distinguishable forms: the 95,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30, p12, and p10, whereas the 85,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30 and p12, but not p10. Additional heterogeneity in these cell surface polyproteins has been observed wtih leukemias induced by exogenous leukemia viruses. Spontaneous leukemia cells of AKR mice invariably express gp70 and the core polyprotein on their cell surface; normal thymocytes of young AKR mice express gp70, but not the core polyprotein on their surface.

Ledbetter, J; Nowinski, R C; Emery, S

1977-01-01

253

Salmon Eggshell Protein Expression: A Marker for Environmental Estrogens  

Microsoft Academic Search

:   A liver complementary DNA expression library from Atlantic salmon (Salmo salar) pretreated with estradiol-17? (E2) was constructed and screened with antibodies raised against salmon eggshell (zona radiata) proteins. Two clones, SalZr2_19\\u000a and SalZr2_23 were sequenced and shown to encode proteins of approximately 50 kDa. SalZr2_23 contains 12 octamer sequence\\u000a lpqr\\/kpa\\/vq repeats also found in SalZr2_19, but only twice. Alignment

Dag O. Oppen-Berntsen; Augustine Arukwe; Fekadu Yadetie; James B. Lorens; Rune Male

1999-01-01

254

Decreased expression of C-erbB-2 and CXCR4 in breast cancer after primary chemotherapy  

PubMed Central

Background Biological molecular markers such as proto-oncogene erbB-2 (HER-2/neu, c-erbB-2), the CXC chemokine receptor 4 (CXCR4), estrogen receptor (ER), Proliferating Cell Nuclear Antigen (PCNA), DNA topoisomerase II (topo II), P-glycoprotein (P-gp) and glutathione S-transferase (GST) were observed for changes after administration of neochemotherapy and whether these protein expression changes were correlated with response to chemotherapy. Methods Sixty-four patients with primary breast cancer who had undergone neo-adjuvant chemotherapy were enrolled in the present study. The expressions of C-erbB-2, CXCR4 and ER-? were measured by immunohistochemistry (IHC) on full tissue sections and on tissue microarrays (TMAs). PCNA, TopoII, P-gp and GST were measured by IHC on TMAs. On the other hand, CXCR4, C-erbB-2 and ER-? expressions were detected using western blot analysis to 16 pairs of fresh preoperative core biopsies. The final surgical specimens were obtained from patients with breast carcinoma who received neo-adjuvant chemotherapy and obtained a partial response (PR). Results Our data demonstrated that the levels of C-erbB-2, CXCR4 and ER-? in patients decreased after they received neo-adjuvant chemotherapy on full tissue sections and on TMAs. The PCNA level was down-regulated after receiving neo-adjuvant chemotherapy, and no significant change was observed for TopoII, P-gp and GST. The levels of C-erbB-2, CXCR4 and ER-? were also down-regulated after neo-adjuvant chemotherapy was administered, as detected by western blot. In addition, the change expressions of C-erbB-2 and CXCR4 in specimens tended to be correlated with pathological change to neo-adjuvant chemotherapy on full tissue sections and on TMAs in a Pearson chi-square analysis. Conclusions As demonstrated in our study, after breast cancer patients were treated with neo-adjuvant systemic therapy, decreased expressions of C-erbB2, ER-? and CXCR4 were observed. Down-regulated expressions of c-erbB-2 and CXCR4 may be a novel mechanism of chemotherapy; the changes of these objective markers may be useful in evaluating the clinical response of neo-adjuvant chemotherapy in breast cancer.

2012-01-01

255

Recombinant Dragline Silk-Like Proteins--Expression and Purification  

PubMed Central

Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification.

Gaines, William A.; Marcotte, William R.

2011-01-01

256

Bcl-XLONG Protein Expression and Phosphorylation in Granulosa Cells  

Microsoft Academic Search

Expression of Bcl-x protein was evaluated in hen ovarian follicles, relative to stage of development, and in cultured granulosa cells after treatment with various apoptosis-suppressing or -inducing agents. Using Western blot analysis, Bcl-XLONG was most frequently ob- served migrating as a doublet with a molecular mass of approximately 28 kDa; the apparent higher molecular mass band of this doublet was

A. L. Johnson; J. T. BRIDGHAM; T. JENSEN

1999-01-01

257

Generation of a recloned transgenic cat expressing red fluorescence protein  

Microsoft Academic Search

Somatic cells from a first-generation red fluorescence protein transgenic cat (first RFP TG cat) were used to produce a recloned RFP transgenic cat (Re-RFP TG cat) (Felis catus) that systemically expressed RFP. A total of 281 RFP cloned embryos were transferred into 13 surrogate mothers (mean=21±7.7 embryos\\/recipient). One surrogate cat was diagnosed pregnant (7.7%) and delivered one live kitten. The

S. J. Cho; J. I. Bang; X. F. Yu; Y. S. Lee; J. H. Kim; J. T. Jeon; S. T. Yee; I. K. Kong

2010-01-01

258

RNA viruses as vectors for the expression of heterologous proteins  

Microsoft Academic Search

RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants.\\u000a Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins.\\u000a Several different schemes are being employed that depend on the genome organization of the virus and on the strategy

Sondra Schlesinger

1995-01-01

259

Protein expression pattern of collagen type XV in mouse cornea  

Microsoft Academic Search

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

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

2004-01-01

260

Plasmids for expression of heterologous proteins in Rhizopus oryzae  

Microsoft Academic Search

Rhizopus oryzae has long been used for enzyme production (e.g., glucoamylase and lipase), organic acid synthesis, and various fermented food applications. In this work, we describe a set of plasmid-based expression vectors that can be used for the production of heterologous proteins in R. oryzae. Three plasmid vectors have been created using either the glucoamylase A (amyA), pyruvate decarboxylase (pdcA),

Jeffrey A. Mertens; Christopher D. Skory; Ashraf S. Ibrahim

2006-01-01

261

Protein tyrosine phosphatases expression during development of mouse superior colliculus  

Microsoft Academic Search

Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system.\\u000a However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated.\\u000a In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate\\u000a seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from

Jacqueline Reinhard; Andrea Horvat-Bröcker; Sebastian Illes; Angelika Zaremba; Piotr Knyazev; Axel Ullrich; Andreas Faissner

2009-01-01

262

Protein phosphorylation in neuronal plasticity and gene expression  

Microsoft Academic Search

Protein kinases and phosphatases are intimately involved in several forms of synaptic plasticity. They play a critical role in the initiation of long-term potentiation and long-term depression, as well as in the induction of genes that permit long-term expression of altered synaptic states. Recent findings demonstrate a central role for the cAMP signaling pathway in the persistent phase of long-term

Howard Schulman

1995-01-01

263

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

264

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

2013-01-15

265

Expression of XPG protein in human normal and tumor tissues  

PubMed Central

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

Aracil, Miguel; Dauffenbach, Lisa M; Diez, Marta Martinez; Richeh, Rana; Moneo, Victoria; Leal, Juan Fernando Martinez; Fernandez, Luis Francisco Garcia; Kerfoot, Christopher A; Galmarini, Carlos M

2013-01-01

266

Phylogeny and expression of carbonic anhydrase-related proteins  

PubMed Central

Background Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate ?-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR. Results We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain, and eye. Conclusions CARP sequences have been strongly conserved between different species, and all three CARPs show high expression in the mouse brain and CARP VIII is also expressed in several other tissues. These findings suggest an important functional role for these proteins in mammals.

2010-01-01

267

Expression pattern of Protein Kinase C ? during mouse embryogenesis  

PubMed Central

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

2013-01-01

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

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

270

Expression cloning of genes encoding human peroxisomal proteins  

SciTech Connect

Numerous metabolic disorders associated with diverse peroxisomal defects have been identified but their molecular characterization has been hampered by difficulties associated with the purification of proteins from this fragile organelle. We have utilized antibodies directed against the C-terminal tripeptide peroxisomal targeting signal to detect hitherto unknown peroxisomal proteins in tissue fractions and to isolate genes encoding peroxisonal proteins from human expression libraries. We immunized rabbits with a peptide conjugate encompassing the C-terminal nine amino acids of rat peroxisomal acyl CoA oxidase. Immunoprecipitation assays using radio-labelled peptide showed that the antibody specifically recognizes the terminal SKL motif as well as C-terminal SHL and SRL but not SHL at an internal position. Affinity-purified antibody was used to probe Western blots of crude and peroxisome-enriched monkey liver preparations and detected 8-10 proteins specifically in the peroxisome fractions. 100 positive clones were identified on screening a human liver cDNA expression library in {lambda}-gt11. Sequence analysis has confirmed the identity of cDNA clones for human acyl CoA oxidase and epoxide hydrolase. Four clones show no sequence identity and their putative role in the human peroxisome is being explored.

Spathaky, J.M.; Tate, A.W.; Cox, T.M. [Univ. of Cambridge (United Kingdom)

1994-09-01

271

Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.  

PubMed

Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. PMID:20347821

Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

2010-03-27

272

Expression of ABC-type transport proteins in human platelets.  

PubMed

We have identified the ATP-binding cassette (ABC) transporter ABCC4 as an active constituent of mediator-storing granules in human platelets. In addition to multidrug resistance protein 4, other ABC-type transport proteins may contribute to platelet secretory function as well as determine intended or adverse effects of drugs. Here, we provide a comprehensive expression profiling of ABC transporters in human platelets based on a novel screening approach by combining the TaqMan low-density array RNA screening platform with a recently developed liquid chromatography/mass spectrometry (MS)/MS method for the simultaneous detection of membrane proteins. Transcripts of 25 ABC transporters were detected and showed differential expression compared with megakaryocytic progenitor cells. On the protein level ABCA7, ABCB4, ABCC1, ABCC3 and ABCC4 were identified by liquid chromatography/MS/MS and localized by immunofluorescence microscopy. Their functions may be related to glutathione and lipid homeostasis, secretion of lipid mediators, cell protection as well as drug transport. PMID:20395880

Niessen, Juliane; Jedlitschky, Gabriele; Grube, Markus; Kawakami, Hirotaka; Kamiie, Junichi; Ohtsuki, Sumio; Schwertz, Hansjörg; Bien, Sandra; Starke, Katharina; Ritter, Christoph; Strobel, Ulrike; Greinacher, Andreas; Terasaki, Tetsuya; Kroemer, Heyo K

2010-06-01

273

Proteomic analysis of the differentially expressed proteins by airborne nanoparticles.  

PubMed

Airborne nanoparticles with thermodynamic diameters less than 56 nm (PM(0.056)) were collected using a Moudi cascade impactor, and the differentially expressed proteins upon exposure to the airborne nanoparticles were identified in human bronchial epithelial cells. More than 600 protein spots were detected on the two-dimensional gel, and the identified 13 of these proteins showed notable changes. Nine were up-regulated and four were down-regulated following treatment with the airborne nanoparticles. Notably, malignant transformation-associated multiple forms of keratins, epigenetic regulation-related MBD1-containing chromatin associated factor 2, epithelial malignancy-related vimentin and exocytosis-related annexin A2 were changed upon exposure to airborne nanoparticle PM(0.056). PMID:21491466

Park, Seul Ki; Jeon, Yu Mi; Son, Bu Soon; Youn, Hyung Sun; Lee, Mi Young

2011-04-14

274

The E4 protein; structure, function and patterns of expression.  

PubMed

The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1(?)E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein's flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1(?)E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1(?)E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1(?)E4, these kinases regulate one of the E1(?)E4 proteins main functions, the association with the cellular keratin network, and eventually also its cleavage by the protease calpain which allows assembly into amyloid-like fibres and reorganisation of the keratin network. Although the E4 proteins of different HPV types appear divergent at the level of their primary amino acid sequence, they share a recognisable modular organisation and pattern of expression, which may underlie conserved functions and regulation. Assembly into higher-order multimers and suppression of cell proliferation are common to all E4 proteins examined. Although not yet formally demonstrated, a role in virus release and transmission remains a likely function for E4. PMID:24016539

Doorbar, John

2013-09-07

275

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

276

Expression and characterization of a lepidopteran general odorant binding protein.  

PubMed

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

Feng, L; Prestwich, G D

1997-05-01

277

Optimizations to achieve high-level expression of cytochrome P450 proteins using Escherichia coli expression systems.  

PubMed

Recombinant expression of membrane-bound cytochrome P450s in bacterial expression systems provide a well-established system capable of producing large yields of catalytically active protein. As the biochemical knowledge regarding cytochrome P450s increases, so does the efficiency of protein expression through various modifications that do not disrupt the functional properties of the protein of interest. Changes such as N-terminal modifications, reduction of secondary mRNA structure, bacterial codon usage, selection of vector and host strain, as well as varying external growth conditions all appear to influence protein expression. Several optimizations are often required for sufficient expression of cytochrome P450s at the desired cellular localization. This review aims to comprehensively summarize and update the significant advances made in membrane protein P450 expression in bacterial expression systems. PMID:23973802

Zelasko, Susan; Palaria, Amrita; Das, Aditi

2013-08-20

278

Expression without boundaries: cell-free protein synthesis in pharmaceutical research.  

PubMed

Proteins are an increasingly important class of new drugs. Pharmaceutical proteins are usually expressed in cell based systems in the development phase and in production, and although cell free methods have recently emerged they have not been used widely for therapeutic protein development or production. Cell free expression methodology is well suited for pharmaceutical protein expression and engineering and will probably become more commonly used in the future. Cell free expression allows protein engineering in high throughput format, flexible strategies for glycosylation and chemical conjugation, and allows easy use of unnatural amino acids as building blocks of proteins. Thus, cell free expression can be used to modify protein solubility, stability, and pharmacokinetics of therapeutic proteins. Likewise, it is potentially useful in protein development for biomaterial matrices, nanoparticles, and vaccines. This review illustrates the potential of cell free expression in pharmaceutical protein research and development while highlighting both advantages and limitations of the method. PMID:22521712

Casteleijn, Marco G; Urtti, Arto; Sarkhel, Sanjay

2012-04-11

279

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

PubMed

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

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

2013-09-18

280

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

281

A new Gateway vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis.  

PubMed

A major obstacle associated with recombinant protein over-expression in Escherichia coli is the production of insoluble inclusion bodies, a problem particularly pronounced with Mycobacterium tuberculosis proteins. One strategy to overcome the formation of inclusion bodies is to use an expression host that is more closely related to the organism from which the proteins are derived. Here we describe methods for efficiently identifying M. tuberculosis proteins that express in soluble form in Mycobacterium smegmatis. We have adapted the M. smegmatis expression vector pYUB1049 to the Gateway cloning system by the addition of att recombination recognition sequences. The resulting vector, designated pDESTsmg, is compatible with our in-house Gateway methods for E. coli expression. A target can be subcloned into pDESTsmg by a simple LR reaction using an entry clone generated for E. coli expression, removing the need to design new primers and re-clone target DNA. Proteins are expressed by culturing the M. smegmatis strain mc(2)4517 in autoinduction media supplemented with Tween 80. The media used are the same as those used for expression of proteins in E. coli, simplifying and reducing the cost of the switch to an alternative host. The methods have been applied to a set of M. tuberculosis proteins that form inclusion bodies when expressed in E. coli. We found that five of eight of these previously insoluble proteins become soluble when expressed in M. smegmatis, demonstrating that this is an efficient salvage strategy. PMID:17949993

Goldstone, Rachael M; Moreland, Nicole J; Bashiri, Ghader; Baker, Edward N; Shaun Lott, J

2007-09-14

282

Prion Protein Expression Regulates Embryonic Stem Cell Pluripotency and Differentiation  

PubMed Central

Cellular prion protein (PRNP) is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB) differentiation in mouse Prnp-null (KO) and WT embryonic stem cell (ESC) lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC) markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin ?v?5) in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel) and SPRN (Shadoo), whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

Miranda, Alberto; Pericuesta, Eva

2011-01-01

283

Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.  

PubMed

Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface. PMID:23894633

Kjellberg, Matti A; Mattjus, Peter

2013-07-24

284

Expression of recombinant green fluorescent protein in Bacillus methanolicus.  

PubMed

Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low-cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50-53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low-cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N-mdh-GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein. PMID:22275315

Nilasari, Dewi; Dover, Nir; Rech, Sabine; Komives, Claire

2012-02-21

285

Protein expression in salivary glands of rats with streptozotocin diabetes  

PubMed Central

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.

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

2009-01-01

286

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

287

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

2012-11-29

288

Evaluation of the Influenza A Replicon for Transient Expression of Recombinant Proteins in Mammalian Cells  

Microsoft Academic Search

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are

Florian Krammer; Jens Pontiller; Christopher Tauer; Dieter Palmberger; Andreas Maccani; Martina Baumann; Reingard Grabherr

2010-01-01

289

MicroRNA Expression in Mouse Oligodendrocytes and Regulation of Proteolipid Protein Gene Expression  

PubMed Central

Over-expression of the major myelin proteolipid protein (PLP) is detrimental to brain development and function and is the most common cause of Pelizaeus-Merzbacher disease. MicroRNAs (miRNA), small non-coding RNAs have been shown to play critical roles in oligodendrocyte lineage. In this study, we sought to investigate whether miRNAs control PLP abundance. To identify candidate miRNAs involved in this regulation, we have examined differentiation-induced changes in the expression of miRNAs in the oligodendroglial cell line, Oli-neu, and in EGFP+ oligodendrocytes ex vivo. We have identified 145 miRNAs that are expressed in oligodendrocyte cell lineage progression. Dicer1 expression decreases in differentiated oligodendrocytes and knock down of Dicer1 results in changes in miRNAs similar to those associated with differentiation. To identify miRNAs that control the PLP expression, we have selected miRNAs whose expression is lower in differentiated vs. undifferentiated Oli-neu cells and that have ? 1 binding site(s) in the PLP 3?UTR. The PLP 3?UTR fused to the luciferase gene reduces the activity of the reporter, suggesting that it negatively regulates message stability or translation. Such suppression is relieved by knock down of miR-20a. Over-expression of miR-20a decreases expression of the endogenous PLP in primary oligodendrocytes and of the reporter gene. Deletion or mutation of the putative binding site for miR-20a in the PLP 3?UTR abrogated such effects. Our data indicate that miRNA expression is regulated by Dicer1 levels in differentiated oligodendrocytes and that miR-20a, a component of the cluster that controls OL cell number, regulates PLP gene expression through its 3? UTR.

Wang, Erming; Cambi, Franca

2012-01-01

290

Expression of the stress proteins, ubiquitin, heat shock protein 72, and myofibrillar protein content after 12 weeks of leg cycling in persons with spinal cord injury  

Microsoft Academic Search

Willoughby DS, Priest JW, Nelson M. Expression of the stress proteins, ubiquitin, heat shock protein 72, and myofibrillar protein content after 12 weeks of leg cycling in persons with spinal cord injury. Arch Phys Med Rehabil 2002;83:649-54. Objective: To determine the effects of leg cycling exercise on ubiquitin (UBI), heat shock protein 72 (HSP-72) mRNA, protein expression, and myofibrillar protein

Darryn S. Willoughby; Joe W. Priest; Matt Nelson

2002-01-01

291

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

292

RNA-binding protein AUF1 represses Dicer expression  

PubMed Central

MicroRNA (miRNA) biogenesis is tightly regulated by numerous proteins. Among them, Dicer is required for the processing of the precursor (pre-)miRNAs into the mature miRNA. Despite its critical function, the mechanisms that regulate Dicer expression are not well understood. Here we report that the RNA-binding protein (RBP) AUF1 (AU-binding factor 1) associates with the endogenous DICER1 mRNA and can interact with several segments of DICER1 mRNA within the coding region (CR) and the 3?-untranslated region (UTR). Through these interactions, AUF1 lowered DICER1 mRNA stability, since silencing AUF1 lengthened DICER1 mRNA half-life and increased Dicer expression, while overexpressing AUF1 lowered DICER1 mRNA and Dicer protein levels. Given that Dicer is necessary for the synthesis of mature miRNAs, the lowering of Dicer levels by AUF1 diminished the levels of miRNAs tested, but not the levels of the corresponding pre-miRNAs. In summary, AUF1 suppresses miRNA production by reducing Dicer production.

Abdelmohsen, Kotb; Tominaga-Yamanaka, Kumiko; Srikantan, Subramanya; Yoon, Je-Hyun; Kang, Min-Ju; Gorospe, Myriam

2012-01-01

293

Expression of autophagy-related proteins in phyllodes tumor  

PubMed Central

Phyllodes tumors (PTs) are classified as fibroepithelial tumors and their histologic grade is determined primarily by the features of the stromal component. In this study, we examined the expression profiles of autophagy-related proteins in the stromal component of PTs and analyzed their clinical implications. We selected 204 human PT samples which were excised and diagnosed at Severance Hospital from 2000 to 2008 and created tissue microarray (TMA) blocks. Immunohistochemical assays for autophagy-related proteins (beclin-1, LC3A, LC3B, and p62) were then performed on these samples. The surgical specimens from higher grade PTs less frequently displayed cytoplasmic expression of beclin-1, LC3A, LC3B, and p62 in the stromal component (p<0.001). In univariate analysis, the following profiles were associated with shorter disease-free survival and overall survival: nuclear beclin-1 positivity in the stromal component (p=0.013 and p=0.044, respectively), LC3A positivity in the stromal component (p<0.001 and p<0.001, respectively), and p62 positivity in the stromal component (p=0.012 and p=0.004, respectively). In conclusion, we determined that increased activity of autophagy-related proteins correlated with a higher histologic grade and poorer prognosis in PTs. These results lead us to conclude that the autophagy activity of the stromal cells plays a key role in the progression of PTs.

Kim, Sang Kyum; Jung, Woo Hee; Koo, Ja Seung

2013-01-01

294

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

Arsenic trioxide (arsenite, As(III)) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As(III) on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As(III) on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As(III)-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As(III) were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As(III) 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 As(III) 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 As(III)-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 As(III) cytotoxicity between these cells. PMID:21945491

Yoshino, Yuta; Yuan, Bo; Kaise, Toshikazu; Takeichi, Makoto; Tanaka, Sachiko; Hirano, Toshihiko; Kroetz, Deanna L; Toyoda, Hiroo

2011-09-16

295

The Expression of Alternative Oxidase and Uncoupling Protein during Fruit Ripening in Mango  

Microsoft Academic Search

The expression of alternative oxidase (Aox) and uncoupling proteins (Ucp) was investigated during ripening in mango (Mangifera indica) and compared with the expression of peroxisomal thiolase, a previously described ripening marker in mango. The multigene family for the Aox in mango was expressed differentially during ripening. Abundance of Aox message and protein both peaked at the ripe stage. Expression of

Michael James Considine; Daniel Oliver Daley; James Whelan

2001-01-01

296

Hepatic expression of multidrug resistance protein 2 in biliary atresia  

PubMed Central

Background Biliary atresia (BA) is an idiopathic inflammatory obliterative cholangiopathy of neonates, leading to progressive biliary cirrhosis. Hepatoportoenterostomy (Kasai procedure) can cure jaundice in 30% to 80% of patients. Postoperative clearance of jaundice is one of the most important factors influencing long-term outcomes of BA patients. Multidrug resistance protein 2 (MRP2) is one of the canalicular export pumps located in hepatocytes; it exports organic anions and their conjugates (e.g., bilirubin) into bile canaliculus. Although MRP2 is an essential transporter for the excretion of bilirubin, its role in the clinical course of BA patients is unclear. The present study investigated the relationship between hepatic MRP2 expression and clinical course in BA patients, with particular emphasis in curing jaundice after hepatoportoenterostomy. Results No significant differences in hepatic MRP2 expression level were observed between BA and controls groups. There was no correlation between MRP2 expression and age at time of surgery in BA and control groups. In BA patients, MRP2 expression level in the jaundice and jaundice-free group did not differ significantly (2.0 × 10-4 vs 3.1 × 10-4, p = 0.094). Although the serum level of total bilirubin just before surgery did not correlate with MRP2 expression level (rs = 0.031, p = 0.914), the serum level of total bilirubin measured at 2 weeks (rs = -0.569, p = 0.034) and 4 weeks after surgery (rs = -0.620, p = 0.018) were significantly correlated with MRP2 expression level. Furthermore, MRP2 expression level was inversely correlated with ratio of change in serum total bilirubin level over 4 weeks (rs = -0.676, p = 0.008), which represents the serum bilirubin level measured at 4 weeks after surgery divided by value just before surgery. There was no correlation between expression level of MRP2 and nuclear receptors, such as retinoid × receptor ?, farnesoid × receptor, pregnane × receptor, or constitutive androstane receptor. Conclusions Hepatic MRP2 expression level was associated with postoperative clearance of jaundice in BA patients, at least within 1 month after hepatoportoenterostomy. This finding suggests that not only morphological appearance of the liver tissue but also the biological status of hepatocytes is important for BA pathophysiology.

2011-01-01

297

Cloning and expression of PCPTP1 encoding protein tyrosine phosphatase.  

PubMed

A novel cDNA encoding PTP (protein tyrosine phosphatase) was cloned from PC12h cells and designated as PCPTP1 (gene encoding PC12 protein Tyr phosphatase). The longest open reading frame (ORF) of this clone encodes a 656-amino-acid (aa) protein with a single PTP catalytic domain. Western blot analysis using a polyclonal Ab (antibody) raised against the cytoplasmic region of PCPTP1 detected two products, a major 65-kDa and minor 42-kDa protein, designated PCPTP1-MFI and PCPTP1-MVQ, respectively, in PC12h cells. These two proteins correspond to the products translated from the second and fifth methionine of PCPTP1, respectively. The bacterially expressed GST::PCPTP1-MVQ fusion protein had phosphatase activity with pNPP (p-nitrophenyl phosphate) as a substrate. Alignment of the aa sequence of PCPTP1-MVQ with those of other PTP showed the highest similarity to STEP and LC-PTP/HePTP, with 54 and 51% identity, respectively. Northern blot analysis showed only one 3.9-kb transcript in PC12h cells, indicating that PCPTP1 corresponds to this 3.9-kb transcript. The 3.9-kb PCPTP1 mRNA was detected in the brain and adrenal gland, but not in other non-neuronal tissues in adult rats. Two other transcripts of 3.3 and 1.7 kb were also detected in brain. NGF (nerve growth factor) and glucocorticoid are known to bimodally regulate the cell fate decision of sympathoadrenal precursors like PC12 cells, with NGF promoting the neuronal phenotype and glucocorticoid promoting the chromaffin phenotype. Still, both agents decreased the level of PCPTP1 mRNA in PC12h cells. Therefore, it is likely that the decrease in the level of PCPTP1 mRNA might be associated or correlated with cell differentiation in PC12h cells. PMID:7557444

Shiozuka, K; Watanabe, Y; Ikeda, T; Hashimoto, S; Kawashima, H

1995-09-11

298

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

299

Quantitative evaluation of the impact of active efflux by p-glycoprotein and breast cancer resistance protein at the blood-brain barrier on the predictability of the unbound concentrations of drugs in the brain using cerebrospinal fluid concentration as a surrogate.  

PubMed

This study investigated the impact of the active efflux mediated by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) at the blood-brain barrier (BBB) on the predictability of the unbound brain concentration (C(u,brain)) by the concentration in the cerebrospinal fluid (CSF) (C(u,CSF)) in rats. C(u,brain) is obtained as the product of the total brain concentration and unbound fraction in the brain (f(u,brain)) determined in vitro in brain slices. Twenty-five compounds, including P-gp and/or Bcrp substrates, were given a constant intravenous infusion, and their plasma, brain, and CSF concentrations were determined. P-gp and/or Bcrp substrates, such as verapamil, loperamide, flavopiridol, genistein, quinidine, dantrolene, daidzein, cimetidine, and pefloxacin, showed a higher CSF-to-brain unbound concentration ratio (K(p,uu,CSF/brain)) compared with non-P-gp and non-Bcrp substrates. K(p,uu,CSF/brain) values of P-gp-specific (quinidine and verapamil) and Bcrp-specific (daidzein and genistein) substrates were significantly decreased in Mdr1a/1b(-/-) and Bcrp(-/-) mice, respectively. Furthermore, consistent with the contribution of P-gp and Bcrp to the net efflux at the BBB, K(p,uu,CSF/brain) values of the common substrates (flavopiridol and erlotinib) were markedly decreased in Mdr1a/1b(-/-)/Bcrp(-/-) mice, but only moderately or weakly in Mdr1a/1b(-/-) mice and negligibly in Bcrp(-/-) mice. In conclusion, predictability of C(u,brain) by C(u,CSF) decreases along with the net transport activities by P-gp and Bcrp at the BBB. C(u,CSF) of non-P-gp and non-Bcrp substrates can be a reliable surrogate of C(u,brain) for lipophilic compounds. PMID:21934030

Kodaira, Hiroshi; Kusuhara, Hiroyuki; Fujita, Takuya; Ushiki, Junko; Fuse, Eiichi; Sugiyama, Yuichi

2011-09-20

300

Regulation of chondrocyte gene expression by osteogenic protein-1  

PubMed Central

Introduction The objective of this study was to investigate which genes are regulated by osteogenic protein-1 (OP-1) in human articular chondrocytes using Affimetrix gene array, in order to understand the role of OP-1 in cartilage homeostasis. Methods Chondrocytes enzymatically isolated from 12 normal ankle cartilage samples were cultured in high-density monolayers and either transfected with OP-1 antisense oligonucleotide in the presence of lipofectin or treated with recombinant OP-1 (100 ng/ml) for 48 hours followed by RNA isolation. Gene expression profiles were analyzed by HG-U133A gene chips from Affimetrix. A cut-off was chosen at 1.5-fold difference from controls. Selected gene array results were verified by real-time PCR and by in vitro measures of proteoglycan synthesis and signal transduction. Results OP-1 controls cartilage homeostasis on multiple levels including regulation of genes responsible for chondrocyte cytoskeleton (cyclin D, Talin1, and Cyclin M1), matrix production, and other anabolic pathways (transforming growth factor-beta (TGF-?)/ bone morphogenetic protein (BMP), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), genes responsible for bone formation, and so on) as well as regulation of cytokines, neuromediators, and various catabolic pathways responsible for matrix degradation and cell death. In many of these cases, OP-1 modulated the expression of not only the ligands, but also their receptors, mediators of downstream signaling, kinases responsible for an activation of the pathways, binding proteins responsible for the inhibition of the pathways, and transcription factors that induce transcriptional responses. Conclusions Gene array data strongly suggest a critical role of OP-1 in human cartilage homeostasis. OP-1 regulates numerous metabolic pathways that are not only limited to its well-documented anabolic function, but also to its anti-catabolic activity. An understanding of OP-1 function in cartilage will provide strong justification for the application of OP-1 protein as a therapeutic treatment for cartilage regeneration and repair.

2011-01-01

301

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

Microsoft Academic Search

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

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

2009-01-01

302

Glycosylated and phosphorylated proteins--expression in yeast and oocytes of Xenopus: prospects and challenges--relevance to expression of thermostable proteins.  

PubMed

Phosphorylation and glycosylation are important posttranslational events in the biosynthesis of proteins. The different degrees of phosphorylation and glycosylation of proteins have been an intriguing phenomenon. Advances in genetic engineering have made it possible to control the degree of glycosylation and phosphorylation of proteins. Structural biology of phosphorylated and glycosylated proteins has been advancing at a much slower pace due to difficulties in using high-resolution NMR studies in solution phase. Major difficulties have arisen from the inherent mobilities of phosphorylated and glycosylated side chains. This paper reviews molecular and structural biology of phosphorylated and glycosylated proteins expressed in eukaryotic expression systems which are especially suited for large-scale production of these proteins. In our laboratory, we have observed that eukaryotic expression systems are particularly suited for the expression of thermostable light-activated proteins, e.g., bacteriorhodopsins and plastocyanins. PMID:11482998

Li, P; Gao, X G; Arellano, R O; Renugopalakrishnan, V

2001-08-01

303

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

PubMed Central

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

Berrade, Luis; Camarero, Julio A.

2013-01-01

304

Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression  

PubMed Central

Drosophila melanogaster S2 cells were co-transfected with plasmid vectors containing the enhanced green fluorescent protein gene (EGFP), under the control of metallothionein promoter (pMt), and the hygromycin selection gene, in view of establishing parameters for optimized gene expression. A protocol of transfection was worked out, leading after hygromycin selection, to ?90% of S2MtEGFP fluorescent cells at day 5 after copper sulfate (CuSO4) induction. As analyzed by confocal microscopy, S2MtEGFP cell cultures were shown to be quite heterogeneous regarding the intensity and cell localization of fluorescence among the EGFP expressing cells. Spectrofluorimetry kinetic studies of CuSO4 induced S2MtEGFP cells showed the EGFP expression at 510 nm as soon as 5 h after induction, the fluorescence increasing progressively from this time to attain values of 4.6 × 105 counts/s after 72 h of induction. Induction with 700 ?M of CuSO4 performed at the exponential phase of the S2MtEGFP culture (106 cells/mL) led to a better performance in terms of cell growth, percent of fluorescent cells and culture intensity of fluorescence. Sodium butyrate (NaBu) treatment of CuSO4 induced S2MtEGFP cell cultures, although leading to a loss of cell culture viability, increased the percent of EGFP expressing cells and sharply enhanced the cell culture fluorescence intensity. The present study established parameters for improving heterologous protein expression in stably transfected Drosophila S2 cells, as assessed by the EGFP expression.

Santos, Mariza G.; Jorge, Soraia A.C.; Brillet, Karl

2007-01-01

305

Ehrlichia chaffeensis Expresses Macrophage- and Tick Cell-Specific 28-Kilodalton Outer Membrane Proteins  

PubMed Central

Ehrlichia chaffeensis, a tick-transmitted rickettsial agent, causes human monocyte/macrophage-tropic ehrlichiosis. In this study, proteomic approaches were used to demonstrate host cell-specific antigenic expression by E. chaffeensis. The differentially expressed antigens include those from the 28-kDa outer membrane protein (p28-Omp) multigene locus. The proteins expressed in infected macrophages are the products of p28-Omp19 and p28-Omp20 genes, whereas in tick cells, the protein expressed is the p28-Omp14 gene product. The differentially expressed proteins are posttranslationally modified by phosphorylation and glycosylation to generate multiple expressed forms. Host cell-specific protein expression is not influenced by growth temperatures and is reversible. Host cell-specific protein expression coupled with posttranslational modifications may be a hallmark for the pathogen's adaptation to a dual-host life cycle and its persistence.

Singu, Vijayakrishna; Liu, Haijie; Cheng, Chuanmin; Ganta, Roman R.

2005-01-01

306

Ehrlichia chaffeensis expresses macrophage- and tick cell-specific 28-kilodalton outer membrane proteins.  

PubMed

Ehrlichia chaffeensis, a tick-transmitted rickettsial agent, causes human monocyte/macrophage-tropic ehrlichiosis. In this study, proteomic approaches were used to demonstrate host cell-specific antigenic expression by E. chaffeensis. The differentially expressed antigens include those from the 28-kDa outer membrane protein (p28-Omp) multigene locus. The proteins expressed in infected macrophages are the products of p28-Omp19 and p28-Omp20 genes, whereas in tick cells, the protein expressed is the p28-Omp14 gene product. The differentially expressed proteins are posttranslationally modified by phosphorylation and glycosylation to generate multiple expressed forms. Host cell-specific protein expression is not influenced by growth temperatures and is reversible. Host cell-specific protein expression coupled with posttranslational modifications may be a hallmark for the pathogen's adaptation to a dual-host life cycle and its persistence. PMID:15618143

Singu, Vijayakrishna; Liu, Haijie; Cheng, Chuanmin; Ganta, Roman R

2005-01-01

307

Constraints imposed by non-functional protein-protein interactions on gene expression and proteome size.  

PubMed

Crowded intracellular environments present a challenge for proteins to form functional specific complexes while reducing non-functional interactions with promiscuous non-functional partners. Here we show how the need to minimize the waste of resources to non-functional interactions limits the proteome diversity and the average concentration of co-expressed and co-localized proteins. Using the results of high-throughput yeast 2-hybrid experiments, we estimate the characteristic strength of non-functional protein-protein interactions. By combining these data with the strengths of specific interactions, we assess the fraction of time proteins spend tied up in non-functional interactions as a function of their overall concentration. This allows us to sketch the phase diagram for baker's yeast cells using the experimentally measured concentrations and subcellular localization of their proteins. The positions of yeast compartments on the phase diagram are consistent with our hypothesis that the yeast proteome has evolved to operate closely to the upper limit of its size, whereas keeping individual protein concentrations sufficiently low to reduce non-functional interactions. These findings have implication for conceptual understanding of intracellular compartmentalization, multicellularity and differentiation. PMID:18682700

Zhang, Jingshan; Maslov, Sergei; Shakhnovich, Eugene I

2008-08-05

308

Expression and Function of Iron-Regulatory Proteins in Retina  

PubMed Central

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

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

2013-01-01

309

Ehrlichia chaffeensis expresses an immunoreactive protein homologous to the Escherichia coli GroEL protein.  

PubMed Central

A clone expressing a 58-kDa protein reactive with human convalescent-phase serum was isolated from a recombinant library of Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis. Sequencing identified two open reading frames, one encoding a 10.3-kDa polypeptide consisting of 94 amino acids and another encoding a 58-kDa polypeptide consisting of 550 amino acids. The sequences of the 10.3- and 58-kDa polypeptides were homologous to those of the Escherichia coli GroES and GroEL heat shock proteins, respectively. Images

Sumner, J W; Sims, K G; Jones, D C; Anderson, B E

1993-01-01

310

Cloning, expression, and assembly of sericin-like protein.  

PubMed

Recombinant sericin proteins of different molecular masses (17.4, 31.9, and 46.5 kDa), based on the 38-amino acid repetitive motif of native sericin, were cloned, expressed, and purified. The recombinant sericin self-assembled during dialysis (starting concentration of 2.5 mg/ml) forming twisted fibers. Circular dichroism and Fourier transform infrared spectroscopy studies demonstrated protein conformational transitions occurred from random coil to beta-sheets during the dialysis. Congo red-stained recombinant sericin fibrils exhibited apple-green birefringence, indicating long-range order in the array of beta-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. > 40 mol % serine), leading to a beta-sheet conformation formed by hydrogen bonding via polar zipper interactions. Analysis of recombinant sericin sequence using Mandel-Gutfreund's (Mandel-Gutfreund, Y., and Gregoret, L. M. (2002) J. Mol. Biol. 323, 453-461) definition of polar and non-polar amino acids showed that the hydrophobicity pattern resembles the most frequent pattern of amyloidogenic proteins, polar amino acid aggregates (PPPPP). Many beta-proteins and peptides are designed to study amyloidogenesis using a polar/non-polar alternating pattern (PNPNPN). Sericin-like proteins or peptides provide an alternative model in terms of hydrophobicity pattern with which to explore questions related to beta-sheet formation and amyloidogenesis. The glue-like property of sericin is attributed to the hydrogen bonding between serine residues of sericin with serine residues in the fibroin structural components of silk fiber. PMID:12963711

Huang, Jia; Valluzzi, Regina; Bini, Elisabetta; Vernaglia, Brian; Kaplan, David L

2003-09-08

311

Expression of equine morbillivirus (EMV) matrix and fusion proteins and their evaluation as diagnostic reagents  

Microsoft Academic Search

Summary.  ?Full-length cDNA clones coding for the matrix (M) and fusion (F) proteins of equine morbillivirus (EMV) were isolated by\\u000a RT-PCR, and expressed in Escherichia coli using two different expression systems. Western blot analysis indicated that the M and F proteins, expressed either by itself\\u000a or as fusion proteins with glutathione S-transferase (GST), were insoluble and degraded after expression. Analysis of

L.-F. Wang; A. R. Gould; P. W. Selleck

1997-01-01

312

Phenylsulfonylfuroxans as modulators of multidrug-resistance-associated protein-1 and P-glycoprotein.  

PubMed

A series of furoxan derivatives were studied for their ability to interact with P-gp and MRP1 transporters in MDCK cells overexpressing these proteins. 3-Phenylsulfonyl substituted furoxans emerged as the most interesting compounds. All of them were capable of inhibiting P-gp, and a few also were capable of inhibiting MRP1. Substituents at the 4-position of 3-phenylsulfonylfuroxan scaffold were able to modulate the selectivity and the intensity of inhibition. In some cases, they reverted MRP1 inhibitor activity, namely, they were capable of potentiating MRP1 dependent efflux. When compounds 16 and 17 were coadministered with doxorubicin, they restored a high degree of the activity of the antibiotic. Preliminary immunoblotting studies carried out on these two compounds indicate that they are capable of nitrating P-gp, which in this form is likely unable to efflux the antibiotic. PMID:20684594

Fruttero, Roberta; Crosetti, Marco; Chegaev, Konstantin; Guglielmo, Stefano; Gasco, Alberto; Berardi, Francesco; Niso, Mauro; Perrone, Roberto; Panaro, Maria Antonietta; Colabufo, Nicola Antonio

2010-08-12

313

Chronic intermittent mechanical stress increases MUC5AC protein expression.  

PubMed

Increased abundance of mucin secretory cells is a characteristic feature of the epithelium in asthma and other chronic airway diseases. We showed previously that the mechanical stresses of airway constriction, both in the intact mouse lung and a cell culture model, activate the epidermal growth factor receptor (EGFR), a known modulator of mucin expression in airway epithelial cells. Here we tested whether chronic, intermittent, short-duration compressive stress (30 cm H(2)O) is sufficient to increase the abundance of MUC5AC-positive cells and intracellular mucin levels in human bronchial epithelial cells cultured at an air-liquid interface. Compressive stress applied for 1 hour per day for 14 days significantly increased the percentage of cells staining positively for MUC5AC protein (22.0 +/- 3.8%, mean +/- SD) relative to unstimulated controls (8.6 +/- 2.6%), and similarly changed intracellular MUC5AC protein levels measured by Western and slot blotting. The effect of compressive stress was gradual, with significant changes in MUC5AC-positive cell numbers evident by Day 7, but required as little as 10 minutes of compressive stress daily. Daily treatment of cells with an EGFR kinase inhibitor (AG1478, 1 muM) significantly but incompletely attenuated the response to compressive stress. Complete attenuation could be accomplished by simultaneous treatment with the combination of AG1478 and a transforming growth factor (TGF)-beta(2) (1 microg/ml)-neutralizing antibody, or with anti-TGF-beta(2) alone. Our findings demonstrate that short duration episodes of mechanical stress, representative of those occurring during bronchoconstriction, are sufficient to increase goblet cell number and MUC5AC protein expression in bronchial epithelial cells in vitro. We propose that the mechanical environment present in asthma may fundamentally bias the composition of airway epithelial lining in favor of mucin secretory cells. PMID:19168703

Park, Jin-Ah; Tschumperlin, Daniel J

2009-01-23

314

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

315

From gene to protein: Prostatic acid phosphatase: Structure and expression of gene and protein*.  

PubMed

A set of classes for medical students is designed to reinforce an understanding of the basic laboratory methods of molecular biology and protein biochemistry in the context of a clinically important problem, prostate gland pathology. Students examine the gene coding for prostatic acid phosphatase and they assay expression of the gene in different lines of prostate cancer cell cultures (LNCaP and PC-3). The three-dimensional structure of the expressed protein is also investigated, in relation to its catalytic function. Students are encouraged to collect data for their experiments and to perform laboratory exercises on their own. The theory and practice should stimulate the students' discussion of various fields of biochemistry and molecular biology. PMID:21706764

Laidler, Piotr; Kuciel, Radoslawa; Duli?ska, Joanna; Gil, Dorota; Mazurkiewicz, Aleksandra; Wróbel, Maria

2004-11-01

316

SSAO/VAP-1 protein expression during mouse embryonic development.  

PubMed

SSAO/VAP-1 is a multifunctional enzyme depending on in which tissue it is expressed. SSAO/VAP-1 is present in almost all adult mammalian tissues, especially in highly vascularised ones and in adipocytes. SSAO/VAP-1 is an amine oxidase able to metabolise various endogenous or exogenous primary amines. Its catalytic activity can lead to cellular oxidative stress, which has been implicated in several pathologies (atherosclerosis, diabetes, and Alzheimer's disease). The aim of this work is to achieve a study of SSAO/VAP-1 protein expression during mouse embryogenesis. Our results show that SSAO/VAP-1 appears early in the development of the vascular system, adipose tissue, and smooth muscle cells. Moreover, its expression is strong in several epithelia of the sensory organs, as well as in the development of cartilage sites. Altogether, this suggests that SSAO/VAP-1 enzyme could be involved in the differentiation processes that take place during embryonic development, concretely in tissue vascularisation. PMID:18729210

Valente, Tony; Solé, Montse; Unzeta, Mercedes

2008-09-01

317

Statins Inhibit Monocyte Chemotactic Protein 1 Expression in Endometriosis  

PubMed Central

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

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

2012-01-01

318

Nuclear genes encoding plastid proteins expressed early in chloroplast development  

SciTech Connect

The overall objective of this grant was to characterize events which occur early in chloroplast biogenesis and to isolate nuclear genes encoding plastid proteins which are expressed during this developmental phase. In addition, the possible requirement of plastid transcription for the expression of the nuclear genes such as rbcS and cab was to be tested. The impetus for this research came from studies of chloroplast biogenesis in barley. We found that plastid DNA copy number was relatively high (120 copies/plastid vs 200 at maximal accumulation) in the meristematic region of the leaf base whereas plastid transcription activity was low in this plastid population. Later in chloroplast development transcription activity increased at least 5 fold per plastid or per template indicating that activation of plastid transcription occurred after most of the build up in plastid DNA per plastid. This suggested that activation of plastid DNA synthesis occurred early in chloroplast development and that nuclear genes involved in this activity must be regulated differently from genes such rbcS or cab which are expressed later in development. 3 refs., 7 figs.

Mullet, J.E.

1991-01-01

319

Expression of p53 protein in pituitary adenomas.  

PubMed

Inactivating mutations of TP53, a tumor suppressor gene, are associated with abnormal cell proliferation. Although p53 expression is common in many human malignancies, p53 protein has seldom been evaluated in pituitary tumors. When detected, the percentage of p53-positive cells is low, and, in general, it is exclusive for invasive lesions. The aim of the present study was to use immunohistochemistry to determine the presence of p53 protein in pituitary adenomas from tumor samples of 163 surgeries performed in 148 patients (40% male, 60% female). In 35% of the cases the adenoma was nonfunctional, while in the others it was associated with PRL, GH and/or ACTH endocrine hypersecretion syndrome. Macroadenomas were observed in 83.2% of the cases with available neuroimage evaluation, of which 28% invaded the cavernous, sphenoid and/or ethmoidal sinus, bone, third ventricle or subfrontal lobe. p53 protein was detected in 2/148 patients (1.3%). Immunohistochemistry was positive for PRL and GH in these cases. Due to the high percentage of invasive pituitary adenomas found in our study, the low frequency of p53 detection suggests that it is inadequate as a routine marker for aggressiveness and as a predictive factor of tumor behavior. PMID:12011941

Oliveira, M C; Marroni, C P; Pizarro, C B; Pereira-Lima, J F; Barbosa-Coutinho, L M; Ferreira, N P

2002-05-01

320

A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins.  

PubMed

We have developed and evaluated a highly parallel protein expression and purification system using ORFs derived from the pathogenic bacterium Streptococcus pneumoniae as a representative test case in conjunction with the Gateway cloning technology. Establishing high throughput protein production capability is essential for genome-wide characterization of protein function. In this study, we focused on protein expression and purification outcomes generated from an expression vector which encodes an NH(2)-terminal hexa-histidine tag and a COOH-terminal S-tag. Purified recombinant proteins were validated by SDS-PAGE, followed by in-gel digestion and identification by MALDI-TOF/TOF analysis. Starting with 1360 sequence-validated destination clones we examined correlation analyses of expression and solubility of a wide variety of recombinant proteins. In total, 428 purified proteins (31%) were recovered in soluble form. We describe a semi-quantitative scoring method using an S-tag assay to improve the throughput and efficiency of expression and solubility studies for recombinant proteins. Given a relatively large dataset derived from proteins representing all functional groups in a microbial genome we correlated various protein characteristics as they relate to protein expression outcomes. PMID:17703947

Kwon, Keehwan; Pieper, Rembert; Shallom, Shamira; Grose, Carissa; Kwon, Erika; Do, Yu; Latham, Saeeda; Alami, Hamid; Huang, Shih-Ting; Gatlin, Christine; Papazisi, Leka; Fleischmann, Robert; Peterson, Scott

2007-07-10

321

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

322

Ubiquitin-Like Protein 5 Positively Regulates Chaperone Gene Expression in the Mitochondrial Unfolded Protein Response  

PubMed Central

Perturbation of the protein-folding environment in the mitochondrial matrix selectively upregulates the expression of nuclear genes encoding mitochondrial chaperones. To identify components of the signal transduction pathway(s) mediating this mitochondrial unfolded protein response (UPRmt), we first isolated a temperature-sensitive mutation (zc32) that conditionally activates the UPRmt in C. elegans and subsequently searched for suppressors by systematic inactivation of genes. RNAi of ubl-5, a gene encoding a ubiquitin-like protein, suppresses activation of the UPRmt markers hsp-60?gfp and hsp-6?gfp by the zc32 mutation and by other manipulations that promote mitochondrial protein misfolding. ubl-5 (RNAi) inhibits the induction of endogenous mitochondrial chaperone encoding genes hsp-60 and hsp-6 and compromises the ability of animals to cope with mitochondrial stress. Mitochondrial morphology and assembly of multi-subunit mitochondrial complexes of biotinylated proteins are also perturbed in ubl-5(RNAi) worms, indicating that UBL-5 also counteracts physiological levels of mitochondrial stress. Induction of mitochondrial stress promotes accumulation of GFP-tagged UBL-5 in nuclei of transgenic worms, suggesting that UBL-5 effects a nuclear step required for mounting a response to the threat of mitochondrial protein misfolding.

Benedetti, Cristina; Haynes, Cole M.; Yang, Yun; Harding, Heather P.; Ron, David

2006-01-01

323

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

324

An In Vitro strategy for the rapid expression of recombinant proteins at low temperatures  

Microsoft Academic Search

In this study, we propose a strategy for recombinant protein expression under conditions of low-temperatures. The low-temperature\\u000a production of recombinant proteins was conducted in a cell-free protein synthesis reaction through the fusion of the N-terminus\\u000a of the protein of interest with that from a temperature-insensitive protein. For instance, while the expression of hlL-2 or\\u000a TNF-? was negligible when the reaction

Ju-Young Byun; Dong-Myung Kim

2008-01-01

325

Combinatorial library approaches for improving soluble protein expression in Escherichia coli.  

PubMed

High-throughput screening methodologies are already used in structural biology to define efficient protein crystallization and expression conditions. Recently, screening approaches have been extended to the optimization of genetic constructs for improved soluble protein expression. With similarities to the directed evolution strategies used in protein engineering, a target gene encoding a poorly expressed protein is mutated by truncation, fragmentation or point mutation. Rare clones with improved protein expression characteristics are then isolated from the random library using a phenotypic screen or selection. This article reviews the progress in this field and provides a general overview of relevant mutation methods, screens and selections. PMID:16369090

Hart, Darren J; Tarendeau, Franck

2005-12-14

326

Variable expression of immunoreactive surface proteins of Propionibacterium acnes.  

PubMed

Despite accumulating data implicating Propionibacterium acnes in a variety of diseases, its precise role in infection remains to be determined. P. acnes antigen-specific CD4(+) T cells are present in early inflamed acne lesions and may be involved in the inflammatory response; however, little is known about the specific antigens involved. In this study, B cell and T cell antigens from P. acnes expression libraries were cloned and evaluated and the four predominant proteins identified were investigated. Two of these antigens share some homology with an M-like protein of Streptococcus equi and have dermatan-sulphate-binding activity (PA-25957 and 5541). The remaining two antigens, PA-21693 and 4687, are similar to the product of the Corynebacterium diphtheriae htaA gene from the hmu ABC transport locus, although only one of these (PA-21693) is encoded within an hmu-like operon and conserved amongst a range of clinical isolates. All four proteins contain an LPXTG motif, although only PA-21693 contains a characteristic sortase-sorting signal. Variation in the expression of PA-4687, 25957 and 5541 is evident amongst clinical isolates and is generated both by frameshifts associated with the putative signal peptide and by variable numbers of repeat regions toward the carboxy-terminus, potentially generating heterogeneity of molecular mass and antigenic variation. In addition, in the case of PA-25957, a frameshift in a C-rich region at the extreme carboxy-terminus eliminates the LPXTG motif in some isolates. For the dermatan-sulphate-binding PA-25957, IgG1 antibody in serum from acne-positive donors was shown to be specific for the amino-terminal region of the protein, which also contains a CD4(+) T cell epitope. In contrast, serum from acne-negative donors shows an IgG2 and IgG3 antibody subclass response to the carboxy-terminal region. These data have implications for the potential role of P. acnes in inflammatory acne and other diseases. PMID:17159220

Lodes, Michael J; Secrist, Heather; Benson, Darin R; Jen, Shyian; Shanebeck, Kurt D; Guderian, Jeffrey; Maisonneuve, Jean-François; Bhatia, Ajay; Persing, David; Patrick, Sheila; Skeiky, Yasir A W

2006-12-01

327

Automated expression and solubility screening of His-tagged proteins in 96-well format  

Microsoft Academic Search

A growing need for sensitive and high-throughput methods for screening the expression and solubility of recombinant proteins exists in structural genomics. Originally, the emergency solution was to use immediately available techniques such as manual lysis of expression cells followed by analysis of protein expression by gel electrophoresis. However, these handmade methods quickly proved to be unfit for the high-throughput demand

Renaud Vincentelli; Stéphane Canaan; Julien Offant; Christian Cambillau; Christophe Bignon

2005-01-01

328

Comparison of Protein and mRNA Expression Evolution in Humans and Chimpanzees  

PubMed Central

Even though mRNA expression levels are commonly used as a proxy for estimating functional differences that occur at the protein level, the relation between mRNA and protein expression is not well established. Further, no study to date has tested whether the evolutionary differences in mRNA expression observed between species reflect those observed in protein expression. Since a large proportion of mRNA expression differences observed between mammalian species appears to have no functional consequences for the phenotype, it is conceivable that many or most mRNA expression differences are not reflected at the protein level. If this is true, then differences in protein expression may largely reflect functional adaptations observed in species phenotypes. In this paper, we present the first direct comparison of mRNA and protein expression differences seen between humans and chimpanzees. We reproducibly find a significant positive correlation between mRNA expression and protein expression differences. This correlation is comparable in magnitude to that found between mRNA and protein expression changes at different developmental stages or in different physiological conditions within one species. Noticeably, this correlation is mainly due to genes with large expression differences between species. Our study opens the door to a new level of understanding of regulatory evolution and poses many new questions that remain to be answered.

Fu, Ning; Drinnenberg, Ines; Kelso, Janet; Wu, Jia-Rui; Paabo, Svante; Zeng, Rong; Khaitovich, Philipp

2007-01-01

329

Multiplexed expression and screening for recombinant protein production in mammalian cells  

Microsoft Academic Search

BACKGROUND: A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great

Susan DJ Chapple; Anna M Crofts; S Paul Shadbolt; John McCafferty; Michael R Dyson

2006-01-01

330

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins  

Microsoft Academic Search

BACKGROUND: Expression of higher eukaryotic genes as soluble, stable recombinant proteins is still a bottleneck step in biochemical and structural studies of novel proteins today. Correct identification of stable domains\\/fragments within the open reading frame (ORF), combined with proper cloning strategies, can greatly enhance the success rate when higher eukaryotic proteins are expressed as these domains\\/fragments. Furthermore, a HTP cloning

Yunjia Chen; Shihong Qiu; Chi-Hao Luan; Ming Luo

2007-01-01

331

Expression of the potato leafroll luteovirus coat protein gene in transgenic potato plants inhibits viral infection  

Microsoft Academic Search

Transgenic potato plants, cultivar Désirée, were produced that contained the coat protein gene of potato leafroll luteovirus (PLRV). The transformed potato plants expressed the PLRV coat protein (CP) RNA sequences but accumulation of coat protein in transgenic tissues could not be detected. Upon inoculation with PLRV, the PLRV CP RNA expressing potato plants showed a reduced rate of virus multiplication.

Frank van der Wilk; Dinie Posthumus-Lutke Willink; Marianne J. Huisman; Harm Huttinga; Rob Goldbach

1991-01-01

332

Comparative Transcriptional and Translational Analysis of Leptospiral Outer Membrane Protein Expression in Response to Temperature  

Microsoft Academic Search

BackgroundLeptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins.

Miranda Lo; Stuart J. Cordwell; Dieter M. Bulach; Ben Adler

2009-01-01

333

High-throughput expression, purification, and characterization of recombinant Caenorhabditis elegans proteins  

Microsoft Academic Search

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more

Raymond Y Huang; Simon J Boulton; Marc Vidal; Steve C Almo; Anne R Bresnick; Mark R Chance

2003-01-01

334

Salivary Proline-rich Proteins: Biochemistry, Molecular Biology, and Regulation of Expression  

Microsoft Academic Search

The proline-rich proteins (PRPs) in mammalian salivary glands are encoded by tissue-specific multigene families whose members have diverged with respect to structure and regulation of expression. PRPs are expressed constitutively in humans, and comprise about [70%] of the total salivary proteins. Families of similar proteins are dramatically increased or induced in parotid and submandibular glands of rats, mice and hamsters

Don M. Carlson

1993-01-01

335

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

336

A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms  

PubMed Central

Green fluorescent protein (GFP) fusion proteins provide a potentially facile tool for identification of well expressed, properly behaved membrane proteins for biochemical and structural study. Here, we present a GFP-expression survey of >300 membrane proteins from 18 bacterial and archaeal extremophiles, organisms expected to be rich sources of membrane proteins having robust biophysical properties. We find that GFP-fusion fluorescence intensity is an excellent indicator of over-expression potential. By employing a follow-up optimization protocol using a suite of non-GFP constructs and different expression temperatures, we obtain 0.5–15 mg L?1 expression levels for 90% of the tested candidate proteins that pass the GFP screen. Evaluation of the results suggests that certain organisms may serve as better sources of well-expressed membrane proteins than others, that the degree to which codon usage matches the expression host is uncorrelated with success rate, and that the combination of GFP screening and expression optimization is essential for producing biochemically tractable quantities of material.

Hammon, Justus; Palanivelu, Dinesh V; Chen, Joy; Patel, Chintan; Minor, Daniel L

2009-01-01

337

Can P-glycoprotein mediate resistance to nilotinib in human leukaemia cells?  

PubMed

The effect of P-glycoprotein (P-gp, ABCB1, MDR1) expression on cell resistance to nilotinib was studied in human leukaemia cells. We used K562/Dox cells overexpressing P-gp and their variants (subclones) with a gradually decreased P-gp expression. These subclones were established by stable transfection of K562/Dox cells with a plasmid vector expressing shRNA targeting the ABCB1 gene. Functional analysis of P-gp using a specific fluorescent probe indicated gradually decreased dye efflux which was proportional to the P-gp expression. We observed that K562/Dox cells overexpressing P-gp contained a significantly reduced intracellular level of nilotinib when compared to their counter partner K562 cells, which do not express P-gp. This effect was accompanied by a decreased sensitivity of the K562/Dox cells to nilotinib. Importantly, cells with downregulated expression of P-gp gradually lost their ability to decrease the intracellular level of nilotinib although they still significantly decreased the intracellular level of daunorubicin (DNR). Accordingly, cells with the reduced expression of P-gp concomitantly failed to provide resistance to nilotinib, however, they exhibited a significant resistance to DNR. Taken together, we demonstrated that the conclusion as to whether P-gp is involved in nilotinib resistance or not strongly depends on its expression at protein level. PMID:23103446

Kosztyu, Petr; Dolezel, Petr; Mlejnek, Petr

2012-10-26

338

[Nuclear organization and expression of milk protein genes].  

PubMed

Milk protein gene expression varies during the pregnancy/lactation cycle under the influence of lactogenic hormones which induce the activation of several transcription factors. Beyond this activation modifying the binding properties of these factors to their consensus sequences, their interactions with DNA is regulated by variations of the chromatin structure. In the nuclei of the mammary epithelial cell, the three dimensional organisation of the chromatin loops, located between matrix attachment regions, is now being studied. The main milk components are organised in supramolecular structures. Milk fat globules are made of a triglyceride core enwrapped by a tripartite membrane originating from various intracellular compartments. The caseins, the main milk proteins, form aggregates: the casein micelles. Their gradual aggregation in the secretory pathway is initiated as soon as from the endoplasmic reticulum. The mesostructures of the milk fat globule and of the casein micelle remain to be elucidated. Our goal is to make some progress into the understanding of the molecular and cellular mechanisms involved in the formation of these milk products. PMID:17151554

Chanat, Eric; Aujean, Etienne; Balteanu, Adrian; Chat, Sophie; Coant, Nicolas; Fontaine, Marie-Louise; Hue-Beauvais, Cathy; Péchoux, Christine; Torbati, Mohammad Bagher Montazer; Pauloin, Alain; Petitbarat, Marie; Devinoy, Eve

2006-01-01

339

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

340

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

341

Bacteriophage T4 DNA replication protein 61. Cloning of the gene and purification of the expressed protein.  

PubMed

In vitro, a bacteriophage T4 primase composed of T4 61 and 41 proteins, catalyzes the formation of pentaribonucleotides used to initiate DNA synthesis on single-stranded DNA. We have determined that cells containing a plasmid with the T4 DNA from 18.68 to 15.05 map units express an activity that substitutes for authentic 61 protein in vitro in catalyzing primer-dependent DNA synthesis with six other T4 DNA replication proteins. This result establishes that this region, genetically assigned to gene 61, is the structural gene for the priming protein. Cells containing a plasmid with gene 61 downstream of the strong phage lambda promoter PL and the antitermination site nutL produce 100-fold more 61 protein than T4-infected cells. We have developed an improved purification procedure which yields 100 mg of homogeneous, active protein from 178 g of these cells. In the plasmid, the T4 DNA downstream of gene 61 expresses a protein of 30,000 daltons. This protein may be the T4 DNA adenine methylase (dam) gene product, since Schlagman and Hattman (Schlagman, S. L. and Hattman, S. (1983) Gene 22, 139-156) have shown that this activity is expressed by plasmids containing T4 DNA from this region. In the PL, nutL vector, the expression of both the 30,000-dalton and 61 proteins is enhanced up to 20-fold by the presence of the phage lambda N protein, a transcription antitermination protein, suggesting that expression of the T4 DNA in the plasmid may be regulated transcriptionally. In addition, in both N+ and N- cells, the level of 61 protein, whose gene is proximal to PL on the plasmid, is lower than that of the product of the promoter distal 30,000-dalton protein gene. This result suggests that, at least in the plasmid construction, the expression of 61 protein may also be regulated after transcription. PMID:2995395

Hinton, D M; Nossal, N G

1985-10-15

342

Construction of pET-32 ? (+) Vector for Protein Expression and Purification  

PubMed Central

The construction of expression vector is a basic tool for biotechnology and production of desired proteins, this article summarized the construction of pET-32 ? (+) vector techniques which are generally used in research laboratories. The procedures include that acquisition of the exogenous DNA fragment for construction of the vector, subcloning the DNA fragment into pET-32 ? (+) expression vector, protein expression in Escherichia coli BL21 (DE3) and protein purification under native conditions in E. coli lysates.

Liu, Zhi-Qiang; Yang, Ping-Chang

2012-01-01

343

Increased Expression of P-Glycoprotein and Doxorubicin Chemoresistance of Metastatic Breast Cancer Is Regulated by miR-298  

PubMed Central

MicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate the expression of a number of genes involved in cancer; therefore, they offer great diagnostic and therapeutic targets. We have developed doxorubicin-resistant and -sensitive metastatic human breast cancer cell lines (MDA-MB-231) to study the chemoresistant mechanisms regulated by miRNAs. We found that doxorubicin localized exclusively to the cytoplasm and was unable to reach the nuclei of resistant tumor cells because of the increased nuclear expression of MDR1/P-glycoprotein (P-gp). An miRNA array between doxorubicin-sensitive and -resistant breast cancer cells showed that reduced expression of miR-298 in doxorubicin-resistant human breast cancer cells was associated with increased expression of P-gp. In a transient transfection experiment, miR-298 directly bound to the MDR1 3? untranslated region and regulated the expression of firefly luciferase reporter in a dose-dependent manner. Overexpression of miR-298 down-regulated P-gp expression, increasing nuclear accumulation of doxorubicin and cytotoxicity in doxorubicin-resistant breast cancer cells. Furthermore, down-regulation of miR-298 increased P-gp expression and induced doxorubicin resistance in sensitive breast cancer cells. In summary, these results suggest that miR-298 directly modulates P-gp expression and is associated with the chemoresistant mechanisms of metastatic human breast cancer. Therefore, miR-298 has diagnostic and therapeutic potential for predicting doxorubicin chemoresistance in human breast cancer.

Bao, Lili; Hazari, Sidhartha; Mehra, Smriti; Kaushal, Deepak; Moroz, Krzysztof; Dash, Srikanta

2012-01-01

344

Statistical lower bounds on protein copy number from fluorescence expression images  

Microsoft Academic Search

Motivation: Fluorescence imaging has become commonplace for quantitatively measuring mRNA or protein expression in cells and tissues. However, such expression data is usually relative—absolute concentrations or molecular copy numbers are typically not known. While this is satisfactory for many applications, for certain kinds of quantitative network modeling and analysis of expression noise, absolute measures of expression are necessary. Results: We

Lee Zamparo; Theodore J. Perkins

2009-01-01

345

Differential protein expression in human corneal endothelial cells cultured from young and older donors  

Microsoft Academic Search

Purpose: To establish a baseline protein fingerprint of cultured human corneal endothelial cells (HCEC), to determine whether the protein profiles exhibit age-related differences, and to identify proteins differentially expressed in HCEC cultured from young and older donors. Methods: Corneas were obtained from five young (<30 years old) and five older donors (>50 years old). HCEC were cultured, and protein was

Cheng Zhu; Ian Rawe; Nancy C. Joyce

2008-01-01

346

Expression of chimeric monomer and dimer proteins on the plasma membrane of mammalian cells  

Microsoft Academic Search

Targeting of proteins to the plasma membrane of cells may be useful for vaccine development, tissue engineering, genetic research, bioseparations, and dis- ease treatment. The ability of different transmembrane domains (TM) to direct a reporter protein (human alpha- feto protein, AFP) to the surface of mammalian cells was examined. High surface expression was achieved with chimeric proteins composed of AFP

Wan-Chih Chou; Kuang-Wen Liao; Yu-Chih Lo; Shu Yaun Jiang; Ming Yang Yeh; Steve R. Roffler

1999-01-01

347

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

348

Expression of the TAR RNA binding protein in human testis  

Microsoft Academic Search

In testis, several RNA binding proteins have been shown to play a role in the translational regulation of specific transcripts. The human protein TRBP (TAR RNA binding protein) is the homologue of the mouse Prbp (Prm-1 RNA binding protein) involved in the protamine mRNA translational delay. TRBP is known to activate the HIV-1 long terminal repeat but this protein has

Jean Pierre Siffroi; Marie Francoise Alfonsi; Georges Guellaen; Jean Pierre Dadoune; Hopital Henri Mondor

2001-01-01

349

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

350

Gene expression and protein localisation of calcyclin, a calcium-binding protein of the S-100 family in fresh neuroblastomas.  

PubMed

Calcyclin gene, a Ca(2+)-binding protein with homology to S-100, has been found to be expressed at different levels in leukaemic cells and in other tumour cells. We recently reported the expression of the gene in human neuroblastoma (NB) cell lines, and suggested a possible role of calcyclin in cell differentiation. To extend our findings, we investigated the expression of the gene in NB cells induced to differentiate by retinoic acid (RA), using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Time-course experiments employing LA-N-5 cells showed that calcyclin mRNA appeared 2 h after RA treatment, long before the cells were blocked in the G1 cell-cycle phase and before the neurite-like structures outgrew from the cell bodies. This suggests the involvement of the gene in the early phase of cell differentiation. Furthermore, we investigated mRNA expression in a series of fresh neuroblastomas. NB tumours showed a heterogeneous pattern of calcyclin expression, although calcyclin seemed to be expressed more frequently in cases with a favourable Shimada histology. We also studied the expression of the protein in formalin fixed and paraffin embedded tissues, by using a specific anticalcyclin antibody. The protein was detected in stromal cells which characterise a more mature histological type, and in nerve sheaths, whereas neuroblasts were negative. The tissue that expressed calcyclin protein showed a Schwann-like differentiation and, unlike S-100 protein, calcyclin was expressed in the perineurium. PMID:7576953

Tonini, G P; Fabretti, G; Kuznicki, J; Massimo, L; Scaruffi, P; Brisigotti, M; Mazzocco, K

1995-01-01

351

Mammalian protein expression noise: scaling principles and the implications for knockdown experiments.  

PubMed

The abundance of a particular protein varies both over time within a single mammalian cell and between cells of a genetically identical population. Here, we investigate the properties of such noisy protein expression in mammalian cells by combining theoretical and experimental approaches. The gamma distribution model is well-known to describe cell-to-cell variability in protein expression in a variety of common scenarios. This model predicts, and experiments show, that when protein levels are manipulated by altering transcription rates or mRNA half-life, protein expression noise, defined as the squared coefficient of variation, is constant. In contrast, we also demonstrate that when protein levels are manipulated by changing protein half-life, as mean levels increase, noise decreases. Thus, in mammalian cells, the scaling relationship between mean protein levels and expression noise depends on how mean levels are perturbed. Therefore it may be important to consider how common experimental manipulations of protein expression affect not only mean levels, but also noise levels. In the context of knockdown experiments, natural cell-to-cell variability in protein expression implies that a particular cell from the knockdown population may have higher protein levels than a cell from the control population. Simulations and experimental data suggest that approximately three-fold knockdown in mean expression levels can reduce such so-called "overlap probability" to less than ~10%. This has implications for the interpretation of knockdown experiments when the readout is a single cell measure. PMID:22990612

Birtwistle, Marc R; von Kriegsheim, Alexander; Dobrzy?ski, Maciej; Kholodenko, Boris N; Kolch, Walter

2012-11-01

352

Expression of outer membrane protein II by gonococci in experimental gonorrhea  

PubMed Central

Gonorrheal urethritis was induced in three males by intraurethral instillation of predominantly pilus+ protein II- gonococci. Virtually all gonococci reisolated from the infected men exhibited protein II+ phenotype. The reisolated gonococci expressed five distinct outer membrane protein II species. Protein IIc+ organisms predominated in urines of all three subjects, but variants expressing this particular protein II were rarely spawned in vitro by input organisms. Protein IIc+ gonococci appeared early in one man's infection; they were joined later by variants that displayed eight other protein II phenotypes, including protein II-. These results show that input protein II- gonococci are supplanted by protein IIc+ variants during incipient gonorrheal urethritis. As infection progresses, a broader variety of protein II+ variants appears.

1988-01-01

353

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

354

Vectors for co-expression of an unrestricted number of proteins.  

PubMed

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five 'pQLink' vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells. PMID:17311810

Scheich, Christoph; Kümmel, Daniel; Soumailakakis, Dimitri; Heinemann, Udo; Büssow, Konrad

2007-02-20

355

Differential effects of opioid agonists on G protein expression in CHO cells expressing cloned human opioid receptors.  

PubMed

Recent evidence indicates that agonist ligands of G protein coupled receptors (GPCR) can activate different signaling systems. Such "agonist-directed" signaling also occurs with opioid receptors. Previous work from our laboratory showed that chronic morphine, but not DAMGO, up-regulates the expression of Galpha12 and that both morphine and DAMGO decreased Galphai3 expression in CHO cells expressing the cloned human mu opioid receptor. In this study, we tested the hypothesis that chronic opioid regulation of G protein expression is agonist-directed. Following a 20h treatment of CHO cells expressing the cloned human mu (hMOR-CHO), delta (hDOR-CHO) or kappa (hKOR-CHO) opioid receptors with various opioid agonists, we determined the expression level of Galpha12 and Galphai3 by Western blots. Among five mu agonists (morphine, etorphine, DADLE, DAMGO, herkinorin) tested with hMOR-CHO cells, only chronic morphine and etorphine up-regulated Galpha12 expression. All five mu agonists decreased Galphai3 expression. Among six delta agonists (SNC80, DPDPE, deltorphin-1, morphine, DADLE, etorphine) tested with hDOR-CHO cells, all six agonists down-regulated Galphai3 expression or moderately up-regulated Galpha12 expression. Among five kappa agonists, ((-)-ethylketocyclazocine, salvinorin A, U69,593, etorphine, (-)-U50,488) tested with hKOR-CHO cells, only chronic (-)-U50,488 and (-)-EKC up-regulated Galpha12 expression. All kappa agonists decreased Galphai3 expression. These data demonstrate that chronic opioid agonist regulation of G protein expression depends not only on the agonist tested, but also on the type of opioid receptor expressed in a common cellular host, providing additional evidence for agonist-directed signaling. PMID:18639745

Xu, Heng; Wang, Xiaoying; Partilla, John S; Bishop-Mathis, Kristen; Benaderet, Tova S; Dersch, Christina M; Simpson, Denise S; Prisinzano, Thomas E; Rothman, Richard B

2008-06-04

356

Menthol diminishes Staphylococcus aureus virulence-associated extracellular proteins expression.  

PubMed

Staphylococcus aureus is a significant human pathogen that is the major cause of a broad spectrum of illnesses, ranging from minor skin infections to life-threatening deep tissue infections and toxinosis. The ability of the organism to cause such a broad range of infections is, to a great extent, attributed to the secretion of a myriad of virulence-related extracellular proteins. Therefore, virulence as a target for antimicrobial chemotherapy has gained great interest. Menthol is a monocyclic terpene alcohol that occurs naturally in plants of the Mentha species lacking anti-S. aureus activity. In this paper, we demonstrate via hemolytic activity assays, tumor necrosis factor release assays, Western blot assays, and real-time reverse transcription-PCR assays that low concentrations of menthol can markedly inhibit the expression of ?-hemolysin, enterotoxins A and B, and toxic shock syndrome toxin 1 in S. aureus. Our results indicate that menthol may be useful in managing S. aureus infections when used in combination with ?-lactam antibiotics, which can often increase S. aureus toxin secretion when used at subinhibitory concentrations. In addition, the menthol basic structure has potential applications in the development of new anti-virulence drugs. PMID:21287163

Qiu, Jiazhang; Luo, Mingjing; Dong, Jing; Wang, Jianfeng; Li, Hongen; Wang, Xiaoliang; Deng, Yanhong; Feng, Haihua; Deng, Xuming

2011-02-02

357

Expression of an uncoupling protein gene homolog in chickens.  

PubMed

An avian uncoupling protein (UCP) gene homolog was recently sequenced from skeletal muscle and was proposed to have a role in thermogenesis in chickens, ducks and hummingbirds. Since mammalian UCP 2 and UCP 3 also appear to have functions associated with energy and substrate partitioning and body weight regulation, the purpose of this study was to further characterize chicken UCP under conditions of nutritional stress and/or leptin administration. Male 3-week-old chickens were starved for 24 or 48 h and then half of each group was refed for an additional 24 h. In a follow-up experiment, chickens were fed or starved for 48 h with or without leptin administration. Feed deprivation increased UCP mRNA expression in skeletal muscle by up to 260% (P<0.001), and in a time-dependent manner in pectoralis muscle. Refeeding for 24 h normalized muscle UCP mRNA levels. Leptin administration had no effect on muscle UCP. Chicken muscle UCP mRNA levels were highly correlated with plasma triglyceride and non-esterified fatty acid (NEFA) concentrations, and with circulating levels of insulin, insulin-like growth factor (IGF)-I and IGF-II. These results suggest that, as in mammals, avian UCP is up-regulated during feed deprivation and is highly correlated with increased fatty acid oxidation and flux into skeletal muscle. PMID:12208305

Evock-Clover, Christina M; Poch, Stephen M; Richards, Mark P; Ashwell, Christopher M; McMurtry, John P

2002-10-01

358

Generation of a recloned transgenic cat expressing red fluorescence protein.  

PubMed

Somatic cells from a first-generation red fluorescence protein transgenic cat (first RFP TG cat) were used to produce a recloned RFP transgenic cat (Re-RFP TG cat) (Felis catus) that systemically expressed RFP. A total of 281 RFP cloned embryos were transferred into 13 surrogate mothers (mean=21+/-7.7 embryos/recipient). One surrogate cat was diagnosed pregnant (7.7%) and delivered one live kitten. The presence of the RFP gene in the mRNA and genomic DNA of the Re-RFP TG cat was confirmed by polymerase chain reaction analyses, and red fluorescence was detected in its internal organs and placental tissue samples. Analysis of nine feline-specific microsatellite loci confirmed that the Re-RFP TG cat was genetically identical to the donor cat. To test whether results such as normality of offspring and a low cloning success were due to epigenetic modifications, global methylation of placenta from the two first cloned RFP TG cats (77.08% and 82.29%) and the Re-RFP TG cat (76.38%) were compared by bisulfite mutagenesis sequencing analysis. In conclusion, although cloning efficiency was low, we demonstrated the successful use of a cloned first RFP TG cat as a donor cat to produce a Re-RFP TG cat. These results may facilitate future developments in biomedical models for human therapeutic applications. PMID:20172599

Cho, S J; Bang, J I; Yu, X F; Lee, Y S; Kim, J H; Jeon, J T; Yee, S T; Kong, I K

2010-02-20

359

Snorkel: An Epitope Tagging System for Measuring the Surface Expression of Membrane Proteins  

PubMed Central

Tags are widely used to monitor a protein’s expression level, interactions, protein trafficking, and localization. Membrane proteins are often tagged in their extracellular domains to allow discrimination between protein in the plasma membrane from that in internal pools. Multipass membrane proteins offer special challenges for inserting a tag since the extracellular regions are often composed of small loops and thus inserting an epitope tag risks perturbing the structure, function, or location of the membrane protein. We have developed a novel tagging system called snorkel where a transmembrane domain followed by a tag is appended to the cytoplasmic C-terminus of the membrane protein. In this way the tag is displayed extracellularly, but structurally separate from the membrane protein. We have tested the snorkel tag system on a diverse panel of membrane proteins including GPCRs and ion channels and demonstrated that it reliably allows for monitoring of the surface expression.

Brown, Michael; Stafford, Lewis J.; Onisk, Dale; Joaquim, Tony; Tobb, Alhagie; Goldman, Larissa; Fancy, David; Stave, James; Chambers, Ross

2013-01-01

360

Potential of the novel antiretroviral drug rilpivirine to modulate the expression and function of drug transporters and drug-metabolising enzymes in vitro.  

PubMed

The objective of this study was to assess the drug-drug interaction potential of the new non-nucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine in vitro. The following were evaluated: P-glycoprotein (P-gp/ABCB1) inhibition by calcein assay; breast cancer resistance protein (BCRP/ABCG2) inhibition by pheophorbide A efflux; and inhibition of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 by 8-fluorescein-cAMP uptake. Inhibition of cytochrome P450 enzymes was assessed using commercially available kits. Substrate characteristics were evaluated by growth inhibition assays in MDCKII cells overexpressing particular ABC transporters. Induction of drug-metabolising enzymes and transporters was quantified by real-time RT-PCR in LS180 cells, and activation of pregnane X receptor (PXR) by a reporter gene assay. Rilpivirine significantly inhibited P-gp (IC(50) = 13.1 ± 6.8 ?mol/L), BCRP (IC(50) = 1.5 ± 0.3 ?mol/L), OATP1B1 (IC(50) = 4.1 ± 1.8 ?mol/L), OATP1B3 (IC(50) = 6.1 ± 0.9 ?mol/L), CYP3A4 (IC(50) = 1.3 ± 0.6 ?mol/L), CYP2C19 (IC(50) = 2.7 ± 0.3 ?mol/L) and CYP2B6 (IC(50) = 4.2 ± 1.6 ?mol/L). Growth inhibition assays indicate that rilpivirine is not a substrate of P-gp, BCRP, or multidrug resistance-associated proteins 1 and 2. In LS180 cells, rilpivirine induced mRNA expression of ABCB1, CYP3A4 and UGT1A3, whereas ABCC1, ABCC2, ABCG2, OATP1B1 and UGT1A9 were not induced. Moreover, rilpivirine was a PXR activator. In conclusion, rilpivirine inhibits and induces several relevant drug-metabolising enzymes and drug transporters, but owing to its low plasma concentrations it is most likely less prone to drug-drug interactions than older NNRTIs. PMID:23428312

Weiss, Johanna; Haefeli, Walter Emil

2013-02-18

361

G-Protein-Coupled Receptor Kinase Activity in Hypertension Increased Vascular and Lymphocyte G-Protein Receptor Kinase2 Protein Expression  

Microsoft Academic Search

Impaired receptor-stimulated adenylyl cyclase activation has been observed in lymphocytes from hypertensive subjects and has been linked to an increase in lymphocyte G-protein receptor kinase-2 (GRK-2) protein expression. However, whether the increase in lymphocyte GRK-2 reflected an increase in vascular GRK-2 was unknown. Therefore, we compared GRK-2 protein expression in lymphocytes and aortas obtained from normotensive Wistar rats, Wistar-Kyoto rats

Robert Gros; Jozef Chorazyczewski; Murray D. Meek; Jeffrey L. Benovic; Stephen S. G. Ferguson; Ross D. Feldman

362

Dietary Protein-Related Changes in Hepatic Transcription Correspond to Modifications in Hepatic Protein Expression in Growing Pigs1  

Microsoft Academic Search

In a previous investigation we showed by expression profiling based on transcription analysis using differential display RT-PCR (DDRT-PCR) and real-time RT-PCR that a soy protein diet (SPI) significantly changes the hepatic transcription pattern compared with a casein diet (CAS). The present study was conducted to determine whether the transcriptional modulation is translated into protein expression. The hepatic mRNA abun- dance

Peter Junghans; Thilo Kaehne; Manfred Beyer; Cornelia C. Metges; Manfred Schwerin

363

Protein Localization and mRNA Expression of Epimorphin in Mouse and Human Kidneys  

Microsoft Academic Search

Epimorphin is a mesenchymal cell surface protein which induces epithelial branching morphogenesis. However, the role of epimorphin in the kidney has not been addressed. In the present study, the localization of epimorphin protein and the expression of its mRNA were investigated in the developing mouse and adult human kidneys using immunohistochemistry and semiquantitative RT-PCR. The in vitro expression of epimorphin

Satoshi Horikoshi; Mutsuko Yoshikawa; Terumi Shibata; Kaoru Takahashi; Isao Shirato; Yasuhiko Tomino

2001-01-01

364

Ethanol rapidly induces steroidogenic acute regulatory protein expression and translocation in rat adrenal gland  

Microsoft Academic Search

Acute ethanol exposure increases GABAergic neuroactive steroids in plasma and brain by releasing these steroids or their precursors from the adrenal glands. The present study showed that ethanol administration rapidly increases the expression of steroidogenic acute regulatory protein (StAR) in the cytosolic and mitochondrial fractions of adrenal glands. The increased StAR protein expression paralleled increases in plasma pregnenolone, progesterone and

Rahul T. Khisti; Sandeep Kumar; A. Leslie Morrow

2003-01-01

365

Expression of heat shock proteins, glutathione peroxidase and catalase in childhood acute lymphoblastic leukemia and nephroblastoma  

Microsoft Academic Search

In this study we analyzed the mRNA expression of the heat shock proteins 27 and 70, and the expression of the radical scavenging enzymes catalase and glutathione peroxidase (GPX) in childhood acute lymphoblastic leukemia (ALL, n = 54) and in nephroblastoma (n = 34). We found a significant positive correlation between both heat shock proteins and also between glutathione peroxidase

G. Stammler; M. Volm

1996-01-01

366

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

ERIC Educational Resources Information Center

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

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

2004-01-01

367

Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii  

Microsoft Academic Search

We have engineered a mutant version of the green fluorescent protein GFP (Cormack et al. Selected for bright fluorescence in E. coli. Gene 1996;173:33–38) for expression in the protozoan parasite Toxoplasma gondii. Although intact GFP was not expressed at any detectable level, GFP fusion proteins could be detected by fluorescence microscopy, flow cytometry (FACS), and immunoblotting. Both extracellular tachyzoites and

Boris Striepen; Cynthia Yingxin He; Mariana Matrajt; Dominique Soldati; David S Roos

1998-01-01

368

Kupffer Cells Are a Dominant Site of Uncoupling Protein 2 Expression in Rat Liver  

Microsoft Academic Search

The mechanisms underlying thermogenesis in liver are not well understood. They may involve proteins related to the mitochondrial uncoupling protein (UCP1) of brown adipocytes. In this paper, it is demonstrated that UCP1 is not expressed in any liver cell type of rat while UCP2, a recently cloned homologue of UCP1, is expressed at a very high level in Kupffer cells

Dominique Larrouy; Patrick Laharrague; Georges Carrera; Nathalie Viguerie-Bascands; Corinne Levi-Meyrueis; Christophe Fleury; Claire Pecqueur; Maryse Nibbelink; Mireille André; Louis Casteilla; Daniel Ricquier

1997-01-01

369

A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production  

Microsoft Academic Search

BACKGROUND: In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a

Lisa D Cabrita; Weiwen Dai; Stephen P Bottomley

2006-01-01

370

Proteomic analysis of differential protein expression in human atherosclerotic plaque progression  

Microsoft Academic Search

In this study, differential protein expression was assessed during human atherosclerotic plaque progression. A multifaceted approach was used in which differential protein expression was studied by two-dimensional (2D) gel electrophoresis and validated in individual patients using western blotting and immunohistochemistry. 2D profiles of whole- mount advanced stable lesions were compared to those of plaques containing a thrombus. Mass spectrometry analysis

Marjo MPC Donners; Monique J Verluyten; Freek G Bouwman; Edwin CM Mariman; Bart Devreese; Frank Vanrobaeys; Jozef van Beeumen; Luc HJM van den Akker; Mat JAP Daemen; Sylvia Heeneman

2005-01-01

371

Expression domains of the Cf1a POU domain protein during Drosophila development  

Microsoft Academic Search

We have used antibodies directed against a unique portion of the Drosophila POU domain protein Cfla to localize its sites of expression in developing embryos. Cfla protein is first detected during germ band extension in the tracheal placodes and in the midline mesectoderm cells. Tracheal expression continues throughout embryonic development, especially in the main longitudinal tracheal trunks. Additional sites of

A. N. Billin; S. J. Poole

1995-01-01

372

Patterns of protein expression in tissues of the killifish, Fundulus heteroclitus and Fundulus grandis  

Microsoft Academic Search

Fundulus is a diverse and widespread genus of small teleost fish of North America. Due to its high tolerance for physiochemical variation (e.g. temperature, oxygen, salinity), Fundulus is a model organism to study physiological and molecular adaptations to environmental stress. The thesis focuses on patterns of protein expression in Fundulus heteroclitus and F. grandis.The patterns of protein expression were investigated

Naga Vijayalaxmi Abbaraju

2011-01-01

373

?-synuclein and ?-synuclein enhance secretion protein production in baculovirus expression vector system.  

PubMed

The baculovirus expression vector system (BEVS) is widely used as a tool for expressing of recombinant proteins in insect cells or larvae. However, the expression level of secretion pathway proteins is often lower than that of cytosolic and nucleus proteins. Thus, we attempted to improve production of secreted proteins by using a secretory alkaline phosphatase-EGFP fusion protein (SEFP)-based bi-cistronic baculovirus vector to identify chaperones that have potential on boosting secreted protein production. As co-expressed SEFP with a chaperone, calreticulin (CALR), it was found that the secreted SEFP enzyme activity can be boosted up to twofold. This result demonstrated the SEFP-based bi-cistronic approach can be used to identify the genes that can enhance secretion protein production in BEVS. Thus, the chaperone activity of ?-synuclein (?-syn) and ?-synuclein (?-syn) was evaluated in cells co-expressed with SEFP and compared that with CALR by analyzing localization, alkaline phosphatase enzyme activity, and mRNA expression levels of SEFP. Our results showed that SEFP enzyme activity from cells co-expressed with both synuclein proteins can be enhanced up to 2.3-fold and this increment was better than that caused by CALR. Moreover, this enhancement might arise from the transcription enhancement or higher RNA stability. By this novel approach, we provided evidences that ?- and ?-syn can enhance secretion proteins production in BEVS. PMID:23314197

Teng, Chao-Yi; Chang, Shou-Lin; Tsai, Meng-Feng; Wu, Tzong-Yuan

2013-01-12

374

Eukaryotic expression system for the incorporation of stable isotopes into proteins  

US Patent & Trademark Office Database

Methods for producing stable isotope-labeled recombinant protein are provided. The methods include isolating a stable isotope-labeled recombinant protein from a Trichoplusia ni larva expressing a recombinant protein, which Trichoplusia ni larva has ingested a food source comprising stable isotope-labeled algae, thereby resulting in incorporation of a stable isotope into the recombinant protein to produce the stable isotope-labeled recombinant protein.

Kobilka; Brian (Palo Alto, CA); Bokoch; Michael (Menlo Park, CA)

2011-12-13

375

An artificial regulatory circuit for stable expression of DNA-binding proteins in a T7 expression system.  

PubMed

We had earlier overproduced the transcription activator protein C of bacteriophage Mu in a phage-T7 expression system. Although we achieved a high level of overproduction, the expression was not consistent. This could be due to the leaky expression of T7 RNA polymerase in the uninduced state. Introduction of pLysS, a plasmid encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase, resulted in consistent, but extremely low production of the C protein. To overcome this problem, we have devised an artificial regulatory circuit to obtain stabilised, consistent overproduction of C protein. The C-binding site was cloned downstream from the transcription start point of T7 lys. Upon induction, the C protein produced binds to its site with a very high affinity, possibly acting as a transcriptional roadblock for lys. This would overcome the inhibitory effect of T7 lysozyme on T7 RNA polymerase. PMID:9185843

Paul, B D; Ramesh, V; Nagaraja, V

1997-04-29

376

Dietary protein-related changes in hepatic transcription correspond to modifications in hepatic protein expression in growing pigs.  

PubMed

In a previous investigation we showed by expression profiling based on transcription analysis using differential display RT-PCR (DDRT-PCR) and real-time RT-PCR that a soy protein diet (SPI) significantly changes the hepatic transcription pattern compared with a casein diet (CAS). The present study was conducted to determine whether the transcriptional modulation is translated into protein expression. The hepatic mRNA abundance of four genes (EP24.16, LC3, NPAP60L, RFC2) that showed diet-related expression in previous DDRT-PCR experiments was analyzed by real-time RT-PCR. Two pigs that showed the most prominent SPI-related changes of transcription and two casein-fed pigs were selected and their hepatic protein pattern was studied comparatively by two-dimensional gel electrophoresis and peptide mass fingerprinting. The two-dimensional protein gel electrophoresis revealed a predominant SPI-associated upregulation of protein expression that corresponded to the results of the mRNA study. Of 380 diet-related protein spots displayed, 215 appeared exclusively or enlarged in the two SPI pigs; 10 of 39 diet-related expressed protein spots extracted could be identified by peptide mass fingerprinting and database search. Compared with the transcriptomics approach, the proteomics approach led in part to the identification of the same diet-associated expressed molecules (plasminogen, trypsin, phospholipase A2, glutathione-S-transferase alpha, retinal binding protein) or at least molecules belonging to the same metabolic pathways (protein and amino acid metabolism, oxidative stress response, lipid metabolism). The present results at the proteome level confirm SPI-related increased oxidative stress response and significant effects on protein biosynthesis already observed at the transcriptome level. PMID:14704291

Junghans, Peter; Kaehne, Thilo; Beyer, Manfred; Metges, Cornelia C; Schwerin, Manfred

2004-01-01

377

P-glycoprotein and breast cancer resistance protein restrict apical-to-basolateral permeability of human brain endothelium to amyloid-beta.  

PubMed

The clearance of amyloid beta (Abeta) from the brain represents a novel therapeutic target for Alzheimer's disease. Conflicting data exist regarding the contribution of adenosine triphosphate-binding cassette transporters to the clearance of Abeta through the blood-brain barrier. Therefore, we investigated whether Abeta could be a substrate for P-glycoprotein (P-gp) and/or for breast cancer resistance protein (BCRP) using a human brain endothelial cell line, hCMEC/D3. Inhibition of P-gp and BCRP increased apical-to-basolateral, but not basolateral-to-apical, permeability of hCMEC/D3 cells to (125)I Abeta 1-40. Our in vitro data suggest that P-gp and BCRP might act to prevent the blood-borne Abeta 1-40 from entering the brain. PMID:19367293

Tai, Leon M; Loughlin, A Jane; Male, David K; Romero, Ignacio A

2009-04-15

378

Evaluation of Limiting Brain Penetration Related to P-glycoprotein and Breast Cancer Resistance Protein Using [ 11 C]GF120918 by PET in Mice  

Microsoft Academic Search

Purpose  GF120918 has a high inhibitory effect on P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). We developed [11C]GF120918 as a positron emission tomography (PET) probe to assess if dual modulation of P-gp and BCRP is useful to evaluate\\u000a brain penetration.\\u000a \\u000a \\u000a \\u000a \\u000a Procedures  PET studies using [11C]GF120918 were conducted on P-gp and\\/or Bcrp knockout mice as well as wild-type mice.\\u000a \\u000a \\u000a \\u000a \\u000a Results  In PET studies,

Kazunori Kawamura; Tomoteru Yamasaki; Fujiko Konno; Joji Yui; Akiko Hatori; Kazuhiko Yanamoto; Hidekatsu Wakizaka; Makoto Takei; Yuichi Kimura; Toshimitsu Fukumura; Ming-Rong Zhang

2011-01-01

379

Oxymatrine inhibits development of morphine-induced tolerance associated with decreased expression of P-glycoprotein in rats.  

PubMed

The effect of oxymatrine on the development of tolerance to the antinociceptive effects of morphine was investigated in rats. The degree of tolerance was assessed using the tail-flick test before and after 6 days of twice daily administration of oxymatrine premorphine (10/20/30 mg/kg). High doses of oxymatrine inhibited the development of morphine tolerance (resembling the effect of 7.5 mg/kg of the NMDA receptor antagonist memantine) while also increasing the antinociceptive effects. A high dose of oxymatrine (30 mg/kg) also significantly inhibited the dramatic increase in expression of morphine-induced P-glycoprotein (P-gp), an ATP-dependent efflux pump acting at the blood-brain barrier, by Western blot analysis. Furthermore, these studies suggest that P-gp modulates the development of morphine tolerance while not affecting the magnitude of the analgesic effect of morphine. These results imply that oxymatrine prevention of the development of tolerance of morphine may be related to a considerable inhibition of P-gp expression. In contrast, the authors' data suggest that the mechanism of oxymatrine enhancement of morphine's analgesic effects is not associated with increase in the level of expression of P-gp. However, they believe that their findings can be used by researchers to develop therapies that will allow patients to take morphine without becoming tolerant of its benefits. PMID:20587445

Li, Yanwei; Yue, Hui; Xing, Yanmin; Sun, Haiyan; Pan, Zhanyu; Xie, Guangru

2010-06-01

380

Fusion order controls expression level and activity of elastin-like polypeptide fusion proteins  

PubMed Central

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin-like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein