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Sample records for p-gp protein expression

  1. Effect of FosPeg® mediated photoactivation on P-gp/ABCB1 protein expression in human nasopharyngeal carcinoma cells.

    PubMed

    Wu, R W K; Chu, E S M; Huang, Z; Xu, C S; Ip, C W; Yow, C M N

    2015-07-01

    Multidrug resistance (MDR) refers to the ability of cancer cells to develop cross resistance to a range of anticancer drugs which are structurally and functionally unrelated. P-glycoprotein (P-gp) is the best studied MDR phenotype in photodynamic therapy (PDT) treated cells. Our pervious study demonstrated that FosPeg® mediated PDT is effective to NPC cell line models. In this in vitro study, the expression of MDR1 gene and its product P-gp in undifferentiated, poorly differentiated and well differentiated human nasopharyngeal carcinoma (NPC) cells were investigated. The influence of P-gp efflux activities on photosensitizer FosPeg® was also examined. Regardless of the differentiation status, PDT tested NPC cell lines all expressed P-gp protein. Results indicated that FosPeg® photoactivation could heighten the expression of MDR1 gene and P-gp transporter protein in a dose dependent manner. Up to 2-fold increase of P-gp protein expression were seen in NPC cells after FosPeg® mediated PDT. Interestingly, our finding demonstrated that FosPeg® mediated PDT efficiency is independent to the MDR1 gene and P-gp protein expression in NPC cells. FosPeg® itself is not the substrate of P-gp transporter protein and no efflux of FosPeg® were observed in NPC cells. Therefore, the PDT efficiency would not be affected even though FosPeg® mediated PDT could induce MDR1 gene and P-gp protein expression in NPC cells. FosPeg® mediated PDT could be a potential therapeutic approach for MDR cancer patients. PMID:25900553

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2006-06-14

    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 microM, which were found to be non-cytotoxic towards the Caco-2 cells, were observed to inhibit P-gp mediated efflux transport of [3H]-digoxin in the cells. The acute inhibitory effect was dependent on the capsaicin concentration and duration of exposure, with abolishment of polarity of [3H]-digoxin transport attained at 50 microM of capsaicin. In contrast, longer term (48 and 72 h) co-incubation of the Caco-2 cells with capsaicin (50 and 100 microM) 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 higher degree of [3H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. The induction of P-gp protein and mRNA levels was also influenced by capsaicin concentration and duration of exposure, with higher expression levels, in particular of the mRNA, seen at higher spice concentrations over prolonged period of incubation. Our data suggest that caution should be exercised when capsaicin is to be consumed with drugs that are P-gp substrates. In particular, the oral bioavailability of these drugs may be influenced by the P-gp status of populations that rely heavily on hot chilli in their diets. PMID:16674925

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

    PubMed

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

    2015-07-01

    Venlafaxine (VLX) could be pumped out of the brain by P-glycoprotein (P-gp). Moreover, the expression of P-gp distributed in blood-brain barrier could be significantly induced by VLX. Thus, P-gp could be considered as the nature barrier for delivering of VLX to the brain. The aim of this study was to investigate whether the efflux function and increased expression of P-gp could be reversed by utilizing solid lipid nanoparticles (SLN). VLX solid lipid nanoparticles (VLX - SLN) were prepared and evaluated. Pharmacokinetics and brain distribution of VLX in different formulations were conducted after oral or intravenous administration. P-gp efflux function to VLX was evaluated by the brain uptake amount of VLX, while P-gp expression was investigated by Western blotting. Results indicated that the entrapment, mean size and zata potential of VLX - SLN was 74.9 ± 3.0 %, 186.3 ± 69.26 nm and -22.8 ± 7.78 mv, respectively. After vein injection of VLX formulations, the brain uptake amount of VLX from VLX - SLN was significantly higher than that of VLX solution, VLX solution with empty SLN (VLX+ empty SLN) and VLX solution with Verapamil (VLX + Ver), respectively. Furthermore, the protein mass of P-gp in VLX - SLN treated group was the lowest among all the investigated groups. These results indicated that SLN could overcome P-gp and achieve brain target by intravenous administration. PMID:25567760

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

    PubMed

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

    2014-05-01

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

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

    PubMed

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

    2008-08-01

    Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 microM, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [(3)H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [(3)H]-digoxin polarized transport attained at 50 microM of piperine. In contrast, prolonged (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 microM) 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 [(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 microg/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. PMID:18417181

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

    SciTech Connect

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

    2008-08-01

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

  9. Effects of Muscone on the Expression of P-gp, MMP-9 on Blood-Brain Barrier Model In Vitro.

    PubMed

    Wang, Guang-Yun; Wang, Ning; Liao, Hua-Ning

    2015-11-01

    Muscone is the main chemical ingredient in Musk which is main crude drug in Tongqiaohuoxue decoction (TQHXD), and TQHXD has a protective effect on damaged neurons, so we hypothesize that muscone can alter blood-brain barrier (BBB) permeability via the modulation of P-glycoprotein (P-gp) and matrix metalloproteinase-9 (MMP-9) expression. In this study, astrocytes (AC) and human umbilical vein endothelial cells (ECV304) were co-cultured to simulate the BBB model in vitro. Leak testing, transmembrane resistance experiments, and BBB-specific enzyme testing were used to test whether the model was successful. Different concentrations of muscone permeating the BBB were detected by gas chromatography (GC). The change of the transendothelial electrical resistance (TEER) on the BBB in vitro after treating with muscone was detected by Millicell-ERS. The protein expression of P-gp, MMP-9 in normal, and oxygen/glucose deprivation (OGD) BBB model was determined by western blotting to inquire that the mechanism of muscone penetrates the BBB model in vitro. The results show that muscone was detected in the lower medium of the BBB model by GC; the values of TEER were no significant difference before and after muscone (8 ?M) was added to the BBB model; the expression of P-gp significantly decreased after the BBB model treatment with muscone (4, 8, and 16 ?M) for 24 h; the expression of P-gp and MMP-9 in different concentrations of muscone groups had different degrees of reduction compared with the BBB in the state of OGD. In conclusion, muscone could permeate the BBB model, and it was associated with the inhibition of P-gp and MMP-9 expression. An understanding of the mechanisms of muscone across the BBB is crucial to the development of therapeutic modalities for cerebral vascular diseases. PMID:25976179

  10. Dasatinib reverses the multidrug resistance of breast cancer MCF-7 cells to doxorubicin by downregulating P-gp expression via inhibiting the activation of ERK signaling pathway

    PubMed Central

    Chen, Ting; Wang, Changyuan; Liu, Qi; Meng, Qiang; Sun, Huijun; Huo, Xiaokui; Sun, Pengyuan; Peng, Jinyong; Liu, Zhihao; Yang, Xiaobo; Liu, Kexin

    2015-01-01

    Multidrug resistance (MDR) is one of the major obstacles to the efficiency of cancer chemotherapy, which often results from the overexpression of drug efflux transporters such as P-glycoprotein (P-gp). In the present study, we determined the effect of dasatinib which was approved for imatinib resistant chronic myelogenous leukemia (CML) and (Ph+) acute lymphoblastic leukemia (ALL) treatment on P-gp-mediated MDR. Our results showed that dasatinib significantly increased the sensitivity of P-gp-overexpressing MCF-7/Adr cells to doxorubicin in MTT assays; thus lead to an enhanced cytotoxicity of doxorubicin in MCF-7/Adr cells. Additionally, dasatinib increased the intracellular accumulation, inhibited the efflux of doxorubicin in MCF-7/Adr cells, and significantly enhanced doxorubicin-induced apoptosis in MCF-7/Adr cells. Further studies showed that dasatinib altered the expression levels of mRNA, protein levels of P-gp, and the phosphorylation of signal–regulated kinase (ERK) both in time-dependent (before 24 h) and dose-dependent manners at concentrations that produced MDR reversals. In conclusion, dasatinib reverses P-gp-mediated MDR by downregulating P-gp expression, which may be partly attributed to the inhibition of ERK pathway. Dasatinib may play an important role in circumventing MDR when combined with other conventional antineoplastic drugs. PMID:25482933

  11. Concomitance of P-gp/LRP Expression with EGFR Mutations in Exons 19 and 21 in Non-Small Cell Lung Cancers

    PubMed Central

    Wei, Hong; Lu, Weipeng; Li, Mei; Zhang, Qiuping

    2016-01-01

    Purpose Traditional chemotherapy is the main adjuvant therapy for the treatment of non-small cell lung cancer (NSCLC). However, the emergence of multi-drug resistance (MDR) has greatly restricted the curative effect of chemotherapy. Therefore, it is necessary to find a method to treat MDR NSCLC clinically. It is worth investigating whether NSCLCs that are resistant to traditional chemotherapy can be effectively treated with tyrosine kinase inhibitors targeting epidermal growth factor receptor (EGFR). Materials and Methods The expression of P-glycoprotein (P-gp) and lung resistance-related protein (LRP) was detected by immunohistochemistry, and mutations in EGFR (exons 19 and 21) and Kirsten rat sarcoma viral oncogene homolog (KRAS) (exon 2) were detected by high-resolution melting analysis (HRMA) of surgical NSCLC specimens from 127 patients who did not undergo traditional chemotherapy or radiotherapy. A Pearson chi-square test was performed to analyze the correlations between the expression of P-gp and LRP and mutations in EGFR and KRAS. Results The expression frequencies of P-gp and LRP were significantly higher in adenocarcinomas from non-smoking patients; the expression frequency of LRP was significantly higher in cancer tissue from female patients. The frequency of EGFR mutations was significantly higher in well to moderately differentiated adenocarcinomas from non-smoking female patients. The frequency of EGFR mutations in the cancers that expressed P-gp, LRP, or both P-gp and LRP was significantly higher than that in cancers that did not express P-gp or LRP. Conclusion NSCLCs expressing P-gp/LRP bear the EGFR mutation in exon 19 or 21 easily. PMID:26632382

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

    PubMed Central

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

    2015-01-01

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

  13. [Effect of tetrandrine combined with daunorubicin on expressions of P21 and P-gp in K562/A02 cells].

    PubMed

    Chen, Bao-An; Li, Jing; Chen, Jian; Gao, Feng; Wu, Ya-Nan; Zhu, Min-Sheng; Ding, Jia-Hua; Gao, Chong; Sun, Xin-Chen; Xu, Wen-Lin; Wang, Xue-Mei

    2009-10-01

    This study was aimed to investigate the reversal effect of Tetrandrine (TET) combined with daunorubicin (DNR) on multidrug resistance (MDR) of K562/A02 cells and its relation to P21, P-gp and their genes so as to provide the new theoretic evidence for clinical use of TET. The experiments were divided into 4 groups: control group (DNR alone), combined 1 group (DNA+0.5 mg/L TET), combined 2 group (DNR+1.0 mg/L TET) and combined 3 group (DNR+2.0 mg/L TET). The expressions of P21, P-gp and mdr-1 gene in K562/A02 cells of different groups were detected by Western blot, flow cytometry and semi-quantitative PCR respectively. The results showed that the expression of P21 was enhanced along with increasing of TET concentration, the expression of P-gp was reduced along with increasing of TET concentration and expression of mdr-1 gene was almost not observed in K562 cells, but the high expression of mdr-1 gene was seen in K562/A02 cells, furthermore, the expression of mdr-1 gene in K562/A02 cells increasingly was reduced along with increasing of TET concentration. It is concluded that the TET possesses the reversal effect on multiple drug resistance of K562/A02 cells with concentration dependence, the reversal effect of TET may be related to up-regulation of P21 expression and down-regulation of P-gp and mdr-1 gene expressions in K562/A02 cells. PMID:19840446

  14. Quantification of proteins by flow cytometry: Quantification of human hepatic transporter P-gp and OATP1B1 using flow cytometry and mass spectrometry.

    PubMed

    Hogg, Karen; Thomas, Jerry; Ashford, David; Cartwright, Jared; Coldwell, Ruth; Weston, Daniel J; Pillmoor, John; Surry, Dominic; O'Toole, Peter

    2015-07-01

    Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter varied considerably in the four hepatocyte samples analysed, ranging from only 6% to 35% of intact and viable cells. The sample with only 6% externally facing transporter was further analysed by confocal microscopy which qualitatively confirmed the low level of transporter in the membrane and the large internal population. Here we prove that flow cytometry is an important tool for future protein analysis as it can not only quantify the number of proteins that a cell express but also identify the number of proteins on the surface and it is easy to apply for routine assays. PMID:25916617

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

    PubMed Central

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

    2013-01-01

    In this study, the peptide sized 21?kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, were isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21 and human colorectal cancer tissue homogenate, resulting in identification of an optimal recombinant Fab clone (Number 29). Further characterization by recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29 by Isopropyl ?-D-1-thiogalactopyranoside (IPTG). After purified by HiTrap Protein L, the specificity of the Fab antibody to P-gp21 was also confirmed. Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope (ALKDKKELEGSGKIAT) comprising residues 883–898 within the transmembrane (TM) domain of human P-gp, but also the binding ability with it was verified. The clinical implication of our results for development of personalized therapy of colorectal cancer will be further studied. PMID:24348182

  16. Evaluation of dual P-gp-BCRP inhibitors as nanoparticle formulation.

    PubMed

    Singh, Manu Smriti; Juvale, Kapil; Wiese, Michael; Lamprecht, Alf

    2015-09-18

    Overcoming multidrug resistance (MDR) in cancer is a major challenge and efforts are on-going to develop inhibitors against the most characterized and ubiquitous MDR transporters: P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1) and breast cancer resistance protein (BCRP). Recently reported, two 4-anilinoquinazolines (compounds 1 and 2), demonstrate potential MDR reversal activity against BCRP and to a lesser extent, P-gp. In this work, we formulated the compounds as polymeric nanoparticles (NPs) and assessed their MDR inhibitory activity in relevant BCRP and P-gp over-expressing cell line models. Particles in the size range 300-365nm with a loading efficiency of 69% (compound 1 NP) and 77% (compound 2 NP) respectively were obtained. BCRP inhibition was observed in Hoechst 33342 and pheophorbide A assays while P-gp inhibition was evaluated in calcein AM and rhodamine-123 assays. In cytotoxicity studies, while BCRP expressing cells showed complete reversal of drug resistance in nearly all treatment groups (both compounds and their respective NP); a higher reversal in NP treated group was obtained as compared with inhibitory compound treated group in P-gp expressing cells. These results demonstrate promising inhibitory activity of both formulations, especially against P-gp expressing cells; which is possibly due to a prolonged presence of encapsulated compounds in NPs and consequently a prolonged sensitization of transmembrane drug transporter. These formulations can therefore be considered as dual-transporter inhibitors and it is imperative to investigate both inhibitors in animal models of MDR owing to the presence of multiple efflux transporters in several cancer models. PMID:25976226

  17. Development of Classification Models for Identifying “True” P-glycoprotein (P-gp) Inhibitors Through Inhibition, ATPase Activation and Monolayer Efflux Assays

    PubMed Central

    Rapposelli, Simona; Coi, Alessio; Imbriani, Marcello; Bianucci, Anna Maria

    2012-01-01

    P-glycoprotein (P-gp) is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external) validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as “true” P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function. PMID:22837672

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

    PubMed

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

    2014-09-01

    Urogenital tract infection with Chlamydia trachomatis is a leading cause of sexually transmitted infections. There is currently no commercially available vaccine against C. trachomatis. The highly conserved plasmid of chlamydiae has been considered to be a virulence factor and the plasmid proteins have important roles in the Chlamydia-specific immune response. This study was designed to evaluate the efficacy of vaccination with plasmid proteins in the prevention of C. muridarum lung infection in a mouse model. C57BL/6N mice were immunised 3 times subcutaneously with recombinant pGP3 or pGP4 and infected with C. muridarum. Immunisation of the mice with recombinant pGP3 or pGP4 protein caused a significantly lower chlamydial burden in the lungs of the infected mice; the lower IFN-? level indicated a reduced extent of inflammation. In vitro or in vivo neutralisation of C. muridarum with sera obtained from immunised mice did not reduce the number of viable C. muridarum in the lungs of mice. However, adoptive transfer of the CD4(+) spleen cells isolated from the immunised mice resulted in a significantly reduced bacterial burden. Our results indicate that it is not the pGP3- and pGP4-specific antibodies, but the CD4(+) cells that are responsible for the protective effect of the immune response to plasmid proteins. PMID:25077421

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-01

    Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. One of the effective approaches to overcome MDR is to use nanoparticle-mediated the gene silence of chemotherapeutic export proteins by RNA interference to increase drug accumulation in drug resistant cancer cells. In this work, a new co-delivery system, DOX-PLGA/PEI/P-gp shRNA nanobubbles (NBs) around 327 nm, to overcome doxorubicin (DOX) resistance in MCF-7 human breast cancer was designed and developed. Positively charged polyethylenimine (PEI) were modified onto the surface of DOX-PLGA NBs through DCC/NHS crosslinking, and could efficiently condense P-gp shRNA into DOX-PLGA/PEI NBs at vector/shRNA weight ratios of 70:1 and above. An in vitro release profile demonstrated an efficient DOX release (more than 80%) from DOX-PLGA/PEI NBs at pH 4.4, suggesting a pH-responsive drug release for the multifunctionalized NBs. Cellular experimental results further showed that DOX-PLGA/PEI/P-gp shRNA NBs could facilitate cellular uptake of DOX into cells and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The IC50 of DOX-PLGA NBs against MCF-7/ADR cells was 2-fold lower than that of free DOX. The increased cellular uptake and nuclear accumulation of DOX delivered by DOX-PLGA/PEI/P-gp shRNA NBs in MCF-7/ADR cells was confirmed by fluorescence microscopy and fluorescence spectrophotometry, and might be owning to the down-regulation of P-gp and reduced the efflux of DOX. The cellular uptake mechanism of DOX-PLGA/PEI/P-gp shRNA NBs indicated that the macropinocytosis was one of the pathways for the uptake of NBs by MCF-7/ADR cells, which was also an energy-dependent process. Furthermore, the in vitro cellular ultrasound imaging suggested that the employment of the DOX-PLGA/PEI/P-gp shRNA NBs could efficiently enhance ultrasound imaging of cancer cells. These results demonstrated that the developed DOX-PLGA/PEI/P-gp shRNA NBs is a potential, safe and efficient theranotic agent for cancer therapy and diagnostics. PMID:26510307

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  3. Long-lived signal peptide of lymphocytic choriomeningitis virus glycoprotein pGP-C.

    PubMed

    Froeschke, Marc; Basler, Michael; Groettrup, Marcus; Dobberstein, Bernhard

    2003-10-24

    Signal peptides (SPs) direct nascent secretory and membrane proteins to the membrane of the endoplasmic reticulum. They are usually cleaved from the nascent polypeptide by signal peptidase and then further proteolytically processed. The SP of the pre-glycoprotein (pGP-C) of the lymphocytic choriomeningitis virus SPGP-C (signal peptide of pGP-C) shows different properties: 1) The SPGP-C is unusually long (58 amino acid residues) and contains two hydrophobic segments interrupted by a lysine residue. 2) The SPGP-C is cleaved only from a subset of pGP-C proteins. A substantial portion of pGP-C accumulates that still contains the SPGP-C.3)The cleaved SPGP-C is rather long-lived (t(1/2) of more than 6 h). 4) The cleaved SPGP-C resides in the membrane and is resistant to digestion with proteinase K even in the presence of detergents, suggesting a very compact structure. 5) SPGP-C accumulates in virus particles. These unusual features of the cleaved SPGP-C suggest that SPGP-C not only targets the nascent pGP-C to the endoplasmic reticulum membrane but also has additional functions in lymphocytic choriomeningitis virus life cycle. PMID:12917426

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

    PubMed

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

    2014-09-01

    Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate if a bovine endothelial/rat astrocyte in vitro BBB co-culture model displayed polarized transport of known efflux transporter substrates. The co-culture model displayed low mannitol permeabilities of 0.95?±?0.1?·?10(-6) cm·s(-1) and high transendothelial electrical resistances of 1,177?±?101 ?·cm(2). Bidirectional transport studies with (3)H-digoxin, (3)H-estrone-3-sulphate and (3)H-etoposide revealed polarized transport favouring the brain-to-blood direction for all substrates. Steady state efflux ratios of 2.5?±?0.2 for digoxin, 4.4?±?0.5 for estrone-3-sulphate and 2.4?±?0.1 for etoposide were observed. These were reduced to 1.1?±?0.08, 1.4?±?0.2 and 1.5?±?0.1, by addition of verapamil (digoxin), Ko143 (estrone-3-sulphate) or zosuquidar?+?reversan (etoposide), respectively. Brain-to-blood permeability of all substrates was investigated in the presence of the efflux transporter inhibitors verapamil, Ko143, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport. PMID:24934296

  5. Stereoselective Regulation of P-gp Activity by Clausenamide Enantiomers in Caco-2, KB/KBv and Brain Microvessel Endothelial Cells

    PubMed Central

    Zhu, Chuan-jiang; Hua, Fang; Zhu, Xiao-lu; Li, Meng; Wang, Hong-xu; Yu, Xiao-ming; Li, Yan

    2015-01-01

    The (?)- and (+)-clausenamide (CLA) enantiomers have different pharmacokinetic effects in animals, but their association with putative stereoselective regulation of P-glycoprotein (P-gp) remains unclear. Using three cells expressing P-gp—Caco-2, KBv and rat brain microvessel endothelial cells(RBMEC), this study investigated the association of CLA enantiomers with P-gp. The results showed that the rhodamine 123 (Rh123) accumulation, an indicator of P-gp activity, in Caco-2, KBv and RBMECs was increased by (?)CLA (1 or 5 ?mol/L) at 8.2%–28.5%, but reduced by (+)CLA at 11.7%–25.9%, showing stereoselectivity in their regulation of P-gp activity. Following co-treatment of these cells with each CLA enantiomer and verapamil as a P-gp inhibitor, the (+)-isomer clearly antagonized the inhibitory effects of verapamil on P-gp efflux, whereas the (?)-isomer had slightly synergistic or additive effects. When higher concentrations (5 or 10 ?mol/L) of CLA enantiomers were added, the stimulatory effects of the (+)-isomer were converted into inhibitory ones, leading to an enhanced intracellular uptake of Rh123 by 24.5%–58.2%; but (?)-isomer kept its inhibition to P-gp activity, causing 30.0%–63.0% increase in the Rh123 uptake. The biphasic effects of (+)CLA were confirmed by CLA uptake in the Caco-2 cells. (+)CLA at 1 ?mol/L had significantly lower intracellular uptake than (?)CLA with a ratio[(?)/(+)] of 2.593, which was decreased to 2.167 and 1.893 after CLA concentrations increased to 2.5 and 5 ?mol/L. Besides, in the non-induced KB cells, (+)CLA(5 ?mol/L) upregulated P-gp expression at 54.5% relative to vehicle control, and decreased Rh123 accumulation by 28.2%, while (?)CLA(5 ?mol/L) downregulated P-gp expression at 15.9% and increased Rh123 accumulation by 18.0%. These results suggested that (?)CLA could be a P-gp inhibitor and (+)CLA could be a modulator with concentration-dependent biphasic effects on P-gp activity, which may result in drug—drug interactions when combined with other P-gp substrate drugs. PMID:26295572

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

    PubMed

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

    2015-09-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    The transport function of P-glycoprotein (P-gp) requires its efficient localization to caveolae, a subset of lipid rafts, and disruption of caveolae suppresses P-gp transport function. However, the regulatory molecules involved in the translocation of P-gp into caveolae remain unknown. In the present study, we showed that c-Src dependent Caveolin-1 phosphorylation promoted the translocation of P-gp into caveolae, resulting in multidrug resistance in adriamycin resistant gastric cancer SGC7901/Adr and breast cancer MCF-7/Adr cells. In a negative feedback loop, the translocation of Cbl-b from the nucleus to the cytoplasm prevented the localization of P-gp to caveolae resulting in the reversal of MDR through the ubiquitination and degradation of c-Src. Clinical data showed a significant positive relationship between Cbl-b expression and survival in P-gp positive breast cancer patients who received anthracycline-based chemotherapy. Our findings identified a new regulatory mechanism of P-gp transport function in multiple drug-resistant gastric and breast cancers. PMID:25788263

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

    PubMed

    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

    2012-10-01

    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

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

    PubMed Central

    Robert-Paganin, Julien; Nonin-Lecomte, Sylvie; Réty, Stéphane

    2012-01-01

    FimX is a large multidomain protein containing an EAL domain and involved in twitching motility in Pseudomonas aeruginosa. We present here two crystallographic structures of the EAL domain of FimX (residues 438–686): one of the apo form and the other of a complex with 5?-pGpG, the reaction product of the hydrolysis of c-di-GMP. In both crystal forms, the EAL domains form a dimer delimiting a large cavity encompassing the catalytic pockets. The ligand is trapped in this cavity by its sugar phosphate moiety. We confirmed by NMR that the guanine bases are not involved in the interaction in solution. We solved here the first structure of an EAL domain bound to the reaction product 5?-pGpG. Though isolated FimX EAL domain has a very low catalytic activity, which would not be significant compared to other catalytic EAL domains, the structure with the product of the reaction can provides some hints in the mechanism of hydrolysis of the c-di-GMP by EAL domains. PMID:23285035

  11. Expression of multidrug resistance proteins in invasive ductal carcinoma of the breast

    PubMed Central

    LI, WEIQUAN; SONG, MAOMIN

    2014-01-01

    Chemotherapy is commonly used for the treatment of breast cancer. However, the resistance to chemotherapeutic agents, often mediated by multidrug resistance (MDR) mechanisms, is a common occurrence. The present study examined the expression of several MDR-related proteins (MRPs) in invasive ductal carcinoma (IDC) of the breast, and assessed their association with clinicopathological variables and their prognostic significance. In addition, immunohistochemistry was used to measure the expression of MRP, p-glycoprotein (P-gp), topoisomerase 2? (Topo2?), thymidylate synthase (TS) and glutathione-S-transferase ? (GST-?) in 156 resected IDCs of the breast. Pearson’s ?2 test and Spearman’s correlation coefficient were used to analyze the association between MDR protein expression and several clinicopathological variables. The association between each of the five MDR proteins was also examined. Furthermore, Kaplan-Meier analysis and Cox regression modeling were used to assess overall survival. The expression of MRP, P-gp, Topo2?, TS and GST-? was detected in 20.5% (32/156), 25.0% (39/156), 84.0% (131/156), 41.7% (65/156) and 41.0% (64/156) of cases examined, respectively. No correlation was identified between MRP and Topo-2? and the clinicopathological variables examined. By contrast, P-gp (?2=20.226; P<0.0001) and GST-? (?2=35.032; P<0.0001) were found to positively correlate with tumor grade. In addition, staining for TS was associated with axillary lymph node metastasis (?2=42.281; P<0.0001). The expression levels of P-gp and GST-? were found to be significantly correlated (r= 0.319; P<0.0001). Furthermore, GST-? expression was elevated in estrogen receptor-negative breast cancer (?2=17.407; P<0.0001). Tumor histological grade, in addition to TS and GST-? expression, were significant predictors of a poor survival outcome. TS and GST-? are consequently useful prognostic biomarkers in IDC, therefore, when establishing a personalized chemotherapeutic plan, the expression of MDR proteins must be considered. PMID:25295098

  12. Isoform I (mdr3) is the major form of P-glycoprotein expressed in mouse brain capillaries. Evidence for cross-reactivity of antibody C219 with an unrelated protein.

    PubMed Central

    Jetté, L; Pouliot, J F; Murphy, G F; Béliveau, R

    1995-01-01

    P-glycoprotein (P-gp) is expressed in various non-cancerous tissues such as the endothelial cells of the blood-brain barrier. We used several monoclonal antibodies (mAbs) and isoform-specific polyclonal antibodies to establish which P-gp isoforms are expressed in isolated mouse brain capillaries. P-gp class I isoform was detected in capillaries with a Western immunoblotting procedure using a specific antiserum. No immunoreactivity was observed with either class II- or class III-specific antisera. Immunoreactivity was observed with mAb C219. However, this antibody detected two distinct immunoreactive proteins (155 and 190 kDa) in the isolated brain capillaries. These two proteins comigrated as a broad band when the samples were submitted to heat prior to gel electrophoresis. The glycoprotein nature of these two antigens was evaluated by their sensitivity to N-glycanase treatment. Following this treatment, the size of the proteins was reduced from 190 and 155 kDa to 180 and 120 kDa, respectively. Triton X-114 phase-partitioning studies showed that the 190 kDa immunoreactive protein was poorly solubilized by Triton X-114, while the 155 kDa protein was partitioned in the detergent-rich phase. In labelling experiments, only the 155 kDa protein was photolabelled with [125I]iodoarylazidoprazosin. These results show that a 190 kDa protein detected by antibody C219 is an antigen unrelated to the three P-gp isoforms presently known. Cross-reactivity of C219 with an unrelated protein emphasizes the fact that more than one antibody should be used in the assessment of P-gp expression in cell lines and tissues. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7848274

  13. Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

    PubMed

    Juuti-Uusitalo, Kati; Vaajasaari, Hanna; Ryhänen, Tuomas; Narkilahti, Susanna; Suuronen, Riitta; Mannermaa, Eliisa; Kaarniranta, Kai; Skottman, Heli

    2012-01-01

    Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE-derived diseases, drug testing and targeted drug therapy. PMID:22272278

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

    PubMed

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

    2015-12-01

    P-glycoprotein (P-gp or ABCB1) belongs to the family of ATP-binding cassette (ABC) transporters responsible for multixenobiotic resistance (MXR) in aquatic organisms. To provide more information of P-gp in shellfish, in this study, complete cDNA of P-gp in three bivalve species including Ruditapes philippinarum, Scapharca subcrenata and Tegillarca granosa were cloned and its expressions in gill, digestive gland, adductor muscle and mantle of the three bivalves were detected after exposure to Prorocentrum lima, a toxogenic dinoflagellate. The complete sequences of R. philippinarum, S. subcrenata and T. granosa P-gp showed high homology with MDR/P-gp/ABCB proteins from other species, having a typical sequence organization as full transporters from the ABCB family. Phylogenetic analyses revealed that the amino acid sequences of P-gp from S. subcrenata and T. granosa had a closest relationship, forming an independent branch, then grouping into the other branch with Mytilus californianus, Mytilus galloprovincialis and Crassostrea gigas. However, P-gp sequences from R. philippinarum were more similar to the homologs from the more distantly related Aplysia californica than to homologs from S. subcrenata and T. granosa, suggesting that bivalves P-gp might have different paralogs. P-glycoprotein expressed in all detected tissues but there were large differences between them. After exposure to P. lima, the expression of P-gp changed in the four tissues in varying degrees within the same species and between different species, but the changes in mRNA and protein level were not always synchronous. PMID:26539802

  15. Amyloid efflux transporter expression at the blood-brain barrier declines in normal aging.

    PubMed

    Silverberg, Gerald D; Messier, Arthur A; Miller, Miles C; Machan, Jason T; Majmudar, Samir S; Stopa, Edward G; Donahue, John E; Johanson, Conrad E

    2010-10-01

    Reduced clearance of amyloid ? peptides (A?) across the blood-brain barrier contributes to amyloid accumulation in Alzheimer disease. Amyloid ? efflux transport is via the endothelial low-density lipoprotein receptor-related protein 1 (LRP-1) and P-glycoprotein (P-gp), whereas A? influx transport is via the receptor for advanced glycation end products. Because age is the major risk factor for developing Alzheimer disease, we measured LRP-1 and P-gp expression and associated transporter expression with A? accumulation in aging rats. Quantitative LRP-1 and P-gp microvessel expression was measured by immunohistochemistry (IHC); LRP-1 and P-gp expression were assessed in microvessel isolates by Western blotting. There was an age-dependent loss of capillary LRP-1 across all ages (3-36 months) by IHC (linear trend p = 0.0004) and between 3 and 20 months by Western blotting (linear trend p < 0.0001). There was a late (30-36 months) P-gp expression loss by IHC (p < 0.05) and Western blotting (p = 0.0112). Loss of LRP-1 correlated with A?42 accumulation (p = 0.0121) and very nearly with A?40 (p = 0.0599) across all ages. Expression of LRP-1 correlated negatively with the expression of receptor for advanced glycation end products (p < 0.0004). These data indicate that alterations in LRP-1 and P-gp expression seem to contribute progressively to A? accumulation in aging. PMID:20838242

  16. The effects of protease inhibitors and nonnucleoside reverse transcriptase inhibitors on p-glycoprotein expression in peripheral blood mononuclear cells in vitro.

    PubMed

    Chandler, Becky; Almond, Lisa; Ford, Jennifer; Owen, Andrew; Hoggard, Patrick; Khoo, Saye; Back, David

    2003-08-15

    Several antiretroviral compounds have been shown to be substrates for the efflux protein P-glycoprotein (P-gp) although few studies have investigated the effects of drug on expression of this protein. Here, an in vitro system has been adopted to investigate the effects of protease inhibitors (PIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) on P-gp expression in peripheral blood mononuclear cells (PBMCs). PBMCs isolated from healthy volunteers were incubated with 10 or 100 microM PI (saquinavir, ritonavir, lopinavir, indinavir, nelfinavir, amprenavir) or 10 microM NNRTI (efavirenz, nevirapine) for 72 hours. Surface P-gp expression was measured by flow cytometry and compared with vehicle-incubated controls. Toxicity was assessed by MTT assay and the effects of each compound were compared between individuals with differing genotypes at position 3435 of exon 26 of MDR1, which was assigned by restriction fragment length polymorphism. Significant increases in median P-gp expression were observed following incubation with 10 microM nelfinavir (10.2 versus 6.7% P-gp-positive cells) and efavirenz (10.0 versus 6.7% P-gp-positive cells). No significant differences in induction were observed between genotypes (CC, CT, TT). Following incubation with 100 microM PI, significant upregulation of P-gp occurred except with amprenavir. However, nelfinavir, ritonavir, and lopinavir caused marked toxicity, indicating that at higher concentrations, the increase in P-gp may be at least partially related to a stress response. These results indicate the potential of some PIs and NNRTIs to induce P-gp expression in PBMCs in vitro. PMID:12902797

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

    PubMed Central

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

    2015-01-01

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

  18. Trametenolic acid B reverses multidrug resistance in breast cancer cells through regulating the expression level of P-glycoprotein.

    PubMed

    Zhang, Qiaoyin; Wang, Junzhi; He, Haibo; Liu, Hongbing; Yan, Ximing; Zou, Kun

    2014-07-01

    Trametenolic acid B (TAB) is the main active composition of Trametes lactinea (Berk.) Pat which possesses antitumor activities. There was no report its antitumor effect through regulating P-glycoprotein (P-gp) so far, due toP-gp over expression is one of the most important mechanisms contributing to the multiple drug resistance phenotype. The present aim was to investigate the effects of TAB on P-gp in multidrug-resistant cells;Paclitaxel-resistant cell line MDA-MB-231/Taxol was established by stepwise exposure for 10 months.MDA-MB-231 cells and MDA-MB-231/Taxol cells were treated with TAB, and their growth was evaluated using MTT assays. Paclitaxel accumulation in the cells was analyzed by high performance liquid chromatogram(HPLC). The activity of P-gp was detected by intracellular accumulation of rhodamine 123 (Rho123), and the protein expression of P-gp was evaluated using western blot. Results indicated that the IC50 of MDA-MB-231/Taxol to paclitaxel (Taxol) was 33 times higher than that of nature MDA-MB-231. TAB increased the intracellular concentration of Taxol and inhibited the activity of P-gp and suppressed the expression of P-gp in MDA-MB-231/Taxol cells. Our present results showed that TAB could reverse Taxol resistance in MDA-MB-231/Taxol cells,mainly inhibiting the activity of P-gp and down-regulating the expression level of P-gp, and then enhancing the accumulation of chemotherapy agents. PMID:25289403

  19. Expression and Localization of P-Glycoprotein, Multidrug Resistance Protein 4, and Breast Cancer Resistance Protein in the Female Lower Genital Tract of Human and Pigtailed Macaque

    PubMed Central

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

    2014-01-01

    Abstract Antiretroviral drug absorption and disposition in cervicovaginal tissue is important for the effectiveness of vaginally or orally administered drug products in preexposure prophylaxis (PrEP) of HIV-1 sexual transmission to women. Therefore, it is imperative to understand critical determinants of cervicovaginal tissue pharmacokinetics. This study aimed to examine the mRNA expression and protein localization of three efflux transporters, P-glycoprotein (P-gp), multidrug resistance-associated protein 4 (MRP4), and breast cancer resistance protein (BCRP), in the lower genital tract of premenopausal women and pigtailed macaques. Along the human lower genital tract, the three transporters were moderately to highly expressed compared to colorectal tissue and liver, as revealed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). In a given genital tract segment, the transporter with the highest expression level was either BCRP or P-gp, while MRP4 was always expressed at the lowest level among the three transporters tested. The immunohistochemical staining showed that P-gp and MRP4 were localized in multiple cell types including epithelial cells and vascular endothelial cells. BCRP was predominantly localized in the vascular endothelial cells. Differences in transporter mRNA level and localization were observed among endocervix, ectocervix, and vagina. Compared to human tissues, the macaque cervicovaginal tissues displayed comparable expression and localization patterns of the three transporters, although subtle differences were observed between the two species. The role of these cervicovaginal transporters in drug absorption and disposition warrants further studies. The resemblance between human and pigtailed macaque in transporter expression and localization suggests the utility of the macaque model in the studies of human cervicovaginal transporters. PMID:24803409

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

    PubMed

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

    2014-03-01

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

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

    PubMed

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

    2014-12-01

    Electroporation (EP) can effectively support the penetration of macromolecules from the extracellular space into cells. Electropores induced by the influence of electromagnetic field generate additional paths of transport for macromolecules. The aim of this study was evaluation of the electroporation effect on doxorubicin transport efficiency to human colon (LoVo and LoVo/DX) and gastric (EPG85-257/P and EPG85-257/RDB) adenocarcinoma cells with overexpression of P-glycoprotein and murine macrophage cell line (P388/D1). In our EP experiments cells were placed into a cuvette with aluminum electrodes and pulsed with five square electric pulses of 1300 V/cm and duration of 50 ?s each. Cells were also treated with low doxorubicin concentration ([DOX]=1.7 ?M). The ultrastructure (TEM) and changes of P-glycoprotein expression of tumor cells subjected to electric field were monitored. The mitochondrial cell function and trypan blue staining were evaluated after 24h. Our results indicate the most pronounced effect of EP with DOX and disturbed ultrastructure in resistant gastric and colon cells with decrease of P-gp expression. Electroporation may be an attractive delivery method of cytostatic drugs in chemotherapy, enabling reduction of drug dose, exposure time and side effects. PMID:24767854

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

    PubMed

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

    2005-04-01

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

  3. Cyclosporin A has low potency as a calcineurin inhibitor in cells expressing high levels of P-glycoprotein.

    PubMed

    Fakata, K L; Elmquist, W F; Swanson, S A; Vorce, R L; Prince, C; Stemmer, P M

    1998-01-01

    Cyclosporin A (CsA) is a widely-used immunosuppressant drug whose therapeutic and toxic actions are mediated through inhibition of calcineurin (CN), a calcium- and calmodulin-dependent phosphatase. Inhibition of CN by CsA requires drug binding to its protein cofactor in the inhibition, cyclophilin. Because cyclophilin is a high affinity target for CsA it is expected that this protein can act as a reservoir for the drug in the cell and may be able to inhibit cellular efflux of CsA. P-glycoprotein (P-gp) is known to increase the rate of CsA efflux from CsA loaded cells but it is not clear if the P-gp drug efflux pump can compete effectively with cyclophilin at therapeutically relevant concentrations of CsA. To test the hypothesis that increased expression of P-gp confers protection against CsA-dependent inhibition of CN phosphatase activity, KB-V cells expressing varying levels of P-gp were analyzed to determine the potency of CsA as a CN inhibitor. When intact cells were treated with CsA, a positive correlation was observed between P-gp expression and resistance to CsA-dependent inhibition of CN: the IC50 is approximately 20-fold higher in the multidrug resistant epidermal carcinoma cell line, KB-V, which expresses P-gp at a high level than in the parental, KB, cell line expressing very low levels of P-gp. The resistance displayed by KB-V cells is abrogated by co-administration of the P-gp inhibitor verapamil, whereas verapamil has no effect on CsA potency in control KB cells. In cell lysates from KB-V cells with different amounts of P-gp CsA exhibits equivalent potency, indicating that the difference in sensitivity to CsA among the cell types requires maintenance of cell integrity. These observations support the view that resistance to CN inhibition by CsA occurs in cells with moderately elevated P-gp activity. Therefore, P-gp activity appears to be an important determinant of CsA cellular specificity for both therapeutic and toxic effects. PMID:9651111

  4. Casein Kinase 2 (CK2)-mediated Phosphorylation of Hsp90? as a Novel Mechanism of Rifampin-induced MDR1 Expression.

    PubMed

    Kim, So Won; Hasanuzzaman, Md; Cho, Munju; Heo, Ye Rang; Ryu, Min-Jung; Ha, Na-Young; Park, Hyun June; Park, Hyung-Yeon; Shin, Jae-Gook

    2015-07-01

    The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms. In this study, we demonstrate that casein kinase 2 (CK2)-mediated phosphorylation of heat shock protein 90? (Hsp90?) and subsequent stabilization of PXR is a key mechanism in the regulation of MDR1 expression. Furthermore, we show that CK2 is directly activated by rifampin. Upon exposure to rifampin, CK2 catalyzes the phosphorylation of Hsp90? at the Ser-225/254 residues. Phosphorylated Hsp90? then interacts with PXR, causing a subsequent increase in its stability, leading to the induction of P-gp expression. In addition, inhibition of CK2 and Hsp90? enhances the down-regulation of PXR and P-gp expression. The results of this study may facilitate the development of new strategies to prevent multidrug resistance and provide a plausible mechanism for acquired drug resistance by CK2-mediated regulation of P-gp expression. PMID:25995454

  5. Multi-drug resistance in a canine lymphoid cell line due to increased P-glycoprotein expression, a potential model for drug-resistant canine lymphoma.

    PubMed

    Zandvliet, M; Teske, E; Schrickx, J A

    2014-12-01

    Canine lymphoma is routinely treated with a doxorubicin-based multidrug chemotherapy protocol, and although treatment is initially successful, tumor recurrence is common and associated with therapy resistance. Active efflux of chemotherapeutic agents by transporter proteins of the ATP-Binding Cassette superfamily forms an effective cellular defense mechanism and a high expression of these transporters is frequently observed in chemotherapy-resistant tumors in both humans and dogs. In this study we describe the ABC-transporter expression in a canine lymphoid cell line and a sub-cell line with acquired drug resistance following prolonged exposure to doxorubicin. This sub-cell line was more resistant to doxorubicin and vincristine, but not to prednisolone, and had a highly increased P-glycoprotein (P-gp/abcb1) expression and transport capacity for the P-gp model-substrate rhodamine123. Both resistance to doxorubicin and vincristine, and rhodamine123 transport capacity were fully reversed by the P-gp inhibitor PSC833. No changes were observed in the expression and function of the ABC-transporters MRP-1 and BCRP. It is concluded that GL-40 cells represent a useful model for studying P-gp dependent drug resistance in canine lymphoid neoplasia, and that this model can be used for screening substances as potential P-gp substrates and their capacity to modulate P-gp mediated drug resistance. PMID:24975508

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

    PubMed Central

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

    2011-01-01

    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. PMID:21660212

  7. Protein expression in liposomes.

    PubMed

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

    1999-08-01

    Compartmentalization is one of the key steps in the evolution of cellular structures and, so far, only few attempts have been made to model this kind of "compartmentalized chemistry" using liposomes. The present work shows that even such complex reactions as the ribosomal synthesis of polypeptides can be carried out in liposomes. A method is described for incorporating into 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC) liposomes the ribosomal complex together with the other components necessary for protein expression. Synthesis of poly(Phe) in the liposomes is monitored by trichloroacetic acid of the (14)C-labelled products. Control experiments carried out in the absence of one of the ribosomal subunits show by contrast no significant polypeptide expression. This methodology opens up the possibility of using liposomes as minimal cell bioreactors with growing degree of synthetic complexity, which may be relevant for the field of origin of life as well as for biotechnological applications. PMID:10425171

  8. Leptospira Protein Expression During Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

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

    2011-01-01

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

  10. EGFR/HER2 inhibitors effectively reduce the malignant potential of MDR breast cancer evoked by P-gp substrates in vitro and in vivo.

    PubMed

    Jin, Yiting; Zhang, Wei; Wang, Hongying; Zhang, Zijing; Chu, Chengyu; Liu, Xiuping; Zou, Qiang

    2016-02-01

    Multidrug resistance (MDR) induced by chemotherapy in breast cancer frequently leads to tumor invasion, metastasis and poor clinical outcome. We preliminarily found that the epidermal growth factor receptor (EGFR) is involved in enhancing the malignant potential of MDR breast cancer cells, but the mechanism remains unclear. In the present study, we demonstrated in vitro and in vivo that EGFR/HER2 promote the invasive and metastatic abilities of MDR breast cancer. More importantly, a new function of EGFR/HER2 inhibitors was revealed for the first time, which could improve the treatment efficacy of breast cancer by reversing the MDR process rather than by inhibiting tumor growth. Firstly, using quantitative real?time PCR and western blot analysis, we found that overexpression of EGFR/HER2 in MCF7/Adr cells upregulated CD147 and MMP2/9 at both the transcription and protein expression levels, which promoted tumor cell migration, as determined using an in vitro invasion assay. Secondly, the upregulated levels of CD147 and MMP2/9 were decreased when EGFR/HER2 activity was inhibited, and therefore tumor invasion was also significantly inhibited. These phenomena were also demonstrated in nude mouse assays. Additionally, in MDR breast cancer patients, we found that overexpression of EGFR and P?gp levels led to shorter overall survival (OS) and disease?free survival (DFS) by IHC assays and Kaplan?Meier survival analysis. In conclusion, EGFR/HER2 play a crucial role in enhancing CD147 and MMP expression to establish favorable conditions for invasion/metastasis in MDR breast cancer. The scope of application of EGFR/HER2 inhibitors may be expanded in EGFR/HER2?positive patients. We suggest that MDR breast cancer patients may benefit from novel therapies targeting EGFR/HER2. PMID:26718028

  11. Liquid Chromatographic Method for Irinotecan Estimation: Screening of P-gp Modulators

    PubMed Central

    Tariq, M.; Negi, L. M.; Talegaonkar, Sushama; Ahmad, F. J.; Iqbal, Zeenat; Khan, A. M.

    2015-01-01

    The present work is aimed to develop a simple, sensitive, robust and reliable HPLC method for the estimation of irinotecan in the physiological media in order to assess the permeability profile of irinotecan, using the everted gut sac, in the presence of various P-gp modulators. Separation was achieved using, C18 column with mobile phase consisting of acetonitrile and 0.045 µM sodium dihydrogen phosphate dihydrate buffer containing ion pair agent heptane sulphonic acid sodium salt (0.0054 µM), pH 3. The flow rate was maintained at 1 ml/min and analysis was performed at 254.9 nm using PDA detector. Calibration data showed an excellent linear relationship between peak-area verses drug concentration (r2, 0.9999). Linearity was found to be in the range of 0.060-10.0 µg/ml. Limits of detection and quantification were found to ~0.020 µg/ml and ~0.060 µg/ml, respectively. The developed method was found to be precise (RSD < 1.5%, for repeatability and <2.55% for intermediate precision, acceptable ranges of precision), accurate (The recovered content of irinotecan in the presence of various P-gp modulators varied from 96.11-101.51%, within acceptable range, 80-120%), specific and robust (% RSD < 2). Developed method has been applied successfully for the evaluation of eleven P-gp modulators from diverse chemical class. PMID:25767314

  12. Forced expression of heat shock protein 27 (Hsp27) reverses P-glycoprotein (ABCB1)-mediated drug efflux and MDR1 gene expression in Adriamycin-resistant human breast cancer cells.

    PubMed

    Kanagasabai, Ragu; Krishnamurthy, Karthikeyan; Druhan, Lawrence J; Ilangovan, Govindasamy

    2011-09-23

    Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor ?B (NF-?B) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-?B (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-?B-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-?B level will be a potential approach to circumvent drug resistance in breast cancer. PMID:21784846

  13. Suppression of P-gp induced multiple drug resistance in a drug resistant gastric cancer cell line by overexpression of Fas

    PubMed Central

    Yin, Fang; Shi, Yong Quan; Zhao, Wei Ping; Xiao, Bing; Miao, Ji Yan; Fan, Dai Ming

    2000-01-01

    AIM: To observe the drug sensitizing effect and related mechanisms of fas gene transduction on human drug-resistant gastric cancer cell SGC7901/VCR (resistant to Vincristine). METHODS: The cell cycle alteration was observed by FACS. The sensitivity of gastric cancer cells to apoptosis was determined by in vitro apoptosis assay. The drug sensitization of cells to several anti-tumor drugs was observed by MTT assay. Immunochemical method was used to show expression of P-gp and Topo II in gastric cancer cells. RESULTS: Comparing to SGC7901 and pBK-SGC7901/VCR, fas-SGC7901/VCR showed decreasing G2 cells and increasing S cells, the G2 phase fraction of pBK-SGC7901/VCR was about 3.0 times that of fas-SGC7901/VCR, but S phase fraction of fas-SGC7901/VCR was about 1.9 times that of pBK-SGC7901/VCR, indicating S phase arrest of fas-SGC7901/VCR. FACS also suggested apoptosis of fas-SGC7901/VCR. fas-SGC7901/VCR was more sensitive to apoptosis inducing agent VM-26 than pBK-SGC7901/VCR. MTT assay showed increased sensitization of fas-SGC7901/VCR to DDP, MMC and 5-FU, but same sensitization to VCR according to pBK-SGC7901/VCR. SGC7901, pBK-SGC7901/VCR and fas-SGC7901/VCR had positively stained Topo II equally. P-gp staining in pBK-SGC7901/VCR was stronger than in SGC7901, but there was little staining of Pgp in fas-SGC7901/VCR. CONCLUSION: fas gene transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree, possibly because of higher sensitization to apoptosis and decreased expression of P-gp. PMID:11819671

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

    PubMed

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

    2014-12-01

    Ammonia is considered to be the main neurotoxin responsible for hepatic encephalopathy resulting from liver failure. Liver failure has been reported to alter expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2) at the blood-brain barrier (BBB). The aim of this study was to investigate whether ammonia is involved in abnormalities of expression and activity of P-gp and Mrp2 at the BBB. Hyperammonemic rats were developed by an intraperitoneal injection of ammonium acetate (NH4 Ac, 4.5 mmol/kg). Results showed that Mrp2 function markedly increased in cortex and hippocampus of rats at 6 h following NH4 Ac administration. Significant increase in function of P-gp was observed in hippocampus of rats. Meanwhile, such alterations were in line with the increase in mRNA and protein levels of P-gp and Mrp2. Significant increase in levels of nuclear amount of nuclear factor-?B (NF-?B) p65 was also observed. Primarily cultured rat brain microvessel endothelial cells (rBMECs) were used for in vitro study. Data indicated that 24 h exposure to ammonia significantly increased function and expression of P-gp and Mrp2 in rBMECs, accompanied with activation of NF-?B. Furthermore, such alterations induced by ammonia were reversed by NF-?B inhibitor. In conclusion, this study demonstrates that hyperammonemia increases the function and expression of P-gp and Mrp2 at the BBB via activating NF-?B pathway. Hyperammonemia, a proverbial main factor responsible for neurocognitive disorder and blood-brain barrier (BBB) dysfunction resulting from liver failure, could increase the expression and activity of P-glycoprotein and multidrug resistance-associated protein 2 (Mrp2) at the BBB both in vivo and in vitro. Furthermore, the NF-?B activation stimulated by hyperammonemia may be the potential mechanism underlying such abnormalities induced by hyperammonemia. PMID:25200138

  15. Distribution and functional activity of P-glycoprotein and multidrug resistance-associated proteins in human brain microvascular endothelial cells in hippocampal sclerosis.

    PubMed

    Kubota, Hisashi; Ishihara, Hideyuki; Langmann, Thomas; Schmitz, Gerd; Stieger, Bruno; Wieser, Heinz-Gregor; Yonekawa, Yasuhiro; Frei, Karl

    2006-03-01

    Multidrug resistance protein, also referred as P-glycoprotein (P-gp, MDR1; ABCB1) and multidrug resistance-associated protein (MRP) 1 (ABCC1) and 2 (ABCC2) are, thus far, candidates to cause antiepileptic drug (AED) resistance epilepsy. In this study, we investigated P-gp, MRP1 and MRP2 expression, localization and functional activity on cryosections and isolated human brain-derived microvascular endothelial cells (HBMEC) from epileptic patients (HBMEC-EPI) with hippocampal sclerosis (HS), as compared with HBMEC isolated from normal brain cortex (HBMEC-CTR). We examined the expression and distribution of three transporters, P-gp, MRP1 and MRP2 on two major parts of the resected tissue, the hippocampus and the parahippocampal gyrus (Gph). P-gp showed diffuse expression not only in endothelium but also by parenchymal cells in both the hippocampus and the Gph. MRP1 labeling was observed in parenchymal cells in the Gph. By contrast, MRP2 was mainly found in endothelium of the hippocampus. P-gp and MRP1 expression in the Gph was relatively high in the patient with long-term seizure history. Quantitative RT-PCR analysis of HBMEC revealed that MDR1, MRP1 as well as MRP5 (ABCC5) and MRP6 (ABCC6) were overexpressed in HBMEC-EPI at the mRNA level. HBMEC from both normal and epilepsy groups displayed protein expression of P-gp, whereas MRP1 and MRP2 were seen only in HBMEC-EPI. Accordingly, it is of particular interest that MRP functional activities were observed in HBMEC-EPI, but not in HBMEC-CTR. Our results suggest that complex MDR expression changes not only in the hippocampus but in the Gph may play a role in AED pharmacoresistance in intractable epilepsy patients with mesial temporal lobe epilepsy (MTLE) by altering the permeability of AEDs across the blood-brain barrier (BBB). PMID:16361082

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

    PubMed Central

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

    2015-01-01

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

  17. Reversing of multidrug resistance breast cancer by co-delivery of P-gp siRNA and doxorubicin via folic acid-modified core-shell nanomicelles.

    PubMed

    Wu, Yang; Zhang, Yu; Zhang, Wei; Sun, Chunlong; Wu, Jianzhong; Tang, Jinhai

    2016-02-01

    Multidrug resistance (MDR) remains one of major limitation for the successful treatment of many cancers including breast cancer. Co-delivery of chemotherapeutic drugs and small interfering RNA (siRNA) has been developed because of its ability to generate synergistic anticancer effects via different mechanisms of action, to reverse MDR and increase the efficacy of chemotherapeutic drugs in cancer therapy. Herein, we employed a kind of efficient multifunctional tumor targeted nanomicelles (PECL3) for the co-delivery of hydrophobic anti-cancer drugs and siRNA. This kind of nanomicelles were constructed by folic acid (FA)-decorated PEG-b-(PCL-g-PEI)-b-PCL triblock copolymers, which were synthesized through "click chemistry" and "ring opening" polymerization. Driven by the "core-shell" structure and the electrostatic interaction, this triblock copolymer could efficiently encapsulate P-glycoprotein (P-gp) siRNA and doxorubicin (DOX). The obtained nanomicelles can prevent renal clearance, RNase degradation and aggregation in circulation. Compared to the non-specific delivery, these FA functionalized nanomicelles could efficiently deliver P-gp siRNA to reducing both P-gp expression levels and IC50 value of the DOX in DOX-resistant breast cancer cells (MCF-7/ADR). Additionally, in vivo results showed that DOX loaded PECL3 (D-PECL3) micelles could reduce toxicity of DOX on nontarget tissues and significantly inhibited MCF-7/ADR tumor growth through encapsulating DOX in the micelles and deliver them to target tumor region. Taken together, these results proof that PECL3 micelles could co-deliver siRNA and drug to inhibit MDR tumor growth. These results suggested that the co-delivery of DOX and siRNA in tumor-targeting nanomicelles could excite synergistic effect of gene therapy and chemotherapy, thus can efficiently reverse MDR cancer and kill the cancer cells. PMID:26655793

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

    PubMed

    Singh, Manu Smriti; Lamprecht, Alf

    2015-01-30

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

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

  20. Protein misinteraction avoidance causes highly expressed proteins to evolve slowly

    E-print Network

    Zhang, Jianzhi

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

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

    PubMed Central

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

    2015-01-01

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

  2. Mechanisms of tetrandrine and 5-bromotetrandrine in reversing multidrug resistance may relate to down-regulation of multidrug resistance associated protein 7 expression.

    PubMed

    Cheng, Jian; Dai, Jing-Ying; Chen, Bao-An; Cai, Xiao-Hui; Wang, Shuai; Gao, Feng

    2012-06-01

    Both tetrandrine (Tet) and 5-bromotetrandrine (BrTet) can effectively reverse P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). The structure of multidrug resistance associated protein 7 (MRP7) has its own specificity and difference compared with other members of the MRP family. This study was aimed to investigate whether Tet and BrTet can inhibit the expression level of MRP7 so as to further look into the mechanisms of the reversal effects of Tet and BrTet on MDR. The inhibitory effects of daunorubicin (DNR) used alone on the proliferation of K562 and K562/A02 cells were evaluated by MTT assay, the IC(50) of DNR and drug resistant folds were calculated. The mRNA level of MRP7 was tested by real-time PCR, and the protein levels of MRP7 and P-gp were tested by Western blot. The DNR accumulation was analyzed by flow cytometry (FCM). The results showed that the resistance of K562/A02 cells to DNR was 23.65-folds of that of K562 cells. After administration of 1 µmol/L Tet or 2 µmol/L BrTet, the mRNA level of MRP7 in the K562/A02 cells decreased to 2% and 12% respectively, and the protein level of MRP7 decreased by 53.2% and 83.7% respectively. The protein level of P-gp decreased by 58.47% and 52.20% in the 1 µmol/L Tet and 2 µmol/L BrTet groups. FCM detection showed that 1 µmol/L Tet and 2 µmol/L BrTet significantly increased the accumulation of DNR in K562/A02 cells by 94.32% and 271% respectively. It is concluded that Tet and BrTet both can reverse MDR in vitro. The mechanisms may be related to the inhibition of MRP7 overexpression and the increase of anticancer drug concentration in cells. At the same molar concentration, the effects of Tet and BrTet in inhibiting the protein level of MRP7 expression do not show significant difference. PMID:22739155

  3. Expression of multiple proteins in transgenic plants

    DOEpatents

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

    2002-01-01

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

  4. Reduced ABCB1 Expression and Activity in the Presence of Acrylic Copolymers

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    2011-01-01

    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. PMID:21269449

  6. HSV-1 Protein Expression Using Recombinant Baculoviruses

    PubMed Central

    Grady, Lorry M.; Bai, Ping; Weller, Sandra K.

    2015-01-01

    The baculovirus expression system is an invaluable method for the expression of Herpes Simplex Virus 1 (HSV-1) proteins. The use of insect cells provides a eukaryotic system for the robust expression of heterologous proteins under control of the baculovirus polyhedrin gene promoter that naturally drives the high expression of the polyhedrin protein. Additionally, insect cells often initiate the necessary posttranslational modifications and/or disulfide-bond formation important for the proper folding of the protein. We and others have successfully expressed and purified several HSV-1 proteins including the polymerase, helicase-primase, single-strand DNA binding protein, and alkaline nuclease. The following protocol is based on 15 years of experience from our laboratory. PMID:24671692

  7. Coevolution of gene expression among interacting proteins

    SciTech Connect

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

    2004-03-01

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

  8. Membrane protein expression: no cells required.

    PubMed

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

    2009-08-01

    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

  9. Downregulation of P-gp, Ras and p-ERK1/2 contributes to the arsenic trioxide-induced reduction in drug resistance towards doxorubicin in gastric cancer cell lines.

    PubMed

    Zhao, Yuan-Yuan; Yu, Li; Liu, Bao-Ling; He, Xin-Jia; Zhang, Bi-Yuan

    2015-11-01

    Multidrug resistance (MDR) to doxorubicin (DOX) limits its effectiveness against tumor cells. Arsenic trioxide (As2O3) has been reported to reduce MDR in various types of cancer, but the mechanisms involving Ras and p-glycoprotein (P-gp) remain to be fully elucidated. The objectives of the present study were to evaluate As2O3 in reversing MDR to DOX, and to identify the association in antitumor activities between the effectiveness of DOX and Ras/phosphorylated (p?) extracellular signal?regulated kinase (ERK)1/2 signaling in SGC7901/ADM and SGC7901/S human gastric cancer cell lines. Cytotoxicity and sensitivity towards As2O3 were assessed using non?toxic and mildly?toxic concentrations (0.1 and 0.5 µM, respectively). The reversing effect of As2O3 on MDR was investigated prior to and following treatment with a cytokine activation of the recombinant human granulocyte colony stimulating factor ERK pathway. The SGC7901/ADM and SGC7901/S cells had the same sensitivity to As2O3. The SGC7901/ADM cells were resistant to DOX and As2O3 treatment reduced the level of resistance to DOX (P<0.01). The expression of P?glycoprotein (P-gp) in the SGC7901/ADM cells was higher than in the SGC7901/S cells (P<0.001). As2O3 treatment decreased the levels of P?gp in a time? and dose?dependent manner (P<0.01). The expression of Ras was higher in the SGC7901/ADM cells than in the SGC7901/S cells, while the expression of p?ERK1/2 remained the same. As2O3 decreased the levels of Ras and p?ERK1/2 (P<0.01). Following pretreatment with rhG?CSF, the levels of Ras and p?ERK1/2 were further decreased (P<0.01). Drug?resistant gastric cancer cells had higher expression levels of P?gp and Ras, but not of p?ERK1/2. Non? and mildly?toxic doses of As2O3 reduced MDR to DOX through Ras/p-ERK1/2 signaling. PMID:26459009

  10. Transient Protein Expression by Agroinfiltration in Lettuce.

    PubMed

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

    2016-01-01

    Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level. PMID:26614281

  11. [Effect of cryptotanshinone on imatinib sensitivity and P-glycoprotein expression of chronic myeloid leukemia cells].

    PubMed

    Ge, Yu-qing; Cheng, Ru-bin; Yang, Bo; Huang, Zhen; Chen, Zhe

    2015-06-01

    Cryptotanshinone (CPT), a lipid soluble active compound in Salvia miltiorrhiza, has a significant inhibitory effect on multiple malignant tumors, e. g. chronic myeloid leukemia (CML) cells and can effectively enhance imatinib's chemotherapeutic effect. However, its functional molecular mechanism remained unclear. In this experiment, the authors conducted a systematic study on the effect of CPT on the imatinib sensitivity and P-glycoprotein (P-gp) expression in CML cells by using CML cells K562 and imatinib persister K562-R. The MTT assays were performed to determine CPT's impact on the inhibitory effect of imatinib. Annexin V-FITC/PI staining analysis was used to detect the changes in the cell apoptosis rate. The active changes in apoptosis regulatory proteins Caspase-3, Caspase-9 and PARP were determined by Western blot. After the cells were pretreated with the gradient concentration of CPT, the expression of P-gp was analyzed by Western blot and flow cytometry. The changes in intracellular concentrations of imatinib were determined by HPLC analysis. The results indicated that the pretreatment with CPT significantly increased the proliferation inhibiting and apoptosis inducing effects of imatinib on K562 and K562-R cells as well as the degradation product expression of pro-apoptotic proteins Caspase-3, Caspase-9 and PARP, with a significant difference with the control group (P < 0.01). However, CPT showed no impact on the P-gp expression in CML cells and the intracellular concentrations of imatinib. In summary, the findings suggested that CPT enhanced the sensitivity of CML cells to imatinib. Its mechanism is not dependent on the inhibition in P-gp expression and the increase in intracellular drug concentration. PMID:26591531

  12. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

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

  13. BACTERIAL PROTEIN EXPRESSION SYSTEM CASCADE (*) is a bacterial protein expression system that provides

    E-print Network

    Lebendiker, Mario

    . ­ Programming therapeutic bacteria expression: antigen expression and display in attenuated pathogenic bacteria terminal promoter and the multiple cloning site for efficient protein expression and purificationALEXa, pALEXb, pALEXc), to facilitate cloning. These vectors also carry an enteroki

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

    PubMed Central

    Liu, Li; Liu, Xiao-Dong

    2014-01-01

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

  15. Expression, purification, and crystallisationof membrane proteins

    NASA Astrophysics Data System (ADS)

    Byrne, Bernadette

    Approximately, 29,000 protein structures are deposited in the Protein Databank (January 2005), but only about 90 of which are independent membrane protein structures. This represents a significant increase in knowledge compared with a matter of only 5 years ago when a mere handful of membrane protein structures were available. Despite the advances, our understanding of the structure-function relationships and mechanism of action of many membrane proteins is still lacking. This is particularly true of many of the more clinically relevant membrane proteins, such as the G-protein-coupled receptors (GPCRs). The GPCRs regulate cellular responses to a wide range of biologically active molecules including hormones and drugs and are thus important targets for therapeutic intervention in a number of disease states. However, the increasing number of membrane protein structures has provided a critical mass of information which has yielded a more rational approach to the process of obtaining diffraction quality crystals. It is the different stages of this process; expression, solubilisation, purification, and crystallisation that will be covered in this lecture.

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

    PubMed

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

    2015-01-01

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

  17. Microgravity alters the expression of salivary proteins.

    PubMed

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

    2014-06-01

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

  18. [Using protein chips to study mechanism underlying reversion of drug resistance in leukemia cells in tetrandrine alone or in combination with droloxifene].

    PubMed

    Chen, Bao-An; Du, Juan; Zhang, Chun-Xiu; Cheng, Jian; Gao, Feng; Lu, Zu-Hong

    2005-12-01

    The aim of this study was to investigate the mechanism to reverse the drug resistance of leukemia cells in tetrandrine (Tet) alone or in combination with droloxifen (Drol) by using protein chips and to lay the theoretical basis for the clinical applications. Three monoclonal antibodies against P-glycoprotein (P-gp), the multidrug resistance-associated protein (MRP1) and the breast cancer resistance protein (BCRP) were immobilized onto the agarose gel film-coated glass slides. Protein chips were prepared respectively from K562/A02 cells cultured for 12, 24 and 48 hours with Tet alone or in combination with Drol. The results showed that Tet alone or in combination with Drol could decrease only the expression of P-gp in a time-dependent manner, the effect for 48 hours as follows: Tet + Drol 82.620 +/- 3.227; Tet alone 86.440 +/- 2.906; Drol alone 87.230 +/- 2.049; control 93.670 +/- 2.748 (P < 0.05). However, down-regulation of P-gp by K562/A02 cells cultured with Tet alone or in combination with Drol began at 24 hours (Tet + Drol 85.270 +/- 3.095; control 93.670 +/- 2.748, P < 0.05). The results were coincident with that of FCM. It is concluded that Tet and Drol can downregulate the expression of P-gp in the time-dependent way. There is a significant difference between Tet alone and Tet combined with Drol at 24 hours (P < 0.05). The expression of MRP1 and BCRP are not closely correlated with the reversal mechanism of Tet and Drol, and which may be involved in the mechanism of this combination to reverse multidrug resistance in leukemia. PMID:16403267

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

    PubMed Central

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

    2013-01-01

    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

  20. Regulation of Mutant p53 Protein Expression

    PubMed Central

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

    2015-01-01

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

  1. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

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

  2. Multidrug-resistance gene 1-type p-glycoprotein (MDR1 p-gp) inhibition by tariquidar impacts on neuroendocrine and behavioral processing of stress.

    PubMed

    Thoeringer, Christoph K; Wultsch, Thomas; Shahbazian, Anaid; Painsipp, Evelin; Holzer, Peter

    2007-01-01

    The multidrug-resistance gene 1-type p-glycoprotein (MDR1 p-gp) is a major gate-keeper at the blood-brain barrier (BBB), protecting the central nervous system from accumulation of toxic xenobiotics and drugs. In addition, MDR1 p-gp has been found to control the intracerebral access of glucocorticoid hormones and thus to modulate the activity of the hypothalamic-pituitary-adrenocortical (HPA) system. In view of the implication of glucocorticoids in the control of behavior, we examined how acute pharmacological inhibition of MDR1 p-gp at the BBB by tariquidar (XR9576; 12 mg/kg, PO) impacts the neuroendocrine and behavioral processing of stress in C57BL/6JIcoHim inbred mice. Inhibition of MDR1 p-gp at the BBB did not alter emotional behavior at baseline. However, mice that were sensitized by water-avoidance stress, a mild psychological stressor, displayed significantly reduced anxiety-related behavior in the elevated plus-maze test when treated with tariquidar. Tariquidar, however, had no effect on stress-coping performance assessed in the forced swim test. Investigating the impact of acute MDR1 p-gp inhibition on the glucocorticoid system, we observed a significant attenuation of the mild stress-induced increase of plasma corticosterone after tariquidar administration. In order to examine whether the anti-anxiety effect of tariquidar in sensitized animals is mediated by glucocorticoids, the animals were treated with corticosterone (1mg/kg, SC) immediately after exposure to water-avoidance stress. Corticosterone caused a significant anxiolytic-like effect in this stress-related anxiety protocol, whereas tariquidar could not further enhance corticosterone's anti-anxiety effects. The current data show for the first time that pharmacological inhibition of MDR1 p-gp at the murine BBB by tariquidar alters emotional behavior and HPA axis activity. By facilitating the entry of corticosterone into the brain, tariquidar enhances feedback inhibition of the HPA system and in this way improves anxiety-related stress processing. These findings highlight a novel approach to the treatment of stress-related affective disorders in humans. PMID:17881135

  3. 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

    Agarwal, Sagar; Elmquist, William F.

    2012-01-01

    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. PMID:22335402

  4. Expressing full-length functional PfEMP1 proteins in the HEK293 expression system.

    PubMed

    Srivastava, Anand; Durocher, Yves; Gamain, Benoît

    2013-01-01

    Due to the A/T-richness of the genome of Plasmodium falciparum, expressing P. falciparum proteins in heterologous expression systems is challenging. In addition, many P. falciparum proteins have high cysteine content and high molecular weight, which further complicates expression of these proteins in heterologous systems. The high molecular weight Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) adhesins expressed on the surface of the infected erythrocytes are among the most difficult proteins to express. Cost reduction in synthetic gene synthesis, as well as improved eukaryotic expression systems, now makes it possible to express such proteins. In this chapter, we describe the construction, production, purification, and functional assessment of the full-length extracellular region of the var2CSA PfEMP1 protein involved in pregnancy-associated malaria (PAM), using a human embryonic kidney (HEK) expression system. PMID:22990788

  5. Experimental validation of in silico target predictions on synergistic protein targets

    E-print Network

    Cortés Ciriano, Isidro; Koutsoukas, Alexios; Abian, Olga; Bender, Andreas; Velazquez-Campoy, Adrian

    2013-03-22

    inhibitors, four of which produce a synergistic effect in HeLa cell cultures in the presence of the aforesaid P-gp inhibitors (two independent samples t-test, pcell cycle (such as Topoisomerase I, Bcl-X and protein kinase C alpha) and for the defense against xenobiotics (such as P-gp 1 and CYP450...

  6. Arabidopsis thaliana SEPALLATA3 protein prokaryotic expression and purification.

    PubMed

    He, Q; Fu, A Y; Zhang, G C; Li, T J; Zhang, J H

    2015-01-01

    SEPALLATA3 (SEP3) can be attributed to E class gene of the ABCE model of floral organ development. In order to reveal how SEP3 proteins form polymers, and the relationship between the polymers and their biological functions, the experiments of Arabidopsis thaliana AtSEP3 protein soluble expression in vitro were performed to construct a vector of prokaryotic expression, and investigate induced expression of recombinant proteins in Escherichia coli cells. The protein soluble expression was analyzed through the aspects of different protein domains, induction time, induction temperature, etc. Different structural domains and expression conditions were screened, and 0.1% IPTG inducing at 22 oC for 15 h was estimated as an optimal expression strategy. The nickel chelating resin was used to purify the protein in size exclusion chromatography (SEC) and the results indicated that AtSEP3 protein was present in the form of tetramer. PMID:26025404

  7. Dexamethasone protects normal human liver cells from apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand by upregulating the expression of P-glycoproteins.

    PubMed

    Zhao, Bo; Xie, Gui-Juan; Li, Rui-Feng; Chen, Qing; Zhang, Xu-Qing

    2015-12-01

    Glucocorticoids are effective for the treatment of acute-on-chronic pre-liver failure, severe chronic hepatitis B and acute liver failure; however, the mechanism underlying the effects of treatment by glucocorticoids remains to be fully elucidated. The role and detailed mechanism of how glucocorticoids prevent liver disease progression can be elucidated by investigating the apoptosis of hepatocytes following glucocorticoid treatment. P?glycoproteins (P?gps) also confer resistance to apoptosis induced by a diverse range of stimuli. Glucocorticoids, particularly dexamethasone (DEX), upregulate the expression of P?gp in several tissues. In the present study, the normal human L?02 liver cell line was used, and techniques, including immunocytochemistry, western blot analysis, flow cytometry and reverse transcription?quantitative polymerase chain reaction analysis were used for determining the expression levels of P?gps, and for evaluating the effect of DEX pretreatment on the expression of P?gps. DEX (1?10 µM) was added to the cell culture media and incubated for 24?72 h. The results revealed that DEX upregulated the mRNA and protein levels of P?gp in a dose? and time?dependent manner. Subsequently, tumor necrosis factor?related apoptosis?inducing ligand (TRAIL) was used for the induction of apoptosis in the cells, followed by a terminal deoxynucleotidyl transferase dUTP nick end labeling assay to assess the apoptotic stages. The results demonstrated that apoptosis in the group of cells, which were pre?treated with DEX was significantly lower than that in the control group. Treatment with tariquidar, a P?gp inhibitor, reduced the anti?apoptotic effects of DEX. These results established that DEX protects normal human liver cells from TRAIL?induced apoptosis by upregulating the expression of P-gp. These observations may be useful for elucidating the mechanism of DEX for preventing the progression of liver disease. PMID:26496964

  8. Contribution of glial cells and pericytes to the mRNA profiles of P-glycoprotein and multidrug resistance-associated proteins in an in vitro model of the blood-brain barrier.

    PubMed

    Berezowski, Vincent; Landry, Christophe; Dehouck, Marie-Pierre; Cecchelli, Roméo; Fenart, Laurence

    2004-08-20

    P-glycoprotein (P-gp) and the multidrug resistance-associated proteins (MRP), whose expression is associated with multidrug resistance, have been recently located in the brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB), without taking into account a possible influence or contribution of glial cells and pericytes. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), the present study analysed the transcriptional expression of P-gp and the seven homologues of MRP transporters in BCECs in solo culture or in an in vitro model of the BBB consisting of a co-culture of BCECs and glial cells. Pericytes, glial cells, isolated brain capillaries and bovine grey matter extracts were also tested. P-gp mRNA, absent in glial cells, was found in brain capillaries and in co-cultured BCECs with an increased signal compared to the in solo culture. No amplification was observed in pericytes or grey matter. While MRP2, MRP3 and MRP7 remained undetected, MRP1, absent in capillaries or grey matter, was amplified in BCECs, glial cells and pericytes. MRP4 gave a low signal in most cultures. MRP5 was ubiquitously expressed, displaying a potent signal in all conditions. In spite of its presence in cultured glial cells, MRP6 mRNA expression appeared to be restricted to BCECs, with the same upregulation in the co-cultured condition as observed with P-gp. Moreover, MRP6 was the only transporter whose endothelial mRNA expression was influenced by the presence of pericytes. The tissue distribution of the expression of these transporters and the contribution of the different cell populations are discussed. PMID:15262198

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

    DOEpatents

    Mayfield, Stephen P. (Cardiff, CA)

    2010-03-16

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

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

    DOEpatents

    Mayfield, Stephen P

    2015-01-13

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

  11. Ecto-5?-nucleotidase expression is associated with the progression of renal cell carcinoma

    PubMed Central

    YU, YI; WANG, WEI; SONG, LEI; HU, WENTAO; DONG, CHI; PEI, HAILONG; ZHOU, GUANGMING; YUE, ZHONGJIN

    2015-01-01

    Renal cell carcinoma (RCC) is a common tissue tumor that occurs across all age groups and has become one of the types of cancer with the fastest increasing incidence. Due to the resistance of RCC chemo- and radiotherapy, surgery is the only currently effective treatment. Therefore, specific markers for the diagnosis and prognosis of RCC are expected to result in novel methods of treatment. Ecto-5?-nucleotidase, also termed cluster of differentiation (CD)73, is a protein that is activated in several types of aggressive cancer and may promote cancer progression. CD73 was examined in the present study to determine the association between the protein and RCC. The expression levels of CD73 in 159 RCC tissue sections and 30 paratumorous normal renal tissue samples obtained from 235 patients that underwent nephrectomy were examined by immunohistochemical staining. By contrast, the expression level of P-glycoprotein (P-gp), a potential prognostic factor in RCC, was also examined in 85 RCC and 13 normal tissue samples. Intense CD73 expression was identified in 75 out of 159 RCC cell membranes compared with normal renal tissues. In contrast, there was high P-gp expression in the blood vessels of 42 out of 85 RCC tissues and there was no significant difference between the P-gp expression identified in RCC cells (34 out of 85) and the cell membrane of normal renal cells (2 out of 13). The expression level of CD73 in RCC cells was significantly associated with tumor type, tumor node metastasis (TNM) stage, and tumor grade. However, the expression of P-gp in RCC cells was only associated with the TNM stage and tumor grade. Using a multivariable Cox regression analysis, it was found that the median survival rate of RCC patients with intense CD73 expression in RCC cells was 62.06±5.35 months, which was drastically shorter compared with rare CD73 expression (103.72±3.67 months). In conclusion, the expression level of CD73 is significantly associated with RCC tumor progression and may serve as a favorable marker for the diagnosis and prognosis of RCC, in addition to being a therapeutic target for the treatment of RCC. PMID:26137095

  12. HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins.

    PubMed

    Yadavalli, Raghavendra; Sam-Yellowe, Tobili

    2015-01-01

    Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed "in vitro human cell free expression systems". The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 µl of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer's Cleft - 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 µl of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford's assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing. PMID:26131624

  13. Proteomics beyond large-scale protein expression analysis.

    PubMed

    Boersema, Paul J; Kahraman, Abdullah; Picotti, Paola

    2015-08-01

    Proteomics is commonly referred to as the application of high-throughput approaches to protein expression analysis. Typical results of proteomics studies are inventories of the protein content of a sample or lists of differentially expressed proteins across multiple conditions. Recently, however, an explosion of novel proteomics workflows has significantly expanded proteomics beyond the analysis of protein expression. Targeted proteomics methods, for example, enable the analysis of the fine dynamics of protein systems, such as a specific pathway or a network of interacting proteins, and the determination of protein complex stoichiometries. Structural proteomics tools allow extraction of restraints for structural modeling and identification of structurally altered proteins on a proteome-wide scale. Other variations of the proteomic workflow can be applied to the large-scale analysis of protein activity, location, degradation and turnover. These exciting developments provide new tools for multi-level 'omics' analysis and for the modeling of biological networks in the context of systems biology studies. PMID:25636126

  14. Protein antigen expression in Escherichia coli for antibody production.

    PubMed

    Rancour, David M; Backues, Steven K; Bednarek, Sebastian Y

    2010-01-01

    Escherichia coli is a frequently used expression system for the generation of protein encoded by genes from diverse kingdoms and, thus, it is well suited for the production of protein antigens for antibody generation. It is a system of choice for many due to factors such as (1) the commercial availability of a vast array of reagents and materials needed for cloning, expression, and purification and (2) the potential high protein yields that can be acquired in a timely and cost-effective manner. This chapter will focus on (1) the general principles to keep in mind when choosing an antigen to express and (2) the use of a modified pGEX vector system (Rancour et al., J. Biol. Chem. 279:54264-54274, 2004) to use in its expression. Simplified protocols are provided for (1) assessing the expression of your protein, (2) testing whether your protein is or is not expressed as a soluble product, (3) performing bulk purifications of soluble or insoluble E. coli-expressed protein to acquire enough to be used for a complete immunization protocol, and (4) an optional procedure for epitope tag removal from your expressed protein of interest in order to avoid the unnecessary and sometimes unwanted production of antibodies against the fusion protein affinity chromatography tag. These four procedures have been used extensively and successfully in our lab as a basis for the production of recombinant protein and subsequent antibody production. PMID:20602203

  15. Pannexin 2 protein expression is not restricted to the CNS

    PubMed Central

    Le Vasseur, Maxence; Lelowski, Jonathan; Bechberger, John F.; Sin, Wun-Chey; Naus, Christian C.

    2014-01-01

    Pannexins (Panx) are proteins homologous to the invertebrate gap junction proteins called innexins (Inx) and are traditionally described as transmembrane channels connecting the intracellular and extracellular compartments. Three distinct Panx paralogs (Panx1, Panx2 and Panx3) have been identified in vertebrates but previous reports on Panx expression and functionality focused primarily on Panx1 and Panx3 proteins. Several gene expression studies reported that Panx2 transcript is largely restricted to the central nervous system (CNS) hence suggesting that Panx2 might serve an important role in the CNS. However, the lack of suitable antibodies prevented the creation of a comprehensive map of Panx2 protein expression and Panx2 protein localization profile is currently mostly inferred from the distribution of its transcript. In this study, we characterized novel commercial monoclonal antibodies and surveyed Panx2 expression and distribution at the mRNA and protein level by real-time qPCR, Western blotting and immunofluorescence. Panx2 protein levels were readily detected in every tissue examined, even when transcriptional analysis predicted very low Panx2 protein expression. Furthermore, our results indicate that Panx2 transcriptional activity is a poor predictor of Panx2 protein abundance and does not correlate with Panx2 protein levels. Despite showing disproportionately high transcript levels, the CNS expressed less Panx2 protein than any other tissues analyzed. Additionally, we showed that Panx2 protein does not localize at the plasma membrane like other gap junction proteins but remains confined within cytoplasmic compartments. Overall, our results demonstrate that the endogenous expression of Panx2 protein is not restricted to the CNS and is more ubiquitous than initially predicted. PMID:25505382

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

    PubMed

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

    2015-01-01

    Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization. PMID:25502200

  17. Protein microarrays for gene expression and antibody screening.

    PubMed

    Lueking, A; Horn, M; Eickhoff, H; Büssow, K; Lehrach, H; Walter, G

    1999-05-15

    Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen-antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor-ligand interactions. PMID:10328771

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

    PubMed

    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

    2011-04-15

    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

  19. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

  20. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

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

  1. Research Report Maternal isolation alters the expression of neural proteins

    E-print Network

    Sokolowski, Marla

    Research Report Maternal isolation alters the expression of neural proteins during development that reverses many of the behavioral deficits, also reverses many of the isolation effects on brain proteins in concentrations and distributions of brain proteins that are involved in normal brain development. When assessed

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

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  4. Functional proteomics of circadian expressed proteins from Chlamydomonas reinhardtii.

    PubMed

    Wagner, Volker; Fiedler, Monika; Markert, Christine; Hippler, Michael; Mittag, Maria

    2004-02-13

    In this study, functional proteomics was successfully applied for the characterization of circadian expressed, basic proteins. For this purpose, we have chosen the green model alga Chlamydomonas reinhardtii since its entire nuclear genome is available and it is ideally suited for biochemical enrichment procedures. Proteins from cells harvested during subjective day and night were heparin affinity purified. They were separated by two-dimensional gel electrophoresis suited for basic proteins and analyzed after tryptic digestion by electrospray ionization mass spectrometry. We can show for the first time that the expressions of a protein disulfide isomerase-like protein and a tetratricopeptide repeat protein change in a circadian manner. Interestingly, both proteins are known to be interaction partners in multiprotein complexes including RNA binding proteins. PMID:14960320

  5. An efficient system for small protein expression and refolding

    PubMed Central

    Cheng, Yuan; Patel, Dinshaw J.

    2015-01-01

    The low expression yield and poor refolding efficiency of small recombinant proteins expressed in Escherichia coli have continued to hinder the large-scale purification of such proteins for structural and biological investigations. A system based on a small fusion partner, the B1 domain of Streptococcal protein G (GB1), was utilized to overcome this problem. We have tested this system on a small cysteine-rich toxin, mutant myotoxin ? (MyoP20G). The highly expressed fusion protein was refolded using an unfolding/refolding protocol. Due to the small size of GB1, we were able to monitor the unfolding/refolding status by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The final product yielded well-resolved NMR spectra, with a topology corresponding to the natural product. We conclude that GB1 not only increases the expression level but also enhances the refolding of small proteins. PMID:15063772

  6. Differential protein expression analysis following olfactory learning in Apis cerana.

    PubMed

    Zhang, Li-Zhen; Yan, Wei-Yu; Wang, Zi-Long; Guo, Ya-Hui; Yi, Yao; Zhang, Shao-Wu; Zeng, Zhi-Jiang

    2015-11-01

    Studies of olfactory learning in honeybees have helped to elucidate the neurobiological basis of learning and memory. In this study, protein expression changes following olfactory learning in Apis cerana were investigated using isobaric tags for relative and absolute quantification (iTRAQ) technology. A total of 2406 proteins were identified from the trained and untrained groups. Among these proteins, 147 were differentially expressed, with 87 up-regulated and 60 down-regulated in the trained group compared with the untrained group. These results suggest that the differentially expressed proteins may be involved in the regulation of olfactory learning and memory in A. cerana. The iTRAQ data can provide information on the global protein expression patterns associated with olfactory learning, which will facilitate our understanding of the molecular mechanisms of learning and memory of honeybees. PMID:26427996

  7. Widening the bottleneck: increasing success in protein expression and purification

    PubMed Central

    Hopkins, Ralph; Esposito, Dominic; Gillette, William

    2010-01-01

    The number of variables at play in the expression and purification of a single protein dwarf those involved in sequencing a genome. Although certain trends are apparent, there is no one-size-fits-all approach to the process of purifying proteins. Thus, whereas numerous genome sequencing projects are providing an overwhelming number of interesting open reading frames for structural biologists to study, fully realizing the potential of this resource is still only a distant hope. We will discuss several current approaches to high throughput expression and purification as well as strategies that have served us well to quickly identify lead protein expression constructs in the context of a core service protein expression and purification laboratory. The use of the baculovirus expression vector system and implementation of a purification screening method will be emphasized. PMID:20650317

  8. The statistics of protein expression ratios for cellular fluorescence studies.

    PubMed

    Smith, Elizabeth M; Mueller, Joachim D

    2012-03-01

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

  9. Protein expression analyses at the single cell level.

    PubMed

    Ohno, Masae; Karagiannis, Peter; Taniguchi, Yuichi

    2014-01-01

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

  10. Cloning technologies for protein expression and purification.

    PubMed

    Hartley, James L

    2006-08-01

    Detailed knowledge of the biochemistry and structure of individual proteins is fundamental to biomedical research. To further our understanding, however, proteins need to be purified in sufficient quantities, usually from recombinant sources. Although the sequences of genomes are now produced in automated factories purified proteins are not, because their behavior is much more variable. The construction of plasmids and viruses to overexpress proteins for their purification is often tedious. Alternatives to traditional methods that are faster, easier and more flexible are needed and are becoming available. PMID:16839756

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

    PubMed

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

    2014-03-01

    Organochlorine (OC) pesticides constitute a major class of persistent and toxic organic pollutants, known to modulate drug-detoxifying enzymes. In the present study, OCs were demonstrated to also alter the activity and expression of human hepatic drug transporters. Activity of the sinusoidal influx transporter OCT1 (organic cation transporter 1) was thus inhibited by endosulfan, chlordane, heptachlor, lindane, and dieldrine, but not by dichlorodiphenyltrichloroethane isomers, whereas those of the canalicular efflux pumps MRP2 (multidrug resistance-associated protein 2) and BCRP (breast cancer resistance protein) were blocked by endosulfan, chlordane, heptachlor, and chlordecone; this latter OC additionally inhibited the multidrug resistance gene 1 (MDR1)/P-glycoprotein (P-gp) activity. OCs, except endosulfan, were next found to induce MDR1/P-gp and MRP2 mRNA expressions in hepatoma HepaRG cells; some of them also upregulated BCRP. By contrast, expression of sinusoidal transporters was not impaired (organic anion-transporting polypeptide (OATP) 1B1 and OATP2B1) or was downregulated (sodium taurocholate co-transporting polypeptide (NTCP) and OCT1). Such regulations of drug transporter activity and expression, depending on the respective nature of OCs and transporters, may contribute to the toxicity of OC pesticides. PMID:24464585

  12. Exocyst Complex Protein Expression in the Human Placenta

    PubMed Central

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

    2014-01-01

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

  13. Major cancer protein amplifies global gene expression

    Cancer.gov

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2006-05-01

    P-glycoprotein (P-gp), the most extensively studied ATP-binding transporter, functions as a biological barrier by extruding toxic substances and xenobiotics out of the cell. This study was carried out to determine the effect of N,N-diethyl-m-toluamide (DEET) and pyridostigmine bromide (PB), alone and in combination, on P-gp expression using Escherichia coli leaky mutant transformed with Mdr1 gene (pT5-7/mdr1), which codes for P-gp or lactose permease (pT5-7/lacY) as negative control. Also, daunomycin (a known P-gp sustrate) was used as a positive control and reserpine (a known P-gp inhibitor) served as a negative control. An in vitro cell-resistant assay was used to monitor the potential of test compounds to interact with P-gp. Following exposure of the cells to pyridostigmine bromide or daunomycin, P-gp conferred significant resistance against both compounds, while reserpine and DEET significantly inhibited the glycoprotein. Cells were grown in the presence of noncytotoxic concentrations of daunomycin, pyridostigmine bromide, reserpine, or DEET, and membrane fractions were examined by Western immunoblotting for expression of P-gp. Daunomycin induced P-gp expression quantitatively more than pyridostigmine bromide, while reserpine and DEET significantly inhibited P-gp expression in cells harboring mdr1. Photoaffinity labeling experiment performed with the P-gp ligand [125I]iodoarylazidoprazosin demonstrated that compounds that induced or inhibited P-gp transport activity also bound to P-gp. DEET was also found to be a potent inhibitor of P-gp-mediated ATPase activity, whereas pyridostigmine bromide increased P-gp ATPase activity. Cells expressing P-gp or lac permease were exposed to pyridostigmine bromide and DEET, alone and in combination. Noncytotoxic concentrations of DEET significantly inhibited P-gp-mediated resistance against pyridostigmine bromide, resulting in a reduction of the number of effective drug interactions with biological targets. An explanation of these results might be that DEET is a third-generation inhibitor of P-gp; it has high potency and specificity for P-gp, it inhibits hydrolysis of ATP, it exerts no appreciable impact on cytochrome P-450 3A4, and it prevents transport of xenobiotics, such as pyridostigmine bromide, out of the cell. This conclusion explains, at least in part, the increased toxicity and bioavailability of pyridostigmine bromide following combined administration with DEET. This study improves our understanding of the basis of chemical interactions with DEET by defining the ability of drugs to interact with P-gp either as inhibitors or substrates, which may in turn lead to altered efficacy or toxicity. PMID:16728371

  16. Green algae as a platform to express therapeutic proteins.

    PubMed

    Lu, Yang; Oyler, George A

    2009-06-01

    Proteins produced by DNA recombinant technology have been playing important roles in modern medicine ever since the first such protein drug was approved by the U.S. Food and Drug Administration about three decades ago. However the inherent high cost of producing recombinant proteins, particularly those produced from mammalian cells, has hampered their broad application. Other protein expression systems that can reduce the cost yet still maintain the high-level therapeutic activities of the recombinant proteins are a top R&D priority. Eukaryotic unicellular green algae cells may provide a good solution to this long-standing challenge. PMID:19772839

  17. Small-scale expression of proteins in E. coli.

    PubMed

    Zerbs, Sarah; Giuliani, Sarah; Collart, Frank

    2014-01-01

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

  18. PATTERNS & PHENOTYPES Abundant and Dynamic Expression of G Protein-

    E-print Network

    Burnstock, Geoffrey

    and ventral white matter of the spinal cord postnatally, demonstrated that many P2Y receptors were likely-activated protein kinases, mediate the effects of P2Y receptors. Thus, activation of the P2Y receptors can causePATTERNS & PHENOTYPES Abundant and Dynamic Expression of G Protein- Coupled P2Y Receptors

  19. Invited Review Functional expression of heterologous proteins in yeast: insights

    E-print Network

    Rao, Rajini

    Invited Review Functional expression of heterologous proteins in yeast: insights into Ca2 signaling of heterologous proteins in yeast: insights into Ca2 signaling and Ca2 -transporting ATPases. Am J Physiol Cell Physiol 287: C580­C589, 2004; 10.1152/ajpcell.00135.2004.-- The baker's yeast Saccharomyces cerevisiae

  20. EMBRYONIC EXPRESSION OF UNCOUPLING PROTEIN 2 GENES IN RAINBOW TROUT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Uncoupling proteins are mitochondrial anion transporters that dissociate respiration from ATP synthesis through proton leaks. Uncoupling protein 2 reportedly plays a role in several physiological processes such as energy partitioning, nutrition and fatty acid metabolism. The mRNA expression of rainb...

  1. Cellular and regional specific changes in multidrug efflux transporter expression during recovery of vasogenic edema in the rat hippocampus and piriform cortex

    PubMed Central

    Kim, Yeon-Jo; Kim, Ji-Eun; Choi, Hui-Chul; Song, Hong-Ki; Kang, Tae-Cheon

    2015-01-01

    In the present study, we investigated the characteristics of drug efflux transporter expressions following status epilepticus (SE). In the hippocampus and piriform cortex (PC), vasogenic edema peaked 3-4 days after SE. The expression of breast cancer resistance protein (BCRP), multidrug resistance protein-4 (MRP4), and p-glycoprotein (p-GP) were decreased 4 days after SE when vasogenic edema was peaked, but subsequently increased 4 weeks after SE. Multidrug resistance protein-1 (MRP1) expression gradually decreased in endothelial cells until 4 weeks after SE. These findings indicate that SE-induced vasogenic edema formation transiently reduced drug efflux pump expressions in endothelial cells. Subsequently, during recovery of vasogenic edema drug efflux pump expressions were differentially upregulated in astrocytes, neuropils, and endothelial cells. Therefore, we suggest that vasogenic edema formation may be a risk factor in pharmacoresistent epilepsy. [BMB Reports 2015; 48(6): 348-353] PMID:25388209

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

    PubMed Central

    2012-01-01

    Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, 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. PMID:22994686

  3. Ubiquitin-dependent proteolysis in yeast cells expressing neurotoxic proteins.

    PubMed

    Braun, Ralf J

    2015-01-01

    Critically impaired protein degradation is discussed to contribute to neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's, and motor neuron diseases. Misfolded, aggregated, or surplus proteins are efficiently degraded via distinct protein degradation pathways, including the ubiquitin-proteasome system, autophagy, and vesicular trafficking. These pathways are regulated by covalent modification of target proteins with the small protein ubiquitin and are evolutionary highly conserved from humans to yeast. The yeast Saccharomyces cerevisiae is an established model for deciphering mechanisms of protein degradation, and for the elucidation of pathways underlying programmed cell death. The expression of human neurotoxic proteins triggers cell death in yeast, with neurotoxic protein-specific differences. Therefore, yeast cell death models are suitable for analyzing the role of protein degradation pathways in modulating cell death upon expression of disease-causing proteins. This review summarizes which protein degradation pathways are affected in these yeast models, and how they are involved in the execution of cell death. I will discuss to which extent this mimics the situation in other neurotoxic models, and how this may contribute to a better understanding of human disorders. PMID:25814926

  4. Ubiquitin-dependent proteolysis in yeast cells expressing neurotoxic proteins

    PubMed Central

    Braun, Ralf J.

    2015-01-01

    Critically impaired protein degradation is discussed to contribute to neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's, and motor neuron diseases. Misfolded, aggregated, or surplus proteins are efficiently degraded via distinct protein degradation pathways, including the ubiquitin-proteasome system, autophagy, and vesicular trafficking. These pathways are regulated by covalent modification of target proteins with the small protein ubiquitin and are evolutionary highly conserved from humans to yeast. The yeast Saccharomyces cerevisiae is an established model for deciphering mechanisms of protein degradation, and for the elucidation of pathways underlying programmed cell death. The expression of human neurotoxic proteins triggers cell death in yeast, with neurotoxic protein-specific differences. Therefore, yeast cell death models are suitable for analyzing the role of protein degradation pathways in modulating cell death upon expression of disease-causing proteins. This review summarizes which protein degradation pathways are affected in these yeast models, and how they are involved in the execution of cell death. I will discuss to which extent this mimics the situation in other neurotoxic models, and how this may contribute to a better understanding of human disorders. PMID:25814926

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

    PubMed Central

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

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

  6. Colorectal cancers differ in respect of PARP-1 protein expression

    E-print Network

    Violetta Sulzyc-Bielicka; Pawel Domagala; Jolanta Hybiak; Anna Majewicz-Broda; Krzysztof Safranow; Wenancjusz Domagala

    2012-07-25

    Recent findings raise the possibility of PARP inhibitor therapy in colorectal cancers(CRCs). However, the extent of PARP-1 protein expression in clinical specimens of CRC is not known. Using immunohistochemistry we assessed PARP-1 protein expression in tissue microarrays of 151 CRCs and its association with the patient's age, sex, Astler-Coller stage, grade and site of the tumor. High PARP nuclear immunoreactivity was found in 68.2% (103/151) of all cases. In turn, 31.8% (48/151)of tumors showed low PARP expression, including 9 (6%) PARP-1 negative CRCs. There was a significant association of PARP-1 expression with the site of CRC and Astler-Coller stage. A high PARP expression was noted in 79.1% of colon vs. 53.9% of rectal tumors (p = 0.001). The mean PARP-1 score was 1.27 times higher in colon vs. rectal cancers (p = 0.009) and it was higher in stage B2 vs. stage C of CRCs (p = 0.018). In conclusion, the level of PARP-1 protein nuclear expression is associated with the tumor site and heterogeneous across clinical specimens of CRC, with the majority of CRCs expressing a high level but minority - low or no PARP-1 expression. These findings may have a clinical significance because the assessment of PARP-1 expression in tumor samples may improve selection of patients with CRC for PARP inhibitor therapy.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2011-11-01

    The effect of bioactive plant natural products on the expression and functional activity of P-glycoprotein (P-gp) is poorly understood. Interactions of bioactive plant-based food and dietary supplements with P-gp can cause significant alteration of pharmacokinetic properties of P-gp substrate drugs when used in combination. This can augment toxicity and/or interfere with the drug's therapeutic outcomes. This study investigated the effects of diverse commonly used plant natural products on the expression and activity of P-gp in human adenocarcinoma cells (LS-180). These natural products included the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (cembratriene), the palm oil-derived ?-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

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

    PubMed Central

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

    2011-01-01

    The effect of bioactive plant natural products on the expression and functional activity of P-glycoprotein (P-gp) is poorly understood. Interactions of bioactive plant-based food and dietary supplements with P-gp can cause significant alteration of pharmacokinetic properties of P-gp substrate drugs when used in combination. This can augment toxicity and/or interfere with the drug’s therapeutic outcomes. This study investigated the effects of diverse commonly used plant natural products on the expression and activity of P-gp in human adenocarcinoma cells (LS-180). These natural products included the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (cembratriene), the palm oil-derived ?-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

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

    PubMed Central

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

    1995-01-01

    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. PMID:7884889

  11. Differential Protein Expression in Congenital and Acquired Cholesteatomas

    PubMed Central

    Kim, Sung Huhn; Choi, Jae Young

    2015-01-01

    Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D) electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5–3), plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5–3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5–3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins may differ from those of acquired cholesteatomas, which originate from retraction pocket epithelia. PMID:26335306

  12. Lentiviral fluorescent protein expression vectors for biotinylation proteomics.

    PubMed

    Riz, Irene; Hawley, Teresa S; Hawley, Robert G

    2011-01-01

    In vivo biotinylation tagging, based on a method in which a protein of interest is tagged with a peptide that is biotinylated in vivo by coexpression of Escherichia coli BirA biotin ligase, has been successfully used for the isolation of protein-protein and protein-DNA complexes in mammalian cells. We describe a modification of this methodology in which cells stably expressing the tagged gene of interest and the BirA gene can be selected by fluorescence-activated cell sorting (FACS). We recently implemented this approach to isolate and characterize proteins associated with TLX1, a homeodomain transcription factor with leukemogenic function. The modified technique utilizes two components: a lentiviral vector coexpressing the gene of interest containing a biotinylation tag on a bicistronic transcript together with a downstream yellow fluorescent protein (YFP) gene; and a second lentiviral vector encoding a fusion protein composed of bacterial BirA linked to the green fluorescent protein (GFP). This FACS-based binary in vivo biotinylation tagging system allows precise control over the levels of BirA-mediated biotinylation as well as the expression of the gene of interest, which is especially important if high-level expression negatively impacts cell growth or viability. PMID:21116996

  13. Selenate specifically sensitizes drug-resistant cancer cells by increasing apoptosis via G2 phase cell cycle arrest without P-GP inhibition.

    PubMed

    Choi, Ae-Ran; Jee Jo, Min; Jung, Myung-Ji; Sik Kim, Hyung; Yoon, Sungpil

    2015-10-01

    The purpose of this study was to identify conditions that will increase the sensitivity of drug-resistant cancer cells. Selenium derivatives have been shown to present anti-cancer properties in the clinic. Currently, selenate, selenite, selenomethionine (SeMet), methyl-selenocysteine (MSC), and methaneselenic acid (MSA) are the most common selenium derivatives used as drugs in humans. Herein, we tested whether these selenium derivatives can sensitize KBV20C cancer cells, which are highly resistant to anti-cancer drugs such as vincristine. All five drugs could sensitize KBV20C cells to the same extent as they sensitized the sensitive parent KB cells, suggesting that selenium-derived drugs can be used for drug-resistant cancer cells. We also observed that these drugs did not inhibit the P-glycoprotein (P-gp) pumping-out ability, suggesting that the sensitization by selenium-derived drugs does not depend on P-gp activity in resistant KBV20C cells. Interestingly, using a cell viability assay, microscopic observation, and Hoechst staining, we found that selenate highly sensitized drug-resistant KBV20C cells by activating the apoptotic pathway, when compared to sensitive KB cells. Furthermore, we investigated why selenate sensitizes resistant KBV20C cells. Selenate-induced toxicity was associated with an increase in G2-phase cell cycle arrest in KBV20C cells, suggesting that the selenate-induced increase in apoptosis resulted from cell cycle arrest in resistant KBV20C cells. Our findings may contribute to the development of selenate-based therapies for patients resistant to cancer drugs. PMID:26134503

  14. Acute leukemia subclassification: a marker protein expression perspective.

    PubMed

    Te Kronnie, Geertruy; Bicciato, Silvio; Basso, Giuseppe

    2004-06-01

    Improved leukemia classification and tailoring of therapy have greatly improved patient outcome particularly for children with acute leukemia (AL). Using immunophenotyping, molecular genetics and cytogenetics the low hanging fruits of biomedical research have been successfully incorporated in routine diagnosis of leukemia subclasses. Future improvements in the classification and understanding of leukemia biology will very likely be more slow and laborious. Recently, gene expression profiling has provided a framework for the global molecular analysis of hematological cancers, and high throughput proteomic analysis of leukemia samples is on the way. Here we consider classification of acute leukemia samples by flow cytometry using the marker proteins of immunophenotyping as a component of the proteome. Marker protein expressions are converted into quantitative expression values and subjected to computational analysis. Quantitative multivariate analysis from panels of marker proteins has demonstrated that marker protein expression profiles can distinguish MLLre from non-MLLre ALL cases and also allow to specifically distinguish MLL/AF4 cases. Potentially, these quantitative expression analyses can be used in clinical diagnosis. Immunophenotypic data collection using flow cytometry is a fast and relatively easily accessible technology that has already been implemented in most centers for leukemia diagnosis and the translation into quantitative expression data sets is a matter of flow cytometer settings and output calibration. However, before application in clinical diagnostics can occur it is crucial that quantitative immunophenotypic data set analysis is validated in independent experiments and in large data sets. PMID:15204097

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

    PubMed Central

    2013-01-01

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

  16. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish

    PubMed Central

    Horstick, Eric J.; Jordan, Diana C.; Bergeron, Sadie A.; Tabor, Kathryn M.; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A.

    2015-01-01

    Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3? untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models. PMID:25628360

  17. Subcellular localization of transiently expressed fluorescent fusion proteins.

    PubMed

    Collings, David A

    2013-01-01

    The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system. PMID:23996319

  18. Protein Co-Expression Network Analysis (ProCoNA)

    SciTech Connect

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

    2013-06-01

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

  19. 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

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

    2011-11-01

    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

  20. Ethnic variability in the expression of hepatic drug transporters: absolute quantification by an optimized targeted quantitative proteomic approach.

    PubMed

    Peng, Kuan-wei; Bacon, James; Zheng, Ming; Guo, Yingying; Wang, Michael Zhuo

    2015-07-01

    Hepatic OATPs 1B1, 1B3 and 2B1, as well as P-gp, play important roles in regulating liver uptake and biliary excretion of drugs. The intrinsic ethnic variability in OATP1B1-mediated hepatic uptake of statins has been proposed to underlie the ethnic variability in plasma exposures of statins between Caucasians and Asians. Using a targeted quantitative proteomic approach, we determined hepatic protein concentrations of OATP1B1, OATP1B3, OATP2B1, P-gp, and PMCA4 (a housekeeping protein) in a panel of human livers (n = 141) and compared protein expression across Caucasian, Asian, African-American, and unidentified donors. Using an optimized protocol that included sodium deoxycholate as a membrane protein solubilizer, the hepatic protein expression levels (mean ± S.D.) of these transporters across all livers were determined to be 15.0 ± 6.0, 16.1 ± 8.1, 4.1 ± 1.3, 0.6 ± 0.2, and 2.4 ± 1.0 fmol/?g of total membrane protein, respectively. The scaling factor was 3.5 mg of total membrane protein in 100 mg of wet liver tissue. OATP1B1 protein expression was significantly associated with the c.388A>G (rs2306283, N130D) single nucleotide polymorphism. When compared across ethnicity, the hepatic expression levels of OATP1B1 and OATP1B3 were unexpectedly higher in Asians relative to Caucasians, suggesting that hepatic OATP expression alone does not explain the increased systemic statin levels in Asians compared with Caucasians. These findings may help improve physiologically based pharmacokinetic modeling to predict statin pharmacokinetic profiles and enable extrapolation of pharmacokinetic data of OATP substrates across ethnic groups. PMID:25926430

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal po...

  2. CyclinD1 protein expressed in pterygia is associated with ?-catenin protein localization

    PubMed Central

    Tung, Jai-Nien; Chiang, Chun-Chi; Tsai, Yi-Yu; Chou, Ying-Yi; Yeh, Kun-Tu; Lee, Huei

    2010-01-01

    Background The Wnt (Wg/Wnt) signaling cascade plays an important role in tumorigenesis. Our previous report indicated that aberrant localization of ?-catenin proteins was a feature of pterygia. Therefore, this study aimed to analyze the association of ?-catenin protein and expression of a downstream gene, cyclin D1, in pterygial tissues. Methods Using immunohistochemistry, ?-catenin and cyclin D1 protein expression was studied, in 150 pterygial specimens and 30 normal conjunctivas. Results Seventy-three (48.7%) and 60 (40.0%) pterygial specimens tested positive for ?-catenin and cyclin D1 protein expression, respectively. Cyclin D1protein expression was significantly higher in ?-catenin-nuclear/cytoplasmic positive groups than in ?-catenin membrane positive and negative groups (p<0.0001). In addition, cyclin D1 expression was significantly higher in the fleshy group than in the atrophic and intermediate groups (p=0.006). Conclusions Our study demonstrated that ?-catenin expressed in nuclei/cytoplasm increases cyclinD1 protein expression, which invokes pterygial cell proliferation. PMID:21179427

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

    PubMed

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

    2015-01-01

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation. PMID:25590335

  4. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia.

    PubMed

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-02-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the ? subunit of the 11S ?? regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S ?2, Rpn10, Rpn13, 11S?, and 11S?) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  5. SPINK 1 Protein Expression and Prostate Cancer Progression

    PubMed Central

    Flavin, Richard; Pettersson, Andreas; Hendrickson, Whitney K.; Fiorentino, Michelangelo; Finn, Stephen; Kunz, Lauren; Judson, Gregory L.; Lis, Rosina; Bailey, Dyane; Fiore, Christopher; Nuttall, Elizabeth; Martin, Neil E.; Stack, Edward; Penney, Kathryn L.; Rider, Jennifer R.; Sinnott, Jennifer; Sweeney, Christopher; Sesso, Howard D.; Fall, Katja; Giovannucci, Edward; Kantoff, Philip; Stampfer, Meir; Loda, Massimo; Mucci, Lorelei A.

    2014-01-01

    Purpose SPINK1 over-expression has been described in prostate cancer and is linked with poor prognosis in many cancers. The objective of this study was to characterize the association between SPINK1 over-expression and prostate cancer specific survival. Experimental Design The study included 879 participants in the US Physicians’ Health Study and Health Professionals Follow–Up Study, diagnosed with prostate cancer (1983 – 2004) and treated by radical prostatectomy. Protein tumor expression of SPINK1 was evaluated by immunohistochemistry on tumor tissue microarrays. Results 74/879 (8%) prostate cancer tumors were SPINK1 positive. Immunohistochemical data was available for PTEN, p-Akt, pS6, stathmin, androgen receptor (AR) and ERG (as a measure of the TMPRSS2:ERG translocation). Compared to SPINK1 negative tumors, SPINK1 positive tumors showed higher PTEN and stathmin expression, and lower expression of AR (p<0.01). SPINK1 over-expression was seen in 47 of 427 (11%) ERG negative samples and in 19 of 427 (4%) ERG positive cases (p=0.0003). We found no significant associations between SPINK1 status and Gleason grade or tumor stage. There was no association between SPINK1 expression and biochemical recurrence (p=0.56). Moreover, there was no association between SPINK1 expression and prostate cancer mortality (there were 75 lethal cases of prostate cancer during a mean of 13.5 years follow-up [HR 0.71 (95% confidence interval 0.29–1.76)]). Conclusions Our results suggest that SPINK1 protein expression may not be a predictor of recurrence or lethal prostate cancer amongst men treated by radical prostatectomy. SPINK1 and ERG protein expression do not appear to be entirely mutually exclusive, as some previous studies have suggested. PMID:24687926

  6. Expression of extracellular matrix proteins in ovarian serous tumors.

    PubMed

    Salani, Ritu; Neuberger, Ilana; Kurman, Robert J; Bristow, Robert E; Chang, Hsueh-Wei; Wang, Tian-Li; Shih, Ie-Ming

    2007-04-01

    The aims of this study were to perform a comprehensive expression analysis of the genes encoding extracellular matrix proteins and to investigate the expression pattern in one of these proteins, syndecan 1, in normal ovarian epithelium as well as benign and malignant ovarian serous tumors. Gene expression of 16 different extracellular matrix proteins was analyzed in ovarian serous tumors based on serial analysis of gene expression database. Semiquantitative reverse transcription-polymerase chain reaction was used to validate the serial analysis of gene expression result from each gene. As compared with normal ovarian surface epithelium, we found overexpression of syndecan 1, collagen type IV alpha 2, elastin microfibril interfase located protein 1, and laminin 5 in ovarian serous carcinomas. Syndecan 1 was selected for further study as it has not been well characterized in ovarian cancer and the syndecan 1 antibody was available for immunohistochemistry. Using a syndecan 1-specific monoclonal antibody, we demonstrated that syndecan 1 was expressed in 30.4% of high-grade serous carcinomas, 29.7% of low-grade carcinomas and serous borderline tumors, but none of benign serous cystadenomas and ovarian surface epithelium. Although both high-grade and low-grade serous carcinomas had a similar percentage of syndecan 1-positive cases, the immunointensity in high-grade carcinoma was significantly higher than that in low-grade carcinomas and serous borderline tumors (P = 0.007). In summary, ovarian carcinomas exhibit up-regulated expression of several extracellular matrix proteins, and syndecan 1 represents a novel tumor-associated marker in ovarian serous carcinomas. PMID:17413980

  7. Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

    PubMed Central

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

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium. PMID:25536421

  8. Beyond protein expression, MOPED goes multi-omics.

    PubMed

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

    2015-01-01

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

  9. Expression sites of two byssal protein genes of Mytilus galloprovincialis.

    PubMed

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

    1996-04-01

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

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

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

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

  11. Over-producing soluble protein complex and validating protein-protein interaction through a new bacterial co-expression system.

    PubMed

    Zeng, Jumei; Zhang, Lei; Li, Yuqing; Wang, Yi; Wang, Mingchao; Duan, Xin; He, Zheng-Guo

    2010-01-01

    Many proteins exert their functions through a protein complex and protein-protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein-protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein-protein interaction of interest in a wide range of biological organisms. PMID:19747546

  12. Expression and Function of Bcl-2 Proteins in Melanoma

    PubMed Central

    Eberle, Jürgen; Hossini, Amir M

    2008-01-01

    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. PMID:19506730

  13. Explanatory chapter: troubleshooting protein expression: what to do when the protein is not soluble.

    PubMed

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Production of soluble protein remains a bottleneck in the biochemistry and structural biology fields. Unfortunately, there is no 'magic bullet' that solves all solubility problems. The following is a protocol to test whether a protein expressed recombinantly is soluble, and possible strategies to circumvent insolubility issues. PMID:24674075

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

    ERIC Educational Resources Information Center

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

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

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

    PubMed

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

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

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

    PubMed Central

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

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  17. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

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

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

  18. 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...

  19. Bayesian Mixture Models for Gene Expression and Protein Profiles

    E-print Network

    Morris, Jeffrey S.

    the clustering im- plicitely defined by the mixture model. Also, see Dahl (2006); Tadesse et al. (2006 approaches that are typical of this literature. In Section 2 we discuss the use of Dirichlet process mixtures28 Bayesian Mixture Models for Gene Expression and Protein Profiles Michele Guindani, Kim-Anh Do

  20. p53 protein expression in central nervous system neoplasms.

    PubMed Central

    Barbareschi, M.; Iuzzolino, P.; Pennella, A.; Allegranza, A.; Arrigoni, G.; Dalla Palma, P.; Doglioni, C.

    1992-01-01

    AIMS: To demonstrate, immunohistochemically, p53 protein expression in a selection of central nervous system tumours; to investigate the relation between p53 expression and that of the proliferation related antigen, PCNA. METHODS: Surgical specimens from 86 central nervous system tumours were routinely fixed, paraffin wax embedded, and immunostained with a monoclonal (PAb 1801) and a policlonal antibody (CM1) p53 protein and a monoclonal antibody against PCNA (PC10). Normal brain samples obtained at necropsy and 10 surgically obtained samples of gliotic brain parenchyma were also immunostained. RESULTS: p53 protein expression was observed in 35 of 86 brain tumours, suggesting frequent p53 gene mutation. p53 protein alterations were associated with all grades of malignancy in tumours displaying solely astrocytic differentiation, with the exception of pilocytic astrocytomas. In those showing oligodendroglial or ependymal differentiation they appeared to be restricted almost to only high grade lesions. No p53 immunoreactivity was observed in normal or gliotic brain tissue; p53 altered expression was not related to the percentage of PCNA labelled cells. CONCLUSIONS: The use of sophisticated gene amplification techniques or highly sensitive immunohistochemical methods might be useful in distinguishing between reactive and neoplastic astrocytic lesions, and in the identification of malignant progression in other non-astrocytic glial tumours. Tumours with very similar histogenetic differentiation features might actually be a genetically heterogeneous group with possible different clinical courses. Images PMID:1355494

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

    SciTech Connect

    Raymond, Amy; Lovell, Scott; Lorimer, Don; Walchli, John; Mixon, Mark; Wallace, Ellen; Thompkins, Kaitlin; Archer, Kimberly; Burgin, Alex; Stewart, Lance

    2009-12-01

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

  2. Expression and Targeting of Secreted Proteins from Chlamydia trachomatis

    PubMed Central

    Bauler, Laura D.

    2014-01-01

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

  3. Universal genetic assay for engineering extracellular protein expression.

    PubMed

    Haitjema, Charles H; Boock, Jason T; Natarajan, Aravind; Dominguez, Miguel A; Gardner, Jeffrey G; Keating, David H; Withers, Sydnor T; DeLisa, Matthew P

    2014-02-21

    A variety of strategies now exist for the extracellular expression of recombinant proteins using laboratory strains of Escherichia coli . However, secreted proteins often accumulate in the culture medium at levels that are too low to be practically useful for most synthetic biology and metabolic engineering applications. The situation is compounded by the lack of generalized screening tools for optimizing the secretion process. To address this challenge, we developed a genetic approach for studying and engineering protein-secretion pathways in E. coli . Using the YebF pathway as a model, we demonstrate that direct fluorescent labeling of tetracysteine-motif-tagged secretory proteins with the biarsenical compound FlAsH is possible in situ without the need to recover the cell-free supernatant. High-throughput screening of a bacterial strain library yielded superior YebF expression hosts capable of secreting higher titers of YebF and YebF-fusion proteins into the culture medium. We also show that the method can be easily extended to other secretory pathways, including type II and type III secretion, directly in E. coli . Thus, our FlAsH-tetracysteine-based genetic assay provides a convenient, high-throughput tool that can be applied generally to diverse secretory pathways. This platform should help to shed light on poorly understood aspects of these processes as well as to further assist in the construction of engineered E. coli strains for efficient secretory-protein production. PMID:24200127

  4. Differentially Expressed Proteins in Malignant and Benign Adrenocortical Tumors

    PubMed Central

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

    2014-01-01

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

  5. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted ?-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the ?-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an ?-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins. PMID:25851716

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

    PubMed Central

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

    1997-01-01

    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 PMID:9252201

  7. Altered expression of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in experimental hepatocellular carcinoma.

    PubMed

    McKillop, I H; Wu, Y; Cahill, P A; Sitzmann, J V

    1998-06-01

    Guanine nucleotide regulatory proteins (G-proteins) play an important role in the onset and progression of malignancy. We hypothesized that alterations in inhibitory G-protein (Gi) expression and/or function may contribute to cellular invasion and formation of hepatocellular carcinoma (HCC). H4IIE hepatoma cells were inoculated directly into the liver parenchyma of ACI strain rats, and membranes were prepared from HCC livers and adjacent nonneoplastic livers 12 days following the initial inoculation. Expression of inhibitory Gialpha proteins was determined by Western blot analysis and changes in the functional activity of these proteins confirmed by pertussis toxin catalyzed ADP ribosylation and adenylyl cyclase activity. Inhibitory Gialpha1, Gialpha1/2, and Gialpha3 protein expression was significantly elevated in HCC when compared to adjacent nonneoplastic liver and sham-operated hepatic tissue. Pertussis toxin catalyzed ADP ribosylation of Gialpha substrates was significantly enhanced in HCC concomitant with increased basal and stimulated adenylyl cyclase activity following uncoupling of Gi-proteins with manganese ions. The role of Gi-proteins in cellular proliferation was confirmed using cultured H4IIE cells and normal hepatocytes. In quiescent H4IIE cells, mastoparan (Gialpha activator) increased [3H] thymidine incorporation and cell growth in a dose-dependent manner, whereas both pertussis toxin (a Gi-protein inhibitor) and 8-bromo-cAMP inhibited mitogenesis. In contrast, in isolated cultured hepatocytes, mastoparan inhibited [3H] thymidine incorporation, while pertussis toxin and 8-bromo-cAMP were mitogenic. We conclude that HCC is associated with marked changes in Gialpha-protein expression in vivo and in vitro, direct activation of which leads to increased mitogenesis in H4IIE cells in vitro. PMID:9572474

  8. Hedyotis diffusa Willd overcomes 5-fluorouracil resistance in human colorectal cancer HCT-8/5-FU cells by downregulating the expression of P-glycoprotein and ATP-binding casette subfamily G member 2

    PubMed Central

    LI, QIONGYU; WANG, XIANGFENG; SHEN, ALING; ZHANG, YUCHEN; CHEN, YOUQIN; SFERRA, THOMAS J.; LIN, JIUMAO; PENG, JUN

    2015-01-01

    Previous studies have demonstrated that Hedyotis diffusa Willd (HDW), a traditional Chinese herbal medicine, exhibits potent anticancer activity in models of colorectal cancer (CRC). Aggressive forms of CRC exhibit resistance to widely used chemotherapeutic drugs, including the antimetabolite, 5-fluorouracil (5-FU); however, less is known with regard to the activity of HDW against 5-FU-resistant cancer. In the present study, the mechanism of action and the potency of ethanol extracts of HDW (EEHDW) were investigated on a multidrug-resistant CRC HCT-8/5-FU cell line. Using an MTT cell proliferation assay, EEHDW treatment was shown to significantly reduce the cell viability of HCT-8/5-FU cells in a dose- and time-dependent manner. Furthermore, EEHDW significantly increased the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine-123, as compared with the untreated controls. To further investigate the molecular mechanisms targeted by EEHDW in the resistant cells, the expression levels of the ABC drug transporter protein, P-glycoprotein (P-gp), and ABC subfamily G member 2 (ABCG2), were analyzed using reverse-transcription polymerase chain reaction and western blot analysis. The mRNA and protein expression levels of P-gp and ABCG2 were reduced in the HCT-8/5-FU cells following EEHDW treatment, indicating that EEHDW inhibits ABCG2-mediated drug resistance by downregulating the expression of ABCG2 and P-gp. Therefore, the potential application of EEHDW as a chemotherapeutic adjuvant represents a promising alternative approach to the treatment of drug-resistant CRC.

  9. Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

    PubMed

    Jung, Yuna; Jung, Hyeim; Lim, Dongbin

    2015-12-01

    Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage ?6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli. PMID:26213264

  10. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    SciTech Connect

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  11. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  12. Manipulating heat shock protein expression in laboratory animals.

    PubMed

    Tolson, J Keith; Roberts, Stephen M

    2005-02-01

    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

  13. Generation of transgenic dogs that conditionally express green fluorescent protein.

    PubMed

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies. PMID:21630415

  14. Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline.

    PubMed

    Hewitt, Stephen N; Choi, Ryan; Kelley, Angela; Crowther, Gregory J; Napuli, Alberto J; Van Voorhis, Wesley C

    2011-09-01

    Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies. PMID:21904041

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

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2015-01-01

    Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins. PMID:17846637

  16. HIV-1 Tat Protein Enhances Expression and Function of Breast Cancer Resistance Protein.

    PubMed

    Zhou, Yancong; Zhang, Kun; Yin, Xiaojie; Nie, Qichang; Ma, Yonggang

    2016-01-01

    ATP binding cassette (ABC) transporters can transfer a variety of antiviral agents from the cytoplasm to body fluid, which results in a reduced intracellular concentration of the drugs. Proteins of HIV-1, e.g., Tat and gp120, altered some types of ABC transporter expression in brain microvascular endothelial cells and astrocytes. However, the effect of Tat on ABC transporters in T lymphocytes is unclear. In this study the status of breast cancer resistance protein (BCRP) in Tat expressing cell lines was examined with real-time PCR and flow cytometry. It was found that HIV-1 Tat protein upregulated BCRP expression and enhanced efflux mediated by BCRP significantly, which could inhibit antiviral drugs from entering infected cells and interfere with the therapeutic effect of HAART. PMID:26367065

  17. Splice Isoforms of Phosducin-like Protein Control the Expression of Heterotrimeric G Proteins*

    PubMed Central

    Gao, Xueli; Sinha, Satyabrata; Belcastro, Marycharmain; Woodard, Catherine; Ramamurthy, Visvanathan; Stoilov, Peter; Sokolov, Maxim

    2013-01-01

    Heterotrimeric G proteins play an essential role in cellular signaling; however, the mechanism regulating their synthesis and assembly remains poorly understood. A line of evidence indicates that the posttranslational processing of G protein ? subunits begins inside the protein-folding chamber of the chaperonin containing t-complex protein 1. This process is facilitated by the ubiquitously expressed phosducin-like protein (PhLP), which is thought to act as a CCT co-factor. Here we demonstrate that alternative splicing of the PhLP gene gives rise to a transcript encoding a truncated, short protein (PhLPs) that is broadly expressed in human tissues but absent in mice. Seeking to elucidate the function of PhLPs, we expressed this protein in the rod photoreceptors of mice and found that this manipulation caused a dramatic translational and posttranslational suppression of rod heterotrimeric G proteins. The investigation of the underlying mechanism revealed that PhLPs disrupts the folding of G? and the assembly of G? and G? subunits, events normally assisted by PhLP, by forming a stable and apparently inactive tertiary complex with CCT preloaded with nascent G?. As a result, the cellular levels of G? and G?, which depends on G? for stability, decline. In addition, PhLPs evokes a profound and rather specific down-regulation of the G? transcript, leading to a complete disappearance of the protein. This study provides the first evidence of a generic mechanism, whereby the splicing of the PhLP gene could potentially and efficiently regulate the cellular levels of heterotrimeric G proteins. PMID:23888055

  18. Efficient expression and purification of biologically active human cystatin proteins.

    PubMed

    Chauhan, Sakshi; Tomar, Raghuvir S

    2016-02-01

    Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease. PMID:26481272

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

    PubMed Central

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

    1995-01-01

    Colorectal tumorigenesis evolves through a series of molecular genetic changes, providing putative markers for tumour progression. This study investigated the relation between expression of the tumour suppressor gene p53 and splice variants v5 and v6 of the cell adhesion molecule CD44 by immunohistochemistry on tissue samples of early adenomas (n = 12), late adneomas (n = 12), Dukes's A and B carcinomas (n = 21), and Dukes's C and D carcinomas (n = 22) and compared these results with expression of these proteins in normal colonic mucosa (n = 17). A statistically significant trend of increasing expression was seen for both p53 (p < 0.005) and CD44 variant exon v6 (p < 0.0005) in subsequent stages of this tumour progression model. High expression of CD44 v5 was seen in most colorectal neoplasms (83%-96%), independent of stage. A statistically significant correlation was present between p53 expression and expression of variant v6 of CD44 (p < 0.01). Both p53 expression and CD44 v6 expression in adenomas increased with the degree of dysplasia (p < 0.05). The results of this study show that mutant p53 protein and variant v6 of the CD44 glycoprotein are markers of tumour progression in colorectal cancer. Images Figure 1 PMID:7534253

  20. The expression and induction of heat shock proteins in molluscs.

    PubMed

    Liu, Dongwu; Chen, Zhiwei

    2013-05-01

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

  1. Expression cloning of genes encoding human peroxisomal proteins

    SciTech Connect

    Spathaky, J.M.; Tate, A.W.; Cox, T.M.

    1994-09-01

    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.

  2. Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene Expression

    PubMed Central

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

    2014-01-01

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

  3. Regulation of RAG-2 protein expression in avian thymocytes.

    PubMed Central

    Ferguson, S E; Accavitti, M A; Wang, D D; Chen, C L; Thompson, C B

    1994-01-01

    The recombinase-activating genes, RAG-1 and RAG-2, have been shown to be necessary to initiate the process of V(D)J recombination during the ontogeny of lymphocytes. While much is known about the end products of this rearrangement process, little is known about the function or regulation of the components of the recombinase system. To this end, we have generated a monoclonal antibody to the chicken RAG-2 protein. Chicken thymocytes were found to express high levels of RAG-2, part of which is phosphorylated. Within thymocytes, RAG-2 is expressed primarily within the nucleus. RAG-2 protein levels are high in the CD4- CD8- and CD4+ CD8+ immature thymocytes but absent at the single-positive CD4+ CD8- or CD4- CD8+ stage of thymocyte development. Mitogenic stimulation of thymocytes with phorbol myristate acetate and ionomycin results in down-regulation of RAG-2 expression. Consistent with these data, in vivo levels of RAG-2 are markedly lower in proliferating thymocytes than in smaller, G0/G1 cells. Down-regulation of RAG-2 expression appears to occur before cells enter S phase, suggesting that RAG-2 function may be limited to noncycling cells. Images PMID:7935443

  4. A Novel Protein Is Lower Expressed in Renal Cell Carcinoma

    PubMed Central

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

    2014-01-01

    Engrailed-2 (EN2) has been identified as a candidate oncogene in breast cancer and prostate cancer. It is usually recognized as a mainly nuclear staining in the cells. However, recent studies showed a cytoplasmic staining occurred in prostate cancer, bladder cancer and clear cell renal cell carcinoma. The inconsistency makes us confused. To clarify the localization and expression of EN2 in renal cell carcinoma, anti-EN2 antibody (ab28731) and anti-EN2 antibody (MAB2600) were used for immunohistochemistry (IHC) respectively. Interestingly, we found that EN2 detected by ab28731 was mainly presented in cytoplasm while EN2 detected by MAB2600 was mainly presented in nucleus. To further investigate the different patterns observed above, lysates from full-length EN2 over expression in HEK293T cells were used to identify which antibody the EN2 molecule bound by western blot. Results showed ab28731 did not react with the lysates. For this reason, the novel specific protein detected by ab28731 was not the EN2 molecule and was named nonEN2. Then using the renal carcinoma tissue microarray and renal tissues, we found that the protein expression levels of nonEN2 in kidney tumor tissues was significantly lower than that in kidney normal tissues (p < 0.05), so was in renal cell lines. Taken together, nonEN2 is lower expressed and may play an important role in renal cell carcinoma. PMID:24786097

  5. Expression of MDR1/P glycoprotein in human sarcomas.

    PubMed Central

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

    1993-01-01

    Conflicting reports of MDR1 gene expression in human tumours are observed according to whether studies are performed at the mRNA or P-glycoprotein level. We have investigated this expression in 22 clinically drug-resistant sarcomas at the mRNA level by Northern blot (NB), Dot blot (DB), in situ hybridisation (ISH), and at the protein level by immunohistochemistry (IHC) using three monoclonal antibodies (MoAbs): C219, JSB1, MRK16. Increased MDR1 mRNA expression was detected by NB, DB, and ISH in 1/22 sarcoma (an Ewing's sarcoma). ISH was perfectly correlated with DB hybridisation and confirmed the expression of tumoral cells alone. Specific staining of 100% of tumoral cells was obtained with the three MoAbs in the same sarcoma. Expression in tumoral cells of 12 other sarcomas was detected with MRK16, and positive staining of stromal cells with both C219 (1/22) and MRK16 (8/22) was observed. This study confirms that MDR1 overexpression occurs in human sarcomas but is not the principal mechanism of drug-resistance. Furthermore, positivity with one antibody does not necessarily imply the presence of P glycoprotein (P-gp) and a disparity may exist between the levels of P-gp and its mRNA in the same sample. So care must be taken in interpreting results and more sensitive techniques such as the polymerase chain reaction (PCR) could prove useful. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7903154

  6. PPARs in the Control of Uncoupling Proteins Gene Expression

    PubMed Central

    Villarroya, Francesc; Iglesias, Roser; Giralt, Marta

    2007-01-01

    Uncoupling proteins (UCPs) are mitochondrial membrane transporters involved in the control of energy conversion in mitochondria. Experimental and genetic evidence relate dysfunctions of UCPs with metabolic syndrome and obesity. The PPAR subtypes mediate to a large extent the transcriptional regulation of the UCP genes, with a distinct relevance depending on the UCP gene and the tissue in which it is expressed. UCP1 gene is under the dual control of PPAR? and PPAR? in relation to brown adipocyte differentiation and lipid oxidation, respectively. UCP3 gene is regulated by PPAR? and PPAR? in the muscle, heart, and adipose tissues. UCP2 gene is also under the control of PPARs even in tissues in which it is the predominantly expressed UCP (eg, the pancreas and liver). This review summarizes the current understanding of the role of PPARs in UCPs gene expression in normal conditions and also in the context of type-2 diabetes or obesity. PMID:17389766

  7. Expression and Localization of Lung Surfactant Proteins in Human Testis

    PubMed Central

    Wagner, Walter; Matthies, Cord; Ruf, Christian; Hartmann, Arndt; Garreis, Fabian; Paulsen, Friedrich

    2015-01-01

    Background Surfactant proteins (SPs) have been described in various tissues and fluids including tissues of the nasolacrimal apparatus, airways and digestive tract. Human testis have a glandular function as a part of the reproductive and the endocrine system, but no data are available on SPs in human testis and prostate under healthy and pathologic conditions. Objective The aim of the study was the detection and characterization of the surfactant proteins A, B, C and D (SP-A, SP-B, SP-C, SP-D) in human testis. Additionally tissue samples affected by testicular cancer were investigated. Results Surfactant proteins A, B, C and D were detected using RT-PCR in healthy testis. By means of Western blot analysis, these SPs were detected at the protein level in normal testis, seminoma and seminal fluid, but not in spermatozoa. Expression of SPs was weaker in seminoma compared to normal testicular tissue. SPs were localized in combination with vimentin immunohistochemically in cells of Sertoli and Leydig. Conclusion Surfactant proteins seem to be inherent part of the human testis. By means of physicochemical properties the proteins appear to play a role during immunological and rheological process of the testicular tissue. The presence of SP-B and SP-C in cells of Sertoli correlates with their function of fluid secretion and may support transportation of spermatozoa. In seminoma the expression of all SP's was generally weaker compared to normal germ cells. This could lead to a reduction of immunomodulatory and rheology processes in the germ cell tumor. PMID:26599233

  8. Identification of differentially expressed serum proteins in gastric adenocarcinoma?

    PubMed Central

    Subbannayya, Yashwanth; Mir, Sartaj Ahmad; Renuse, Santosh; Manda, Srikanth S.; Pinto, Sneha M.; Puttamallesh, Vinuth N.; Solanki, Hitendra Singh; Manju, H.C.; Syed, Nazia; Sharma, Rakesh; Christopher, Rita; Vijayakumar, M.; Kumar, K.V. Veerendra; Prasad, T.S. Keshava; Ramaswamy, Girija; Kumar, Rekha V.; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu–Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. Biological significance Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well classified patient samples would prove useful in identifying novel blood based biomarkers for gastric cancers. This article is part of a Special Issue entitled: Proteomics in India. PMID:25952687

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

    PubMed Central

    2013-01-01

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

  10. Protein expression and characterization of SEP3 from Arabidopsis thaliana.

    PubMed

    Shi, Q; Zhou, J; Wang, P; Lin, X; Xu, Y

    2015-01-01

    SEPALLATA (SEP) MADS-box genes play crucial roles in the regulation of floral growth and development. They are required for the specification of sepals, petals, stamens, and carpels as well as for floral determinacy. SEPs perform their functions through the formation of homo- or hetero-polymers, which are the molecular basis of floral quartets. In vitro assays indicated that SEP3 forms a tetramer after binding to DNA, but it is unclear whether DNA binding induces the tetramer, because SEP3 is often reported to form a dimer. Here, we analyzed the oligomeric status of SEP3 domains in the absence of the DNA-binding MADS-box domain. The truncated SEP3 was constructed as a fusion protein and expressed in prokaryotic cells. The purified protein fragment displayed as a tetramer in the size exclusion chromatographic column, and a glutaraldehyde cross-linking assay demonstrated that the protein contained a dimer unit. Yeast two-hybrid tests further verified that the fragments form homologous polymers in vivo, and that the K domain is involved in tetramer formation. Current results imply that the SEP3 protein regulates the formation of flower meristems using the tetramer as a unit, and that the DNA-binding MADS-box is dispensable for polymer formation. The C-terminal region does not contribute to homo-tetramer formation, but it may be reserved to glue other proteins. PMID:26505403

  11. Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein.

    PubMed

    Ma, Q; Wang, R; Yang, C S; Lu, A Y

    1990-12-01

    A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase [NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)] mRNA was expressed in Escherichia coli. The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR). Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction. The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites. E. coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor. The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M. After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase. The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis. It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase. The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm. Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme. Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid. The remaining 19 amino acid residues at the NH2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol. PMID:1703398

  12. Multidrug resistance gene (P-glycoprotein) expression in the human fetus.

    PubMed Central

    van Kalken, C.; Giaccone, G.; van der Valk, P.; Kuiper, C. M.; Hadisaputro, M. M.; Bosma, S. A.; Scheper, R. J.; Meijer, C. J.; Pinedo, H. M.

    1992-01-01

    P-glycoprotein, a transmembrane protein associated with multidrug resistance in cancer cells, is also expressed in normal tissues. To get more insight into the physiologic role of mdr1/P-glycoprotein, we investigated its expression in human fetal tissues after 7 to 38 weeks of gestation by an immunohistochemical technique, using three different monoclonal antibodies, and by a sensitive RNAse protection assay. Expression of mdr1-mRNA could already be demonstrated in the embryonal phase of human development, after 7 weeks of gestation. Comparing the adult with the fetal tissue distribution, differences were found in specific organs, such as adrenal, intestine, respiratory epithelium, and brain capillaries. In the fetal zone cells of the fetal adrenal cortex no staining was observed by immunohistochemistry, whereas the definitive zone showed increasing expression throughout gestation. Prenatal intestine did not show staining of the epithelium, although definite mdr1-mRNA expression was observed in late specimens. Interestingly, respiratory epithelium of main bronchi and pharynx, not expressing P-gp in adults, did stain positive. Expression of P-gp in brain capillaries was not observed before the third trimester of pregnancy, whereas in kidney and liver, mdr1-mRNA expression and staining for P-glycoprotein were detected in early fetal life (11 to 14 weeks). These findings suggest a pivotal role of P-glycoprotein in physiology of various organs already in early phases of human development and may help to identify its physiologic substrates. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1359789

  13. High-throughput Protein Expression Generator Using a Microfluidic Platform

    PubMed Central

    Michaely, Efrat; Gerber, Doron

    2012-01-01

    Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions. The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology. PMID:22951599

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

    E-print Network

    Beebe, David J.

    their molecular structures and functions. A variety of protein expression (biological synthesis) systems have been systems.2­10 One is the batch process, in which the protein synthesis machinery and other reagents This approach leads to higher protein expression yield than the batch process because protein synthesis

  15. Expression of recombinant green fluorescent protein in Bacillus methanolicus.

    PubMed

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

    2012-01-01

    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

  16. Glycolipid Transfer Protein Expression Is Affected by Glycosphingolipid Synthesis

    PubMed Central

    Kjellberg, Matti A.; Mattjus, Peter

    2013-01-01

    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

  17. Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

    PubMed

    Kjellberg, Matti A; Mattjus, Peter

    2013-01-01

    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

  18. Simplifying protein expression with ligation-free, traceless and tag-switching Venuka Durani a

    E-print Network

    Magliery, Thomas J.

    independent cloning Traceless tagging a b s t r a c t Synthetic biology and genome-scale protein work both Available online 21 June 2012 Keywords: Plasmid Protein expression Co-expression Protease cleavage Ligation require rapid and efficient cloning, expression and purification. Tools for co-expression of multiple

  19. High-throughput protein expression using a combination of ligation-independent cloning (LIC) and infrared fluorescent protein (IFP) detection.

    PubMed

    Dortay, Hakan; Akula, Usha Madhuri; Westphal, Christin; Sittig, Marie; Mueller-Roeber, Bernd

    2011-01-01

    Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here. PMID:21541323

  20. A novel copper-regulated promoter system for expression of heterologous proteins in Schizosaccharomyces pombe

    E-print Network

    Labbé, Simon

    A novel copper-regulated promoter system for expression of heterologous proteins fostered the development and utilization of expression systems for gene function analysis. Using -X, which regulate the expression of heterologous genes as a function of copper availability

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

    PubMed Central

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

    2014-01-01

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

  2. Simvastatin enhances bone morphogenetic protein receptor type II expression

    SciTech Connect

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2006-01-06

    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.

  3. Construction of expression vectors for protein production in Gluconobacter oxydans.

    PubMed

    Kallnik, Verena; Meyer, Maria; Deppenmeier, Uwe; Schweiger, Paul

    2010-12-01

    The characteristic ability of Gluconobacter oxydans to incompletely oxidize numerous sugars, sugar acids, polyols, and alcohols has been exploited in several biotechnological processes, for example vitamin C production. The genome sequence of G. oxydans 621H is known but molecular tools are needed for the characterization of putative proteins and for the improvement of industrial strains by heterologous and homologous gene expression. To this end, promoter regions for the genes encoding G. oxydans ribosomal proteins L35 and L13 were introduced into the broad-host-range plasmid pBBR1MCS-2 to construct two new expression vectors for gene expression in Gluconobacter spp. These vectors were named pBBR1p264 and pBBR1p452, respectively, and have many advantages over current vectors for Gluconobacter spp. The uidA gene encoding ?-D-glucuronidase was inserted downstream of the promoter regions and these promoter-reporter fusions were used to assess relative promoter strength. The constructs displayed distinct promoter strengths and strong (pBBR1p264), moderate (pBBR1p452) and weak (pBBR1MCS-2 carrying the intrinsic lac promoter) promoters were identified. PMID:20969898

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

    PubMed Central

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

    2011-01-01

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

  5. An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli

    PubMed Central

    Chhetri, Gaurav; Kalita, Parismita; Tripathi, Timir

    2015-01-01

    Bacterial cells can be engineered to express non-native genes, resulting in the production of, recombinant proteins, which have various biotechnological and pharmaceutical applications. In eukaryotes, such as yeast or mammalian cells, which have large genomes, a higher recombinant protein expression can be troublesome. Comparatively, in the Escherichia coli (E. coli) expression system, although the expression is induced with isopropyl ?-d-1-thiogalactopyranoside (IPTG), studies have shown low expression levels of proteins. Irrespective of the purpose of protein production, the production process requires the accomplishment of three individual factors: expression, solubilization and purification. Although several efforts, including changing the host, vector, culture parameters of the recombinant host strain, co-expression of other genes and changing of the gene sequences, have been directed towards enhancing recombinant protein expression, the protein expression is still considered as a significant limiting step. Our protocol explains a simple method to enhance the recombinant protein expression that we have optimized using several unrelated proteins. It works with both T5 and T7 promoters. This protocol can be used to enhance the expressions of most of the proteins. The advantages of this technique are presented below:•It produces several fold increase in the expression of poorly expressed, less expressed or non-expressed recombinant proteins.•It does not employ any additional component such as chaperones, heat shock proteins or co-expression of other genes.•In addition to being inexpensive, easy to manage, universal, and quick to perform, the proposed method does not require any commercial kits and, can be used for various recombinant proteins expressed in the E. coli expression system. PMID:26629417

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

    PubMed Central

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

    2015-01-01

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

  7. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

    SciTech Connect

    Yamakoshi, Takako; Makino, Teruhiko; Ur Rehman, Mati; Yoshihisa, Yoko; Sugimori, Michiya; Shimizu, Tadamichi

    2013-03-01

    Highlights: ? Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ? Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ? TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ? TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ? The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.

  8. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters

    PubMed Central

    Hanna, Eileen Marie; Zaki, Nazar; Amin, Amr

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present “DyCluster”, a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster. PMID:26641660

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

    SciTech Connect

    Wu, Jiawen; Peng, Dungeng; Voehler, Markus; Sanders, Charles R.; Center for Structural Biology, Vanderbilt University ; Li, Jun; Tennessee Valley Healthcare System – Nashville VA

    2013-10-11

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

  10. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment. PMID:26297626

  11. Transcriptome analysis of a CHO cell line expressing a recombinant therapeutic protein treated with inducers of protein expression.

    PubMed

    Fomina-Yadlin, Dina; Mujacic, Mirna; Maggiora, Kathy; Quesnell, Garrett; Saleem, Ramsey; McGrew, Jeffrey T

    2015-10-20

    The search for specific productivity (qP) determinants in Chinese hamster ovary (CHO) cells has been the focus of the biopharmaceutical cell line engineering efforts aimed at creating "super-producer" cell lines. In this study, we evaluated the impact of small-molecule inducers and temperature shift on recombinant protein production, and used transcriptomic analysis to define gene-phenotype correlations for qP in our biological system. Next-generation RNA Sequencing (RNA-Seq) analysis revealed that each individual inducer (caffeine, hexamethylene bisacetamide (HMBA) and sodium butyrate (NaBu)) or a combination treatment had a distinct impact on the gene expression program of the RANK-Fc cell line. Temperature shift to 31°C impacted inducer action with respect to transcriptional changes and phenotypic cell line parameters. We showed that inducer treatment was able to increase expression level of the Fc- fusion mRNA and the selectable marker mRNA from 16% up to 45% of total mRNA in the cell. We further demonstrated that qP exhibited a strong positive linear correlation to transcript levels of both the RANK-Fc fusion protein and the dihydrofolate reductase (DHFR) selectable marker. In fact, these were 2 out of 7 transcripts with significant positive correlation to qP at both temperatures. Many more transcripts were anti- correlated to qP, and gene set enrichment analysis (GSEA) revealed that those were involved in cell cycle progression, transcription, mRNA processing, translation and protein folding. Therefore, we postulate that the transcript level of the recombinant protein is a major qP determinant in our biological system, while downregulation of routine activity within the cell is necessary to divert cellular resources towards recombinant protein production. PMID:26325199

  12. Stochastic modeling of gene expression, protein modification, and polymerization

    E-print Network

    Mugler, Andrew

    2015-01-01

    Many fundamental cellular processes involve small numbers of molecules. When numbers are small, fluctuations dominate, and stochastic models, which account for these fluctuations, are required. In this chapter, we describe minimal stochastic models of three fundamental cellular processes: gene expression, protein modification, and polymerization. We introduce key analytic tools for solving each model, including the generating function, eigenfunction expansion, and operator methods, and we discuss how these tools are extended to more complicated models. These analytic tools provide an elegant, efficient, and often insightful alternative to stochastic simulation.

  13. Human protein factory for converting the transcriptome into an in vitro-expressed proteome,.

    PubMed

    Goshima, Naoki; Kawamura, Yoshifumi; Fukumoto, Akiko; Miura, Aya; Honma, Reiko; Satoh, Ryohei; Wakamatsu, Ai; Yamamoto, Jun-ichi; Kimura, Kouichi; Nishikawa, Tetsuo; Andoh, Taichi; Iida, Yuki; Ishikawa, Kumiko; Ito, Emi; Kagawa, Naoko; Kaminaga, Chie; Kanehori, Kei-ichi; Kawakami, Bunsei; Kenmochi, Kiyokazu; Kimura, Rie; Kobayashi, Miki; Kuroita, Toshihiro; Kuwayama, Hisashi; Maruyama, Yukio; Matsuo, Kiyoshi; Minami, Kazuyoshi; Mitsubori, Mariko; Mori, Masatoshi; Morishita, Riyo; Murase, Atsushi; Nishikawa, Akira; Nishikawa, Shigemichi; Okamoto, Toshihiko; Sakagami, Noriko; Sakamoto, Yutaka; Sasaki, Yukari; Seki, Tomoe; Sono, Saki; Sugiyama, Akio; Sumiya, Tsuyoshi; Takayama, Tomoko; Takayama, Yukiko; Takeda, Hiroyuki; Togashi, Takushi; Yahata, Kazuhide; Yamada, Hiroko; Yanagisawa, Yuka; Endo, Yaeta; Imamoto, Fumio; Kisu, Yasutomo; Tanaka, Shigeo; Isogai, Takao; Imai, Jun-ichi; Watanabe, Shinya; Nomura, Nobuo

    2008-12-01

    Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research. PMID:19054851

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

    PubMed

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

    2013-12-15

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

  15. Introduction of unnatural amino acids into proteins using expressed protein ligation.

    PubMed

    Ayers, B; Blaschke, U K; Camarero, J A; Cotton, G J; Holford, M; Muir, T W

    1999-01-01

    Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins. PMID:10685045

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  17. Differential expression of red cell proteins in hemoglobinopathy.

    PubMed

    Chakrabarti, Abhijit; Bhattacharya, Dipankar; Basu, Avik; Basu, Sumanta; Saha, Sutapa; Halder, Suchismita

    2011-02-01

    Red blood cell proteome has not been studied well until recently, as the large abundance of hemoglobin posed challenge to the detection of other cytosolic proteins in the linear dynamic range. However, in the last couple of years, due to emergence of various novel hemoglobin depletion strategies and more state-of-the-art detection techniques, a number of works on erythrocyte proteome have appeared in the literature. As a result, we now have much deeper information about both the membrane as well as the cytosolic proteins of erythrocytes. In this review, we have discussed the role of red cell proteome on the two most well-studied hemoglobin disorders, sickle cell disease and thalassemia, emphasizing on the differential expression of the redox regulator proteins and chaperones, in particular. We have also touched upon the importance of the association of the varying levels of hemoglobin variants, particularly HbE on the clinical manifestation of composite diseases like HbE? thalassemia. PMID:21246741

  18. Expression of Water Channel Proteins in Mesembryanthemum crystallinum1

    PubMed Central

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

    2000-01-01

    We have characterized transcripts for nine major intrinsic proteins (MIPs), some of which function as water channels (aquaporins), from the ice plant Mesembryanthemum crystallinum. To determine the cellular distribution and expression of these MIPs, oligopeptide-based antibodies were generated against MIP-A, MIP-B, MIP-C, or MIP-F, which, according to sequence and functional characteristics, are located in the plasma membrane (PM) and tonoplast, respectively. MIPs were most abundant in cells involved in bulk water flow and solute flux. The tonoplast MIP-F was found in all cells, while signature cell types identified different PM-MIPs: MIP-A predominantly in phloem-associated cells, MIP-B in xylem parenchyma, and MIP-C in the epidermis and endodermis of immature roots. Membrane protein analysis confirmed MIP-F as tonoplast located. MIP-A and MIP-B were found in tonoplast fractions and also in fractions distinct from either the tonoplast or PM. MIP-C was most abundant but not exclusive to PM fractions, where it is expected based on its sequence signature. We suggest that within the cell, MIPs are mobile, which is similar to aquaporins cycling through animal endosomes. MIP cycling and the differential regulation of these proteins observed under conditions of salt stress may be fundamental for the control of tissue water flux. PMID:10806230

  19. Functions of BET proteins in erythroid gene expression.

    PubMed

    Stonestrom, Aaron J; Hsu, Sarah C; Jahn, Kristen S; Huang, Peng; Keller, Cheryl A; Giardine, Belinda M; Kadauke, Stephan; Campbell, Amy E; Evans, Perry; Hardison, Ross C; Blobel, Gerd A

    2015-04-30

    Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members. PMID:25696920

  20. Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane.

    PubMed

    Fantappiè, Ornella; Sassoli, Chiara; Tani, Alessia; Nosi, Daniele; Marchetti, Serena; Formigli, Lucia; Mazzanti, Roberto

    2015-06-01

    Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells. PMID:25691007

  1. Probing Gene Expression in Live Cells, One Protein Molecule at a Time

    E-print Network

    Li, Tiejun

    Probing Gene Expression in Live Cells, One Protein Molecule at a Time Ji Yu,1 * Jie Xiao,1 molecules in individual Escherichia coli cells. A fusion protein of a fast-maturing yellow fluorescent protein (YFP) and a membrane- targeting peptide was expressed under a repressed condition. The membrane

  2. A proposal for the physiological significance of mdr1 and Bcrp1/Abcg2 gene expression in normal tissue regeneration and after cancer therapy.

    PubMed

    Israeli, David; Ziaei, Simin; Gonin, Patrick; Garcia, Luis

    2005-01-01

    Cellular multi-drug resistance (MDR), which often develops in cancer cells of patients subjected to anti-cancer treatment, remains a significant barrier to successful cancer therapy. One of the principal causes of cellular MDR development is an increased expression of ABC-transporter genes such as mdr1 and Bcrp1/Abcg2. Despite many years of intensive research, the natural biological role of mdr1 in the context of cancer has remained elusive. Some hints about this role came, however, from an observation that P-gp, the mdr1 encoded protein, is expressed widely in stem cells and from the discovery that P-gp possesses an anti-apoptotic activity independently of exogenous drug application. Here, we discuss our own and other groups' recently published works and propose an integrated view of mdr1 and Bcrp1/Abcg2 activity during tissue regeneration in normal tissues as part of a stress-induced regeneration genetic program and in cancerous tissues in response to cancer therapy. PMID:15498591

  3. CMV-driven expression of green uorescent protein (GFP) in male germ cells of transgenic mice

    E-print Network

    Museo Nacional de Ciencias Naturales

    CMV-driven expression of green ¯uorescent protein (GFP) in male germ cells of transgenic mice if the expression of green ¯uorescent protein (GFP) during sperma- togenesis can compromise the fertility of transgenic animals, we have produced mouse transgenic lines expressing GFP in the testis under the control

  4. Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy.

    PubMed

    Duwé, Sam; De Zitter, Elke; Gielen, Vincent; Moeyaert, Benjamien; Vandenberg, Wim; Grotjohann, Tim; Clays, Koen; Jakobs, Stefan; Van Meervelt, Luc; Dedecker, Peter

    2015-10-27

    "Smart fluorophores", such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ?70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"- and "off"-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins. PMID:26308583

  5. [Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it].

    PubMed

    Weng, Daihui; Lei, Yingfeng; Dong, Yangchao; Han, Peijun; Ye, Chuantao; Yang, Jing; Wang, Yuan; Yin, Wen

    2015-12-01

    To construct the plasmid expressing the fusion protein of Dengue virus type 2 (DENV2) nonstructural protein 3 (NS3) with affinity tag, and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification (TAP) assay. Methods Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments, after amplified by PCR with DENV2 cDNA as template, were digested and cloned into the mammalian eukaryotic expression vector pCI-SF with the tandem affinity tag (FLAG-StrepII). The recombinant pCI-NS3-SF was transiently transformed by Lipofectamine(TM) 2000 into HEK293T cells, and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay. Results The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system. Conclusion TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3. PMID:26648287

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin

    PubMed Central

    Round, Ekaterina; Shevchenko, Vitaly; Gushchin, Ivan; Polovinkin, Vitaly; Borshchevskiy, Valentin; Gordeliy, Valentin

    2015-01-01

    Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. PMID:26046789

  8. The Up-Regulation of Ribosomal Proteins Further Regulates Protein Expression Profile in Female Schistosoma japonicum after Pairing

    PubMed Central

    Sun, Jun; Li, Chen; Wang, Suwen

    2015-01-01

    Background Pairing of Schistosoma males and females leads to and maintains female sexual maturation. However, the mechanism by which pairing facilitates sexual maturation of females is not clear. An increasing body of evidence suggests that ribosomal proteins have regulatory rather than constitutive roles in protein translation. Methodology/Principal Findings To investigate the effect of ribosome regulation on female sex maturation, Solexa and iTRAQ techniques were used to analyze the relationship between ribosomal gene or protein expression and sexual development of Schistosoma females. In the present study, considerably higher number of ribosomal genes or proteins were found to be differentially expressed in paired 23-day-old females. Moreover, mature female-specific proteins associated with egg production, such as ferritin-1 heavy chain and superoxide dismutase, were selectively highly expressed in paired females, rather than higher level of protein synthesis of all transcripts compared with those in unpaired 23-day-old females. Furthermore, other developmental stages were utilized to investigate different expression pattern of ribosomal proteins in females by analysing 18-day-old female schistosomula from single- or double-sex infections to determine the relationship between ribosomal protein expression pattern and development. Results showed that undeveloped 18-day-old females from single- and double-sex infections, as well as 23-day-old unpaired females, possessed similar ribosomal protein expression patterns, which were distinct from those in 23-day-old paired females. Conclusions/Significance Our findings reveal that the pairing of females and males triggers a specialized ribosomal protein expression profile which further regulates the protein profile for sexual maturation in Schistosoma japonicum, based on its gene expression profile. PMID:26070205

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

    PubMed Central

    Mbikay, M; Seidah, N G; Chrétien, M

    2001-01-01

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

  10. Expression of epithelial adhesion proteins and integrins in chronic inflammation.

    PubMed Central

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

    1995-01-01

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

  11. Expression of the Transcriptional Repressor Protein Kid-1 Leads to the Disintegration of the Nucleolus*

    E-print Network

    Witzgall, Ralph - Naturwissenschaftliche Fakultät III

    Expression of the Transcriptional Repressor Protein Kid-1 Leads to the Disintegration, Massachusetts 02129 The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two a biochemical and functional analysis of the Kid-1 protein in transfected cells. The full-length Kid-1 protein

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

    E-print Network

    Mootha, Vamsi K.

    , with uncoupling protein 3L having greater effects in whole yeast and a smaller effect on the state 4 respiration of effects of uncoupling protein 3L and uncoupling protein 1 in whole yeast versus isolated mitochondriaAssessment of uncoupling activity of uncoupling protein 3 using a yeast heterologous expression

  13. Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans.

    PubMed

    Mai, Yong-Zhan; Li, Yan-Wei; Li, Rui-Jun; Li, Wei; Huang, Xia-Zi; Mo, Ze-Quan; Li, An-Xing

    2015-06-30

    Cryptocaryoniasis is a severe disease of farmed marine fish caused by the parasitic ciliate Cryptocaryon irritans. This disease can lead to considerable economic loss, but studies on proteins linked to disease development and antigenic proteins for vaccine development have been relatively scarce to date. In this study, 53 protein spots with differential abundance, representing 12 proteins, were identified based on a pair-wise comparison among theronts, trophonts, and tomonts. Meanwhile, 33 protein spots that elicited serological responses in rabbits were identified, representing 9 proteins. In addition, 27 common antigenic protein spots reacted with grouper anti-sera, representing 10 proteins. Most of the identified proteins were involved in cytoskeletal and metabolic pathways. Among these proteins, actin and ?-tubulin appeared in all three developmental stages with differences in molecular weights and isoelectric points; 4 proteins (vacuolar ATP synthase catalytic subunit ?, mcm2-3-5 family protein, 26S proteasome subunit P45 family protein and dnaK protein) were highly expressed only in theronts; while protein kinase domain containing protein and heat shock protein 70 showed high levels of expression only in trophonts and tomonts, respectively. Moreover, actin was co-detected with 3 rabbit anti-sera while ?-tubulin, V-type ATPase ? subunit family protein, heat shock protein 70, mitochondrial-type hsp70, and dnaK proteins showed immunoreactivity with corresponding rabbit anti-sera in theronts, trophonts, and tomonts. Furthermore, ?-tubulin, the metabolic-related protein enolase, NADH-ubiquinone oxidoreductase 75 kDa subunit, malate dehydrogenase, as well as polypyrimidine tract-binding protein, glutamine synthetase, protein kinase domain containing protein, TNFR/NGFR cysteine-rich region family protein, and vacuolar ATP synthase catalytic subunit ?, were commonly detected by grouper anti-sera. Therefore, these findings could contribute to an understanding of the differences in gene expression and phenotypes among the different stages of parasitic infection, and might be considered as a source of candidate proteins for disease diagnosis and vaccine development. PMID:25997646

  14. Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli.

    PubMed

    Rocha, T S; Tramuta, C; Catania, S; Matucci, A; Giuffrida, M G; Baro, C; Profiti, M; Bertolotti, L; Rosati, S

    2015-04-01

    Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression in Escherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples of Mycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications. PMID:25667423

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

    PubMed

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

    2015-04-01

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

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

    PubMed Central

    WANG, HAO; WANG, CHEN; ZHANG, LEI; LU, YINGHUA; DUAN, QIANGLIN; GONG, ZHU; LIANG, AIBIN; SONG, HAOMING; WANG, LEMIN

    2015-01-01

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

  17. Protein Expression Profiles Characterize Distinct Features of Mouse Cerebral Cortices at Different Developmental Stages

    PubMed Central

    Zhang, Haijun; Kawase-Koga, Yoko; Sun, Tao

    2015-01-01

    The proper development of the mammalian cerebral cortex requires precise protein synthesis and accurate regulation of protein expression levels. To reveal signatures of protein expression in developing mouse cortices, we here generate proteomic profiles of cortices at embryonic and postnatal stages using tandem mass spectrometry (MS/MS). We found that protein expression profiles are mostly consistent with biological features of the developing cortex. Gene Ontology (GO) and KEGG pathway analyses demonstrate conserved molecules that maintain cortical development such as proteins involved in metabolism. GO and KEGG pathway analyses further identify differentially expressed proteins that function at specific stages, for example proteins regulating the cell cycle in the embryonic cortex, and proteins controlling axon guidance in the postnatal cortex, suggesting that distinct protein expression profiles determine biological events in the developing cortex. Furthermore, the STRING network analysis has revealed that many proteins control a single biological event, such as the cell cycle regulation, through cohesive interactions, indicating a complex network regulation in the cortex. Our study has identified protein networks that control the cortical development and has provided a protein reference for further investigation of protein interactions in the cortex. PMID:25915664

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

    SciTech Connect

    Saisho, Kenji; Fukuhara, Atsunori; Yasuda, Tomoko; Sato, Yoshifumi; Fukui, Kenji; Iwahashi, Hiromi; Imagawa, Akihisa; Hatta, Mitsutoki; Shimomura, Iichiro; Yamagata, Kazuya

    2009-11-06

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

  19. An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

    PubMed

    Linhoff, Michael W; Laurén, Juha; Cassidy, Robert M; Dobie, Frederick A; Takahashi, Hideto; Nygaard, Haakon B; Airaksinen, Matti S; Strittmatter, Stephen M; Craig, Ann Marie

    2009-03-12

    Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia. PMID:19285470

  20. An Unbiased Expression Screen for Synaptogenic Proteins Identifies the LRRTM Protein Family as Synaptic Organizers

    PubMed Central

    Linhoff, Michael W.; Laurén, Juha; Cassidy, Robert M.; Dobie, Frederick A.; Takahashi, Hideto; Nygaard, Haakon B.; Airaksinen, Matti S.; Strittmatter, Stephen M.; Craig, Ann Marie

    2009-01-01

    Summary A delineation of the molecular basis of synapse development is crucial for understanding brain function. Co-cultures of neurons with transfected fibroblastoid cells have been used to demonstrate the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the co-culture assay using custom-made full-length cDNA libraries. Re-isolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is far from complete. We identify LRRTM1 as a novel transmembrane protein capable of inducing presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially-induced clustering of LRRTMs mediates postsynaptic differentiation in dendrites. We generate LRRTM1 -/- mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the recently reported linkage of LRRTM1 to handedness and schizophrenia. PMID:19285470

  1. Cell-specific expression of the carrot EP2 lipid transfer protein gene.

    PubMed Central

    Sterk, P; Booij, H; Schellekens, G A; Van Kammen, A; De Vries, S C

    1991-01-01

    A cDNA corresponding to a 10-kD protein, designated extracellular protein 2 (EP2), that is secreted by embryogenic cell cultures of carrot was obtained by expression screening. The derived protein sequence and antisera against heterologous plant lipid transfer proteins identified the EP2 protein as a lipid transfer protein. Protein gel blot analysis showed that the EP2 protein is present in cell walls and conditioned medium of cell cultures. RNA gel blot analysis revealed that the EP2 gene is expressed in embryogenic cell cultures, the shoot apex of seedlings, developing flowers, and maturing seeds. In situ hybridization showed expression of the EP2 gene in protoderm cells of somatic and zygotic embryos and transient expression in epidermis cells of leaf primordia and all flower organs. In the shoot apical meristem, expression is found in the tunica and lateral zone. In maturing seeds, the EP2 gene is expressed in the outer epidermis of the integument, the seed coat, and the pericarp epidermis, as well as transiently in between both mericarps. Based on the extracellular location of the EP2 protein and the expression pattern of the encoding gene, we propose a role for plant lipid transfer proteins in the transport of cutin monomers through the extracellular matrix to sites of cutin synthesis. PMID:1822991

  2. Thymocytes Express the golli Products of the Myelin Basic Protein Gene and Levels of Expression Are Stage Dependent1

    E-print Network

    Bongarzone, Ernesto R.

    of the myelin basic protein gene have been shown to be expressed in mouse thymus and brain. The full repertoire in the thymus. The results showed that MTS5 cortical/MTS10 medullary epithelial cells and NLDC145 dendritic at the protein and mRNA level. The presence of golli products in the early developing thymus in mouse and human

  3. Depression of Protein Synthetic Capacity Due to Cloned-Gene Expression in E. coli

    E-print Network

    Wood, Thomas K.

    Depression of Protein Synthetic Capacity Due to Cloned-Gene Expression in E. coli Thomas K. Wood capaci- ties for heterologous protein production. As seen with this and other systems, cloned the trans- lational capacity of the cell at high levels of cloned-gene expression. The results suggest

  4. A Nucleus-Encoded Chloroplast Protein Regulated by Iron Availability Governs Expression of the Photosystem I

    E-print Network

    A Nucleus-Encoded Chloroplast Protein Regulated by Iron Availability Governs Expression in the chloroplast and the nucleus. Chloroplast gene expression is subjected to anterograde control by a battery of nucleus- encoded proteins that are imported in the chloroplast, where they mostly intervene

  5. Maternal low protein diet and postnatal high fat diet increases adipose imprinted gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maternal and postnatal diet can alter Igf2 gene expression and DNA methylation. To test whether maternal low protein and postnatal high fat (HF) diet result in alteration in Igf2 expression and obesity, we fed obese-prone Sprague-Dawley rats 8% (LP) or 20% (NP) protein for 3 wk prior to breeding and...

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

    ERIC Educational Resources Information Center

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

    2004-01-01

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

  7. Global Survey of Organ and Organelle Protein Expression in Mouse: Combined

    E-print Network

    Morris, Quaid

    Resource Global Survey of Organ and Organelle Protein Expression in Mouse: Combined Proteomic Organs and organelles represent core biologi- cal systems in mammals, but the diversity in protein of the expressed tissue and organelle proteomes of a leading model mammal. INTRODUCTION Elucidation of gene

  8. Heat shock protein expression enhances heat tolerance of reptile embryos

    PubMed Central

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

    2014-01-01

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

  9. Heat shock protein expression enhances heat tolerance of reptile embryos.

    PubMed

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

    2014-09-22

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

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

    SciTech Connect

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

    1997-07-01

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

  11. Impact of Adenovirus E4-ORF3 Oligomerization and Protein Localization on Cellular Gene Expression

    PubMed Central

    Vink, Elizabeth I.; Zheng, Yueting; Yeasmin, Rukhsana; Stamminger, Thomas; Krug, Laurie T.; Hearing, Patrick

    2015-01-01

    The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation. PMID:25984715

  12. Impact of Adenovirus E4-ORF3 Oligomerization and Protein Localization on Cellular Gene Expression.

    PubMed

    Vink, Elizabeth I; Zheng, Yueting; Yeasmin, Rukhsana; Stamminger, Thomas; Krug, Laurie T; Hearing, Patrick

    2015-05-01

    The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation. PMID:25984715

  13. Protein kinase Cµ plays an essential role in hypertonicity-induced heat shock protein 70 expression

    PubMed Central

    Lim, Yun-Sook; Lee, Jae-Seon; Huang, Tai-Qin

    2008-01-01

    Heat shock protein 70 (HSP70), which evidences important functions as a molecular chaperone and anti-apoptotic molecule, is substantially induced in cells exposed to a variety of stresses, including hypertonic stress, heavy metals, heat shock, and oxidative stress, and prevents cellular damage under these conditions. However, the molecular mechanism underlying the induction of HSP70 in response to hypertonicity has been characterized to a far lesser extent. In this study, we have investigated the cellular signaling pathway of HSP70 induction under hypertonic conditions. Initially, we applied a variety of kinase inhibitors to NIH3T3 cells that had been exposed to hypertonicity. The induction of HSP70 was suppressed specifically by treatment with protein kinase C (PKC) inhibitors (Gö6976 and GF109203X). As hypertonicity dramatically increased the phosphorylation of PKCµ, we then evaluated the role of PKCµ in hypertonicity-induced HSP70 expression and cell viability. The depletion of PKCµ with siRNA or the inhibition of PKCµ activity with inhibitors resulted in a reduction in HSP70 induction and cell viability. Tonicity-responsive enhancer binding protein (TonEBP), a transcription factor for hypertonicity-induced HSP70 expression, was translocated rapidly into the nucleus and was modified gradually in the nucleus under hypertonic conditions. When we administered treatment with PKC inhibitors, the mobility shift of TonEBP was affected in the nucleus. However, PKCµ evidenced no subcellular co-localization with TonEBP during hypertonic exposure. From our results, we have concluded that PKCµ performs a critical function in hypertonicity-induced HSP70 induction, and finally cellular protection, via the indirect regulation of TonEBP modification. PMID:19116445

  14. Distinct protein expression and activity of transglutaminases found in different epidermal tumors.

    PubMed

    Karashima, Tadashi; Furumura, Minao; Ishii, Norito; Ohyama, Bungo; Saruta, Hiroshi; Natsuaki, Yohei; Nakama, Takekuni; Ohata, Chika; Tsuruta, Daisuke; Hitomi, Kiyotaka; Hashimoto, Takashi

    2014-06-01

    We investigated protein expression and in situ activity of transglutaminases (TGs) in normal skin and various epidermal neoplasms. In normal skin, TG1 protein expression and TG activity were found at keratinocyte cell membranes in upper epidermis and granular layer, respectively. In seborrhoeic keratosis, TG1 protein was expressed evenly throughout tumors, while TG activity increased in gradient fashion from lower tumor area to cornified layer. In squamous cell carcinoma, TG1 protein was expressed at inner side of cell membranes, whereas TG activity was found in cytoplasm predominantly at horn pearls. In basal cell carcinoma, weak TG activity was found in cytoplasm of all tumor cells without the presence of TG1 protein. Immunoblotting and in situ activity assays using specific substrate peptides confirmed that TG2, but not TG1, contributed to the TG activity. These results suggested that different expression and activation of TGs may contribute to characteristics of the skin tumors. PMID:24750515

  15. Molecular cloning and expression of a novel klotho-related protein.

    PubMed

    Yahata, K; Mori, K; Arai, H; Koide, S; Ogawa, Y; Mukoyama, M; Sugawara, A; Ozaki, S; Tanaka, I; Nabeshima, Y; Nakao, K

    2000-01-01

    Klotho protein is a novel beta-glucosidase-like protein produced predominantly in the kidney. The klotho mouse, which genetically lacks klotho gene expression, manifests various systemic phenotypes resembling aging. In the present study we succeeded in isolating a novel human protein structurally related to klotho protein. The protein possesses one beta-glucosidase-like domain and is 42% identical with klotho protein at the amino acid level. Unlike klotho protein, it possesses neither a signal sequence nor a transmembrane domain, suggesting that it is a cytosolic protein, and thus was termed cytosolic beta-glucosidase-like protein-1 (cBGL1). By Northern blot analysis cBGL1 mRNA was expressed most abundantly in the liver, followed by the small intestine, colon, spleen, and kidney. When klotho and cBGL1 gene expression was examined in renal cell carcinoma tissues, both klotho and cBGL1 mRNA levels in tumors were lower than those in nontumor regions, suggesting that renal epithelial cells may lose klotho and cBGL1 gene expression during the course of malignant transformation. In conclusion, we describe the primary structure and gene expression of a novel protein related to klotho protein. PMID:11043382

  16. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    NASA Astrophysics Data System (ADS)

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-03-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.

  17. Virus-Derived Vectors for the Expression of Multiple Proteins in Plants.

    PubMed

    Saxena, Pooja; Thuenemann, Eva C; Sainsbury, Frank; Lomonossoff, George P

    2016-01-01

    This chapter constitutes a practical guide to using the "pEAQ" vector series for transient or stable expression of one or more protein(s) in Nicotiana benthamiana plants. The pEAQ vectors are a series of small binary vectors designed for controlled expression of multiple proteins in plants. To achieve high levels of expression, an expression system based on translational enhancement by the untranslated regions of RNA-2 from cowpea mosaic virus (CPMV), named CPMV-HT, is used. The expression vector pEAQ-HT combines the user-friendly pEAQ plasmid with CPMV-HT to provide a system for high-level expression of proteins in plants. PMID:26614280

  18. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    PubMed Central

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-01-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development. PMID:24626130

  19. NCI: SBIR & STTR - Find Funding - Contracts - 269 Development of Novel Protein Expression Technologies for Glycosylated Cancer Related Proteins

    Cancer.gov

    The purpose of this initiative is to provide support for the development of novel technologies for the expression of cancer-related glycosylated proteins. Many proteins become post-translationally modified (PTM) during the “secretory process” which involves of a journey from their site of synthesis in the rough endoplasmic reticulum (ER), through the Golgi apparatus and then to various cellular and extracellular destinations.

  20. Expression, phosphorylation, and glycosylation of CNS proteins in aversive operant conditioning associated memory in Lymnaea stagnalis.

    PubMed

    Silverman-Gavrila, L B; Senzel, A G; Charlton, M P; Feng, Z-P

    2011-07-14

    Long-term memory formation requires "de novo" expression and post-translational modification of many proteins. Understanding the temporal and spatial regulatory pattern of these proteins is fundamental to decoding the molecular basis of learning and memory. We characterized changes in expression, phosphorylation, and glycosylation of CNS proteins after operant conditioning in pond snail Lymnaea stagnalis. The phosphorylation and the glycosylation levels of proteins, measured by the ratio of Pro-Q Diamond (phosphoproteins) or Pro-Q Emerald (glycoproteins) vs. SYPRO-Ruby (total proteins) signals, increased during memory formation. Proteins whose modulation of phosphorylation might be involved in learning and memory were identified by mass spectrometry (MS) and are associated with cytoskeleton, glutamine cycle, energy metabolism, G-protein signaling, neurotransmitter release regulation, iron transport, protein synthesis, and cell division. Phosphorylation of actin increased during memory formation. To identify proteins whose expression levels changed in long-term memory formation we used two-dimensional difference gel electrophoresis followed by MS. The up-regulated proteins are mostly associated with lipoprotein and cholesterol metabolism, protein synthesis and degradation, cytoskeleton, nucleic acid synthesis, and energy supply. The down-regulated proteins are enzymes of aspartic acid metabolism involved in regulation of protein synthesis. Our proteomic analyses have revealed a number of candidate proteins associated with memory formation. These findings provide new directions for further investigation into the signaling networks required for memory formation and consolidation. PMID:21530618

  1. Human chorionic gonadotropin promotes expression of protein absorption factors in the intestine of goldfish (Carassius auratus).

    PubMed

    Zhou, Y; Hao, G; Zhong, H; Wu, Q; Lu, S Q; Zhao, Q; Liu, Z

    2015-01-01

    Protein use is crucial for the ovulation and spawning of fish. Currently, limited information is available regarding the expression of protein absorption factors during the breeding seasons of teleosts and thus how various proteins involved in this process is not well-understood. The expression of CDX2, CREB, gluatamate dehydrogenase, LAT2, aminopeptidase N, PepT1, and SP1 were significantly elevated from the non-breeding season to the breeding season in female goldfish, and all proteins except PepT1 and SP1 were elevated in male goldfish. Injection of human chorionic gonadotropin upregulated the expression of all proteins except for aminopeptidase N in female goldfish and SP1 in male goldfish, suggesting a luteinizing hormone-inductive effect on protein absorption factors. Protein use in the intestine is increased during the breeding seasons as a result of increased luteinizing hormone. PMID:26345757

  2. High-throughput cloning and expression of integral membrane proteins in Escherichia coli.

    PubMed

    Bruni, Renato; Kloss, Brian

    2013-01-01

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

  3. [Expression of mannose-binding lectin associated protein 19 (MAp19) in HeLa cells].

    PubMed

    Li, Ting; Li, Ping; Jia, Tianjun; Jia, Xiaohui; Zhao, Xia; Cheng, Yongting

    2016-01-01

    Objective To construct an eukaryotic expression vector of mannose-binding lectin associated protein 19 (MAp19) and further express MAp19 fusion proteins. Methods MAp19 gene fragment was amplified by PCR. The eukaryotic expression vector pcDNA3.1/Myc-His A-MAp19 was constructed by gene cloning technology, identified by restriction enzyme digestion and further confirmed by sequencing. The recombined plasmid was then transfected into HeLa cells by liposome-mediated method, and the positive clones with vector pcDNA3.1/Myc-His A-MAp19 were selected by G418. Location of MAp19 fusion proteins in the transfected HeLa cells was observed under the fluorescence microscope, and the expression levels of MAp19 fusion proteins were detected by Western blotting. Results The recombined vector pcDNA3.1/Myc-His A-MAp19 was successfully constructed. The result of DNA sequencing was in accordance with NCBI data bank. By G418 selecting, we obtained the HeLa cells which could stably express exogenous MAp19 fusion proteins. The protein was located in the cytoplasm. Western blotting also suggested that the MAp19 was secretory protein. Conclusion The vector expressing MAp19 has been prepared successfully, and can express the target protein (MAp19) in the eukaryotic cells (HeLa cells). PMID:26728378

  4. Cloning and expression of Toxoplasma gondii dense granular protein 4 (GRA4) in Pichia pastoris.

    PubMed

    Lau, Y L; Hasan, M T; Thiruvengadam, G; Idris, M M; Init, I

    2010-12-01

    GRA4 of Toxoplasma gondii has been shown to prompt IgG, IgM and IgA responses in previous studies and is thus considered one of the major immunogenic proteins from T. gondii that can be used for both diagnostics purposes and vaccine development. This study seeks to clone and express the GRA4 in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. In order to achieve this, the gene was cloned into the pPICZ? A expression vector, which was then incorporated into the P. pastoris genome via insertional integration for expression of the recombinant protein, under the AOX1 promoter. The antigen was expressed along with the prepro sequence of the ?-factor of yeast so that it could be excreted out of the P. pastoris cells and obtained from the medium. Upon SDS-PAGE analysis it was found that the recombinant protein was expressed optimally as a 40 kDa protein after 96 hours of induction with 0.75% of methanol. The expressed GRA4 protein showed discrepancy in size with the calculated molecular mass. This may be attributed to the various posttranslational modifications including glycosylation and phosphorylation. Despite the difference in molecular weight, the recombinant protein was able to detect toxoplasmosis in Western blot format. The recombinant GRA4 was expressed with an intact polyhistidine-tag, which could be used for future purification of the antigen. PMID:21399595

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

    PubMed

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-02-01

    Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. PMID:26494603

  7. Expression of Proteins Linked to Poor Ovarian Cancer Outcome

    Cancer.gov

    Scientists have established the presence of certain proteins in ovarian cancer tissues and have linked these proteins to poor survival rates in women with advanced stages of the disease. The study, led by scientists at the National Cancer Institute (NCI),

  8. A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

    NASA Astrophysics Data System (ADS)

    Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

    2000-05-01

    RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

  9. Expression of synthetic human tropoelastin (hTE) protein in Nicotiana tabacum.

    PubMed

    Abdelghani, Mona; El-Heba, Ghada A Abu; Abdelhadi, Abdelhadi A; Abdallah, Naglaa A

    2015-01-01

    Plant molecular farming (PMF) is an important growing prospective approach in plant biotechnology; it includes production of recombinant pharmaceutical and industrial proteins in large quantities from engineered plants. Elastin is a major protein component of tissues that require elasticity, it helps keep skin smooth as it stretches to allow normal. Elastin is used as a raw material for the cosmetic industry. In this work, we aimed to use plant as a bioreactor for the expression and production of the full human tropoelastin protein. Agrobacterium- mediated transient expression system into Nicotiana tabacum using syringe agroinfiltration was used to provide fast and convenient way to produce recombinant proteins with greater expression overall the plant leaf. This study aimed to establish an efficient and rapid system for transiently expression and production of human recombinant tropoelastin protein in transgenic N. tabacum plants. Modified elastin (ELN) gene was biosynthesized and cloned into pCambia1390 vector to be used into N. tabacum agroinfilteration. Optimization of codon usage for the human tropoelastin gene, without changing the primary structure of the protein was carried out to ensure high expression in tobacco plants. The obtained data proved that the 5(th) day post-infiltration is the optimum interval to obtain the maximum production of our recombinant protein. Southern blot analysis was able to detect 2175 bp fragment length representing the ELN orf (open reding frame). On the other hand, ELN -expression within plant's tissue was visualized by RT-PCR during the period 3-10 days post agroinfiltration. At the protein level, western and ELISA confirmed the expression of recombinant tropoelastin protein. Western blot analysis detected the tropoelastin protein as parent band at ?70 kDa from freshly extracted protein, while two degraded bands of ?55 and ?45 kDa, representing a pattern of tropoelastin were appeared with frozen samples. This study showed that biosynthetic ELN gene was successfully expressed into N. tabacum leaves using agroinfiltration technique. PMID:25984768

  10. Differential expression of Yes-associated protein and phosphorylated Yes-associated protein is correlated with expression of Ki-67 and phospho-ERK in colorectal adenocarcinoma.

    PubMed

    Kim, Dong-Hoon; Kim, Seok-Hyung; Lee, Ok-Jun; Huang, Song-Mei; Kwon, Ju-Lee; Kim, Jin Man; Kim, Ji-Yeon; Seong, In Ock; Song, Kyu Sang; Kim, Kyung-Hee

    2013-11-01

    Yes-associated protein (YAP) is a transcriptional co-activator and functions as a nuclear downstream effector of the Hippo pathway. Differential expression of YAP and phosphorylated Yes-associated protein (pYAP), which are involved in the expression of Ki-67 and phosphorylated extracellular signal-regulated kinase (pERK) in colorectal adenocarcinoma (CRAC), is not clear. Herein, we hypothesized that nuclear expression of YAP could predict cell proliferation and poor prognosis, while cytoplasmic expression of pYAP would show a reverse correlation with cell proliferation. Paraffin-embedded samples from 144 CRAC patients were studied using immunohistochemistry for YAP, pYAP, Ki-67 and pERK. Frozen samples from 20 CRAC patients were examined for YAP mRNA in tumor and non-tumor tissues, using quantitative real-time PCR. High nuclear YAP expression coincided with high Ki-67 expression (P=0.002). The high nuclear YAP expression group tended to display a poor overall and disease-free survival (P=0.089 and P=0.089, respectively), but YAP mRNA levels in the 20 CRAC tissues were not significantly different in comparison with the 20 non-tumor tissues (P=0.929). We observed an inverse correlation between high cytoplasmic pYAP expression and high Ki-67 expression (P=0.001). Nuclear pERK expression was positively correlated with nuclear YAP expression, but negatively correlated with cytoplasmic pYAP expression (P=0.017 and P=0.020, respectively). Activated nuclear YAP and inactivated cytoplasmic pYAP in CRAC showed a positive correlation with Ki-67 and nuclear pERK expression, suggesting that the expression of YAP and pYAP is a possible predictor of tumor cell proliferation and prognosis in CRAC. PMID:23673988

  11. Induction of cells differentiation and ABC transporters expression by a myco-estrogen, zearalenone, in human choriocarcinoma cell line (BeWo).

    PubMed

    Prouillac, Caroline; Videmann, Bernadette; Mazallon, Michelle; Lecoeur, Sylvaine

    2009-09-19

    The mycotoxin zearalenone, produced by Fusarium species, is a worldwide contaminant of concern in cereals and other plant products. Due to its estrogenic activity, zearalenone (ZEA) is known to have toxicological effect in animals on reproductive system and the placental transfer of ZEA was suggested by in vivo studies. Although passive diffusion is the principal transport mechanism across the placenta, several carrier-mediated transport protein such as ABC transporter (P-gp, MRP1, MRP2, BCRP) have been identified in the placenta. In this work, we have investigated the effect of ZEA on trophoblast differentiation and ABC transporter expression by using an in vitro model of transplacental barrier, the BeWo cell line. In the presence of 10 microM ZEA morphological (syncytium formation) and biochemical (hCG secretion) differentiation of BeWo cells were observed after a 48h exposure. Results were compared to 17beta-estradiol (E2) and an inducer of syncytialisation (forskolin). The influence of cell differentiation and ZEA exposure on expression profiles of major ABC transporters was investigated in BeWo cells: expression of mRNA MRP1, MRP2 and BCRP was induced after 24h of ZEA exposure. Induction of P-gp, MRP1, and MRP2 protein was observed after 48h of ZEA exposure. Similar results were obtained after forskolin exposure. Our study reported for the first time the implication of a food contaminant in biological effect and ABC transporter expression modulation in human choriocarcinoma cells. PMID:19580841

  12. Maternal Protein Restriction Affects Postnatal Growth and the Expression of Key Proteins Involved in Lifespan Regulation in Mice

    PubMed Central

    Chen, Jian-Hua; Martin-Gronert, Malgorzata S.; Tarry-Adkins, Jane; Ozanne, Susan E.

    2009-01-01

    We previously reported that maternal protein restriction in rodents influenced the rate of growth in early life and ultimately affected longevity. Low birth weight caused by maternal protein restriction followed by catch-up growth (recuperated animals) was associated with shortened lifespan whereas protein restriction and slow growth during lactation (postnatal low protein: PLP animals) increased lifespan. We aim to explore the mechanistic basis by which these differences arise. Here we investigated effects of maternal diet on organ growth, metabolic parameters and the expression of insulin/IGF1 signalling proteins and Sirt1 in muscle of male mice at weaning. PLP mice which experienced protein restriction during lactation had lower fasting glucose (P?=?0.038) and insulin levels (P?=?0.046) suggesting improved insulin sensitivity. PLP mice had higher relative weights (adjusted by body weight) of brain (P?=?0.0002) and thymus (P?=?0.031) compared to controls suggesting that enhanced functional capacity of these two tissues is beneficial to longevity. They also had increased expression of insulin receptor substrate 1 (P?=?0.021) and protein kinase C zeta (P?=?0.046). Recuperated animals expressed decreased levels of many insulin signalling proteins including PI3 kinase subunits p85? (P?=?0.018), p110? (P?=?0.048) and protein kinase C zeta (P?=?0.006) which may predispose these animals to insulin resistance. Sirt1 protein expression was reduced in recuperated offspring. These observations suggest that maternal protein restriction can affect major metabolic pathways implicated in regulation of lifespan at a young age which may explain the impact of maternal diet on longevity. PMID:19308256

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Orabona, Emanuele; De Stefano, Luca; Ferry, Mike; Hasty, Jeff; di Bernardo, Mario; di Bernardo, Diego

    2014-01-01

    We describe an innovative experimental and computational approach to control the expression of a protein in a population of yeast cells. We designed a simple control algorithm to automatically regulate the administration of inducer molecules to the cells by comparing the actual protein expression level in the cell population with the desired expression level. We then built an automated platform based on a microfluidic device, a time-lapse microscopy apparatus, and a set of motorized syringes, all controlled by a computer. We tested the platform to force yeast cells to express a desired fixed, or time-varying, amount of a reporter protein over thousands of minutes. The computer automatically switched the type of sugar administered to the cells, its concentration and its duration, according to the control algorithm. Our approach can be used to control expression of any protein, fused to a fluorescent reporter, provided that an external molecule known to (indirectly) affect its promoter activity is available. PMID:24831205

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

    USGS Publications Warehouse

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

    2011-01-01

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

  16. Expression of membrane proteins in the eyes of transgenic Drosophila melanogaster.

    PubMed

    Hackmann, Yvonne; Joedicke, Lisa; Panneels, Valérie; Sinning, Irmgard

    2015-01-01

    In recent years, improved protein expression and crystallization strategies, as well as advanced synchrotron radiation sources and crystallographic tools considerably increased the number of crystal structures of integral membrane proteins from higher eukaryotes. However, seen as a proportion of the total number of candidate proteins, these achievements still appear meager, reflecting the huge effort that is often required to obtain high-level and functional expression of eukaryotic membrane proteins. Besides bacteria, yeast, insect, or mammalian cells are frequently used for heterologous expression, but despite considerable investments in time, labor, and money, there are numerous drawbacks to these systems. Are there other strategies that allow for an effective, large-scale production of functional membrane proteins? This chapter describes the expression of proteins in photoreceptor cells of transgenic Drosophila as an easily accessible, versatile alternative. We present step-by-step protocols starting from the cloning of the target gene into a suitable vector for fly eye expression and ending with the harvest of transgenic Drosophila and subsequent protein purification from the eye. Our examples span a number of eukaryotic membrane proteins from different classes-including receptors, transporters, channels, and enzymes-that were successfully expressed without further optimization. The protocols provided here are robust and straightforward to follow even without prior experience in Drosophila work. PMID:25857784

  17. Optimized expression, solubilization and purification of nuclear inclusion protein b of cardamom mosaic virus.

    PubMed

    Jebasingh, T; Jacob, T; Shah, M; Das, D; Krishnaswamy, S; Usha, R

    2008-04-01

    All RNA viruses encode an RNA-dependent RNA polymerase (RdRP) that is required for replication of the viral genome. Nuclear inclusion b (NIb) gene codes for the RdRp in Potyviridae viruses. In this study, expression, solubilization and purification of NIb protein of Cardamom mosaic virus (CdMV) is reported. The objective of the present study was to express and purify the NIb protein of CdMV on a large scale for structural characterization, as the structure of the RdRp from a plant virus is yet to be determined. However, the expression of NIb protein with hexa-histidine tag in Escherichia coli led to insoluble aggregates. Out of all the approaches [making truncated versions to reduce the size of protein; replacing an amino acid residue likely to be involved in hydrophobic intermolecular interactions with a hydrophilic one; expressing the protein along with chaperones; expression in Origami cells for proper disulphide bond formation, in E. coli as a fusion with maltose-binding protein (MBP) and in Nicotiana tabacum] to obtain the RdRp in a soluble form, only expression in E. coli as a fusion with MBP and its expression in N. tabacum were successful. The NIb expressed in plant or as a fusion with MBP in E. coli can be scaled up for further work. PMID:21086722

  18. Mutation Hotspots Associate with Gene Expression, Signaling Pathways, Protein Domains and Drug Response - Theo Knijnenburg, TCGA Scientific Symposium 2015

    Cancer.gov

    Home News and Events Multimedia Library Videos Mutation Hotspots Associate with Gene Expression, Signaling Pathways, Protein Domains and Drug Respo Mutation Hotspots Associate with Gene Expression, Signaling Pathways, Protein Domains and Drug Response

  19. P-glycoprotein involvement in cuticular penetration of [14C]thiodicarb in resistant tobacco budworms.

    PubMed

    Lanning, C L; Ayad, H M; Abou-Donia, M B

    1996-06-01

    Pesticides have been shown to interact with the multidrug resistance protein associated with cancer chemotherapy, P-glycoprotein (P-gp). P-gp, therefore, has also been implicated in the development of pesticide resistance. The purpose of this study was to characterize the effect P-gp has on the accumulation of the carbamate pesticide, thiodicarb. For these studies, resistant tobacco budworm larvae, expressing four times the P-gp as susceptible larvae, were pretreated with the P-gp inhibitor, quinidine, and challenged topically with thiodicarb. Quinidine enhanced thiodicarb toxicity in a dose-dependent manner, with mortality in the presence of P-gp inhibition increased up to 33%. Quinidine treatment increased [14C]thiodicarb accumulation 2- to 3-fold as compared to thiodicarb treatment alone. This study suggests that P-gp contributes to quinidine synergism of thiodicarb toxicity and suggests that P-gp may be involved in cuticular resistance to pesticides. PMID:8644124

  20. Evolutionary Divergence of Gene and Protein Expression in the Brains of Humans and Chimpanzees.

    PubMed

    Bauernfeind, Amy L; Soderblom, Erik J; Turner, Meredith E; Moseley, M Arthur; Ely, John J; Hof, Patrick R; Sherwood, Chet C; Wray, Gregory A; Babbitt, Courtney C

    2015-08-01

    Although transcriptomic profiling has become the standard approach for exploring molecular differences in the primate brain, very little is known about how the expression levels of gene transcripts relate to downstream protein abundance. Moreover, it is unknown whether the relationship changes depending on the brain region or species under investigation. We performed high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses on two regions of the human and chimpanzee brain: The anterior cingulate cortex and caudate nucleus. In both brain regions, we found a lower correlation between mRNA and protein expression levels in humans and chimpanzees than has been reported for other tissues and cell types, suggesting that the brain may engage extensive tissue-specific regulation affecting protein abundance. In both species, only a few categories of biological function exhibited strong correlations between mRNA and protein expression levels. These categories included oxidative metabolism and protein synthesis and modification, indicating that the expression levels of mRNA transcripts supporting these biological functions are more predictive of protein expression compared with other functional categories. More generally, however, the two measures of molecular expression provided strikingly divergent perspectives into differential expression between human and chimpanzee brains: mRNA comparisons revealed significant differences in neuronal communication, ion transport, and regulatory processes, whereas protein comparisons indicated differences in perception and cognition, metabolic processes, and organization of the cytoskeleton. Our results highlight the importance of examining protein expression in evolutionary analyses and call for a more thorough understanding of tissue-specific protein expression levels. PMID:26163674

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

    PubMed

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

    2015-02-01

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

  2. Clinical significance of migration and invasion inhibitor protein expression in non-small-cell lung cancer

    PubMed Central

    WANG, XINHUA; LIU, HONGLING; WANG, XIAOYU; AN, YUZHI

    2014-01-01

    Migration and invasion inhibitor protein (MIIP) was initially identified in a yeast two-hybrid screen. Recently, MIIP has emerged as a key protein in regulating cell migration and invasion. However, the MIIP expression profile in non-small-cell lung cancer (NSCLC) has not been analyzed. In the present study, MIIP mRNA expression levels were evaluated using the SYBR Green quantitative real-time polymerase chain reaction method in 37 NSCLC specimens and matched normal tissue samples. MIIP protein expression in a further 94 NSCLC specimens was examined with immunohistochemistry. Patient survival data were collected retrospectively, and the association between MIIP protein expression and the five-year overall survival rate was evaluated. The results revealed that MIIP mRNA and protein expression were downregulated in cancer tissues, as compared with the matched normal tissues. MIIP expression levels were significantly associated with pathology and tumor stage, with reduced MIIP mRNA expression levels detected in advanced tumor stage samples. Furthermore, patients with MIIP-positive protein expression had an improved prognosis as compared with those patients with MIIP-negative protein expression, with five-year survival rates of 41.7 and 22.4%, respectively (Kaplan-Meier, log-rank, P=0.028). A significant association between MIIP protein expression and improved prognosis was also demonstrated using univariate and multivariate analyses (P=0.033 and P=0.040, respectively). These results suggest that MIIP may have a potential role in the pathogenesis of NSCLC and also confirm that MIIP is a putative tumor-suppressor gene. Therefore, MIIP may be identified as a functional genetic marker of NSCLC development and prognosis, and may be an attractive therapeutic target for the treatment of lung cancer. PMID:25360165

  3. Expression Atlas update—an integrated database of gene and protein expression in humans, animals and plants

    PubMed Central

    Petryszak, Robert; Keays, Maria; Tang, Y. Amy; Fonseca, Nuno A.; Barrera, Elisabet; Burdett, Tony; Füllgrabe, Anja; Fuentes, Alfonso Muñoz-Pomer; Jupp, Simon; Koskinen, Satu; Mannion, Oliver; Huerta, Laura; Megy, Karine; Snow, Catherine; Williams, Eleanor; Barzine, Mitra; Hastings, Emma; Weisser, Hendrik; Wright, James; Jaiswal, Pankaj; Huber, Wolfgang; Choudhary, Jyoti; Parkinson, Helen E.; Brazma, Alvis

    2016-01-01

    Expression Atlas (http://www.ebi.ac.uk/gxa) provides information about gene and protein expression in animal and plant samples of different cell types, organism parts, developmental stages, diseases and other conditions. It consists of selected microarray and RNA-sequencing studies from ArrayExpress, which have been manually curated, annotated with ontology terms, checked for high quality and processed using standardised analysis methods. Since the last update, Atlas has grown seven-fold (1572 studies as of August 2015), and incorporates baseline expression profiles of tissues from Human Protein Atlas, GTEx and FANTOM5, and of cancer cell lines from ENCODE, CCLE and Genentech projects. Plant studies constitute a quarter of Atlas data. For genes of interest, the user can view baseline expression in tissues, and differential expression for biologically meaningful pairwise comparisons—estimated using consistent methodology across all of Atlas. Our first proteomics study in human tissues is now displayed alongside transcriptomics data in the same tissues. Novel analyses and visualisations include: ‘enrichment’ in each differential comparison of GO terms, Reactome, Plant Reactome pathways and InterPro domains; hierarchical clustering (by baseline expression) of most variable genes and experimental conditions; and, for a given gene-condition, distribution of baseline expression across biological replicates. PMID:26481351

  4. Expression Atlas update-an integrated database of gene and protein expression in humans, animals and plants.

    PubMed

    Petryszak, Robert; Keays, Maria; Tang, Y Amy; Fonseca, Nuno A; Barrera, Elisabet; Burdett, Tony; Füllgrabe, Anja; Fuentes, Alfonso Muñoz-Pomer; Jupp, Simon; Koskinen, Satu; Mannion, Oliver; Huerta, Laura; Megy, Karine; Snow, Catherine; Williams, Eleanor; Barzine, Mitra; Hastings, Emma; Weisser, Hendrik; Wright, James; Jaiswal, Pankaj; Huber, Wolfgang; Choudhary, Jyoti; Parkinson, Helen E; Brazma, Alvis

    2016-01-01

    Expression Atlas (http://www.ebi.ac.uk/gxa) provides information about gene and protein expression in animal and plant samples of different cell types, organism parts, developmental stages, diseases and other conditions. It consists of selected microarray and RNA-sequencing studies from ArrayExpress, which have been manually curated, annotated with ontology terms, checked for high quality and processed using standardised analysis methods. Since the last update, Atlas has grown seven-fold (1572 studies as of August 2015), and incorporates baseline expression profiles of tissues from Human Protein Atlas, GTEx and FANTOM5, and of cancer cell lines from ENCODE, CCLE and Genentech projects. Plant studies constitute a quarter of Atlas data. For genes of interest, the user can view baseline expression in tissues, and differential expression for biologically meaningful pairwise comparisons-estimated using consistent methodology across all of Atlas. Our first proteomics study in human tissues is now displayed alongside transcriptomics data in the same tissues. Novel analyses and visualisations include: 'enrichment' in each differential comparison of GO terms, Reactome, Plant Reactome pathways and InterPro domains; hierarchical clustering (by baseline expression) of most variable genes and experimental conditions; and, for a given gene-condition, distribution of baseline expression across biological replicates. PMID:26481351

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

    PubMed

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

    2012-06-15

    We treated detonator-explosion-induced craniocerebral injury in rabbits with hyperbaric oxygen 1-24 hours post-injury. Expression of the apoptosis-regulating protein cytochrome c, the pro-apoptotic protein Bax and the apoptosis marker caspase-3 in the tissues surrounding the area of injury was significantly reduced, while that of the anti-apoptotic protein Bcl-2 was significantly increased. Our findings indicate that the curative effects of early hyperbaric oxygen on cortical cell apoptosis is associated with suppression of cytochrome c release from mitochondria. This mechanism underlies the observed reduction in Bax expression and upregulation of Bcl-2 expression. PMID:25657662

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

    PubMed Central

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

    2012-01-01

    We treated detonator-explosion-induced craniocerebral injury in rabbits with hyperbaric oxygen 1-24 hours post-injury. Expression of the apoptosis-regulating protein cytochrome c, the pro-apoptotic protein Bax and the apoptosis marker caspase-3 in the tissues surrounding the area of injury was significantly reduced, while that of the anti-apoptotic protein Bcl-2 was significantly increased. Our findings indicate that the curative effects of early hyperbaric oxygen on cortical cell apoptosis is associated with suppression of cytochrome c release from mitochondria. This mechanism underlies the observed reduction in Bax expression and upregulation of Bcl-2 expression. PMID:25657662

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

    DOEpatents

    Mayfield, Stephen (Cardiff, CA)

    2000-01-01

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

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

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

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

  9. Receptor-associated protein promotes t-PA expression, reduces PAI-1 expression and improves neurorecovery after acute ischemic stroke.

    PubMed

    Li, Dan-Dong; Pang, Hong-Gang; Song, Jin-Ning; Zhao, Yong-Lin; Zhang, Bin-Fei; Ma, Xu-Dong; Sun, Peng

    2015-03-15

    Receptor-associated protein (RAP) is a receptor antagonist that inhibits ligand interactions with the receptors that belong to the low density lipoprotein receptor gene family. The low-density lipoprotein receptor-related protein 1 (LRP1) has a crucial role in regulating tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) expression. Furthermore, the functional balance of these two proteins is directly associated with the initiation and development of cerebral ischemic stroke. In the present study, the effect of RAP post-treatment was investigated in a rat autologous thromboembolic model. The expression and activity of t-PA and PAI-1 were detected and the neurological function was tested. The results suggest that post-treatment with RAP is able to improve neurorecovery after ischemic stroke by decreasing vascular damage and regulating t-PA and PAI-1 expressions. Post-treatment with RAP promotes t-PA expression, suppresses PAI-1 expression, significantly improves functional outcomes and decreases the amount of TUNEL-positive cells. RAP-treated rats show lower intracranial hemoglobin levels and a smaller ischemic zone. In conclusion, post-treatment with RAP regulates t-PA and PAI-1 expressions and thereby contributes to the improvement of functional outcomes after cerebral ischemia. Our findings strongly suggest that RAP may be of value in neurorecovery after stroke. PMID:25702149

  10. Expression and immunological characterization of cardamom mosaic virus coat protein displaying HIV gp41 epitopes.

    PubMed

    Damodharan, Subha; Gujar, Ravindra; Pattabiraman, Sathyamurthy; Nesakumar, Manohar; Hanna, Luke Elizabeth; Vadakkuppattu, Ramanathan D; Usha, Ramakrishnan

    2013-05-01

    The coat protein of cardamom mosaic virus (CdMV), a member of the genus Macluravirus, assembles into virus-like particles when expressed in an Escherichia coli expression system. The N and C-termini of the coat protein were engineered with the Kennedy peptide and the 2F5 and 4E10 epitopes of gp41 of HIV. The chimeric proteins reacted with sera from HIV positive persons and also stimulated secretion of cytokines by peripheral blood mononuclear cells from these persons. Thus, a system based on the coat protein of CdMV can be used to display HIV-1 antigens. PMID:23668610

  11. Differential protein expression in the nucleus accumbens of high and low active mice.

    PubMed

    Ferguson, David P; Dangott, Lawrence J; Vellers, Heather L; Schmitt, Emily E; Lightfoot, J Timothy

    2015-09-15

    Physical inactivity is associated with the development of a variety of chronic illnesses. Literature has shown that physical activity is genetically regulated; however there is limited information on the mechanisms that influence this process with existing studies primarily focused on genomic and/or transcription association studies. There have been no studies to determine differential protein expression in the nucleus accumbens, the brain site thought to be involved in activity regulation, between high and low active animals. We compared the global nucleus accumbens proteome signature from known high- and low-active mice and identified seven differentially expressed proteins. Low active mice generally over expressed proteins associated with neural stress (Stress 70 protein and V type proton ATPase catalytic subunit A), and the high-active mice over expressed proteins associated with metabolism (creatine kinase B, succinyl-CoA ligase). Previously suggested mechanisms associated with activity regulation in the nucleus accumbens have centered on dopamine receptor 1 and endocannabinoid receptor 1. However, these proteins and the associated pathways were not differentially expressed between high and low active mice. In conclusion, protein expression must be determined as part of the effort to identify involved mechanisms in regulating activity and there appears to be separate nucleus accumbens proteome signatures associated with high- and low-active mice. PMID:26008157

  12. Physiological concentration of amino acids regulates insulin-like-growth-factor-binding protein 1 expression.

    PubMed Central

    Jousse, C; Bruhat, A; Ferrara, M; Fafournoux, P

    1998-01-01

    Protein undernutrition is characterized by growth failure in young growing animals. Current evidence suggests that biosynthesis of insulin-like growth factor (IGF)-I and IGF-binding protein 1 (IGFBP-1) are key control points for nutritional regulation of growth. Here we examined the role of amino acid limitation in regulating the IGFBP-1 expression in the hepatic cell line. Our data show that leucine limitation strongly induces IGFBP-1 without affecting IGF-I and IGF-II expression in human HepG2 cells and in isolated rat hepatocytes. Depletion of arginine, cystine and all essential amino acids leads to induction of IGFBP-1 mRNA and protein expression in a dose-dependent manner. IGFBP-1 expression is significantly induced by leucine concentration in the range of that observed in the blood of rats fed a low-protein diet or in humans affected by kwashiorkor. Moreover, treatment of HepG2 cells with amino acids at a concentration reproducing the amino acid concentration found in portal blood of rats fed a low-protein diet leads to a significantly higher expression of IGFBP-1. These data represent the first demonstration that an amino acid limitation, as occurs during dietary protein deficiency, induces IGFBP-1 expression in hepatic cells. Therefore, amino acids by themselves can play, in concert with hormones, an important role in the control of gene expression. PMID:9693114

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

    PubMed Central

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

    2013-01-01

    We sought to evaluate the relationship between cell division and protein expression when using commercial poly(ethylenimine) (PEI)-based polyplexes. The membrane dye PKH26 was used to assess cell division, and cyan fluorescent protein (CFP) was used to monitor protein expression. When analyzed at the whole population level, a greater number of cells divided than expressed protein, regardless of the level of protein expression observed, giving apparent consistency with the hypothesis that protein expression requires cells to pass through mitosis in order for the transgene to overcome the nuclear membrane. However, when the polyplex-exposed population was evaluated for the amount of division in the protein-expressing subpopulation, it was observed that substantial amounts of expression had occurred in the absence of division. Indeed, in HeLa S3 cells, this represented the majority of expressing cells. Of interest, the doubling time for both cell lines was slowed by ~2-fold upon exposure to polyplexes. This change was not altered by the origin of the plasmid DNA (pDNA) transgene promoter (cytomegalovirus (CMV) or elongation factor-1 alpha (EF1?)). Gene expression arrays in polyplex-exposed HeLa S3 cells showed upregulation of cell cycle arrest genes and downregulation of genes related to mitosis. Chemokine, interleukin, and toll-like receptor genes were also upregulated, suggesting activation of proinflammatory pathways. In summary, we find evidence that a cell division-independent expression pathway exists, and that polyplex exposure slows cell division and increases inflammatory response. PMID:23458572

  14. Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

    2016-01-01

    Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ?2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include ?-enolase, ?-catenin, 14-3-3 ?, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

  15. Utility of proteomics techniques for assessing protein expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. Utility of proteomics techniques for assessing protein expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteomic technologies are currently used as an effective analytical tool for examining modifications in protein profiles for assessing the bio-safety of genetically modified (GM) crop organisms. Understanding the natural variation of soybean seed proteins is necessary to evaluate potential uninten...

  17. Proteomic analysis of Lawsonia intracellularis reveals expression of outer membrane proteins during infection.

    PubMed

    Watson, Eleanor; Alberdi, M Pilar; Inglis, Neil F; Lainson, Alex; Porter, Megan E; Manson, Erin; Imrie, Lisa; Mclean, Kevin; Smith, David G E

    2014-12-01

    Lawsonia intracellularis is the aetiological agent of the commercially significant porcine disease, proliferative enteropathy. Current understanding of host-pathogen interaction is limited due to the fastidious microaerophilic obligate intracellular nature of the bacterium. In the present study, expression of bacterial proteins during infection was investigated using a mass spectrometry approach. LC-ESI-MS/MS analysis of two isolates of L. intracellularis from heavily-infected epithelial cell cultures and database mining using fully annotated L. intracellularis genome sequences identified 19 proteins. According to the Clusters of Orthologous Groups (COG) functional classification, proteins were identified with roles in cell metabolism, protein synthesis and oxidative stress protection; seven proteins with putative or unknown function were also identified. Detailed bioinformatic analyses of five uncharacterised proteins, which were expressed by both isolates, identified domains and motifs common to other outer membrane-associated proteins with important roles in pathogenesis including adherence and invasion. Analysis of recombinant proteins on Western blots using immune sera from L. intracellularis-infected pigs identified two proteins, LI0841 and LI0902 as antigenic. The detection of five outer membrane proteins expressed during infection, including two antigenic proteins, demonstrates the potential of this approach to interrogate L. intracellularis host-pathogen interactions and identify novel targets which may be exploited in disease control. PMID:25457368

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

    PubMed

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

    2014-10-01

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

  19. Latitudinal variation in protein expression after heat stress in the salt marsh mussel Geukensia demissa.

    PubMed

    Fields, Peter A; Cox, Kelly M; Karch, Kelly R

    2012-11-01

    Individuals of a broadly distributed species often experience significantly different environmental conditions depending on location. For example, the mussel Geukensia demissa occurs intertidally from the Gulf of St. Lawrence to central Florida; within this range, northern populations are exposed to temperatures cold enough to freeze the tissue, whereas southern populations can experience temperatures approaching the species' upper lethal limit. Thus, G. demissa provides an ideal system with which to study physiological variation in conspecifics occurring across a broad latitudinal range. We collected G. demissa at five sites from Maine to Florida, encompassing a range of 1900 km, and have used a proteomic approach to describe how protein expression varies in individuals from the different locations. We acclimated individuals from each site to common conditions (18°C) for 4 weeks, and exposed a subset of these to acute heat stress (40°C). We separated gill proteins using two-dimensional gel electrophoresis and quantified abundances of the resulting protein spots. Among mussels acclimated to 18°C protein, expression profiles were more similar among individuals from the same site than among sites, but there was no discernible correlation with latitude. In contrast, after acute heat stress, protein expression among mussels from different locations varied substantially, with 31 of 448 proteins changing in abundance in the northernmost (Maine) group, compared with 5-11 proteins in the four southern groups. Identification of 27 of these proteins revealed five functional clusters: chaperones, cytoskeletal proteins, oxidative stress proteins, regulatory proteins, and a translation initiation factor. Across these functional categories, the two northernmost groups, Maine and New York, showed the greatest number of proteins that changed significantly in abundance, as well as the greatest fold-change in abundance for many of the proteins. We conclude that the northern populations of G. demissa are physiologically distinct from the southern groups, and that the differences in protein-expression profiles are consistent with greater sensitivity to heat stress to the north. PMID:22641835

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

    SciTech Connect

    Takahashi, Nobuhiko; Division of Gastroenterology and Hematology Yoshizaki, Takayuki; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka

    2011-11-11

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

  1. GONADAL STEROIDS REGULATED THE EXPRESSION OF GLIAL FIBRILLARY ACIDIC PROTEIN IN THE ADULT MALE RAT HIPPOCAMPUS

    EPA Science Inventory

    This study demonstrates that gonadal steroids (estradiol, testosterone, dihydrotestosterone) can inhibit the expression of glial fibrillary acidic protein and it MRNA in the adult male rat brain. esticular hormones may influence the activity of astrocytes in the intact and lesion...

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

    E-print Network

    Menolascina, Filippo

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

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

    SciTech Connect

    Ito, Keisuke; Sugawara, Taishi; Shiroishi, Mitsunori; Tokuda, Natsuko; Kurokawa, Azusa; Misaka, Takumi; Makyio, Hisayoshi; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro; Nomura, Norimichi; Murata, Takeshi; Abe, Keiko; Iwata, So

    2008-07-11

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

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

    E-print Network

    Lindquist, Susan

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

  5. Alkbh8 Regulates Selenocysteine-Protein Expression to Protect against Reactive Oxygen Species Damage

    E-print Network

    Endres, Lauren

    Environmental and metabolic sources of reactive oxygen species (ROS) can damage DNA, proteins and lipids to promote disease. Regulation of gene expression can prevent this damage and can include increased transcription, ...

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

    PubMed Central

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

    2012-01-01

    Brain cancer is a devastating disease. Despite extensive research, treatment of brain tumors has been largely ineffective and the diagnosis of brain cancer remains uniformly fatal. Failure of brain cancer treatment may be in part due to limitations in drug delivery, influenced by the ABC drug efflux transporters P-gp and BCRP at the blood-brain and blood-tumor barriers, in brain tumor cells, as well as in brain tumor stem-like cells. P-gp and BCRP limit various anti-cancer drugs from entering the brain and tumor tissues, thus rendering chemotherapy ineffective. To overcome this obstacle, two strategies – targeting transporter regulation and direct transporter inhibition – have been proposed. In this review, we focus on these strategies. We first introduce the latest findings on signaling pathways that could potentially be targeted to down-regulate P-gp and BCRP expression and/or transport activity. We then highlight in detail the new paradigm of P-gp and BCRP working as a “cooperative team of gatekeepers” at the blood-brain barrier, discuss its ramifications for brain cancer therapy, and summarize the latest findings on dual P-gp/BCRP inhibitors. Finally, we provide a brief summary with conclusions and outline the perspectives for future research endeavors in this field. PMID:21827403

  7. Breast cancer resistance protein and P-glycoprotein in brain cancer: two gatekeepers team up.

    PubMed

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

    2011-01-01

    Brain cancer is a devastating disease. Despite extensive research, treatment of brain tumors has been largely ineffective and the diagnosis of brain cancer remains uniformly fatal. Failure of brain cancer treatment may be in part due to limitations in drug delivery, influenced by the ABC drug efflux transporters P-gp and BCRP at the blood-brain and blood-tumor barriers, in brain tumor cells, as well as in brain tumor stem-like cells. P-gp and BCRP limit various anti-cancer drugs from entering the brain and tumor tissues, thus rendering chemotherapy ineffective. To overcome this obstacle, two strategies - targeting transporter regulation and direct transporter inhibition - have been proposed. In this review, we focus on these strategies. We first introduce the latest findings on signaling pathways that could potentially be targeted to down-regulate P-gp and BCRP expression and/or transport activity. We then highlight in detail the new paradigm of P-gp and BCRP working as a "cooperative team of gatekeepers" at the blood-brain barrier, discuss its ramifications for brain cancer therapy, and summarize the latest findings on dual P-gp/BCRP inhibitors. Finally, we provide a brief summary with conclusions and outline the perspectives for future research endeavors in this field. PMID:21827403

  8. Expression and affinity purification of recombinant proteins from plants

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  9. Expression of Translationally Controlled Tumor Protein in Human Kidney and in Renal Cell Carcinoma

    PubMed Central

    Ambrosio, Maria R.; Rocca, Bruno J.; Barone, Aurora; Onorati, Monica; Mundo, Lucia; Crivelli, Filippo; Di Nuovo, Franca; De Falco, Giulia; del Vecchio, Maria T.; Tripodi, Sergio A.; Tosi, Piero

    2015-01-01

    Translationally controlled tumor protein is a multifaceted protein involved in several physiological and biological functions. Its expression in normal kidney and in renal carcinomas, once corroborated by functional data, may add elements to elucidate renal physiology and carcinogenesis. In this study, translationally controlled tumor protein expression was evaluated by quantitative real time polymerase chain reaction and western blotting, and its localization was examined by immunohistochemistry on 84 nephrectomies for cancer. In normal kidney protein expression was found in the cytoplasm of proximal and distal tubular cells, in cells of the thick segment of the loop of Henle, and in urothelial cells of the pelvis. It was also detectable in cells of renal carcinoma with different pattern of localization (membranous and cytoplasmic) depending on tumor histotype. Our data may suggest an involvement of translationally controlled tumor protein in normal physiology and carcinogenesis. However, functional in vitro and in vivo studies are needed to verify this hypothesis. PMID:26425551

  10. Generation and immunogenicity of Japanese encephalitis virus envelope protein expressed in transgenic rice.

    PubMed

    Wang, Yuanyuan; Deng, Hanqing; Zhang, Xiaobo; Xiao, Hailin; Jiang, Yunbo; Song, Yunfeng; Fang, Liurong; Xiao, Shaobo; Zhen, Yonglian; Chen, Huanchun

    2009-03-01

    Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 microg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine. PMID:19166811

  11. Chemokines derived from soluble fusion proteins expressed in Escherichia coli are biologically active

    SciTech Connect

    Magistrelli, Giovanni; Gueneau, Franck; Muslmani, Machadiya; Ravn, Ulla; Kosco-Vilbois, Marie; Fischer, Nicolas . E-mail: nfischer@novimmune.com

    2005-08-26

    Chemokines are a class of low molecular weight proteins that are involved in leukocytes trafficking. Due to their involvement in recruiting immune cells to sites of inflammation, chemokines, and chemokine receptors have become an attractive class of therapeutic targets. However, when expressed in Escherichia coli chemokines are poorly soluble and accumulate in inclusion bodies. Several purification methods have been described but involve time-consuming refolding, buffer exchange, and purification steps that complicate expression of these proteins. Here, we describe a simple and reliable method to express chemokines as fusions to the protein NusA. The fusion proteins were largely found in the soluble fraction and could be readily purified in a single step. Proteolytic cleavage was used to obtain soluble recombinant chemokines that were found to be very active in a novel in vitro chemotaxis assays. This method could be applied to several {alpha} and {beta} human chemokines, suggesting that it is generally applicable to this class of proteins.

  12. Chronic Alcohol Consumption Leads to a Tissue Specific Expression of Uncoupling Protein-2

    PubMed Central

    Graw, Jan A.; von Haefen, Clarissa; Poyraz, Deniz; Möbius, Nadine; Sifringer, Marco; Spies, Claudia D.

    2015-01-01

    Uncoupling proteins (UCPs) are anion channels that can decouple the mitochondrial respiratory chain. "Mild uncoupling" of internal respiration reduces free radical production and oxidative cell stress. Chronic alcohol consumption is a potent inducer of oxidative stress in multiple tissues and regulates UCP-2 and -4 expression in the brain. To analyse the impact of chronic alcohol intake on UCP-2 expression in tissues with high endogenous UCP-2 contents, male Wistar rats (n=34) were treated with a 12-week 5% alcohol diet. In the lungs and the spleen of rats with a chronic alcohol diet cytochrome c release from mitochondria was significantly increased. Both organs did not show any altered gene and protein expression of UCP-2. Different to cerebral tissue chronic alcohol consumption has no regulatory effect on UCP-2 gene and protein expression in organs with a high endogenous UCP-2 content. Therefore, chronic alcohol consumption leads to a tissue specific expression of UCP-2. PMID:26664262

  13. Changes in protein expression in maturing equine testis: a quantitative DIGE analysis 

    E-print Network

    Roper-Foo, Pilar

    2011-01-11

    -Foo_Final_Thesis_withappendix-FINAL-VERSION.pdf.txt Content-Type text/plain; charset=ISO-8859-1 CHANGES IN PROTEIN EXPRESSION IN MATURING EQUINE TESTIS: A QUANTITATIVE DIGE ANALYSIS Major: Genetics April 2009 Submitted to the Office of Undergraduate Research Texas A...&M University in partial fulfillment of the requirements for the designation as UNDERGRADUATE RESEARCH SCHOLAR A Senior Scholars Thesis by PILAR ROPER-FOO CHANGES IN PROTEIN EXPRESSION IN MATURING EQUINE TESTIS: A QUANTITATIVE DIGE ANALYSIS...

  14. Prokaryotical expression of structural and non-structural proteins of hepatitis G virus

    PubMed Central

    Xia, Ning-Shao; Yang, Hai-Jie; Zhang, Jun; Lin, Chang-Qing; Wang, Ying-Bin; Wang, Juan; Zhan, Mei-Yun; Ng, MH

    2001-01-01

    AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSE T and (or) pGEX, and expressed in E. coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis. PMID:11819846

  15. Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

    PubMed Central

    Parikh, Amit; Kumar, Devanand; Chawla, Yogesh; Kurthkoti, Krishna; Khan, Shazia; Varshney, Umesh

    2013-01-01

    Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research. PMID:23315736

  16. Tumor redox metabolism correlation with the expression level of red fluorescent protein

    NASA Astrophysics Data System (ADS)

    Sha, Shuang; Wang, Anle; Lin, Qiaoya; Zhang, Zhihong

    2015-03-01

    The redox metabolism is variable and complicated with the progress of tumor development. Whether the tumor redox state will affect the exogenous gene expression or not, are still not clear now . To investigate the relationship between tumor endogenous redox state and the exogenous gene expression level, a far red fluorescent protein fRFP was used to monitor tumor cells proliferation and as an exogenous protein expression in tumors. NADH (nicotinamide adenine dinucleotide) and Fp (flavin protein) are two important coenzymes in the mitochondria respiratory chain, which can be as a standard representation for redox metabolism state. Three tumor subcutaneous models (melanoma, human pancreatic carcinoma and nasopharyngeal carcinoma) were used to observe their redox state and protein expression by our home-made redox scanner. The results showed that the distribution of fRFP fluorescent protein expression in the inner tumor regions are heterogeneous, and the fluorescent intensity of fRFP and the fluorescent intensity of NADH have high correlation. In addition, we also found the linear coefficient in three tumors are different, the value of coefficient is (R2 = 0.966 and R2 = 0.943) in melanoma, (R2 = 0.701 and R2 = 0.942) in human pancreatic carcinoma, and (R2 = 0.994) in nasopharyngeal carcinoma, respectively. From these results, we consider that the exogenous protein expression of fRFP in tumor had some relationship with the tumor redox state of NADH.

  17. Conserved lamin A protein expression in differentiated cells in the earthworm Eudrilus eugeniae.

    PubMed

    Kalidas, Ramamoorthy M; Raja, Subramanian Elaiya; Mydeen, Sheik Abdul Kader Nagoor Meeran; Samuel, Selvan Christyraj Johnson Retnaraj; Durairaj, Selvan Christyraj Jackson; Nino, Gopi D; Palanichelvam, Karuppaiah; Vaithi, Arumugaswami; Sudhakar, Sivasubramaniam

    2015-09-01

    Lamin A is an intermediate filament protein found in most of the differentiated vertebrate cells but absent in stem cells. It shapes the skeletal frame structure beneath the inner nuclear membrane of the cell nucleus. As there are few studies of the expression of lamin A in invertebrates, in the present work, we have analyzed the sequence, immunochemical conservation and expression pattern of lamin A protein in the earthworm Eudrilus eugeniae, a model organism for tissue regeneration. The expression of lamin A has been confirmed in E. eugeniae by immunoblot. Its localization in the nuclear membrane has been observed by immunohistochemistry using two different rabbit anti-sera raised against human lamin A peptides, which are located at the C-terminus of the lamin A protein. These two antibodies detected 70?kDa lamin A protein in mice and a single 65?kDa protein in the earthworm. The Oct-4 positive undifferentiated blastemal tissues of regenerating earthworm do not express lamin A, while the Oct-4 negative differentiated cells express lamin A. This pattern was also confirmed in the earthworm prostate gland. The present study is the first evidence for the immunochemical identification of lamin A and Oct-4 in the earthworm. Along with the partial sequence obtained from the earthworm genome, the present results suggest that lamin A protein and its expression pattern is conserved from the earthworm to humans. PMID:25858151

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

    NASA Astrophysics Data System (ADS)

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

    2005-07-01

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

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

    PubMed

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

    2015-03-01

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

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

    SciTech Connect

    Yinghui Fu; Friedman, D.L.; Richards, S.; Pearlman, J.A.; Gibbs, R.A.; Pizzuti, A.; Perryman, M.B.; Fenwick, R.G. Jr.; Caskey, C.T. ); Ashizawa, Tetsuo Veterans Administration Medical Center, Houston, TX ); Scarlato, G. )

    1993-04-09

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

  1. AP-1 elements and TCL1 protein regulate expression of the gene encoding protein tyrosine phosphatase PTPROt in leukemia

    PubMed Central

    Zanesi, Nicola; Datta, Jharna; Roy, Satavisha; Kutay, Huban; Checovich, Allyn M.; Kaou, Mohamed; Zhong, Yiming; Johnson, Amy J.; Lucas, David M.; Heerema, Nyla A.; Hagan, John; Mo, Xiaokui; Jarjoura, David; Byrd, John C.; Croce, Carlo M.

    2011-01-01

    We previously demonstrated that the gene encoding PTPROt, the truncated form of protein tyrosine phosphatase receptor type O expressed predominantly in hematopoietic cells, is a candidate tumor suppressor and is down-regulated in chronic lymphocytic leukemia (CLL). Here, we show that PTPROt expression is significantly reduced in CD19+ spleen B cells from E?-T cell leukemia 1 (TCL1) transgenic mice relative to the wild-type mice. Strikingly, as much as a 60% decrease in PTPROt expression occurs at 7 weeks independently of promoter methylation. To elucidate the potential mechanism for this early suppression of PTPROt in these mice, we explored the role of activating protein-1 (AP-1) in its expression. We first demonstrate that AP-1 activation by 12-O-tetradecanoylphorbol-13-acetate induces PTPROt expression with concurrent recruitment of c-fos and c-jun to its promoter. The PTPROt promoter is also responsive to over- and underexpression of AP-1, confirming the role of AP-1 in PTPROt expression. Next, we demonstrate that TCL1 can repress the PTPROt promoter by altering c-fos expression and c-jun activation state. Finally, using primary CLL cells we have shown an inverse relationship between TCL1 and PTPROt expression. These findings further substantiate the role of TCL1 in PTPROt suppression and its importance in the pathogenesis of CLL. PMID:22001392

  2. An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability.

    PubMed

    Fan, Junping; Heng, Jie; Dai, Shuyan; Shaw, Neil; Zhou, Bei; Huang, Bo; He, Zheng; Wang, Ya; Jiang, Taijiao; Li, Xuemei; Liu, Zhijie; Wang, Xianping; Zhang, Xuejun C

    2011-07-01

    Membrane proteins account for about 30% of the genomes sequenced to date and play important roles in a variety of cellular functions. However, determining the three-dimensional structures of membrane proteins continues to pose a major challenge for structural biologists due to difficulties in recombinant expression and purification. We describe here a high throughput pipeline for Escherichia coli based membrane protein expression and purification. A ligation-independent cloning (LIC)-based vector encoding a C-terminal green fluorescence protein (GFP) tag was used for cloning in a high throughput mode. The GFP tag facilitated expression screening in E. coli through both cell culture fluorescence measurements and in-gel fluorescence imaging. Positive candidates from the GFP screening were subsequently sub-cloned into a LIC-based, GFP free vector for further expression and purification. The expressed, C-terminal His-tagged membrane proteins were purified via membrane enrichment and Ni-affinity chromatography. Thermofluor technique was applied to screen optimal buffers and detergents for the purified membrane proteins. This pipeline has been successfully tested for membrane proteins from E. coli and can be potentially expanded to other prokaryotes. PMID:21354311

  3. Ethylene Regulates Monomeric GTP-Binding Protein Gene Expression and Activity in Arabidopsis1

    PubMed Central

    Moshkov, Igor E.; Mur, Luis A.J.; Novikova, Galina V.; Smith, Aileen R.; Hall, Michael A.

    2003-01-01

    Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction. PMID:12692329

  4. Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions.

    PubMed

    Narayanan, Anoop; Ridilla, Marc; Yernool, Dinesh A

    2011-01-01

    A major rate-limiting step in determining structures of membrane proteins is heterologous protein production. Toxicity often associated with rapid overexpression results in reduced biomass along with low yields of target protein. Mitigation of toxic effects was achieved using a method we call "restrained expression," a controlled reduction in the frequency of transcription initiation by exploiting the infrequent transitions of Lac repressor to a free state from its complex with the lac-operator site within a T7lac promoter that occur in the absence of the inducer isopropyl ?-D-1-thiogalactopyranoside. In addition, production of the T7 RNA polymerase that drives transcription of the target is limited using the tightly regulated arabinose promoter in Escherichia coli strain BL21-AI. Using this approach, we can achieve a 200-fold range of green fluorescent protein expression levels. Application to members of a family of ion pumps results in 5- to 25-fold increases in expression over the benchmark BL21(DE3) host strain. A viral ion channel highly toxic to E. coli can also be overexpressed. In comparative analyses, restrained expression outperforms commonly used E. coli expression strategies. The mechanism underlying improved target protein yield arises from minimization of protein aggregation and proteolysis that reduce membrane integrity and cell viability. This study establishes a method to overexpress toxic proteins. PMID:21031485

  5. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

    PubMed Central

    Goehring, April; Lee, Chia-Hsueh; Wang, Kevin H.; Michel, Jennifer Carlisle; Claxton, Derek P.; Baconguis, Isabelle; Althoff, Thorsten; Fischer, Suzanne; Garcia, K. Christopher; Gouaux, Eric

    2014-01-01

    Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in over-expression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol we show how to use small-scale transient transfection and fluorescence-detection, size-exclusion chromatography (FSEC) experiments using a GFP-His8 tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI? (N-acetylglucosaminyltransferase I-negative) cells in suspension culture, and over-express the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl), for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks. PMID:25299155

  6. Expression of p16 protein in infantile hemangioma

    PubMed Central

    WANG, SHI; ZHENG, WEI

    2015-01-01

    The present study aimed to investigate the expression and significance of p16 in the occurrence, development and regression of infantile hemangioma (IH). The expression of p16 was examined in proliferating, involuting hemangioma and normal tissues using immunohistochemical techniques. The expression of p16 was significantly lower in proliferating hemangioma than in involuting hemangioma, and was significantly lower in the involuting hemangioma than in normal tissues. Significant differences were found between the three groups (P<0.05). The results indicate that p16 may be important in the regression of IH endothelial cells and in anti-angiogenesis. There is a certain association between p16 expression and the regression of hemangioma. This provides a theoretical basis for the further study of the pathological mechanisms of p16 in hemangioma and potential gene therapies that may treat this disease.

  7. Evaluation of Affinity-Tagged Protein Expression Strategies using Local and Global Isotope Ratio Measurements

    SciTech Connect

    Hervey, IV, William Judson; Khalsa-Moyers, Gurusahai K; Lankford, Patricia K; Owens, Elizabeth T; McKeown, Catherine K; Lu, Tse-Yuan S; Foote, Linda J; Morrell-Falvey, Jennifer L; McDonald, W Hayes; Pelletier, Dale A; Hurst, Gregory {Greg} B

    2009-01-01

    Protein enrichments of engineered, affinity-tagged (or bait ) fusion proteins with interaction partners are often laden with background, non-specific proteins, due to interactions that occur in vitro as an artifact of the technique. Furthermore, the in vivo expression of the bait protein may itself affect physiology or metabolism. In this study, intrinsic affinity purification challenges were investigated in a model protein complex, DNA-dependent RNA polymerase (RNAP), encompassing chromosome- and plasmid-encoding strategies for bait proteins in two different microbial species: Escherichia coli and Rhodopseudomonas palustris. Isotope ratio measurements of bait protein expression strains relative to native, wild-type strains were performed by liquid chromatography tandem mass spectrometry (LC-MS-MS) to assess bait protein expression strategies in each species. Authentic interacting proteins of RNAP were successfully discerned from artifactual co-isolating proteins by the isotopic differentiation of interactions as random or targeted (I-DIRT) method (A. J. Tackett et al. J. Proteome Res. 2005, 4 (5), 1752-1756). To investigate broader effects of bait protein production in the bacteria, we compared proteomes from strains harboring a plasmid that encodes an affinity-tagged subunit (RpoA) of the RNAP complex with the corresponding wild-type strains using stable isotope metabolic labeling. The ratio of RpoA abundance in plasmid strains versus wild type was 0.8 for R. palustris and 1.7 for E. coli. While most other proteins showed no appreciable difference, proteins significantly increased in abundance in plasmid-encoded bait-expressing strains of both species included the plasmid encoded antibiotic resistance protein, GenR and proteins involved in amino acid biosynthesis. Together, these local, complex-specific and more global, whole proteome isotopic abundance ratio measurements provided a tool for evaluating both in vivo and in vitro effects of plasmid-encoding strategies for bait protein expression. This approach has the potential for enabling discovery of protein-protein interactions among the growing number of sequenced microbial species without the need for development of chromosomal insertion systems.

  8. Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity

    PubMed Central

    Lindinger, Peter W.; Christe, Martine; Eberle, Alex N.; Kern, Beatrice; Peterli, Ralph; Peters, Thomas; Jayawardene, Kamburapola J.I.; Fearnley, Ian M.; Walker, John E.

    2015-01-01

    Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender. PMID:26217759

  9. Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity.

    PubMed

    Lindinger, Peter W; Christe, Martine; Eberle, Alex N; Kern, Beatrice; Peterli, Ralph; Peters, Thomas; Jayawardene, Kamburapola J I; Fearnley, Ian M; Walker, John E

    2015-09-01

    Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender. PMID:26217759

  10. Codon Usage in Signal Sequences Affects Protein Expression and Secretion Using Baculovirus/Insect Cell Expression System

    PubMed Central

    Tao, Shiheng; Chen, Hongying

    2015-01-01

    By introducing synonymous mutations into the coding sequences of GP64sp and FibHsp signal peptides, the influences of mRNA secondary structure and codon usage of signal sequences on protein expression and secretion were investigated using baculovirus/insect cell expression system. The results showed that mRNA structural stability of the signal sequences was not correlated with the protein production and secretion levels, and FibHsp was more tolerable to codon changes than GP64sp. Codon bias analyses revealed that codons for GP64sp were well de-optimized and contained more non-optimal codons than FibHsp. Synonymous mutations in GP64sp sufficiently increased its average codon usage frequency and resulted in dramatic reduction of the activity and secretion of luciferase. Protein degradation inhibition assay with MG-132 showed that higher codon usage frequency in the signal sequence increased the production as well as the degradation of luciferase protein, indicating that the synonymous codon substitutions in the signal sequence caused misfolding of luciferase instead of slowing down the protein production. Meanwhile, we found that introduction of more non-optimal codons into FibHsp could increase the production and secretion levels of luciferase, which suggested a new strategy to improve the production of secretory proteins in insect cells. PMID:26697848

  11. A Hybrid Approach to Protein Differential Expression in Mass Spectrometry-Based Proteomics

    SciTech Connect

    Wang, Xuan; Anderson, Gordon A.; Smith, Richard D.; Dabney, Alan R.

    2012-04-19

    Motivation: Quantitative mass spectrometry-based proteomics involves statistical inference on protein abundance, based on the intensities of each protein's associated spectral peaks. However, typical MS-based proteomics data sets have substantial proportions of missing observations, due at least in part to censoring of low intensities. This complicates intensity-based differential expression analysis. Results: We outline a statistical method for protein differential expression, based on a simple Binomial likelihood. By modeling peak intensities as binary, in terms of 'presence/ absence,' we enable the selection of proteins not typically amendable to quantitative analysis; e.g., 'one-state' proteins that are present in one condition but absent in another. In addition, we present an analysis protocol that combines quantitative and presence/ absence analysis of a given data set in a principled way, resulting in a single list of selected proteins with a single associated FDR.

  12. Author's personal copy Protein expression following heat shock in the nervous system of Locusta migratoria

    E-print Network

    Robertson, Meldrum

    Author's personal copy Protein expression following heat shock in the nervous system of Locusta 9 August 2011 Keywords: Heat shock Nervous system Hsp70 Locusta migratoria Proteomics of Hsp70 transcript or protein in the nervous system. We compared Hsp70 increase following HS

  13. Papillomavirus Capsid Protein Expression in Escherichia coli: Purification and Assembly of HPV11

    E-print Network

    Harrison, Stephen C.

    Papillomavirus Capsid Protein Expression in Escherichia coli: Purification and Assembly of HPV11 proteins of human papillomavirus (HPV) types 11 and 16 were puri®ed and analyzed for structural integrity for vaccine design. # 2001 Academic Press Keywords: human papillomavirus; virus assembly; virus capsids

  14. Absolute protein expression profiling estimates the relative contributions of transcriptional and

    E-print Network

    (observed peptide count) by learned probabilities for identifying the peptides. APEX abundances agree a method for large-scale absolute protein expression measurements (APEX) and apply it to estimate and Escherichia coli proteomes. APEX relies upon correcting each protein's mass spectrometry sampling depth

  15. Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing.

    PubMed

    Yan, Chunlan; Wu, Wei; Li, Haiyan; Zhang, Guanglin; Duerksen-Hughes, Penelope J; Zhu, Xinqiang; Yang, Jun

    2010-04-01

    Benzo[a]pyrene (BaP) is a potent pro-carcinogen generated from the combustion of fossil fuel and cigarette smoke. Previously, using a proteomic approach, we have shown that BaP can induce changes in the expression of many cellular proteins, including transcription regulators. In the present study, using a similar approach, we examined the nuclear protein response to BaP in HeLa cells and found that BaP treatment caused expression changes in many nuclear proteins. Twenty-four of these proteins were successfully identified, several of which are involved in the alternative splicing of mRNA, DNA replication, recombination, and repair. The changed expression levels were further confirmed by immunoblot analysis using specific antibodies for two proteins, Lamin A and mitotic checkpoint protein Bub3. The nuclear localization of these two proteins was also confirmed by confocal microscopy. To determine whether alternative splicing was activated following BaP treatment, we examined Fas and CD44, two genes previously shown to be targets of alternative splicing in respond to DNA damage. While no significant activation of alternative splicing was observed for Fas, CD44 splicing variants were found after BaP treatment. Together, these data show that DNA damage induces dramatic changes in nuclear protein expression, and that alternative splicing might be involved in the cellular response to DNA damage. PMID:20097212

  16. Contrast Ultrasound Imaging Does Not AffectHeatShockProtein70Expression

    E-print Network

    Illinois at Urbana-Champaign, University of

    Contrast Ultrasound Imaging Does Not AffectHeatShockProtein70Expression in Cholesterol-Fed Rabbit of Nutritional Sciences (B.W.S., J.W.E., W.D.O.), and Departments of Statistics protein. Methods--Male New Zealand White rabbits (n = 32) were fed a standard chow diet (n = 4) or a 1

  17. A high-throughput protein expression system in Pichia pastoris using a newly developed episomal vector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe here the construction of a Gateway-compatible vector, pBGP1-DEST, for rapid and convenient preparation of expression plasmids for production of secretory proteins in Pichia pastoris. pBGP1-DEST directs the synthesis of a fusion protein consisting of the N-terminal signal and pro-sequence...

  18. Concordance of gene expression in human protein complexes reveals tissue specificity

    E-print Network

    specificity to groups of disease-linked genes and proteins is therefore in order. Recently, a strategyConcordance of gene expression in human protein complexes reveals tissue specificity and pathology June 22, 2013; Accepted July 4, 2013 ABSTRACT Disease-causing variants in human genes usually lead

  19. Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis (2D-GE) on whey samples from a group of 8 cows prior to and 18 hours after infection with Escherichia coli, and to identify differentially expressed milk proteins by peptide seq...

  20. Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement

    NASA Astrophysics Data System (ADS)

    Zhang, Yuanxing

    Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

  1. Expression of muscovy duck parvovirus capsid proteins (VP2 and VP3) in a baculovirus expression system and demonstration of immunity induced by the recombinant proteins.

    PubMed

    Le Gall-Reculé, G; Jestin, V; Chagnaud, P; Blanchard, P; Jestin, A

    1996-09-01

    The gene encoding the muscovy duck parvovirus (DPV)-strain 89384 capsid proteins VP2 and VP3 was cloned in a baculovirus expression system and expressed in insect cells. The recombinant proteins were found to react with specific anti-DPV serum by Western blotting and to be located in the nucleus of insect cells (Sf9) as shown by immunofluorescence. Empty virus-like particles (VLPs) identical in size and appearance to DPV virions were observed by electron microscopy. The antigenicity and immunogenicity of the recombinant proteins were evaluated by ELISA and seroneutralization. Immunization of 3-week-old muscovy ducklings induced anti-DPV antibodies; neutralizing antibody titres were consistent with those observed in ducklings inoculated with a commercial inactivated vaccine. The way to develop these promising results is discussed. PMID:8811015

  2. Foxn1 Protein Expression in the Developing, Aging, and Regenerating Thymus.

    PubMed

    Rode, Immanuel; Martins, Vera C; Küblbeck, Günter; Maltry, Nicole; Tessmer, Claudia; Rodewald, Hans-Reimer

    2015-12-15

    The forkhead box N1 (Foxn1) protein is the key regulator of thymic epithelial cell (TEC) development, yet how Foxn1 functions remains largely unknown. All mature TECs arise from Foxn1-expressing progenitors/immature TECs and it is widely assumed that TECs as a whole are defined by Foxn1 expression. However, data on the Foxn1 protein are virtually lacking. In this study, we developed novel tools to visualize Foxn1 protein expression at single-cell resolution. We generated Foxn1 knock-in mice expressing a C-terminal hemagglutinin-tagged Foxn1 protein, and a cytometry-grade monoclonal anti-Foxn1 Ab. We evaluated Foxn1 expression patterns in TEC subsets and its dynamics during normal thymus development, aging, injury, and regeneration. Upon challenges, upregulation of Foxn1 was a common feature of thymus regeneration, but the timing of Foxn1 expression changed and the responding TEC subsets depended on the type of treatment. Whereas dexamethasone and recombinant human fibroblast growth factor 7 promoted expansion of Foxn1(+)Ly51(+)CD80(-) TECs, castration led to expansion of Foxn1(+)Ly51(-)CD80(+) TECs. Collectively, Foxn1 expression is highly heterogeneous in the normal thymus, with large fractions of Foxn1(low) or Foxn1(-) TECs accumulating with age. Furthermore, Foxn1 expression is responsive to perturbations. PMID:26538393

  3. Dissecting the expression dynamics of RNA-binding proteins in posttranscriptional regulatory networks

    E-print Network

    Babu, M. Madan

    in pathological conditions. disease posttranslational modifications protein noise regulation systems biology Gene expression is a highly regulated process and is controlled at several levels. In eukaryotes, control of gene within the cell. However, a much less understood level of gene expression regulation, which occurs

  4. Human ?-Defensin Expression Is Not Dependent on CCAAT/Enhancer Binding Protein-? in a Murine Model

    PubMed Central

    Glenthøj, Andreas; Dahl, Sara; Larsen, Maria T.; Cowland, Jack B.; Borregaard, Niels

    2014-01-01

    Specific granule deficiency (SGD) is a rare congenital disorder characterized by recurrent infections. The disease is caused by inactivating mutations of the CCAAT/enhancer binding protein-? (C/EBP-?) gene. As a consequence, specific and gelatinase granules lack most matrix proteins. Furthermore, azurophil granules contain diminished amounts of their most abundant proteins, ?-defensins, also known as human neutrophil peptides (HNPs). In accordance with this, in vitro models have demonstrated induction of HNPs by C/EBP-?. Since mice do not express myeloid defensins, they cannot per se be used to characterize the role of C/EBP-? in controlling HNP expression in vivo. We therefore crossed a transgenic HNP-1-expressing mouse with the Cebpe-/- mouse to study the in vivo significance of C/EBP-? for HNP-1 transcription and expression. Surprisingly, neither expression nor processing of HNP-1 was affected by lack of C/EBP-? in these mice. Transduction of C/EBP-? into primary bone marrow cells from HNP-1 mice induced some HNP-1 expression, but not to levels comparable to expression human cells. Taken together, our data infer that the HNP-1 of the transgenic mouse does not show an expression pattern equivalent to endogenous secondary granule proteins. This limits the use of these transgenic mice as a model for human conditions. PMID:24658030

  5. Effect of Chemically-Induced, Cloned-Gene Expression on Protein Synthesis in Emcoh

    E-print Network

    Wood, Thomas K.

    Effect of Chemically-Induced, Cloned-Gene Expression on Protein Synthesis in Emcoh Thomas K. Wood investigated the metabolic limitations of cloned-gene expression in bacterial cells (for over-production of p cloned-geneexpression p-galactosidase INTRODUCTION As the biotechnology market matures, there is an in

  6. Using Biological Networks in Protein Function Prediction and Gene Expression Analysis

    E-print Network

    Wong, Limsoon

    and gene expression profile analysis that are based on biological networks. 1 Introduction Present for analyzing biological data over the past decade. These networks can be roughly categorized into the followingUsing Biological Networks in Protein Function Prediction and Gene Expression Analysis Limsoon Wong

  7. Amylase and cyclic amp receptor protein expression in human diabetic parotid glands

    E-print Network

    Terasaki, Mark

    Amylase and cyclic amp receptor protein expression in human diabetic parotid glands Monica Piras1 and molecular changes that occur in salivary glands during diabetes. METHODS: Biopsy samples of parotid glands gland samples. In type 1 diabetes amylase expression was greater than in non-diabetic glands, whereas

  8. The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

    PubMed

    Mlynek, Georg; Lehner, Anita; Neuhold, Jana; Leeb, Sarah; Kostan, Julius; Charnagalov, Alexej; Stolt-Bergner, Peggy; Djinovi?-Carugo, Kristina; Pinotsis, Nikos

    2014-06-01

    Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner. PMID:24647677

  9. Priming of protein expression in the defence response of Zantedeschia aethiopica to Pectobacterium carotovorum.

    PubMed

    Luzzatto-Knaan, Tal; Kerem, Zohar; Doron-Faigenboim, Adi; Yedidia, Iris

    2014-05-01

    The defence response of Zantedeschia aethiopica, a natural rhizomatous host of the soft rot bacterium Pectobacterium carotovorum, was studied following the activation of common induced resistance pathways—systemic acquired resistance and induced systemic resistance. Proteomic tools were used, together with in?vitro quantification and in?situ localization of selected oxidizing enzymes. In total, 527 proteins were analysed by label-free mass spectrometry (MS) and annotated against the National Center for Biotechnology Information (NCBI) nonredundant (nr) protein database of rice (Oryza sativa). Of these, the fore most differentially expressed group comprised 215 proteins that were primed following application of methyl jasmonate (MJ) and subsequent infection with the pathogen. Sixty-five proteins were down-regulated following MJ treatments. The application of benzothiadiazole (BTH) increased the expression of 23 proteins; however, subsequent infection with the pathogen repressed their expression and did not induce priming. The sorting of primed proteins by Gene Ontology protein function category revealed that the primed proteins included nucleic acid-binding proteins, cofactor-binding proteins, ion-binding proteins, transferases, hydrolases and oxidoreductases. In line with the highlighted involvement of oxidoreductases in the defence response, we determined their activities, priming pattern and localization in?planta. Increased activities were confined to the area surrounding the pathogen penetration site, associating these enzymes with the induced systemic resistance afforded by the jasmonic acid signalling pathway. The results presented here demonstrate the concerted priming of protein expression following MJ treatment, making it a prominent part of the defence response of Z.?aethiopica to P.?carotovorum. PMID:24822269

  10. Oscillatory kinetics of gene expression: Protein conversion and slow mRNA transport

    SciTech Connect

    Zhdanov, V. P.

    2009-06-15

    The negative feedback between mRNA and regulatory-protein production may result in oscillations in the kinetics of gene expression if the mRNA-protein interplay includes protein conversion. Using a mean-field kinetic model, we show that such oscillations can be amplified due to limitations of the mRNA transport between the nucleus and cytoplasm. This effect may be dramatic for the mRNA population in the nucleus.

  11. SLocX: Predicting Subcellular Localization of Arabidopsis Proteins Leveraging Gene Expression Data.

    PubMed

    Ryngajllo, Malgorzata; Childs, Liam; Lohse, Marc; Giorgi, Federico M; Lude, Anja; Selbig, Joachim; Usadel, Björn

    2011-01-01

    Despite the growing volume of experimentally validated knowledge about the subcellular localization of plant proteins, a well performing in silico prediction tool is still a necessity. Existing tools, which employ information derived from protein sequence alone, offer limited accuracy and/or rely on full sequence availability. We explored whether gene expression profiling data can be harnessed to enhance prediction performance. To achieve this, we trained several support vector machines to predict the subcellular localization of Arabidopsis thaliana proteins using sequence derived information, expression behavior, or a combination of these data and compared their predictive performance through a cross-validation test. We show that gene expression carries information about the subcellular localization not available in sequence information, yielding dramatic benefits for plastid localization prediction, and some notable improvements for other compartments such as the mitochondrion, the Golgi, and the plasma membrane. Based on these results, we constructed a novel subcellular localization prediction engine, SLocX, combining gene expression profiling data with protein sequence-based information. We then validated the results of this engine using an independent test set of annotated proteins and a transient expression of GFP fusion proteins. Here, we present the prediction framework and a website of predicted localizations for Arabidopsis. The relatively good accuracy of our prediction engine, even in cases where only partial protein sequence is available (e.g., in sequences lacking the N-terminal region), offers a promising opportunity for similar application to non-sequenced or poorly annotated plant species. Although the prediction scope of our method is currently limited by the availability of expression information on the ATH1 array, we believe that the advances in measuring gene expression technology will make our method applicable for all Arabidopsis proteins. PMID:22639594

  12. The choriocarcinoma cell line BeWo: syncytial fusion and expression of syncytium-specific proteins.

    PubMed

    Orendi, Kristina; Gauster, Martin; Moser, Gerit; Meiri, Hamutal; Huppertz, Berthold

    2010-11-01

    Fusion of the trophoblast-derived choriocarcinoma cell line BeWo can be triggered by forskolin. BeWo cells are regularly used as a cell culture model to mimic in vivo syncytialisation of placental villous trophoblast. The ? subunit of human chorionic gonadotropin (CGB), placental alkaline phosphatase as well as placental protein 13 (PP13, LGALS13) are exclusively expressed in the syncytiotrophoblast of the human placenta, and CGB is commonly used as a marker of syncytial differentiation. Here we tested the hypothesis that syncytial fusion precedes CGB and LGALS13 expression in trophoblast-derived BeWo cells. BeWo cells were cultured for 48?h in the presence or absence of forskolin and varying concentrations of H-89, a protein kinase A inhibitor that interferes with the forskolin-mediated pathway of syncytial fusion. LGALS13 and CGB expression were quantified by DELFIA and real-time PCR. Cell fusion was determined by morphological analysis and cell counting after immunofluorescence staining. In forskolin-stimulated BeWo cells that were hindered to fuse by treatment with H-89, levels of CGB protein expression were not altered, while LGALS13 protein and mRNA expression decreased significantly to control levels without forskolin. The LGALS13 protein expression data coincided with a significant decrease in syncytial fusion, while CGB protein expression was unaffected by rates of cell fusion and proliferation. We postulate that CGB protein expression is not necessarily linked to syncytial fusion, and thus CGB should be used with great caution as a marker of BeWo cell fusion. PMID:20696850

  13. Right ventricular protein expression profile in end-stage heart failure

    PubMed Central

    Su, Yan Ru; Chiusa, Manuel; Brittain, Evan; Hemnes, Anna R.; Absi, Tarek S.; Lim, Chee Chew

    2015-01-01

    Abstract Little is known about the right ventricular (RV) proteome in human heart failure (HF), including possible differences compared to the left ventricular (LV) proteome. We used 2-dimensional differential in-gel electrophoresis (pH: 4–7, 10–150 kDa), followed by liquid chromatography tandem mass spectrometry, to compare the RV and LV proteomes in 12 explanted human hearts. We used Western blotting and multiple-reaction monitoring for protein verification and RNA sequencing for messenger RNA and protein expression correlation. In all 12 hearts, the right ventricles (RVs) demonstrated differential expression of 11 proteins relative to the left ventricles (LVs), including lesser expression of CRYM, TPM1, CLU, TXNL1, and COQ9 and greater expression of TNNI3, SAAI, ERP29, ACTN2, HSPB2, and NDUFS3. Principal-components analysis did not suggest RV-versus-LV proteome partitioning. In the nonischemic RVs (n = 6), 7 proteins were differentially expressed relative to the ischemic RVs (n = 6), including increased expression of CRYM, B7Z964, desmin, ANXA5, and MIME and decreased expression of SERPINA1 and ANT3. Principal-components analysis demonstrated partitioning of the nonischemic and ischemic RV proteomes, and gene ontology analysis identified differences in hemostasis and atherosclerosis-associated networks. There were no proteomic differences between RVs with echocardiographic dysfunction (n = 8) and those with normal function (n = 4). Messenger RNA and protein expression did not correlate consistently, suggesting a major role for RV posttranscriptional protein expression regulation. Differences in contractile, cytoskeletal, metabolic, signaling, and survival pathways exist between the RV and the LV in HF and may be related to the underlying HF etiology and differential posttranscriptional regulation. PMID:26401249

  14. Right ventricular protein expression profile in end-stage heart failure.

    PubMed

    Su, Yan Ru; Chiusa, Manuel; Brittain, Evan; Hemnes, Anna R; Absi, Tarek S; Lim, Chee Chew; Di Salvo, Thomas G

    2015-09-01

    Little is known about the right ventricular (RV) proteome in human heart failure (HF), including possible differences compared to the left ventricular (LV) proteome. We used 2-dimensional differential in-gel electrophoresis (pH: 4-7, 10-150 kDa), followed by liquid chromatography tandem mass spectrometry, to compare the RV and LV proteomes in 12 explanted human hearts. We used Western blotting and multiple-reaction monitoring for protein verification and RNA sequencing for messenger RNA and protein expression correlation. In all 12 hearts, the right ventricles (RVs) demonstrated differential expression of 11 proteins relative to the left ventricles (LVs), including lesser expression of CRYM, TPM1, CLU, TXNL1, and COQ9 and greater expression of TNNI3, SAAI, ERP29, ACTN2, HSPB2, and NDUFS3. Principal-components analysis did not suggest RV-versus-LV proteome partitioning. In the nonischemic RVs (n = 6), 7 proteins were differentially expressed relative to the ischemic RVs (n = 6), including increased expression of CRYM, B7Z964, desmin, ANXA5, and MIME and decreased expression of SERPINA1 and ANT3. Principal-components analysis demonstrated partitioning of the nonischemic and ischemic RV proteomes, and gene ontology analysis identified differences in hemostasis and atherosclerosis-associated networks. There were no proteomic differences between RVs with echocardiographic dysfunction (n = 8) and those with normal function (n = 4). Messenger RNA and protein expression did not correlate consistently, suggesting a major role for RV posttranscriptional protein expression regulation. Differences in contractile, cytoskeletal, metabolic, signaling, and survival pathways exist between the RV and the LV in HF and may be related to the underlying HF etiology and differential posttranscriptional regulation. PMID:26401249

  15. MUC-1 mucin protein expression in B-cell lymphomas.

    PubMed

    Teruya-Feldstein, Julie; Donnelly, Gerard B; Goy, Andre; Hegde, Abhijith; Nanjangud, Gouri; Qin, Jing; Thaler, Howard; Gilles, Frederic; Dyomin, Vadim G; Lloyd, Kenneth O; Zelenetz, Andrew D; Houldsworth, Jane; Chaganti, R S K

    2003-03-01

    We have recently shown that MUC1, mapped to the chromosomal band 1q21, is rearranged or amplified in 15% of B-cell lymphomas and that rearrangement led to over-expression of MUC-1 mucin in a case of diffuse large B-cell lymphoma (DLBCL). To determine the incidence of MUC-1 mucin expression and its clinical significance in B-cell lymphomas, we investigated a panel of 113 cases by immunohistochemistry (IHC). MUC-1 mucin expression was detected in the majority of cases (92.9%), with moderate to high levels noted in 50.4% of all histologic subsets comprising DLBCL (82 cases), follicular lymphoma (FL) (15 cases), FL with transformation to DLBCL (4 cases), and other B-cell lymphomas (12 cases). No statistically significant correlation was found between MUC-1 mucin expression and MUC1 genomic status (amplification/rearrangement) evaluated by Southern blot analysis, and 1q21 abnormality by karyotypic analysis. For all cases, MUC-1 mucin expression correlated with a previous history of lymphoma (p=0.003). PMID:12610353

  16. Characterization of hepatitis C virus structural proteins with a recombinant baculovirus expression system.

    PubMed

    Hsu, H H; Donets, M; Greenberg, H B; Feinstone, S M

    1993-05-01

    We cloned and expressed the sequences encoding the structural proteins of the hepatitis C virus in a baculovirus eukaryotic expression system. Four recombinant constructs expressed sufficient hepatitis C virus-specific proteins in insect cell culture to allow analysis of protein cleavage, glycosylation and immunoreactivity. Using immunoblot analysis, we detected a 22-kD protein corresponding to the hepatitis C virus capsid protein cleaved from a larger precursor. Recombinant constructs encoding the presumptive envelope (E1) protein produced products ranging from 30 to 35 kD, whereas constructs encoding the presumptive E2/NS1 protein expressed products ranging in size from 68 to 73 kD. The recombinant envelope proteins were glycosylated, as shown by sensitivity to endoglycosidase F digestion, whereas the capsid was not. We examined the immunoreactivity of these recombinant proteins using sera from 50 patients chronically infected with HCV. Forty-seven of 50 of these sera contained antibodies against the capsid, 14 (28%) also had antibodies against E1 and at least 5 (10%) had antibody against E2/NS1. Forty-seven of 50 sera (94%) were viremic, as determined on hepatitis C virus polymerase chain reaction. The three sera that were hepatitis C virus polymerase chain reaction negative did not have envelope antibodies, whereas all sera that had envelope antibodies were also hepatitis C virus polymerase chain reaction positive. Thus antibodies to baculovirus-expressed hepatitis C virus structural proteins, including E1 and E2/NS1, are found in the presence of viremia. PMID:8387945

  17. Expression of a Recombinant Elastin-Like Protein in Pichia pastoris

    PubMed Central

    Sallach, Rory E.; Conticello, Vincent P.; Chaikof, Elliot L.

    2009-01-01

    The translation of highly repetitive gene sequences is often associated with reduced levels of protein expression and may be prone to mutational events. In this report, we describe a modified concatemerization strategy to construct a gene with enhanced sequence diversity that encodes a highly repetitive elastin-like protein polymer for expression in Pichia pastoris. Specifically, degenerate oligonucleotides were used to create a monomer library, which after concatemerization yielded a genetically nonrepetitive DNA sequence that encoded identical pentapeptide repeat sequences. By limiting genetic repetition, the risk of genetic deletions, rearrangements, or premature termination errors during protein synthesis is minimized. PMID:19827084

  18. Temozolomide Treatment for Pediatric Refractory Anaplastic Ependymoma with Low MGMT Protein Expression.

    PubMed

    Komori, Kazutoshi; Yanagisawa, Ryu; Miyairi, Yosuke; Sakashita, Kazuo; Shiohara, Masaaki; Fujihara, Ikuko; Morita, Daisuke; Nakamura, Tomohiko; Ogiso, Yoshifumi; Sano, Kenji; Shirahata, Mitsuaki; Fukuoka, Kohei; Ichimura, Koichi; Shigeta, Hiroaki

    2016-01-01

    The benefit of postoperative chemotherapy for anaplastic ependymoma remains unknown. We report two pediatric patients with refractory anaplastic ependymoma treated with temozolomide (TMZ). We did not detect O(6) -methylguanine-DNA methyltransferase (MGMT) promoter methylation in tumor samples; however, MGMT protein expression was low. With TMZ treatment, one patient had a 7-month complete remission; the other, stable disease for 15 months. Three other patients did not respond to TMZ; two had high and one low MGMT expression, and two showed no MGMT promoter methylation. These findings suggest that TMZ may be effective for pediatric refractory anaplastic ependymoma with low MGMT protein expression. Pediatr Blood Cancer © 2015 Wiley Periodicals, Inc. PMID:26305586

  19. Tissue-specific expression of a soybean hypersensitive-induced response (HIR) protein gene promoter.

    PubMed

    Koellhoffer, Jessica P; Xing, Aiqiu; Moon, Bryan P; Li, Zhongsen

    2015-02-01

    A Glycine max gene encoding a putative protein similar to hypersensitive-induced response proteins (HIR) was identified as a gene with preferred expressions in flowers and developing seeds by whole transcriptome gene expression profiling. Its promoter gm-hir1 was cloned and revealed to strongly express a fluorescence reporter gene primarily in integuments, anther tapetum, and seed coat with unique tissue-specificity. Expression in the inner integument was apparent prior to pollination, while expression in the outer integument started to develop from the micropylar end outward as the embryo matured. A 5'-deletion study showed that the promoter can be truncated to 600 bp long relative to the translation start site without affecting expression. A positive regulatory element was identified between 600 and 481 bp that controls expression in the inner integument, with no noticeable effect on expression in the outer integument or tapetum. Additionally, removal of the 5'UTR intron had no effect on levels or location of gm-hir1 expression while truncation to 370 bp resulted in a complete loss of expression suggesting that elements controlling both the outer integument and tapetum expression are located within the 481-370 bp region. PMID:25501569

  20. Ttyh1 protein is expressed in glia in vitro and shows elevated expression in activated astrocytes following status epilepticus.

    PubMed

    Wiernasz, Elzbieta; Kaliszewska, Aleksandra; Brutkowski, Wojciech; Bednarczyk, Joanna; Gorniak, Malgorzata; Kaza, Beata; Lukasiuk, Katarzyna

    2014-12-01

    In a previous study, we showed that Ttyh1 protein is expressed in neurons in vitro and in vivo in the form of punctuate structures, which are localized to neuropil and neuronal somata. Herein, we provide the first description of Ttyh1 protein expression in astrocytes, oligodendrocytes and microglia in vitro. Moreover, using double immunofluorescence, we show Ttyh1 protein expression in activated astrocytes in the hippocampus following amygdala stimulation-induced status epilepticus. We demonstrate that in migrating astrocytes in in vitro wound model Ttyh1 concentrates at the edges of extending processes. These data suggest that Ttyh1 not only participates in shaping neuronal morphology, as previously described, but may also play a role in the function of activated glia in brain pathology. To localize Ttyh1 expression in the cellular compartments of neurons and astrocytes, we performed in vitro double immunofluorescent staining using markers for the following subcellular structures: endoplasmic reticulum (GRP78), Golgi apparatus (GM130), clathrin-coated vehicles (clathrin), early endosomes (Rab5 and APPL2), recycling endosomes (Rab11), trans-Golgi network (TGN46), endoplasmic reticulum membrane (calnexin), late endosomes and lysosomes (LAMP1) and synaptic vesicles (synaptoporin and synaptotagmin 1). We found that Ttyh1 is present in the endoplasmic reticulum, Golgi apparatus and clathrin-coated vesicles (clathrin) in both neurons and astrocytes and also in late endosomes or lysosomes in astrocytes. The presence of Ttyh1 was negligible in early endosomes, recycling endosomes, trans-Golgi network, endoplasmic reticulum membrane and synaptic vesicles. PMID:25316497

  1. Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications

    PubMed Central

    Mohammadzadeh, Sara; Khabiri, Alireza; Roohvand, Farzin; Memarnejadian, Arash; Salmanian, Ali Hatef; Ajdary, Soheila; Ehsani, Parastoo

    2014-01-01

    Background: Hepatitis C virus (HCV) is major cause of liver cirrhosis in humans. HCV capsid (core) protein (HCVcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. Plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. Although invention of transgenic (stable) tobacco plants expressing HCVcp with proper antigenic properties was recently reported, no data for “transient-expression” that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for HCVcp. Objectives: The purpose of this study was to design a highly codon-optimized HCVcp gene for construction of an efficient transient-plant expression system for production of HCVcp with proper antigenic properties in a regional tobacco plant (Iranian Jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco. Materials and Methods: A codon-optimized gene encoding the Kozak sequence, 6xHis-tag, HCVcp (1-122) and KDEL peptide in tandem (from N- to C-terminal) was designed and inserted into potato virus-X (PVX) and classic pBI121 binary vectors in separate cloning reactions. The resulted recombinant plasmids were transferred into Agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. The effect of gene silencing suppressor P19 protein derived from tomato bushy stunt virus on the expression yield of HCVcp by each construct was also evaluated by co-infiltration in separate groups. The expressed HCVcp was evaluated by dot and western blotting and ELISA assays. Results: The codon-optimized gene had an increased adaptation index value (from 0.65 to 0.85) and reduced GC content (from 62.62 to 51.05) in tobacco and removed the possible deleterious effect of “GGTAAG” splice site in native HCVcp. Blotting assays via specific antibodies confirmed the expression of the 15 kDa HCVcp. The expression level of HCVcp was enhanced by 4-5 times in P19 co-agroinfiltrated plants with better outcomes for PVX, compared to pBI121 vector (0.022% versus 0.019% of the total soluble protein). The plant-derived HCVcp (pHCVcp) could properly identify the HCVcp antibody in HCV-infected human sera compared to Escherichia coli-derived HCVcp (eHCVcp), indicating its potential for diagnostic/immunization applications. Conclusions: By employment of gene optimization strategies, use of viral-based vectors and suppression of plant-derived gene silencing effect, efficient transient expression of HCVcp in tobacco with proper antigenic properties could be possible. PMID:25598788

  2. Atrazine Affects Phosphoprotein and Protein Expression in MCF-10A Human Breast Epithelial Cells

    PubMed Central

    Huang, Peixin; Yang, John; Song, Qisheng; Sheehan, David

    2014-01-01

    Atrazine, a member of the 2-chloro-s-triazine family of herbicides, is the most widely used pesticide in the world and often detected in agriculture watersheds. Although it was generally considered as an endocrine disruptor, posing a potential threat to human health, the molecular mechanisms of atrazine effects remain unclear. Using two-dimensional gel electrophoresis, we identified a panel of differentially expressed phosphoproteins and total proteins in human breast epithelial MCF-10A cells after being exposed to environmentally relevant concentrations of atrazine. Atrazine treatments for 6 h resulted in differential expression of 4 phosphoproteins and 8 total-proteins as compared to the control cells (>1.5-fold, p < 0.05). MALDI-TOF MS/MS analysis revealed that the differentially expressed proteins belong to various cellular compartments (nucleus, cytosol, membrane) and varied in function, including those regulating the stress response such as peroxiredoxin I, HSP70 and HSP27; structural proteins such as tropomyosin and profilin 1; and oncogenesis proteins such as ANP32A. Six of the 12 identified proteins were verified by quantitative PCR for their transcript levels. The most up-regulated phosphoprotein by atrazine treatment, ANP32A, was further analyzed for its expression, distribution and cellular localization using Western blot and immunocytochemical approaches. The results revealed that ANP32 expression after atrazine treatment increased dose and time dependently and was primarily located in the nucleus. This study may provide new evidence on the potential toxicity of atrazine in human cells. PMID:25275270

  3. Reduced expression of exocytotic proteins caused by anti-cholinesterase pesticides in Brachionus calyciflorus (Rotifera: Monogononta).

    PubMed

    Pérez-Legaspi, I A; Rico-Martínez, R; Quintanar, J L

    2015-08-01

    The organophosphate and carbamate pesticides methyl-parathion and carbaryl have a common action mechanism: they inhibit acetylcholinesterase enzyme by blocking the transmission of nerve impulses. However, they can alter the expression of exocytotic membrane proteins (SNARE), by modifying release of neurotransmitters and other substances. This study evaluated the adverse effects of the pesticides methyl-parathion and carbaryl on expression of SNARE proteins: Syntaxin-1, Syntaxin-4 and SNAP-23 in freshwater rotifer Brachionus calyciflorus. Protein expression of these three proteins was analyzed before and after exposure to these two pesticides by Western Blot. The expression of Syntaxin-1, Syntaxin-4 and SNAP-23 proteins in B. calyciflorussignificantly decreases with increasing concentration of either pesticides. This suggests that organophosphates and carbamates have adverse effects on expression of membrane proteins of exocytosis by altering the recognition, docking and fusion of presynaptic and vesicular membranes involved in exocytosis of neurotransmitters. Our results demonstrate that the neurotoxic effect of anticholinesterase pesticides influences the interaction of syntaxins and SNAP-25 and the proper assembly of the SNARE complex. PMID:26465735

  4. Small structural differences of targeted anti-tumor toxins result in strong variation of protein expression.

    PubMed

    Gilabert-Oriol, Roger; Thakur, Mayank; Weise, Christoph; Dernedde, Jens; von Mallinckrodt, Benedicta; Fuchs, Hendrik; Weng, Alexander

    2013-09-01

    Targeted anti-tumor toxins consist of a toxic functional moiety that is chemically linked or recombinantly fused to a cell-directing ligand. Ribosome-inactivating proteins (RIPs), especially type I RIPs such as saporin or dianthin, are commonly used as toxin components. Although expression of type I RIP-based fusion proteins is well reported, the achievement of higher protein yields in heterologous expression systems through innovative strategies is of major interest. In the present study, the targeted toxins (his)saporin-EGF (SE) and (his)dianthin-EGF (DE) were expressed as fusion proteins under identical expression conditions. However, the total amount of DE was nearly two-times higher than SE. The identity of the heterologously expressed targeted toxins was confirmed by mass spectrometric studies. Their biological specific activity, monitored in real time, was almost equal. Sequence alignment shows 84% identity and a structural comparison revealed five major differences, two of which affect the secondary structure resulting in a loop (SE) to ?-strand (DE) conversion and one introduces a gap in SE (after position 57). In conclusion, these structural variations resulted in different protein expression levels while codon usage and toxicity to bacteria were excluded as a cause. Minor structural differences identified in this study may be considered responsible for the protection of DE from bacterial proteases and therefore may serve as a lead to modify certain domains in type I RIP-based targeted toxins. PMID:23867360

  5. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated.

    PubMed

    Chung, Daehwan; Young, Jenna; Bomble, Yannick J; Vander Wall, Todd A; Groom, Joseph; Himmel, Michael E; Westpheling, Janet

    2015-01-01

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure and function of this important enzyme in biomass deconstruction. PMID:25799047

  6. Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

    PubMed Central

    Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

    1998-01-01

    A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

  7. Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins

    PubMed Central

    Saritas-Yildirim, Banu; Pliner, Hannah A.; Ochoa, Angelica; Silva, Elena M.

    2015-01-01

    Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development. PMID:26327321

  8. Expression and subcellular localisation of poly(A)-binding proteins 

    E-print Network

    Burgess, Hannah

    2010-11-24

    Poly(A)-binding proteins (PABPs) are important regulators of mRNA translation and stability. In mammals four cytoplasmic PABPs with a similar domain structure have been described - PABP1, tPABP, PABP4 and ePABP. The ...

  9. Expression determinants of mammalian argonaute proteins in mediating gene silencing

    E-print Network

    Kay, Mark A.

    . INTRODUCTION Argonaute (Ago) proteins are essential for one of the last steps in the microRNA (miRNA typically commences with RNA polymerase II-mediated transcription of primary microRNA transcripts (pri-micro at Chapel Hill, Chapel Hill, NC, USA Received August 6, 2011; Accepted December 8, 2011 ABSTRACT RNA

  10. Circadian clock protein cryptochrome regulates the expression of proinflammatory cytokines.

    PubMed

    Narasimamurthy, Rajesh; Hatori, Megumi; Nayak, Surendra K; Liu, Fei; Panda, Satchidananda; Verma, Inder M

    2012-07-31

    Chronic sleep deprivation perturbs the circadian clock and increases susceptibility to diseases such as diabetes, obesity, and cancer. Increased inflammation is one of the common underlying mechanisms of these diseases, thus raising a hypothesis that circadian-oscillator components may regulate immune response. Here we show that absence of the core clock component protein cryptochrome (CRY) leads to constitutive elevation of proinflammatory cytokines in a cell-autonomous manner. We observed a constitutive NF-?B and protein kinase A (PKA) signaling activation in Cry1(-/-);Cry2(-/-) cells. We further demonstrate that increased phosphorylation of p65 at S276 residue in Cry1(-/-);Cry2(-/-) cells is due to increased PKA signaling activity, likely induced by a significantly high basal level of cAMP, which we detected in these cells. In addition, we report that CRY1 binds to adenylyl cyclase and limits cAMP production. Based on these data, we propose that absence of CRY protein(s) might release its (their) inhibition on cAMP production, resulting in elevated cAMP and increased PKA activation, subsequently leading to NF-?B activation through phosphorylation of p65 at S276. These results offer a mechanistic framework for understanding the link between circadian rhythm disruption and increased susceptibility to chronic inflammatory diseases. PMID:22778400

  11. Elevated Fis Expression Enhances Recombinant Protein Production in

    E-print Network

    Chen, Wilfred

    ,1 Tin Htay,1 Pauli Kallio,2 Wilfred Chen1 1 Department of Chemical Engineering, University_Chen@qmail.ucr.edu; 2 Institute of Biotechnology, ETH-Honggerberg, CH-8093, Zurich, Switzerland. Received 18 October 1996; accepted 14 February 1997 Abstract: A genetic strategy to enhance recombinant protein production

  12. Characterization of giardin protein expression during encystation of Giardia duodenalis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Giardia duodenalis trophozoites attach to the gut surface by means of a ventral disk that contains various giardin proteins that appear to be important to VD structural integrity. One approach to preventing giardiasis is to stimulate giardin-specific antibodies and thereby block trophozoite attachme...

  13. Steroidogenic Acute Regulatory Protein Expression in the Central Nervous System

    PubMed Central

    King, Steven R.; Stocco, Douglas M.

    2011-01-01

    Locally produced neurosteroids are proposed to have many functions in the central nervous system. The identification of the steroidogenic acute regulatory protein in steroid-producing neural cells provides a new tool to understand the sites, regulation, and importance of their synthesis. PMID:22649383

  14. JAZ (Znf346), a SIRT1-interacting protein, protects neurons by stimulating p21 (WAF/CIP1) protein expression.

    PubMed

    Mallick, Sathi; D'Mello, Santosh R

    2014-12-19

    SIRT1, a class III histone deacetylase, protects neurons in various models of neurodegenerative diseases. We previously described that neuroprotection by SIRT1 is independent of its catalytic activity. To elucidate how SIRT1 protects neurons, we performed a mass spectrometric screen to find SIRT1-interacting proteins. One of the proteins identified was JAZ (Znf346), a member of a new class of Cys-2-His-2 zinc finger proteins. To investigate the significance of JAZ in the regulation of neuronal survival, we overexpressed it in neurons. We found that JAZ protects cerebellar granule neurons against potassium deprivation-induced death and cortical neurons from death resulting from oxidative stress. JAZ also protects neurons against toxicity induced by mutant huntingtin and mutant ataxin-1 expression. Although expression of endogenous JAZ does not change in neurons primed to die, knockdown of its expression promotes death of otherwise healthy neurons. In contrast to its protective effect in neurons, overexpression of JAZ in different cell lines promotes death. We find that JAZ suppresses cell cycle progression, thereby explaining its contrasting effect in postmitotic neurons versus proliferating cell lines. Although not affecting the expression of several cyclins, overexpression of JAZ stimulates expression of p21 (WAF1/CIP1), a cell cycle inhibitor known to have neuroprotective effects. Results of chromatin immunoprecipitation and transcriptional assays indicate that the stimulatory effect of JAZ on p21 expression is mediated at the transcriptional level. Furthermore, knockdown of p21 expression inhibits the neuroprotective effect of JAZ. Together, our results suggest that JAZ protects neurons by inhibiting cell cycle re-entry through the transcriptional stimulation of p21 expression. PMID:25331946

  15. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

    PubMed Central

    Besser, Stefanie; Sicker, Marit; Marx, Grit; Winkler, Ulrike; Eulenburg, Volker; Hülsmann, Swen; Hirrlinger, Johannes

    2015-01-01

    GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type. PMID:26076353

  16. Functional evaluation of candidate ice structuring proteins using cell-free expression systems.

    PubMed

    Brödel, A K; Raymond, J A; Duman, J G; Bier, F F; Kubick, S

    2013-02-10

    Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications. PMID:23195406

  17. Gene expression profiles and protein–protein interaction network analysis in AIDS patients with HIV-associated encephalitis and dementia

    PubMed Central

    Shityakov, Sergey; Dandekar, Thomas; Förster, Carola

    2015-01-01

    Central nervous system dysfunction is an important cause of morbidity and mortality in patients with human immunodeficiency virus type 1 (HIV-1) infection and acquired immunodeficiency virus syndrome (AIDS). Patients with AIDS are usually affected by HIV-associated encephalitis (HIVE) with viral replication limited to cells of monocyte origin. To examine the molecular mechanisms underlying HIVE-induced dementia, the GSE4755 Affymetrix data were obtained from the Gene Expression Omnibus database and the differentially expressed genes (DEGs) between the samples from AIDS patients with and without apparent features of HIVE-induced dementia were identified. In addition, protein–protein interaction networks were constructed by mapping DEGs into protein–protein interaction data to identify the pathways that these DEGs are involved in. The results revealed that the expression of 1,528 DEGs is mainly involved in the immune response, regulation of cell proliferation, cellular response to inflammation, signal transduction, and viral replication cycle. Heat-shock protein alpha, class A member 1 (HSP90AA1), and fibronectin 1 were detected as hub nodes with degree values >130. In conclusion, the results indicate that HSP90A and fibronectin 1 play important roles in HIVE pathogenesis. PMID:26604827

  18. The adenovirus E1B 19-kilodalton protein stimulates gene expression by increasing DNA levels.

    PubMed Central

    Herrmann, C H; Mathews, M B

    1989-01-01

    In transient expression assays, the adenovirus E1B 19-kilodalton (19K) tumor antigen increases expression from viral promoters and the promoter for the cellular 70-kilodalton heat shock protein (hsp70). To study the mechanism of this effect, we constructed HeLa cell lines that contain stably integrated copies of the 19K gene. Compared with a 19K- control cell line, 19K+ cells produced a significantly higher level of expression from every promoter introduced into the cells by transfection. The 19K protein also increased expression of an RNA polymerase III-transcribed gene but did not affect the level of expression of the endogenous hsp70 gene. The rate of transcription from transfected promoters, as measured by a nuclear run-on assay, was higher in the 19K+ cells than in the 19K- control cells. Furthermore, the level of plasmid DNA remained higher in the 19K+ cell line, suggesting that the 19K protein stabilizes transfected plasmid DNA. The elevated DNA levels seemed to account in full for the increased transcription. The role of the 19K protein in increasing gene expression during viral infection was found to be due to a replication-dependent increase in viral DNA levels. Thus, the 19K protein activates transcription indirectly by producing a higher level of viral or plasmid DNA. The DNA stabilization function of the 19K protein is probably related to the protective role of the 19K protein during viral infection and represents the first example of a viral oncogene product that modulates gene expression by regulating viral and plasmid DNA levels. Images PMID:2531284

  19. Expression patterns of protein kinase D 3 during mouse development

    PubMed Central

    Ellwanger, Kornelia; Pfizenmaier, Klaus; Lutz, Sylke; Hausser, Angelika

    2008-01-01

    Background The PKD family of serine/threonine kinases comprises a single member in Drosophila (dPKD), two isoforms in C. elegans (DKF-1 and 2) and three members, PKD1, PKD2 and PKD3 in mammals. PKD1 and PKD2 have been the focus of most studies up to date, which implicate these enzymes in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, immune responses, apoptosis and cell proliferation. Concerning PKD3, a role in the formation of vesicular transport carriers at the trans-Golgi network (TGN) and in basal glucose transport has been inferred from in vitro studies. So far, however, the physiological functions of the kinase during development remain unknown. Results We have examined the expression pattern of PKD3 during the development of mouse embryos by immunohistochemistry. Using a PKD3 specific antibody we demonstrate that the kinase is differentially expressed during organogenesis. In the developing heart a strong PKD3 expression is constantly detected from E10 to E16.5. From E12.5 on PKD3 is increasingly expressed in neuronal as well as in the supporting connective tissue and in skeletal muscles. Conclusion The data presented support an important role for PKD3 during development of these tissues. PMID:18439271

  20. Coat protein expression strategy of oat blue dwarf virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oat blue dwarf virus (OBDV) was the first marafivirus (family Tymoviridae) to be sequenced and for which an infectious clone has been reported. Although sequence data are now available for multiple marafiviruses, precise details of the expression strategy of these viruses remain undocumented. Tran...

  1. Ultrafast surface hydration dynamics and expression of protein functionality: ?-Chymotrypsin

    PubMed Central

    Pal, Samir Kumar; Peon, Jorge; Zewail, Ahmed H.

    2002-01-01

    We report studies of hydration dynamics at the surface of the enzyme protein bovine pancreatic ?-chymotrypsin. The probe is the well known 1-anilinonaphthalene-8-sulfonate, which binds selectively in the native state of the protein, not the molten globule, as shown by x-ray crystallography. With femtosecond time resolution, we examined the hydration dynamics at two pHs, when the protein is physiologically in the inactive state (pH 3.6) or the active state (pH 6.7); the global structure and the binding site remain the same. The hydration correlation function, C(t), whose decay is governed by the rotational and translational motions of water molecules at the site, shows the behavior observed in this laboratory for other proteins, Subtilisin Carlsberg and Monellin, using the intrinsic amino acid tryptophan as a probe for surface hydration. However, the time scales and amplitudes vary drastically at the two pHs. For the inactive protein state, C(t) decays with an ultrafast component, close to bulk-type behavior, but 50% of the C(t) decays at a much slower rate, ? = 43 ps. In contrast, for the active state, the ultrafast component becomes dominant (90%) and the slow component changes to a faster decay, ? = 28 ps. These results indicate that in the active state water molecules in the hydration layer around the site have a high degree of mobility, whereas in the inactive state the water is more rigidly structured. For the substrate–enzyme complex, the function and dynamics at the probe site are correlated, and the relevance to the enzymatic action is clear. PMID:12427971

  2. Construction and expression of prokaryotic expression vectors fused with genes of Magnaporthe oryzae effector proteins and mCherry.

    PubMed

    Yang, Y Q; Wang, H; Liang, M L; Yan, J L; Liu, L; Li, C Y; Yang, J

    2015-01-01

    The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors. PMID:26400311

  3. Application of Cydia pomonella expressed sequence tags: Identification and expression of three general odorant binding proteins in codling moth.

    PubMed

    Garczynski, Stephen F; Coates, Brad S; Unruh, Thomas R; Schaeffer, Scott; Jiwan, Derick; Koepke, Tyson; Dhingra, Amit

    2013-10-01

    The codling moth, Cydia pomonella, is one of the most important pests of pome fruits in the world, yet the molecular genetics and the physiology of this insect remain poorly understood. A combined assembly of 8?341 expressed sequence tags was generated from Roche 454 GS-FLX sequencing of eight tissue-specific cDNA libraries. Putative chemosensory proteins (12) and odorant binding proteins (OBPs) (18) were annotated, which included three putative general OBP (GOBP), one more than typically reported for other Lepidoptera. To further characterize CpomGOBPs, we cloned cDNA copies of their transcripts and determined their expression patterns in various tissues. Cloning and sequencing of the 698?nt transcript for CpomGOBP1 resulted in the prediction of a 163 amino acid coding region, and subsequent RT-PCR indicated that the transcripts were mainly expressed in antennae and mouthparts. The 1?289 nt (160 amino acid) CpomGOBP2 and the novel 702 nt (169 amino acid) CpomGOBP3 transcripts are mainly expressed in antennae, mouthparts, and female abdomen tips. These results indicate that next generation sequencing is useful for the identification of novel transcripts of interest, and that codling moth expresses a transcript encoding for a new member of the GOBP subfamily. PMID:23956229

  4. Cigarette smoke induces the expression of Notch3, not Notch1, protein in lung adenocarcinoma

    PubMed Central

    CHENG, ZHENSHUN; TAN, QIUYUE; TAN, WEIJUN; ZHANG, LI

    2015-01-01

    The aim of the present study was to determine the effect of cigarette smoke on the expression of Notch proteins in lung adenocarcinoma (LAC). Protein expression levels of Notch1 and Notch3 were analyzed using immunohistochemistry in 102 human LAC specimens. Of these, 52 were obtained from smokers and 50 from non-smokers. In addition, cigarette smoke extract (CSE) at varying concentrations (1, 2.5 and 5%) was administered to A549 cells. The expression of Notch1 and Notch3 protein was then detected by western blot analysis at different time points (0, 8, 24 and 48 h). Of the 102 LAC specimens, 42 (41.2%) were positive for Notch1 and 63 (61.8%) were positive for Notch3. There was no significant difference in the level of Notch1 expression between smokers and non-smokers with LAC (P>0.05). The positive rate and staining intensity of Notch3 expression were increased in the smokers compared with the non-smokers (P<0.05). The expression of Notch3 protein in A549 cells increased in a time- and dose-dependent manner following treatment with CSE, whilst the expression of Notch1 protein appeared stable. The results suggested that cigarette smoke was able to induce the expression of Notch3, not Notch1, protein in LAC. The data revealed an upregulation of Notch3 in LAC following cigarette smoke exposure. Such findings may provide a novel therapeutic target for the treatment of LAC. PMID:26622547

  5. Expression, immunolocalization and serodiagnostic value of a myophilin-like protein from Schistosoma japonicum

    PubMed Central

    Peng, Hailin; Song, Kai; Huang, Chengyu; Ye, Sai; Song, Huaiguang; Hu, Wei; Han, Zeguang; McManus, Donald P.; Zhao, Guoping; Zhang, Qinghua

    2008-01-01

    The cDNA of a Schistosoma japonicum myophilin-like protein was cloned, sequenced, and expressed in Escherichia coli as a recombined protein (rSj myophilin-like protein), and the protein was purified by affinity chromatography. The deduced amino acid sequences of the Sj myophilin-like protein showed significant homology to myophilin, calponin, Np22 and Mp20. Northern blot and RT-PCR analyzes revealed expression of the Sj myophilin-like protein mRNA in eggs, sporocysts, cercariae, hepatic schistosomula and adult worms. Confocal fluorescence microscopy localized the native protein to the muscle of the adult worm. In schistosome-infected rabbits, the rSj myophilin-like protein antibody level, assessed by ELISA, was elevated after infection but was reduced after praziquantel treatment. In humans, the myophilin-like protein antibody level was evaluated by ELISA in sera from 33 non-infected humans and 61 schistosomiasis patients; the results showed a highly significant difference between the two groups with a sensitivity of 57.4%. Taken together, the myophilin-like protein may prove useful for monitoring the therapeutic effect of praziquantel rather than in serodiagnosis of schistosomiasis. PMID:18329020

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

    PubMed Central

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

    2013-01-01

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

  7. A protein tagging system for signal amplification in gene expression and fluorescence imaging

    PubMed Central

    Tanenbaum, Marvin E.; Gilbert, Luke A.; Qi, Lei S.; Weissman, Jonathan S.; Vale, Ronald D.

    2014-01-01

    Summary Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a novel protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and in controlling biological outputs. PMID:25307933

  8. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    NASA Astrophysics Data System (ADS)

    Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

    2011-06-01

    Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

  9. A protein-tagging system for signal amplification in gene expression and fluorescence imaging.

    PubMed

    Tanenbaum, Marvin E; Gilbert, Luke A; Qi, Lei S; Weissman, Jonathan S; Vale, Ronald D

    2014-10-23

    Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs. PMID:25307933

  10. Lambda foo: a lambda phage vector for the expression of foreign proteins.

    PubMed Central

    Maruyama, I N; Maruyama, H I; Brenner, S

    1994-01-01

    This work describes a lambda phage expression system, lambda foo, that produces foreign proteins fused to the surface of the virus particle. The lambda foo vector has multiple cloning sites for the insertion of a foreign DNA fragment and color selection for recombinants. Foreign proteins are fused to the C terminus of a truncated phage tail protein, pV, by a peptide linker. Conditional chain termination allows the assembly and fusion of multisubunit proteins. We have attached the complete Escherichia coli beta-galactosidase and the plant Bauhinia purpurea agglutinin by cloning their genes into the vector. The constructs express functionally active proteins on the phage particle surface and have been purified by affinity chromatography with an antibody for beta-galactosidase and a mucin as a ligand for Bauhinia purpurea agglutinin. Images PMID:8058794

  11. Soluble expression of proteins correlates with a lack of positively-charged surface

    NASA Astrophysics Data System (ADS)

    Chan, Pedro; Curtis, Robin A.; Warwicker, Jim

    2013-11-01

    Prediction of protein solubility is gaining importance with the growing use of protein molecules as therapeutics, and ongoing requirements for high level expression. We have investigated protein surface features that correlate with insolubility. Non-polar surface patches associate to some degree with insolubility, but this is far exceeded by the association with positively-charged patches. Negatively-charged patches do not separate insoluble/soluble subsets. The separation of soluble and insoluble subsets by positive charge clustering (area under the curve for a ROC plot is 0.85) has a striking parallel with the separation that delineates nucleic acid-binding proteins, although most of the insoluble dataset are not known to bind nucleic acid. Additionally, these basic patches are enriched for arginine, relative to lysine. The results are discussed in the context of expression systems and downstream processing, contributing to a view of protein solubility in which the molecular interactions of charged groups are far from equivalent.

  12. Probing Gene Expression in Live Cells, One Protein Molecule at a Time

    NASA Astrophysics Data System (ADS)

    Yu, Ji; Xiao, Jie; Ren, Xiaojia; Lao, Kaiqin; Xie, X. Sunney

    2006-03-01

    We directly observed real-time production of single protein molecules in individual Escherichia coli cells. A fusion protein of a fast-maturing yellow fluorescent protein (YFP) and a membrane-targeting peptide was expressed under a repressed condition. The membrane-localized YFP can be detected with single-molecule sensitivity. We found that the protein molecules are produced in bursts, with each burst originating from a stochastically transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. The quantitative study of low-level gene expression demonstrates the potential of single-molecule experiments in elucidating the workings of fundamental biological processes in living cells.

  13. The Effect of ?-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris

    PubMed Central

    Lin-Cereghino, Geoff P.; Stark, Carolyn M.; Kim, Daniel; Chang, Jennifer; Shaheen, Nadia; Poerwanto, Hansel; Agari, Kimiko; Moua, Pachai; Low, Lauren K.; Tran, Namphuong; Huang, Amy D.; Nattestad, Maria; Oshiro, Kristin T.; Chang, John William; Chavan, Archana; Tsai, Jerry W.; Lin-Cereghino, Joan

    2013-01-01

    The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae ?-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the ?-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of ?-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. PMID:23454485

  14. Effect of thermal stress on protein expression in the mussel Mytilus galloprovincialis Lmk.

    PubMed

    González-Riopedre, M; Novás, A; Dobaño, E; Ramos-Martínez, J I; Barcia, R

    2007-07-01

    The exposure of organisms to stressing agents may affect the level and pattern of protein expression. Certain proteins with an important role in protein homeostasis and in the tolerance to stress, known as stress proteins, are especially affected. Different tissues and cells show a range of sensitivities to stress, depending on the habitat to which organisms have adapted. The response of different tissues and cells from the mussel Mytilus galloprovincialis Lmk. to heat shock has been studied in this work using different exposure times and temperatures. During the assays, protein expression was observed to vary depending on the tissue studied, the temperature or the exposure time used. But maybe the most prominent thing is the different response obtained from the cultured haemocytes and those freshly obtained from stressed mussels, which makes us think that the extraction procedure is the main cause of the response of non-cultured cells, although the haemolymph may contain components that modulate haemocyte response. PMID:17462933

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

    PubMed Central

    Av-Gay, Yossef; Jamil, Sarwat; Drews, Steven J.

    1999-01-01

    PknB is a member of the newly discovered eukaryotic-like protein serine/threonine kinase (PSTK) family of proteins. The pknB gene was cloned and expressed in Escherichia coli. The active recombinant protein was purified and shown to be reactive with antiphosphoserine antibodies, as well as with antibodies to the phosphorylated eukaryotic Ser/Thr kinases mitogen-activated protein kinase kinase 3 and 6, P38, and Creb. In vitro kinase assays demonstrated that PknB is a functional kinase that is autophosphorylated on serine/threonine residues and is also able to phosphorylate the peptide substrate myelin basic protein. Analysis of pknB expression in Mycobacterium tuberculosis indicates the presence of pknB mRNA in (i) organisms grown in vitro in bacteriological media, (ii) a murine macrophage in vitro infection model, and (iii) in vivo alveolar macrophages from a patient with tuberculosis. PMID:10531215

  16. Serum Immune-Related Proteins are Differentially Expressed during Hibernation in the American Black Bear

    PubMed Central

    Chow, Brian A.; Donahue, Seth W.; Vaughan, Michael R.; McConkey, Brendan; Vijayan, Mathilakath M.

    2013-01-01

    Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (?25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included ?2-macroglobulin, complement components C1s and C4, immunoglobulin ? and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, ?2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears. PMID:23825529

  17. Computational classification models for predicting the interaction of drugs with P-glycoprotein and breast cancer resistance protein.

    PubMed

    Eri?, S; Kalini?, M; Ili?, K; Zloh, M

    2014-01-01

    P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) are two members of the adenosine triphosphate (ATP) binding cassette (ABC) family of transporters which function as membrane efflux transporters and display considerable substrate promiscuity. Both are known to significantly influence the absorption, distribution and elimination of drugs, mediate drug-drug interactions and contribute to multiple drug resistance (MDR) of cancer cells. Correspondingly, timely characterization of the interaction of novel leads and drug candidates with these two transporters is of great importance. In this study, several computational classification models for prediction of transport and inhibition of P-gp and BCRP, respectively, were developed based on newly compiled and critically evaluated experimental data. Artificial neural network (ANN) and support vector machine (SVM) ensemble based models were explored, as well as knowledge-based approaches to descriptor selection. The average overall classification accuracy of best performing models was 82% for P-gp transport, 88% for BCRP transport, 89% for P-gp inhibition and 87% for BCRP inhibition, determined across an array of different test sets. An analysis of substrate overlap between P-gp and BCRP was also performed. The accuracy, simplicity and interpretability of the proposed models suggest that they could be of significant utility in the drug discovery and development settings. PMID:25435255

  18. Construction and Analysis of the Protein-Protein Interaction Networks Based on Gene Expression Profiles of Parkinson's Disease

    PubMed Central

    Rakshit, Hindol; Rathi, Nitin; Roy, Debjani

    2014-01-01

    Background Parkinson's Disease (PD) is one of the most prevailing neurodegenerative diseases. Improving diagnoses and treatments of this disease is essential, as currently there exists no cure for this disease. Microarray and proteomics data have revealed abnormal expression of several genes and proteins responsible for PD. Nevertheless, few studies have been reported involving PD-specific protein-protein interactions. Results Microarray based gene expression data and protein-protein interaction (PPI) databases were combined to construct the PPI networks of differentially expressed (DE) genes in post mortem brain tissue samples of patients with Parkinson's disease. Samples were collected from the substantia nigra and the frontal cerebral cortex. From the microarray data, two sets of DE genes were selected by 2-tailed t-tests and Significance Analysis of Microarrays (SAM), run separately to construct two Query-Query PPI (QQPPI) networks. Several topological properties of these networks were studied. Nodes with High Connectivity (hubs) and High Betweenness Low Connectivity (bottlenecks) were identified to be the most significant nodes of the networks. Three and four-cliques were identified in the QQPPI networks. These cliques contain most of the topologically significant nodes of the networks which form core functional modules consisting of tightly knitted sub-networks. Hitherto unreported 37 PD disease markers were identified based on their topological significance in the networks. Of these 37 markers, eight were significantly involved in the core functional modules and showed significant change in co-expression levels. Four (ARRB2, STX1A, TFRC and MARCKS) out of the 37 markers were found to be associated with several neurotransmitters including dopamine. Conclusion This study represents a novel investigation of the PPI networks for PD, a complex disease. 37 proteins identified in our study can be considered as PD network biomarkers. These network biomarkers may provide as potential therapeutic targets for PD applications development. PMID:25170921

  19. Conserved role for the Dachshund protein with Drosophila Pax6 homolog Eyeless in insulin expression

    PubMed Central

    Okamoto, Naoki; Nishimori, Yuka; Nishimura, Takashi

    2012-01-01

    Members of the insulin family peptides have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. The Drosophila genome encodes seven insulin-like peptide genes, dilp1–7, and the most prominent dilps (dilp2, dilp3, and dilp5) are expressed in brain neurosecretory cells known as “insulin-producing cells” (IPCs). Although these dilps are expressed in the same cells, the expression of each dilp is regulated independently. However, the molecular mechanisms that regulate the expression of individual dilps in the IPCs remain largely unknown. Here, we show that Dachshund (Dac), which is a highly conserved nuclear protein, is a critical transcription factor that specifically regulates dilp5 expression. Dac was strongly expressed in IPCs throughout development. dac loss-of-function analyses revealed a severely reduced dilp5 expression level in young larvae. Dac interacted physically with the Drosophila Pax6 homolog Eyeless (Ey), and these proteins synergistically promoted dilp5 expression. In addition, the mammalian homolog of Dac, Dach1/2, facilitated the promoting action of Pax6 on the expression of islet hormone genes in cultured mammalian cells. These observations indicate the conserved role of Dac/Dach in controlling insulin expression in conjunction with Ey/Pax6. PMID:22308399

  20. Fusion protein strategy to increase expression and solubility of hypervariable region of VP2 protein of infectious bursal disease virus in Escherichia coli.

    PubMed

    Sedighzadeh, Sahar Sadat; Shamsara, Mehdi; Shahpiri, Azar

    2012-10-01

    Infectious bursal disease is one of the most important viral diseases in the young chickens. VP2 protein is the major host protective immunogen of the virus. A hypervariable region is present in VP2 protein (hvVP2) that contains immunodominant epitops. The high hydrophobicity of hvVP2 region causes protein aggregation in Escherichia coli (E. coli). The objective of the present study was to improve the expression and the solubility of the hvVP2 protein in E. coli. The effects of fusion partners on the solubility of hvVP2 protein were studied. The protein was expressed in forms of unfused and N-terminally fused to GST and NusA. The results showed that the unfused hvVP2 protein was expressed in very low level. But, N-terminally fused hvVP2 protein to GST (glutathione-S-transferase) and NusA (N utilization substance A) showed significantly enhanced protein expression. The fusion of GST and hvVP2 was produced in aggregated form while in the presence of NusA, the hvVP2 protein was expressed in a soluble form. The NusA-hvVP2 protein was detected by a neutralizing monoclonal antibody, 1A6, in antigen-capture ELISA. In conclusion, the NusA protein is a suitable fusion partner to improve expression and solubility of the hvVP2 protein in E. coli. PMID:22865249

  1. Interaction of drugs of abuse and maintenance treatments with human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2).

    PubMed

    Tournier, Nicolas; Chevillard, Lucie; Megarbane, Bruno; Pirnay, Stéphane; Scherrmann, Jean-Michel; Declèves, Xavier

    2010-08-01

    Drug interaction with P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) may influence its tissue disposition including blood-brain barrier transport and result in potent drug-drug interactions. The limited data obtained using in-vitro models indicate that methadone, buprenorphine, and cannabinoids may interact with human P-gp; but almost nothing is known about drugs of abuse and BCRP. We used in vitro P-gp and BCRP inhibition flow cytometric assays with hMDR1- and hBCRP-transfected HEK293 cells to test 14 compounds or metabolites frequently involved in addiction, including buprenorphine, norbuprenorphine, methadone, ibogaine, cocaine, cocaethylene, amphetamine, N-methyl-3,4-methylenedioxyamphetamine, 3,4-methylenedioxyamphetamine, nicotine, ketamine, Delta9-tetrahydrocannabinol (THC), naloxone, and morphine. Drugs that in vitro inhibited P-gp or BCRP were tested in hMDR1- and hBCRP-MDCKII bidirectional transport studies. Human P-gp was significantly inhibited in a concentration-dependent manner by norbuprenorphine>buprenorphine>methadone>ibogaine and THC. Similarly, BCRP was inhibited by buprenorphine>norbuprenorphine>ibogaine and THC. None of the other tested compounds inhibited either transporter, even at high concentration (100 microm). Norbuprenorphine (transport efflux ratio approoximately 11) and methadone (transport efflux ratio approoximately 1.9) transport was P-gp-mediated; however, with no significant stereo-selectivity regarding methadone enantiomers. BCRP did not transport any of the tested compounds. However, the clinical significance of the interaction of norbuprenorphine with P-gp remains to be evaluated. PMID:19887017

  2. Screening and expression of selected taxonomically conserved and unique hypothetical proteins in Burkholderia pseudomallei K96243

    NASA Astrophysics Data System (ADS)

    Akhir, Nor Azurah Mat; Nadzirin, Nurul; Mohamed, Rahmah; Firdaus-Raih, Mohd

    2015-09-01

    Hypothetical proteins of bacterial pathogens represent a large numbers of novel biological mechanisms which could belong to essential pathways in the bacteria. They lack functional characterizations mainly due to the inability of sequence homology based methods to detect functional relationships in the absence of detectable sequence similarity. The dataset derived from this study showed 550 candidates conserved in genomes that has pathogenicity information and only present in the Burkholderiales order. The dataset has been narrowed down to taxonomic clusters. Ten proteins were selected for ORF amplification, seven of them were successfully amplified, and only four proteins were successfully expressed. These proteins will be great candidates in determining the true function via structural biology.

  3. InsectDirectTM Protein Expression & Purification System

    E-print Network

    Lebendiker, Mario

    by the tightly controlled T7lac promoter. Insect Cell Expression Vectors * pIEx-7 is available only as an Ek/LIC vector. The pIEx-7 Ek/LIC Vector Kit includes Linearized Ek/LIC Vector; T4 DNA Polymerase, LIC; and controls. Tb: thrombin; Ek: enterokonase pIEx-1, pIEx-2, and pIEx-3 vectors are also available in an Ek/LIC

  4. Differential expression of protein kinase C isoforms in coronary arteries of diabetic mice lacking the G-protein G?11

    PubMed Central

    2010-01-01

    Background Diabetes mellitus counts as a major risk factor for developing atherosclerosis. The activation of protein kinase C (PKC) is commonly known to take a pivotal part in the pathogenesis of atherosclerosis, though the influence of specific PKC isozymes remains unclear. There is evidence from large clinical trials suggesting excessive neurohumoral stimulation, amongst other pathways leading to PKC activation, as a central mechanism in the pathogenesis of diabetic heart disease. The present study was therefore designed to determine the role of Gq-protein signalling via G?11 in diabetes for the expression of PKC isozymes in the coronary vessels. Methods The role of G?11 in diabetes was examined in knockout mice with global deletion of G?11 compared to wildtype controls. An experimental type 1-diabetes was induced in both groups by injection of streptozotocin. Expression and localization of the PKC isozymes ?, ?II, ?, ?, and ? was examined by quantitative immunohistochemistry. Results 8 weeks after induction of diabetes a diminished expression of PKC ? was observed in wildtype animals. This alteration was not seen in G?11 knockout animals, however, these mice showed a diminished expression of PKC?. Direct comparison of wildtype and knockout control animals revealed a diminished expression of PKC ? and ? in G?11 knockout animals. Conclusion The present study shows that expression of the nPKCs ? and ? in coronary vessels is under control of the g-protein G?11. The reduced expression of PKC ? that we observed in coronary arteries from G?11-knockout mice compared to wildtype controls upon induction of diabetes could reduce apoptosis and promote plaque stability. These findings suggest a mechanism that may in part underlie the therapeutic benefit of RAS inhibition on cardiovascular endpoints in diabetic patients. PMID:21190563

  5. Matrix metalloproteinase 2 (MMP2) protein expression and laryngeal cancer prognosis: a meta analysis

    PubMed Central

    Liu, Rong-Rong; Li, Ming-Dong; Li, Te; Tan, Yun; Zhang, Min; Chen, Ji-Chuan

    2015-01-01

    Objective: To investigate the protein expression of matrix metalloproteinase 2 (MMP2) and its clinical significance in laryngeal cancer. Methods: A comprehensive search for the related literature published in China and other countries was conducted in a variety of databases, including MEDLINE, Embase, China Academic Journals Full-text Database, Wanfang Data and VIP Database. A total of seven case-control studies were included in the final systematic assessment. A meta-analysis software program was used to statistically analyze the raw data from each study for the calculation of the pooled odds ratio (OR) and 95% confidence interval (95% CI). Results: The meta-analysis indicated that, compared with normal laryngeal tissue, the MMP2 protein was highly expressed in the laryngeal cancer tissue [OR=21.67; 95% CI: 11.61-40.43; P<0.001]. Compared with highly differentiated laryngeal cancer, the MMP2 protein expression level was higher in the moderately and poorly differentiated laryngeal cancers [OR=0.25; 95% CI: 0.13-0.46; P<0.001]. Compared with laryngeal cancers without lymph node metastasis, the laryngeal cancers with lymph node metastasis exhibited a greatly elevated MMP2 protein expression [OR=0.25; 95% CI: 0.14-0.46; P<0.001]. Conclusion: High protein expression levels of MMP2 may play an important role in the tumorigenesis, progression and prognosis of laryngeal cancer. PMID:25932160

  6. One third of dynamic protein expression profiles can be predicted by simple rate equations

    PubMed Central

    Tchourine, Konstantine; Poultney, Christopher S.; Wang, Li; Silva, Gustavo M.; Manohar, Sandhya; Mueller, Christian L.; Bonneau, Richard; Vogel, Christine

    2014-01-01

    Cells respond to environmental stimuli with expression changes at both the mRNA and protein level, and a plethora of known and unknown regulators affect synthesis and degradation rates of the resulting proteins. To investigate the major principles of gene expression regulation in dynamic systems, we estimated protein synthesis and degradation rates from parallel time series data of mRNA and protein expression and tested the degree to which expression changes can be modeled by a set of simple linear differential equations. Examining three published datasets for yeast responding to diamide, rapamycin, and sodium chloride treatment, we find that almost one-third of genes can be well-modeled, and the estimated rates assume realistic values. Prediction quality is linked to low measurement noise and the shape of the expression profile. Synthesis and degradation rates do not correlate within one treatment, consistent with their independent regulation. When performing robustness analyses of the rate estimates, we observed that most genes adhere to one of two major modes of regulation, which we term synthesis- and degradation-independent regulation. These two modes, in which only one of the rates has to be tightly set, while the other one can assume various values, offer an efficient way for the cell to respond to stimuli and re-establish proteostasis. We experimentally validate degradation-independent regulation under oxidative stress for the heatshock protein Ssa4. PMID:25111754

  7. RNA Protein Granules Modulate tau Isoform Expression and Induce Neuronal Sprouting*

    PubMed Central

    Moschner, Katharina; Sündermann, Frederik; Meyer, Heiko; da Graca, Abel Pereira; Appel, Neele; Paululat, Achim; Bakota, Lidia; Brandt, Roland

    2014-01-01

    The neuronal microtubule-associated protein Tau is expressed in different variants, and changes in Tau isoform composition occur during development and disease. Here, we investigate a potential role of the multivalent tau mRNA-binding proteins G3BP1 and IMP1 in regulating neuronal tau expression. We demonstrate that G3BP1 and IMP1 expression induces the formation of structures, which qualify as neuronal ribonucleoprotein (RNP) granules and concentrate multivalent proteins and mRNA. We show that RNP granule formation leads to a >30-fold increase in the ratio of high molecular weight to low molecular weight tau mRNA and an ?12-fold increase in high molecular weight to low molecular weight Tau protein. We report that RNP granule formation is associated with increased neurite formation and enhanced process growth. G3BP1 deletion constructs that do not induce granule formation are also deficient in inducing neuronal sprouting or changing the expression pattern of tau. The data indicate that granule formation driven by multivalent proteins modulates tau isoform expression and suggest a morphoregulatory function of RNP granules during health and disease. PMID:24755223

  8. Calcium affecting protein expression in longan under simulated acid rain stress.

    PubMed

    Pan, Tengfei; Li, Yongyu; Ma, Cuilan; Qiu, Dongliang

    2015-08-01

    Longan (Dimocarpus longana Lour. cv. Wulongling) of uniform one-aged seedlings grown in pots were selected to study specific proteins expressed in leaves under simulated acid rain (SiAR) stress and exogenous Ca(2+) regulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that there was a protein band specifically expressed under SiAR of pH 2.5 stress for 15 days with its molecular weight of about 23 kD. A 17 kD protein band specifically expressed after SiAR stress 5 days. Compared with pH 2.5, the pH 3.5 of SiAR made a less influence to protein expression. Two-dimensional electrophoresis (2-DE) results showed that six new specific proteins including C4 (20.2 kD pI 6.0), F (24 kD pI 6.35), B3 (22.3 kD pI 6.35), B4 (23.5 kD pI 6.5), C5 (21.8 kD pI 5.6), and C6 (20.2 kD pI 5.6) specifically expressed. C4 always expressed during SiAR stress. F expressed under the stress of pH 2.5 for 15 days and expressed in all pH SiAR stress for 20 days. The expression of proteins including B3, C5, and C6 was related to pH value and stress intensity of SiAR. The expression of B4 resulted from synergistic effects of SiAR and Ca. The expression of G1 (Mr 19.3 kD, pI 4.5), G2 (Mr 17.8 kD, pI 4.65), G3 (Mr 16.6 kD, pI 4.6), and G4 (Mr 14.7 kD, pI 4.4) enhanced under the treatment of 5 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM chlorpromazine (CPZ). These proteins showed antagonistic effects and might be relative to the Ca-calmodulin (Ca-CaM) system of longan in response to SiAR stress. PMID:25893616

  9. Aberrant expression of DNA damage response proteins is associated with breast cancer subtype and clinical features

    PubMed Central

    Guler, Gulnur; Himmetoglu, Cigdem; Jimenez, Rafael E.; Geyer, Susan M.; Wang, Wenle P.; Costinean, Stefan; Pilarski, Robert T.; Morrison, Carl; Suren, Dinc; Liu, Jianhua; Chen, Jingchun; Kamal, Jyoti; Shapiro, Charles L.

    2013-01-01

    Landmark studies of the status of DNA damage checkpoints and associated repair functions in preneoplastic and neoplastic cells has focused attention on importance of these pathways in cancer development, and inhibitors of repair pathways are in clinical trials for treatment of triple negative breast cancer. Cancer heterogeneity suggests that specific cancer subtypes will have distinct mechanisms of DNA damage survival, dependent on biological context. In this study, status of DNA damage response (DDR)-associated proteins was examined in breast cancer subtypes in association with clinical features; 479 breast cancers were examined for expression of DDR proteins ?H2AX, BRCA1, pChk2, and p53, DNA damage-sensitive tumor suppressors Fhit and Wwox, and Wwox-interacting proteins Ap2?, Ap2?, ErbB4, and correlations among proteins, tumor subtypes, and clinical features were assessed. In a multivariable model, triple negative cancers showed significantly reduced Fhit and Wwox, increased p53 and Ap2? protein expression, and were significantly more likely than other subtype tumors to exhibit aberrant expression of two or more DDR-associated proteins. Disease-free survival was associated with subtype, Fhit and membrane ErbB4 expression level and aberrant expression of multiple DDR-associated proteins. These results suggest that definition of specific DNA repair and checkpoint defects in subgroups of triple negative cancer might identify new treatment targets. Expression of Wwox and its interactor, ErbB4, was highly significantly reduced in metastatic tissues vs. matched primary tissues, suggesting that Wwox signal pathway loss contributes to lymph node metastasis, perhaps by allowing survival of tumor cells that have detached from basement membranes, as proposed for the role of Wwox in ovarian cancer spread. PMID:21069451

  10. Regulators of Expression of the Oligopeptide Permease A Proteins of Borrelia burgdorferi?

    PubMed Central

    Medrano, Melisa S.; Ding, Yanpeng; Wang, Xing-Guo; Lu, Peng; Coburn, Jenifer; Hu, Linden T.

    2007-01-01

    Borrelia burgdorferi undergoes an infectious cycle that requires adaptation to different hosts and marked differences in environment. B. burgdorferi copes with its different environments by regulating the expression of proteins required for survival in specific settings. The B. burgdorferi oligopeptide permease (Opp) is one of only a few transporters encoded by the B. burgdorferi genome. Opp proteins in other bacteria serve multiple environmental adaptation functions. B. burgdorferi appears to broaden the usage of this transporter by utilizing five different substrate binding proteins (OppA proteins) that interact with the integral membrane components of the transporter. Expression of the OppA proteins is individually regulated and may play different roles in adaptation to host environments. Very little is known about the mechanisms used by B. burgdorferi to regulate the expression of different OppA proteins. Here we show that the alternative sigma factors, RpoS and RpoN, regulate the expression of oppA5 but not that of other oppA genes. Using a reporter assay with Escherichia coli and gel shift binding assays, we also show that the B. burgdorferi BosR/Fur homologue interacts with the oppA4 promoter and that another candidate transcription factor, EbfC, interacts with the oppA5 promoter. Binding to the promoters was confirmed by gel shift assays. Expression of BosR/Fur in its different hosts does appear to parallel the expression of oppA4. A better understanding of the factors involved in gene regulation in B. burgdorferi will help to identify coregulated proteins that may cooperate to allow the organism to survive in a specific environment. PMID:17237172

  11. Expression of L1 protein correlates with cluster of differentiation 24 and integrin ?1 expression in gastrointestinal stromal tumors

    PubMed Central

    DU, YUE; ZHANG, HAIHONG; JIANG, ZHONGMIN; HUANG, GUOWEI; LU, WENLI; WANG, HESHENG

    2015-01-01

    The present study examined 66 cases of gastrointestinal stromal tumors (GISTs), 20 cases of smooth muscle tumors, 20 cases of schwannomas and 20 cases of normal gastric tissues in order to analyze the expression of L1, cluster of differentiation (CD)24 and integrin ?1 by immunohistochemical staining. Patients were subjected to follow-up, and survival data were evaluated. L1 expression was detected in 57.6% of GIST cases; this was a significantly higher percentage compared with that found in the smooth muscle tumor cases or the normal control group. CD24 and integrin ?1 were also expressed at significantly higher levels in the GIST cases than in the normal control group, although no significant difference was found in the expression levels of these proteins in smooth muscle tumor or schwannoma cases. These higher levels of L1 and integrin ?1 expression were associated with an increased risk of invasive GIST, and were significantly positively correlated with Ki-67 expression. CD24 expression was not associated with the risk of GIST invasion or Ki-67 expression. There were positive correlations between L1, CD24 and integrin ?1 expression; however, these had no significant association with patient survival. Therefore, L1 alone or in conjunction with CD24 (L1 + CD24), or integrin ?1 (L1 + integrin ?1) can be considered a valuable indicator for the differential diagnosis of GIST. Furthermore, L1 and integrin ?1 can be used alone or in combination to evaluate the biological behavior of GISTs. Future studies are required to evaluate the prognostic value of these markers. PMID:26137113

  12. Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

    PubMed

    Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

    2015-09-01

    The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine. PMID:26435147

  13. LAPTM5: A novel lysosomal-associated multispanning membrane protein preferentially expressed in hematopoietic cells

    SciTech Connect

    Adra, C.N.; Zhu, Shaochun; Ko, Jone-Long

    1996-07-15

    While a large body of knowledge about cell membrane proteins exists, much less is known about the repertoire and function of integral membrane proteins of intracellular organelles. In looking for novel classes of genes that are functionally important to hematopoietic cells, we have cloned the cDNA for a gene preferentially expressed in adult hematopoietic tissues. During embryonic development the gene is expressed in both hematopoietic and nonhematopoietic tissues. In cell lines the gene is expressed specifically in hematopoietic lineages, whereas in normal adult tissues the mRNA is preferentially detected at high levels in lymphoid and myeloid tissues. The predicted protein is a pentaspanner with no homology to known genes and conserved across evolution. Immunocytological and cell fractionation studies with a specific antibody revealed a protein localizing in lysosomes. The gene, provisionally named LAPTM5, maps to chromosome 1p34. The expression pattern of the gene together with preliminary evidence that the protein interacts with ubiquitin indicates that the protein may have a special functional role during embryogenesis and in adult hematopoietic cells. 53 refs., 9 figs.

  14. Isolation of nuclear proteins from flax (Linum usitatissimum L.) seed coats for gene expression regulation studies

    PubMed Central

    2012-01-01

    Background While seed biology is well characterized and numerous studies have focused on this subject over the past years, the regulation of seed coat development and metabolism is for the most part still non-elucidated. It is well known that the seed coat has an essential role in seed development and its features are associated with important agronomical traits. It also constitutes a rich source of valuable compounds such as pharmaceuticals. Most of the cell genetic material is contained in the nucleus; therefore nuclear proteins constitute a major actor for gene expression regulation. Isolation of nuclear proteins responsible for specific seed coat expression is an important prerequisite for understanding seed coat metabolism and development. The extraction of nuclear proteins may be problematic due to the presence of specific components that can interfere with the extraction process. The seed coat is a rich source of mucilage and phenolics, which are good examples of these hindering compounds. Findings In the present study, we propose an optimized nuclear protein extraction protocol able to provide nuclear proteins from flax seed coat without contaminants and sufficient yield and quality for their use in transcriptional gene expression regulation by gel shift experiments. Conclusions Routinely, around 250 ?g of nuclear proteins per gram of fresh weight were extracted from immature flax seed coats. The isolation protocol described hereafter may serve as an effective tool for gene expression regulation and seed coat-focused proteomics studies. PMID:22230709

  15. Trm9-Catalyzed tRNA Modifications Regulate Global Protein Expression by Codon-Biased Translation

    PubMed Central

    Deng, Wenjun; Babu, I. Ramesh; Su, Dan; Yin, Shanye; Begley, Thomas J.; Dedon, Peter C.

    2015-01-01

    Post-transcriptional modifications of transfer RNAs (tRNAs) have long been recognized to play crucial roles in regulating the rate and fidelity of translation. However, the extent to which they determine global protein production remains poorly understood. Here we use quantitative proteomics to show a direct link between wobble uridine 5-methoxycarbonylmethyl (mcm5) and 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modifications catalyzed by tRNA methyltransferase 9 (Trm9) in tRNAArg(UCU) and tRNAGlu(UUC) and selective translation of proteins from genes enriched with their cognate codons. Controlling for bias in protein expression and alternations in mRNA expression, we find that loss of Trm9 selectively impairs expression of proteins from genes enriched with AGA and GAA codons under both normal and stress conditions. Moreover, we show that AGA and GAA codons occur with high frequency in clusters along the transcripts, which may play a role in modulating translation. Consistent with these results, proteins subject to enhanced ribosome pausing in yeast lacking mcm5U and mcm5s2U are more likely to be down-regulated and contain a larger number of AGA/GAA clusters. Together, these results suggest that Trm9-catalyzed tRNA modifications play a significant role in regulating protein expression within the cell. PMID:26670883

  16. Differential soluble protein expression between Trichomonas vaginalis isolates exhibiting low and high virulence phenotypes.

    PubMed

    Cuervo, Patrícia; Cupolillo, Elisa; Britto, Constança; González, Luis Javier; E Silva-Filho, Fernando Costa; Lopes, Letícia Coutinho; Domont, Gilberto Barbosa; De Jesus, Jose Batista

    2008-04-30

    A comparative analysis of proteomic maps of long-term grown and fresh clinical Trichomonas vaginalis isolates exhibiting low and high virulence phenotypes, respectively, was performed using two-dimensional gel electrophoresis and mass spectrometry. Of 29 protein spots differentially expressed between the isolates, 19 were over-expressed in the isolate exhibiting high virulence phenotype: proteins associated with cytoskeletal dynamics, such as coronin and several isoforms of actin, as well as proteins involved in signal transduction, protein turnover, proteolysis, and energetic and polyamine metabolisms were identified. Some malate dehydrogenase, fructose-1,6-bisphosphate aldolase and ornithine cyclodeamidase isoforms were exclusively expressed by the highly virulent isolate. During interaction assays with VEC, parasites exhibiting high virulence phenotype rapidly adhered and switched to amoeboid forms. In contrast, low adhesion and no morphological transformation were observed in parasites displaying low virulence phenotype. Our findings demonstrate that expression of specific proteins by high and low virulence parasites could be associated with the ability of each isolate to undergo morphological transformation and interact with host cells. Such data represent an important step towards understanding of the complex interaction network of proteins that participate in the mechanism of pathogenesis of this protozoan. PMID:18541479

  17. Brain-Expressed X-linked (BEX) proteins in human cancers.

    PubMed

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

    2015-12-01

    The Brain-Expressed X-linked (BEX) family proteins are comprised of five human proteins including BEX1, BEX2, BEX3, BEX4 and BEX5. BEX family proteins are expressed in a wide range of tissues and are known to play a role in neuronal development. Recent studies suggest a role of BEX family proteins in cancers. BEX1 expression is lost in a subgroup of patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Expression of BEX1 controls cell surface receptor signaling and restores imatinib response in resistant cells. BEX2 is overexpressed in a group of breast cancer patients and also in gliomas. Increased BEX2 expression led to enhanced NF-?B signaling as well as cell proliferation. Although BEX2 acts as tumor promoter in a subset of breast cancer, BEX3 expression displayed an opposite role. Overexpression of BEX3 resulted in inhibition of tumor formation in breast cancer mouse xenograft models. The role of BEX4 and BEX5 in cancer has not yet been defined. Collectively this suggests that BEX family members have distinct roles in cancers. While BEX1 and BEX3 act as tumor suppressors, BEX2 seems to act as an oncogene. PMID:26408910

  18. Ontogeny of tight junction protein expression in the ovine cerebral cortex during development.

    PubMed

    Sadowska, G B; Ahmedli, N; Chen, X; Stonestreet, B S

    2015-12-01

    Tight junctions of the blood-brain barrier are composed of transmembrane and associated cytoplasmic proteins. The transmembrane claudin proteins form the primary seal between endothelial cells and junctional adhesion molecules (JAMs) regulate tight junction formation. We have previously shown that claudin-1, claudin-5, zonula occludens (ZO)-1, and ZO-2 exhibit differential developmental regulation from 60% of gestation up to maturity in adult sheep. The purpose of the current study was to examine developmental changes in claudin-3, -12, and JAM-A protein expression in cerebral cortices of fetuses at 60%, 80%, and 90% gestation, and in newborn and adult sheep. We also examined correlations between changes in endogenous cortisol levels and tight junction protein expression in cerebral cortices of the fetuses. Claudin-3, -12 and JAM-A expressions were determined by Western immunoblot. Claudin-3 and -12 were lower (P<0.01) at 60%, 80%, 90% and in newborns than in adults, and JAM-A was lower in adults than in fetuses at 80% and 90% gestation. Claudin-3 expression demonstrated a direct correlation with increasing plasma cortisol levels (r=0.60, n=15, P<0.02) in the fetuses. We conclude that: claudin-3, -12 and JAM-A are expressed as early as 60% of gestation in ovine cerebral cortices, exhibit differential developmental regulation, and that increasing endogenous glucocorticoids modulate claudin-3 expression in the fetus. PMID:26424381

  19. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    PubMed Central

    Gao, Meili; Li, Yongfei; Xue, Xiaochang; Wang, Xianfeng; Long, Jiangang

    2012-01-01

    Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed. PMID:23093835

  20. Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins.

    PubMed

    Leuzinger, Kahlin; Dent, Matthew; Hurtado, Jonathan; Stahnke, Jake; Lai, Huafang; Zhou, Xiaohong; Chen, Qiang

    2013-01-01

    Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education. PMID:23913006

  1. Versatile co-expression of graft-protective proteins using 2A-linked cassettes

    PubMed Central

    Fisicaro, Nella; Londrigan, Sarah L.; Brady, Jamie L.; Salvaris, Evelyn; Nottle, Mark B.; O’Connell, Philip J.; Robson, Simon C.; d’Apice, Anthony J. F.; Lew, Andrew M.; Cowan, Peter J.

    2013-01-01

    Background Expression of multiple graft-protective proteins targeted to different locations (i.e., intracellular, cell surface, and secreted) has become an increasingly important goal in xenotransplantation. The 2A “ribosome skip” signal is used as a linker to enable transgene co-expression, but some studies have shown that post-translational modification and trafficking of 2A-linked proteins may be adversely affected depending on their position relative to 2A. We tested whether several relevant proteins, subject to a range of processing and localization mechanisms, could be efficiently co-expressed using the 2A system. Methods Six expression cassettes were constructed, each containing up to four 2A-linked open reading frames, encoding combinations of human CD55, thrombomodulin (TBM), CD39, CTLA4-Ig and hygromycin resistance. Each linker incorporated a furin cleavage site to remove the carboxy-terminal extension that remains on upstream proteins after 2A processing. The cassettes were used to produce vectors for transfection, adenoviral transduction and transgenesis. Expression was detected by flow cytometry and/or Western blotting. Results All proteins were expressed in the appropriate location following transient transfection of COS-7 cells, irrespective of the number of linked genes. The percentage of stable transfectants expressing a linked gene was increased 10-fold (from 4–5% to 58–67%) by incorporating the hygromycin resistance gene into the cassette. Stable transfection of transgenic GalT KO pig fibroblasts with a hygromycin- TBM-CD39 construct resulted in surface expression of both TBM and CD39 by the majority of hygromycin-resistant cells. Expression was maintained after flow cytometric sorting and expansion. Adenoviral transduction of NIT-1 mouse insulinoma cells with a TBM-CD39 construct resulted in strong expression of both genes on the cell surface. Mice transgenic for 3-gene (CD55- TBM-CD39) or 4-gene (CD55- TBM-CTLA4Ig-CD39) constructs expressed all genes except CD55. Conclusions These results confirm the versatility of the 2A system, and demonstrate that careful construct design can minimize potential problems with post-translational modification and trafficking. In addition, incorporation of a selection marker into the 2A-linked chain can dramatically increase the proportion of stable transfectants expressing proteins of interest. This provides a powerful method for the rapid modification of existing genetically modified pigs. PMID:21496119

  2. Diversity of Histologic Patterns and Expression of Cytoskeletal Proteins in Canine Skeletal Osteosarcoma.

    PubMed

    Nagamine, E; Hirayama, K; Matsuda, K; Okamoto, M; Ohmachi, T; Kadosawa, T; Taniyama, H

    2015-09-01

    Osteosarcoma (OS), the most common bone tumor, includes OS of the head (OSH) and appendicular OS (OSA). In dogs, it is classified into 6 histologic subtypes: osteoblastic, chondroblastic, fibroblastic, telangiectatic, giant cell, and poorly differentiated. This study investigated the significance of the histologic classification relevant to clinical outcome and the histologic and immunohistochemical relationships between pleomorphism and expression of cytoskeletal proteins in 60 cases each of OSH and OSA. Most neoplasms exhibited histologic diversity, and 64% of OS contained multiple subtypes. In addition to the above 6 subtypes, myxoid, round cell, and epithelioid subtypes were observed. Although the epithelioid subtypes were observed in only OSH, no significant difference in the frequency of other subtypes was observed. Also, no significant relevance was observed between the clinical outcome and histologic subtypes. Cytokeratin (CK) was expressed in both epithelioid and sarcomatoid tumor cells in various subtypes, and all CK-positive tumor cells also expressed vimentin. Vimentin and ?-smooth muscle actin (SMA) were expressed in all subtypes. A few SMA-positive spindle-shaped tumor cells exhibited desmin expression. Glial fibrillary acidic protein-positive tumor cells were observed in many subtypes, and some of these cells showed neurofilament expression. Although OSH exhibited significantly stronger immunoreactivity for SMA than OSA, no significant difference in other cytoskeletal proteins was observed. Some tumor cells had cytoskeletal protein expression compatible with the corresponding histologic subtypes, such as CK in the epithelioid subtype and SMA in the fibroblastic subtype. Thus, canine skeletal OS is composed of pleomorphic and heterogenous tumor cells as is reflected in the diversity of histologic patterns and expression of cytoskeletal proteins. PMID:25770040

  3. Expression of Agrobacterium Homolog Genes Encoding T-complex Recruiting Protein under Virulence Induction Conditions

    PubMed Central

    Yang, Jing; Wu, Meixia; Zhang, Xin; Guo, Minliang; Huang, Zhiwei

    2015-01-01

    The proteins encoded by three Agrobacterial genes, atu5117, atu4860, and atu4856, are highly homologous to each other in amino acid sequence. All three proteins can bind to VirD2 and are named VBP1, VBP2, and VBP3 (VirD2-binding protein), respectively. VBP is involved in T-DNA transfer by recruiting the T-complex from the cytosol to the polar transport apparatus T4SS (type IV secretion system) and is defined as the “T-complex recruiting protein.” However, it remains unknown how these three homologous genes co-exist in a relatively small prokaryotic genome. To understand whether these three homologous genes are expressed differentially under virulence induction conditions, we examined the effects of virulence induction conditions, including various pH values, temperatures and acetosyringone (AS, an effective virulence inducer to Agrobacterium tumefaciens) concentrations, on the expression of the three VBP-encoding genes. Our data showed that vbp1 (atu5117) and vbp3 (atu4856) maintained constant expression under the tested induction conditions, whereas the expression of vbp2 (atu4860) was affected by the conditions. Culture conditions favorable to the expression of vbp2 differed from the reported induction conditions for other virulence proteins. In particular, the pH value was a crucial factor for the expression of vbp2. In addition, the deletion of vbp1 affected the expression of vbp2. Taken together, these results suggest that the mechanisms regulating the expression of these three homologous genes are different from the virulence induction mechanism and that VBP homologs are presumably involved in other biological processes in addition to T-complex recruitment. PMID:26696988

  4. A Comparative Approach to Understanding Tissue-Specific Expression of Uncoupling Protein 1 Expression in Adipose Tissue

    PubMed Central

    Shore, Andrew; Emes, Richard D.; Wessely, Frank; Kemp, Paul; Cillo, Clemente; D’Armiento, Maria; Hoggard, Nigel; Lomax, Michael A.

    2012-01-01

    The thermoregulatory function of brown adipose tissue (BAT) is due to the tissue-specific expression of uncoupling protein 1 (UCP1) which is thought to have evolved in early mammals. We report that a CpG island close to the UCP1 transcription start site is highly conserved in all 29 vertebrates examined apart from the mouse and xenopus. Using methylation sensitive restriction digest and bisulfite mapping we show that the CpG island in both the bovine and human is largely un-methylated and is not related to differences in UCP1 expression between white and BAT. Tissue-specific expression of UCP1 has been proposed to be regulated by a conserved 5? distal enhancer which has been reported to be absent in marsupials. We demonstrate that the enhancer, is also absent in five eutherians as well as marsupials, monotremes, amphibians, and fish, is present in pigs despite UCP1 having become a pseudogene, and that absence of the enhancer element does not relate to BAT-specific UCP1 expression. We identify an additional putative 5? regulatory unit which is conserved in 14 eutherian species but absent in other eutherians and vertebrates, but again unrelated to UCP1 expression. We conclude that despite clear evidence of conservation of regulatory elements in the UCP1 5? untranslated region, this does not appear to be related to species or tissues-specific expression of UCP1. PMID:23293654

  5. Dexamethasone increases aquaporin-2 protein expression in ex vivo inner medullary collecting duct suspensions

    PubMed Central

    Chen, Minguang; Cai, Hui; Klein, Janet D.; Laur, Oskar; Chen, Guangping

    2015-01-01

    Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and plays an important role in the maintenance of body water homeostasis. Excessive glucocorticoid as often seen in Cushing's syndrome causes water retention. However, whether and how glucocorticoid regulates AQP2 remains unclear. In this study, we examined the direct effect of dexamethasone on AQP2 protein expression and activity. Dexamethasone increased AQP2 protein abundance in rat inner medullary collecting duct (IMCD) suspensions. This was confirmed in HEK293 cells transfected with AQP2 cDNA. Cell surface protein biotinylation showed an increase of dexamethasone-induced cell membrane AQP2 expression and this effect was blocked by glucocorticoid receptor antagonist RU486. Functionally, dexamethasone treatment of oocytes injected with an AQP2 cRNA increased water transport activity as judged by cell rupture time in a hypo-osmotic solution (66 ± 13 s in dexamethasone vs. 101 ± 11 s in control, n = 15). We further found that dexamethasone treatment reduced AQP2 protein degradation, which could result in an increase of AQP2 protein. Interestingly, dexamethasone promoted cell membrane AQP2 moving to less buoyant lipid raft submicrodomains. Taken together, our data demonstrate that dexamethasone promotes AQP2 protein expression and increases water permeability mainly via inhibition of AQP2 protein degradation. The increase in AQP2 activity promotes water reabsorption, which may contribute to glucocorticoid-induced water retention and hypertension. PMID:26578982

  6. dbDEPC 2.0: updated database of differentially expressed proteins in human cancers

    PubMed Central

    He, Ying; Zhang, Menghuan; Ju, Yuanhu; Yu, Zhonghao; Lv, Daqing; Sun, Han; Yuan, Weilan; He, Fei; Zhang, Jianshe; Li, Hong; Li, Jing; Wang-Sattler, Rui; Li, Yixue; Zhang, Guoqing; Xie, Lu

    2012-01-01

    A large amount of differentially expressed proteins (DEPs) have been identified in various cancer proteomics experiments, curation and annotation of these proteins are important in deciphering their roles in oncogenesis and tumor progression, and may further help to discover potential protein biomarkers for clinical applications. In 2009, we published the first database of DEPs in human cancers (dbDEPCs). In this updated version of 2011, dbDEPC 2.0 has more than doubly expanded to over 4000 protein entries, curated from 331 experiments across 20 types of human cancers. This resource allows researchers to search whether their interested proteins have been reported changing in certain cancers, to compare their own proteomic discovery with previous studies, to picture selected protein expression heatmap across multiple cancers and to relate protein expression changes with aberrance in other genetic level. New important developments include addition of experiment design information, advanced filter tools for customer-specified analysis and a network analysis tool. We expect dbDEPC 2.0 to be a much more powerful tool than it was in its first release and can serve as reference to both proteomics and cancer researchers. dbDEPC 2.0 is available at http://lifecenter.sgst.cn/dbdepc/index.do. PMID:22096234

  7. Hypobaric hypoxia-mediated protein expression in plasma of susceptible & tolerant rats

    PubMed Central

    Kumar, Santosh; Sharma, Priyanka; Bansal, Anju; Sharma, Prakash C.; Aggarwal, Kamal K.

    2014-01-01

    Background & objectives: Low availability of oxygen at high altitudes has a great impact on the human life processes. There is a widespread interest and need to find out protein(s) that are possibly involved in mediating tolerance to hypobaric hypoxia. We undertook this study to identify and characterize protein expression in plasma of hypoxia susceptible and tolerant rats. Methods: Male albino Sprague Dawley rats were segregated into susceptible and tolerant groups on the basis of their gasping time when exposed to simulated hypobaric hypoxia of 32,000 ft (9,754 m) at 32°C. Comparative proteome profiling of blood plasma of hypoxia susceptible and tolerant individuals was performed using 2-dimentional (2-D) gel electrophoresis. Results: Three proteins with higher expression levels were selected separately from tolerant and susceptible samples. Characterization of these proteins from tolerant sample using MALDI-TOF/TOF and MASCOT search indicated their homology with two different super-families viz. NADB-Rossmann superfamily (Rab GDP dissociation inhibitor ?) and Transferrin superfamily (two Serotransferrins), having potential role in imparting tolerance against hypoxia. Three high level upregulated proteins were characterized from blood plasma of hypoxia susceptible animals showing similarity with threonine tRNA ligase (mitochondrial), carbohydrate sulphotransferase 7 and aspartate tRNA ligase (cytoplasmic) that play a role in ATP binding, carbohydrate metabolism and protein biosynthesis, respectively. Interpretation & conclusions: Our results indicated that rats segregated into hypoxia sensitive and tolerant based on their gasping time showed differential expression of proteins in blood plasma. Characterization of these differentially expressed proteins will lead to better understanding of molecular responses occurring during hypoxia and subsequently development of biomarkers for categorization of hypoxia susceptible and tolerant individuals. PMID:25758574

  8. The acidity of protein fusion partners predominantly determines the efficacy to improve the solubility of the target proteins expressed in Escherichia coli.

    PubMed

    Su, Yu; Zou, Zhurong; Feng, Shuying; Zhou, Pei; Cao, Lijuan

    2007-05-01

    Maximization of the soluble protein expression in Escherichia coli (E. coli) via the fusion expression strategy is usually preferred for academic, industrial and pharmaceutical purposes. In this study, a set of distinct protein fusion partners were comparatively evaluated to promote the soluble expression of two target proteins including the bovine enterokinase largely prone to aggregation and the green fluorescent protein with moderate native solubility. Within protein attributes that are putatively involved in protein solubility, the protein acidity was of particular concern. Our results explicitly indicated the protein fusion partners with a stronger acidity remarkably exhibited a higher capacity to enhance the solubility of the target proteins. Among them, msyB, an E. coli acidic protein that suppresses the mutants lacking function of protein export, was revealed as an excellent protein fusion partner with the distinguished features including high potential to enhance protein solubility, efficient expression, relatively small size and the origin of E. coli itself. In principle, our results confirmed the modified solubility model of Wilkinson-Harrison and especially deepened understanding its essence. Meanwhile, the roles of other parameters such as protein hydrophilicity in solubility enhancement were discussed, a guideline to design or search an optimum protein solubility enhancer was also proposed. PMID:17374413

  9. Unsupervised Clustering of Subcellular Protein Expression Patterns in High-Throughput Microscopy Images Reveals Protein Complexes and Functional Relationships between Proteins

    PubMed Central

    Handfield, Louis-François; Chong, Yolanda T.; Simmons, Jibril; Andrews, Brenda J.; Moses, Alan M.

    2013-01-01

    Protein subcellular localization has been systematically characterized in budding yeast using fluorescently tagged proteins. Based on the fluorescence microscopy images, subcellular localization of many proteins can be classified automatically using supervised machine learning approaches that have been trained to recognize predefined image classes based on statistical features. Here, we present an unsupervised analysis of protein expression patterns in a set of high-resolution, high-throughput microscope images. Our analysis is based on 7 biologically interpretable features which are evaluated on automatically identified cells, and whose cell-stage dependency is captured by a continuous model for cell growth. We show that it is possible to identify most previously identified localization patterns in a cluster analysis based on these features and that similarities between the inferred expression patterns contain more information about protein function than can be explained by a previous manual categorization of subcellular localization. Furthermore, the inferred cell-stage associated to each fluorescence measurement allows us to visualize large groups of proteins entering the bud at specific stages of bud growth. These correspond to proteins localized to organelles, revealing that the organelles must be entering the bud in a stereotypical order. We also identify and organize a smaller group of proteins that show subtle differences in the way they move around the bud during growth. Our results suggest that biologically interpretable features based on explicit models of cell morphology will yield unprecedented power for pattern discovery in high-resolution, high-throughput microscopy images. PMID:23785265

  10. Expression of the mouse PR domain protein Prdm8 in the developing central nervous system.

    PubMed

    Komai, Tae; Iwanari, Hiroko; Mochizuki, Yasuhiro; Hamakubo, Takao; Shinkai, Yoichi

    2009-10-01

    It was first shown in the PR (PRDI-BF1 and RIZ homology) domain family proteins that the PR domain has homology to the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain, a catalytic domain of the histone lysine methyltransferases. Recently, there are many reports that the PR domain proteins have important roles in development and/or cell differentiation. In this report, we show the expression patterns of one of the mouse PR domain proteins, Prdm8, in the developing central nervous system. In the developing retina, Prdm8 expression was detected in postmitotic neurons in the inner nuclear layer and the ganglion cell layer, and its expression became restricted predominantly to the rod bipolar cells when retinogenesis was completed. In the developing spinal cord, Prdm8 was expressed first in the progenitor populations of ventral interneurons and motor neurons, and later in a subpopulation of interneurons. In the developing brain, Prdm8 expression was observed in postmitotic neurons in the intermediate zone and the cortical plate. In the postnatal brain, Prdm8 was expressed mainly in layer 4 neurons of the cerebral cortex. These results show that Prdm8 expression is tightly regulated in a spatio-temporal manner during neural development and mainly restricted to postmitotic neurons, except in the spinal cord. PMID:19616129

  11. Redox Protein Expression Predicts Radiotherapeutic Response in Early-Stage Invasive Breast Cancer Patients

    SciTech Connect

    Woolston, Caroline M.; Al-Attar, Ahmad; Storr, Sarah J.; Ellis, Ian O.; Morgan, David A.L.; Martin, Stewart G.

    2011-04-01

    Purpose: Early-stage invasive breast cancer patients have commonly undergone breast-conserving surgery and radiotherapy. In a large majority of these patients, the treatment is effective; however, a proportion will develop local recurrence. Deregulated redox systems provide cancer cells protection from increased oxidative stress, such as that induced by ionizing radiation. Therefore, the expression of redox proteins was examined in tumor specimens from this defined cohort to determine whether such expression could predict response. Methods and Materials: The nuclear and cytoplasmic expression of nine redox proteins (glutathione, glutathione reductase, glutaredoxin, glutathione peroxidase 1, 3, and 4, and glutathione S-transferase-{theta}, -{pi}, and -{alpha}) was assessed using conventional immunohistochemistry on a tissue microarray of 224 tumors. Results: A high cytoplasmic expression of glutathione S-transferase-{theta} significantly correlated with a greater risk of local recurrence (p = .008) and, when combined with a low nuclear expression (p = .009), became an independent predictive factor (p = .002) for local recurrence. High cytoplasmic expression of glutathione S-transferase-{theta} also correlated with a worse overall survival (p = .009). Low nuclear and cytoplasmic expression of glutathione peroxidase 3 (p = .002) correlated with a greater risk of local recurrence and was an independent predictive factor (p = .005). These proteins did not correlate with tumor grade, suggesting their function might be specific to the regulation of oxidative stress rather than alterations of tumor phenotype. Only nuclear (p = .005) and cytoplasmic (p = .001) expression of glutathione peroxidase 4 correlated with the tumor grade. Conclusions: Our results support the use of redox protein expression, namely glutathione S-transferase-{theta} and glutathione peroxidase 3, to predict the response to radiotherapy in early-stage breast cancer patients. If incorporated into routine diagnostic tests, they have the potential to aid clinicians in their stratification of patients into more tailored treatment regimens. Future targeted therapies to these systems might improve the efficacy of reactive oxygen species-inducing therapies, such as radiotherapy.

  12. [Expression and antitumor activity of fusion protein RGD-TRAIL in Pichia pastoris].

    PubMed

    Wang, Xiao-fei; Chen, Shu-zhen

    2015-05-01

    To compare the activity of RGD-TRAIL in different expression systems, RGD-TRAIL in both Escherichia coli (E.coli) and Pichia pastoris was constructed and expressed. In vitro activity of RGD-TRAIL from Pichia pastoris expression system was also analyzed. Genetic engineering techniques were used to construct recombinant plasmid pET30-rgd-trail and pHBM-rgd-trail. The recombinant protein RGD-TRAIL was purified with Ni ion affinity chromatography after induction. MTT assay, ELISA, scratch wound healing, transwell migration assay and Hoechst 33342 staining were performed to detect the effects of RGD-TRAIL on proliferation, binding activity, migration and apoptosis. The expression of apoptosis-associated proteins was detected by Western blotting. Recombinant protein RGD-TRAIL was successfully expressed in a form of inclusion body in E.coli, while expressed secretorily in Pichia pastoris. It possessed more potent cytotoxicity than RGD-TRAIL in E.coli by MTT assay. The RGD-TRAIL expressed by Pichia pastoris showed powerful binding affinity with cancer cells expressing ?(v), DR4, DR5 and highly potent cytotoxicity through inducing apoptosis of cancer cells. Nuclear fragmentation was examined by Hoechst 33342 staining. Cleaved PARP and caspase-3 were also detected after incubation with RGD-TRAIL. Additionally, RGD-TRAIL inhibited migration significantly in A549 and HT1080 cells. The results demonstrate that Pichia pastoris expression system is more suitable for the recombinant protein RGD-TRAIL. Its binding affinity and antitumor activity might make RGD-TRAIL a promising candidate for cancer therapy. PMID:26234135

  13. Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

    PubMed Central

    O'Ryan, Liam; Rimington, Tracy; Cant, Natasha; Ford, Robert C.

    2012-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel. PMID:22433465

  14. Detecting differential protein expression in large-scale population proteomics

    SciTech Connect

    Ryu, Soyoung; Qian, Weijun; Camp, David G.; Smith, Richard D.; Tompkins, Ronald G.; Davis, Ronald W.; Xiao, Wenzhong

    2014-06-17

    Mass spectrometry-based high-throughput quantitative proteomics shows great potential in clinical biomarker studies, identifying and quantifying thousands of proteins in biological samples. However, methods are needed to appropriately handle issues/challenges unique to mass spectrometry data in order to detect as many biomarker proteins as possible. One issue is that different mass spectrometry experiments generate quite different total numbers of quantified peptides, which can result in more missing peptide abundances in an experiment with a smaller total number of quantified peptides. Another issue is that the quantification of peptides is sometimes absent, especially for less abundant peptides and such missing values contain the information about the peptide abundance. Here, we propose a Significance Analysis for Large-scale Proteomics Studies (SALPS) that handles missing peptide intensity values caused by the two mechanisms mentioned above. Our model has a robust performance in both simulated data and proteomics data from a large clinical study. Because varying patients’ sample qualities and deviating instrument performances are not avoidable for clinical studies performed over the course of several years, we believe that our approach will be useful to analyze large-scale clinical proteomics data.

  15. A convenient and general expression platform for the production of secreted proteins from human cells.

    PubMed

    Aydin, Halil; Azimi, Farshad C; Cook, Jonathan D; Lee, Jeffrey E

    2012-01-01

    Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) (1,2), baculovirus-infected insect (S. frugiperda or T. ni) cells (3), and cell-free in vitro translation systems (2,4) have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins (5-9). However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories. Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein (5,10). In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines. PMID:22872008

  16. Maternal age effects on myometrial expression of contractile proteins, uterine gene expression, and contractile activity during labor in the rat

    PubMed Central

    Elmes, Matthew; Szyszka, Alexandra; Pauliat, Caroline; Clifford, Bethan; Daniel, Zoe; Cheng, Zhangrui; Wathes, Claire; McMullen, Sarah

    2015-01-01

    Advanced maternal age of first time pregnant mothers is associated with prolonged and dysfunctional labor and significant risk of emergency cesarean section. We investigated the influence of maternal age on myometrial contractility, expression of contractile associated proteins (CAPs), and global gene expression in the parturient uterus. Female Wistar rats either 8 (YOUNG n = 10) or 24 (OLDER n = 10) weeks old were fed laboratory chow, mated, and killed during parturition. Myometrial strips were dissected to determine contractile activity, cholesterol (CHOL) and triglycerides (TAG) content, protein expression of connexin-43 (GJA1), prostaglandin-endoperoxide synthase 2 (PTGS2), and caveolin 1 (CAV-1). Maternal plasma concentrations of prostaglandins PGE2, PGF2?, and progesterone were determined by RIA. Global gene expression in uterine samples was compared using Affymetrix Genechip Gene 2.0 ST arrays and Ingenuity Pathway analysis (IPA). Spontaneous contractility in myometrium exhibited by YOUNG rats was threefold greater than OLDER animals (P < 0.027) but maternal age had no significant effect on myometrial CAP expression, lipid profiles, or pregnancy-related hormones. OLDER myometrium increased contractile activity in response to PGF2?, phenylephrine, and carbachol, a response absent in YOUNG rats (all P < 0.002). Microarray analysis identified that maternal age affected expression of genes related to immune and inflammatory responses, lipid transport and metabolism, steroid metabolism, tissue remodeling, and smooth muscle contraction. In conclusion YOUNG laboring rat myometrium seems primed to contract maximally, whereas activity is blunted in OLDER animals and requires stimulation to meet contractile potential. Further work investigating maternal age effects on myometrial function is required with focus on lipid metabolism and inflammatory pathways. PMID:25876907

  17. Expression of a cDNA encoding the wheat CM16 protein in Escherichia coli.

    PubMed

    Lullien-Pellerin, V; Gavalda, S; Joudrier, P; Gautier, M F

    1994-06-01

    The wheat kernel CM16 protein, a subunit of the heterotetrameric insect alpha-amylase inhibitor that has been involved in the technological quality of wheat-products, was produced in Escherichia coli. Cloning of the cDNA part encoding the mature protein in a pET expression plasmid, under the control of a promoter for the bacteriophage T7 RNA polymerase, allows the synthesis of large amounts of the CM16 protein in the bacteria. Upon induction with isopropyl thiogalactopyranoside the recombinant protein accumulates in insoluble inclusion bodies. Solubilization with 6 M urea containing 0.5 mM dithiothreitol, followed by slow elimination of the denaturing agents by step dialysis, results in a significant recovery of the recombinant protein in a soluble, monomeric form. Characterization of the protein was done by automated Edman degradation and total amino acid determination. The recombinant protein in comparison with the one isolated from wheat exhibits a Met extension at the N-terminus that was introduced in the construction for translation initiation. The CM16 protein produced in this manner has the advantage over wheat purified protein of not being contaminated with other proteins from the same family and constitutes adequate material for further analysis of the technological properties of the protein in wheat-derived products. PMID:7950364

  18. Expression and characterization of a synthetic protein C activator in Pichia pastoris.

    PubMed

    Kunes, Yune Zhang; Sanz, María-Cruz; Tumanova, Irina; Birr, Cynthia A; Shi, Philip Q; Bruguera, Pau; Ruiz, Juan A; Sánchez-Martínez, Demetrio

    2002-12-01

    Protein C activators are proteases that activate protein C in the mammalian coagulation system. A reptilian protein C activator is a critical component in current functional assays for protein C, its cofactor protein S, as well as for the overall status of the protein C pathway. We have constructed a synthetic gene for a protein C activator, based on a published snake-venom polypeptide sequence. This recombinant protein C activator was expressed in Pichia pastoris as a secreted glycoprotein (ILPCA) using the AOX1 promoter and the alpha-factor signal sequence. A fermentation protocol was developed that produced about 150 mg/L biologically active ILPCA secreted in the fermented broth. A two-step purification scheme was devised to purify ILPCA to approximately 80% purity. The ILPCA produced has an apparent molecular weight of approximately 68 kDa and a deglycosilated molecular weight of 28 kDa. Steady-state kinetic analysis reveals that ILPCA activates purified human protein C with a K(m) of 77 nM and a k(cat) of 0.39 s(-1). In conclusion, ILPCA is a recombinant protein that can be produced reliably and in large quantities under controlled manufacturing conditions, activates protein C, and can be used in coagulation assays as an alternative to native venom preparations. PMID:12460764

  19. Differential protein expression in the susceptible and resistant Myzus persicae (Sulzer) to imidacloprid.

    PubMed

    Meng, JianYu; Zhang, ChangYu; Chen, XingJiang; Cao, Yi; Shang, ShengHua

    2014-10-01

    Myzus persicae, a serious economic agricultural pest, has developed resistance to imidacloprid (IMI), which was widely used to control this aphid worldwide. To gain a better understanding of the mechanisms of IMI resistance in M. persicae, we carried out a comparative proteomic analysis. Total proteins of the IMI-susceptible and resistant strains were extracted and separated by two-dimensional gel electrophoresis. More than 1300 protein spots were reproducibly detected, including 14 that were more abundant and 14 less abundant. Mass spectrometry analysis and database searching helped us to identify 25 differentially abundant proteins. The identified proteins were categorized into several functional groups including signal transduction, RNA processing, protein processing, transport processing, stress response, metabolisms, and cytoskeleton structure, etc. This study is the first analysis of differentially expressed proteins in IMI-susceptible and resistant M. Persicae, and gives new insights into the mechanisms of IMI resistance in M. persicae. PMID:25307459

  20. Proteomic Study of Differential Protein Expression in Mouse Lung Tissues after Aerosolized Ricin Poisoning

    PubMed Central

    Guo, Zhendong; Han, Chao; Du, Jiajun; Zhao, Siyan; Fu, Yingying; Zheng, Guanyu; Sun, Yucheng; Zhang, Yi; Liu, Wensen; Wan, Jiayu; Qian, Jun; Liu, Linna

    2014-01-01

    Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning. PMID:24786090

  1. A novel expression system for production of soluble prion proteins in E. coli

    PubMed Central

    2012-01-01

    Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies. PMID:22233534

  2. Prokaryotic Expression of Truncated S1 Protein of Porcine Epidemic Diarrhea Virus and Production of Monoclonal Antibodies to Recombinant Protein.

    PubMed

    Wang, Jinliang; Chen, Jinlong; Wei, Feng; Dong, Yankai; Zhu, Lichuang; Han, Wenyu; Wang, Leyi; Shen, Zhiqiang

    2015-10-01

    Monoclonal antibodies are known to have several applications in clinical diagnosis and therapy. In the present study, the truncated S1 gene, encoding the exterior of the viral spike protein of porcine epidemic diarrhea virus (PEDV), was subcloned into prokaryotic expression vector pET32a (+) and expressed as a recombinant protein in Escherichia coli BL21(DE3). Female BALB/c mice were immunized with the purified recombinant truncated S1 protein, and three monoclonal antibodies (MAb designated as E3, G8, and G9) against the truncated S1 protein obtained by hydridoma technique. Further characterization demonstrated that the three MAbs (E2, G8, and G9) belong to IgG1 subclass and have different affinities (G9?>?G8?>?E3). Furthermore, all of the three MAbs reacted with PEDV in the fluorescent antibody assay. Our study suggests that purified truncated S1 protein and the three developed MAbs could be useful in the development of a diagnostic assay for anti-PEDV antibodies and PEDV antigen, respectively. PMID:26492620

  3. Association of brominated proteins and changes in protein expression in the rat kidney with subcarcinogenic to carcinogenic doses of bromate

    SciTech Connect

    Kolisetty, Narendrababu; Bull, Richard J.; Muralidhara, Srinivasa; Costyn, Leah J.; Delker, Don A.; Guo, Zhongxian; Cotruvo, Joseph A.; Fisher, Jeffrey W.; Cummings, Brian S.

    2013-10-15

    The water disinfection byproduct bromate (BrO{sub 3}{sup ?}) produces cytotoxic and carcinogenic effects in rat kidneys. Our previous studies demonstrated that BrO{sub 3}{sup ?} caused sex-dependent differences in renal gene and protein expression in rats and the elimination of brominated organic carbon in their urine. The present study examined changes in renal cell apoptosis and protein expression in male and female F344 rats treated with BrO{sub 3}{sup ?} and associated these changes with accumulation of 3-bromotyrosine (3-BT)-modified proteins. Rats were treated with 0, 11.5, 46 and 308 mg/L BrO{sub 3}{sup ?} in drinking water for 28 days and renal sections were prepared and examined for apoptosis (TUNEL-staining), 8-oxo-deoxyguanosine (8-oxoG), 3-BT, osteopontin, Kim-1, clusterin, and p-21 expression. TUNEL-staining in renal proximal tubules increased in a dose-related manner beginning at 11.5 mg BrO{sub 3}{sup ?}/L in female rats and 46 mg/L in males. Increased 8-oxoG staining was observed at doses as low as 46 mg/L. Osteopontin expression also increased in a dose-related manner after treatment with 46 mg/L, in males only. In contrast, Kim-1 expression increased in a dose-related manner in both sexes, although to a greater extent in females at the highest dose. Clusterin and p21 expression also increased in a dose-related manner in both sexes. The expression of 3-BT-modified proteins only increased in male rats, following a pattern previously reported for accumulation of ?-2{sub u}-globulin. Increases in apoptosis in renal proximal tubules of male and female rats at the lowest doses suggest a common mode of action for renal carcinogenesis for the two sexes that is independent of ?-2{sub u}-globulin nephropathy. - Highlights: • Bromate induced nephrotoxicity in both male and female rats by similar mechanisms. • Apoptosis was seen in both male and female rats at the lowest doses tested. • Bromate-induced apoptosis correlated to 8-oxo-deoxyguanosine formation. • Bromate increased the level of 3-bromotyrosine-modified proteins in male rats only. • These data identify possible novel mechanisms for bromate-induced nephrotoxicity.

  4. Inducible protein expression in Drosophila Schneider 2 cells using the lac operator-repressor system.

    PubMed

    Wakiyama, Motoaki; Muramatsu, Reiko; Kaitsu, Yoko; Ikeda, Mariko; Yokoyama, Shigeyuki

    2011-12-01

    Schneider line 2 cells, derived from Drosophila melanogaster, can be used as a highly versatile gene expression system. Two powerful promoters derived from the actin5C (Ac5) and metallothionein (Mtn) genes are available. The Mtn promoter can be used for the inducible expression of heterologous proteins unsuitable for constitutive expression. However, to circumvent using CuSO(4) or CdCl(2) as inducers of the Mtn promoter, we created a modified Ac5 promoter, Ac5LacO, in which two short lac operator sequences are embedded. Expression from the Ac5LacO promoter was regulated with co-expression of the lac repressor and IPTG. More than 25-fold induction of firefly luciferase expression was achieved in transient transfection experiments. Furthermore, we demonstrated that the lac operator-repressor regulatory system functioned in chromosomally integrated cell lines. PMID:21826399

  5. Negative regulation of parathyroid hormone-related protein expression by steroid hormones

    SciTech Connect

    Kajitani, Takashi; Tamamori-Adachi, Mimi; Okinaga, Hiroko; Chikamori, Minoru; Iizuka, Masayoshi; Okazaki, Tomoki

    2011-04-15

    Highlights: {yields} Steroid hormones repress expression of PTHrP in the cell lines where the corresponding nuclear receptors are expressed. {yields} Nuclear receptors are required for suppression of PTHrP expression by steroid hormones, except for androgen receptor. {yields} Androgen-induced suppression of PTHrP expression appears to be mediated by estrogen receptor. -- Abstract: Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here we studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor {alpha}, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.

  6. Isolation of mitochondria from liver and extraction of total RNA and protein: analyses of microRNA and protein expressions.

    PubMed

    Borralho, Pedro M; Steer, Clifford J; Rodrigues, Cecília M P

    2015-01-01

    Several studies have indicated the presence of microRNAs (miRNAs) within mitochondria, although the origin, as well as the biological function of these mitochondrially located miRNAs is largely unknown. The identification and significance of this subcellular localization is gaining increasing relevance to the pathogenesis of certain disease states. Here we describe the isolation of highly purified mitochondria from rat liver by differential centrifugation, followed by RNAse A treatment to eliminate contaminating RNA. The coupled extraction of total RNA and protein is a more efficient design for allowing the downstream evaluation of miRNA and protein expression in mitochondria. PMID:25308484

  7. Molecular Characterization and Expression Profiling of the Protein Disulfide Isomerase Gene Family in Brachypodium distachyon L

    PubMed Central

    Zhu, Jiantang; Yin, Guangjun; Li, Xiaohui; Hu, Yingkao; Li, Jiarui; Yan, Yueming

    2014-01-01

    Protein disulfide isomerases (PDI) are involved in catalyzing protein disulfide bonding and isomerization in the endoplasmic reticulum and functions as a chaperone to inhibit the aggregation of misfolded proteins. Brachypodium distachyon is a widely used model plant for temperate grass species such as wheat and barley. In this work, we report the first molecular characterization, phylogenies, and expression profiles of PDI and PDI-like (PDIL) genes in B. distachyon in different tissues under various abiotic stresses. Eleven PDI and PDIL genes in the B. distachyon genome by in silico identification were evenly distributed across all five chromosomes. The plant PDI family has three conserved motifs that are involved in catalyzing protein disulfide bonding and isomerization, but a different exon/intron structural organization showed a high degree of structural differentiation. Two pairs of genes (BdPDIL4-1 and BdPDIL4-2; BdPDIL7-1 and BdPDIL7-2) contained segmental duplications, indicating each pair originated from one progenitor. Promoter analysis showed that Brachypodium PDI family members contained important cis-acting regulatory elements involved in seed storage protein synthesis and diverse stress response. All Brachypodium PDI genes investigated were ubiquitously expressed in different organs, but differentiation in expression levels among different genes and organs was clear. BdPDIL1-1 and BdPDIL5-1 were expressed abundantly in developing grains, suggesting that they have important roles in synthesis and accumulation of seed storage proteins. Diverse treatments (drought, salt, ABA, and H2O2) induced up- and down-regulated expression of Brachypodium PDI genes in seedling leaves. Interestingly, BdPDIL1-1 displayed significantly up-regulated expression following all abiotic stress treatments, indicating that it could be involved in multiple stress responses. Our results provide new insights into the structural and functional characteristics of the plant PDI gene family. PMID:24747843

  8. Expression in Pichia pastoris and immunological evaluation of a truncated Dengue envelope protein.

    PubMed

    Valdés, Iris; Hermida, Lisset; Zulueta, Aída; Martín, Jorge; Silva, Ricardo; Alvarez, Mayling; Guzmán, María G; Guillén, Gerardo

    2007-01-01

    Among the Dengue virus structural proteins, the Envelope glycoprotein is the most important because of its antigenic characteristics. In this work, the E protein from Dengue-2 virus truncated at the C-terminus region was successfully expressed in Pichia pastoris. The E2trunc gene was cloned under the AOX1 promoter from P. pastoris and the signal peptide of the sucrose invertase gene from Saccharomyces cerevisiae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression revealed the presence of a protein with the expected size, which was completely associated to the insoluble fraction after cellular disruption. The recombinant N-glycosylated protein reacted with two conformational antibodies against Dengue-2, indicating a proper folding of it. In addition, it was able to induce antiviral antibodies after mice immunization. PMID:17401146

  9. Identification, cloning, and expression of the major capsid protein gene of human herpesvirus 6.

    PubMed Central

    Littler, E; Lawrence, G; Liu, M Y; Barrell, B G; Arrand, J R

    1990-01-01

    DNA sequence analysis of part of the human herpesvirus 6 (HHV-6) genome led to the identification of an open reading frame with amino acid sequence homology to the major capsid proteins (MCP) of other HHVs. DIAGON analysis showed that the closest homology was with human cytomegalovirus. Plasmids were constructed which were shown to express the HHV-6 MCP as either the entire open reading frame or as portions of it, and the recombinant-produced proteins were used to raise antisera. The antisera were shown by immunofluorescence to react with HHV-6-infected lymphoblastoid cells and in Western blots with a 135-kilodalton protein specific to HHV-6-infected cells. The recombinant protein expressed from the entire HHV-6 MCP gene was detected only weakly in Western blot assays with normal HHV-6-positive human sera as a probe. Images PMID:2153237

  10. Screening for Differentially Expressed Proteins Relevant to the Differential Diagnosis of Sarcoidosis and Tuberculosis

    PubMed Central

    Hu, Yang; Wang, Liu-Sheng; Zhou, Ying; Li, Qiu-Hong; Li, Yan; Du, Yu-Kui; He, Xian; Li, Nan; Yin, Zhao-Fang; Wei, Ya-Ru; Weng, Dong; Li, Hui-Ping

    2015-01-01

    Background In this study, we sought to identify differentially expressed proteins in the serum of patients with sarcoidosis or tuberculosis and to evaluate these proteins as markers for the differential diagnosis of sarcoidosis and sputum-negative tuberculosis. Methods Using protein microarrays, we identified 3 proteins exhibiting differential expression between patients with sarcoidosis and tuberculosis. Elevated expression of these proteins was verified using the enzyme-linked immunosorbent assay (ELISA) and was further confirmed by immunohistochemistry. Receiver operating characteristic (ROC) curve, logistic regression analysis, parallel, and serial tests were used to evaluate the diagnostic efficacy of the proteins. Results Intercellular Adhesion Molecule 1(ICAM-1) and leptin were screened for differentially expressed proteins relevant to sarcoidosis and tuberculosis. Using ROC curves, we found that ICAM-1 (cutoff value: 57740 pg/mL) had an area under the curve (AUC), sensitivity, and specificity of 0.718, 62.3%, and 79.5% respectively, while leptin (cutoff value: 1193.186 pg/mL) had an AUC, sensitivity, and specificity of 0.763, 88.3%, and 65.8%, respectively. Logistic regression analysis revealed that the AUC, sensitivity, and specificity of combined leptin and ICAM-1 were 0.787, 89.6%, and 65.8%, respectively, while those of combined leptin, ICAM-1, and body mass index (BMI) were 0.837, 90.9%, and 64.4%, respectively, which had the greatest diagnostic value. Parallel and serial tests indicated that the BMI-leptin parallel with the ICAM-1 serial was the best diagnostic method, achieving a sensitivity and specificity of 86.5% and 73.1%, respectively. Thus, our results identified elevated expression of ICAM-1 and leptin in serum and granulomas of sarcoidosis patients. Conclusio