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Sample records for channel protein expression

  1. Protein expression of G-protein inwardly rectifying potassium channels (GIRK in breast cancer cells

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    Plummer Howard K

    2006-08-01

    Full Text Available Abstract Background Previous data from our laboratory has indicated that a functional link exists between the G-protein-coupled inwardly rectifying potassium (GIRK channel and the beta-adrenergic receptor pathway in breast cancer cell lines, and these pathways were involved in growth regulation of these cells. Alcohol is an established risk factor for breast cancer and has been found to open GIRK. In order to further investigate GIRK channels in breast cancer and possible alteration by ethanol, we identified GIRK channel protein expression in breast cancer cells. Results Cell pellets were collected and membrane protein was isolated to determine GIRK protein expression. GIRK protein was also analyzed by immuno-precipitation. GIRK protein was over-expressed in cells by transfection of GIRK plasmids. Gene expression studies were done by real-time RT-PCR. GIRK protein expression was identified in breast cancer cell lines. Expression of GIRK1 at the indicated molecular weight (MW (62 kDa was seen in cell lines MDA-MB-453 and ZR-75-1. In addition, GIRK1 expression was seen at a lower MW (40–42 kDa in MDA-MB-361, MDA-MB-468, MCF-7, ZR-75-1, and MDA-MB-453 cell lines. To prove the lower MW protein was GIRK1, MDA-MB-453 cells were immuno-precipitated. GIRK2 expression was seen in MDA-MB-468, MCF-7, and ZR-75-1 and was variable in MDA-MB-453, while GIRK4 protein expression was seen in all six cell lines tested. This is the first report indicating GIRK protein expression in breast cancer cells. To determine functionality, MDA-MB-453 cells were stimulated with ethanol. Decreased GIRK1 protein expression levels were seen after treatment with 0.12% ethanol in MDA-MB-453 breast cancer cells. Serum-free media decreased GIRK protein expression, possibly due to lack of estrogen in the media. Transfection of GIRK1 or GIRK4 plasmids increased GIRK1 protein expression and decreased gene expression in MDA-MB-453 breast cancer cells. Conclusion Our data indicates

  2. Do cysteine residues regulate transient receptor potential canonical type 6 (TRPC6) channel protein expression?

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Krueger, Katharina;

    2012-01-01

    The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed that patie......The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed...... that patients with chronic renal failure had significantly elevated homocysteine levels and TRPC6 mRNA expression levels in monocytes compared to control subjects. We further observed that administration of homocysteine or acetylcysteine significantly increased TRPC6 channel protein expression compared...

  3. Expression of G-protein inwardly rectifying potassium channels (GIRKs in lung cancer cell lines

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    Schuller Hildegard M

    2005-08-01

    Full Text Available Abstract Background Previous data from our laboratory has indicated that there is a functional link between the β-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1 in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. Methods GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU assay. Results GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with β2-adrenergic antagonist ICI 118,551 (100 μM daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the β-adrenergic agonist isoproterenol (10 μM decreased growth rates in these cells. The GIRK inhibitor U50488H (2 μM also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2 was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4 mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4 mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein

  4. Expression of G-protein inwardly rectifying potassium channels (GIRKs) in lung cancer cell lines

    International Nuclear Information System (INIS)

    Previous data from our laboratory has indicated that there is a functional link between the β-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU) assay. GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC) cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with β2-adrenergic antagonist ICI 118,551 (100 μM) daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the β-adrenergic agonist isoproterenol (10 μM) decreased growth rates in these cells. The GIRK inhibitor U50488H (2 μM) also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2) was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4) mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4) mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein expression was not seen in any non-SCLC cells

  5. Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity.

    Science.gov (United States)

    Londino, James D; Lazrak, Ahmed; Jurkuvenaite, Asta; Collawn, James F; Noah, James W; Matalon, Sadis

    2013-05-01

    The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

  6. Development of heart failure is independent of K+ channel-interacting protein 2 expression

    DEFF Research Database (Denmark)

    Speerschneider, Tobias; Grubb, Søren; Metoska, Artina;

    2013-01-01

    Abstract  Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K(+) channel-interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss of the...... before and every 2 weeks after the operation. Ten weeks post-surgery, surface ECG was recorded and we paced the heart in vivo to induce arrhythmias. Afterwards, tissue from the left ventricle was used for immunoblotting. Time courses of HF development were comparable in TAC-operated WT and KChIP2...

  7. Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

    Science.gov (United States)

    Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

    2016-04-01

    p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. PMID:26721627

  8. Expression of hNav1.8 sodium channel protein in affected nerves of patients with trigeminal neuralgia

    Institute of Scientific and Technical Information of China (English)

    ZHU Ling-lan; JIANG Xiao-zhong; ZHAO Yun-fu; LI Yu-li; HE Jin

    2004-01-01

    Objective: To explore the pathogenesis of trigeminal neuralgia (TN) and to provide a new target for the drug treatment of TN by studying the expression of tetrodotoxin-resistant hNavl. 8 sodium channel protein in affected nerves of patients with TN. Methods: Twelve affected inferior alveolar nerves were obtained from patients with idiopathic TN, to whom the drug therapy was not effective. As negative control, one normal inferior alveolar nerve was obtained from patients who accepted the combined radical neck dissection with glossectomy and mandibulectomy. One muscle sample was obtained as normal control. One dorsal root ganglion from rat was as positive control. These tissues and prepared hNav1.8 antibody were conducted immunohistochemistry response. Results: hNavl. 8 channel protein was expresses in all the 12 specimens of the affected nerves of patients with TN, but not in the muscle sample and the normal inferior alveolar nerve. Conclusion:The abnormal expression of hNavl. 8 channel protein in the affected nerves of patients with TN may play an impo~nt role in the pathogenesis of TN.

  9. Expression of tetraspan protein CD63 activates protein-tyrosine kinase (PTK) and enhances the PTK-induced inhibition of ROMK channels.

    NARCIS (Netherlands)

    Lin, D.; Kamsteeg, E.J.; Zhang, Y.; Jin, Y.; Sterling, H.; Yue, P.; Roos, M.; Duffield, A.; Spencer, J.; Caplan, M.; Wang, W.H.

    2008-01-01

    In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase alpha (RPTPalpha) are expressed in the cortical collecting duct and outer medulla collecting duct. Immu

  10. CFTR anion channel modulates expression of human transmembrane mucin MUC3 through the PDZ protein GOPC.

    Science.gov (United States)

    Pelaseyed, Thaher; Hansson, Gunnar C

    2011-09-15

    The transmembrane mucins in the enterocyte are type 1 transmembrane proteins with long and rigid mucin domains, rich in proline, threonine and serine residues that carry numerous O-glycans. Three of these mucins, MUC3, MUC12 and MUC17 are unique in harboring C-terminal class I PDZ motifs, making them suitable ligands for PDZ proteins. A screening of 123 different human PDZ domains for binding to MUC3 identified a strong interaction with the PDZ protein GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). This interaction was mediated by the C-terminal PDZ motif of MUC3, binding to the single GOPC PDZ domain. GOPC is also a binding partner for cystic fibrosis transmembrane conductance regulator (CFTR) that directs CFTR for degradation. Overexpression of GOPC downregulated the total levels of MUC3, an effect that was reversed by introducing CFTR. The results suggest that CFTR and MUC3 compete for binding to GOPC, which in turn can regulate levels of these two proteins. For the first time a direct coupling between mucins and the CFTR channel is demonstrated, a finding that will shed further light on the still poorly understood relationship between cystic fibrosis and the mucus phenotype of this disease.

  11. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

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    McClafferty Heather

    2005-01-01

    Full Text Available Abstract Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP

  12. Relationship between expression of muscle-specific uncoupling protein 2 messenger RNA and genetic selection toward growth in channel catfish.

    Science.gov (United States)

    Kobayashi, Y; Peterson, B C; Waldbieser, G C

    2015-04-01

    This study tested the hypothesis that increased growth in channel catfish is associated with expression of the genes that code for uncoupling proteins (UCP) 2 and 3, members of the mitochondrial channel proteins involved in nutrient sensing and metabolism. The specific objective was to contrast the levels of UCP2 messenger RNA (mRNA) in fast vs slow growing catfish as well as in fed vs fasted catfish. Two distinct UCP2 transcripts were identified and named UCP2a and UCP2b, respectively. Nucleotide and amino acid sequence of catfish UCP2s were highly similar to UCP2 and other UCPs from other fish and mammals (>75%). Expression of UCP2a mRNA was detectable at very low levels in various metabolically active tissues, whereas the expression of UCP2b mRNA was readily detectable in the muscle and heart. In a 21-wk feeding study, fish that grew faster had a greater percent body fat at the end of the study (P muscle was increased (P growth and associated fat accumulation appears to be independent of muscle UCP2b mRNA expression and UCP2b-mediated mechanisms.

  13. Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels.

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    Nielsen, S; Marples, D; Birn, H; Mohtashami, M; Dalby, N O; Trimble, M; Knepper, M

    1995-01-01

    Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant association of both AQP2 and VAMP2 in the same vesicles (P < 0.0001). Furthermore, the presence of AQP2 in vesicles immunoisolated with anti-VAMP2 antibodies was confirmed by immunoblotting. In conclusion, VAMP2, a component of the neuronal SNARE complex, is expressed in vesicles carrying AQP2, suggesting a role in vasopressin-regulated vesicle trafficking of AQP2

  14. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

    Science.gov (United States)

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

    2016-08-01

    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease.

  15. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation.

    Science.gov (United States)

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

    2016-01-01

    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease. PMID:27488468

  16. Channel properties of Nax expressed in neurons.

    Directory of Open Access Journals (Sweden)

    Masahito Matsumoto

    Full Text Available Nax is a sodium-concentration ([Na+]-sensitive Na channel with a gating threshold of ~150 mM for extracellular [Na+] ([Na+]o in vitro. We previously reported that Nax was preferentially expressed in the glial cells of sensory circumventricular organs including the subfornical organ, and was involved in [Na+] sensing for the control of salt-intake behavior. Although Nax was also suggested to be expressed in the neurons of some brain regions including the amygdala and cerebral cortex, the channel properties of Nax have not yet been adequately characterized in neurons. We herein verified that Nax was expressed in neurons in the lateral amygdala of mice using an antibody that was newly generated against mouse Nax. To investigate the channel properties of Nax expressed in neurons, we established an inducible cell line of Nax using the mouse neuroblastoma cell line, Neuro-2a, which is endogenously devoid of the expression of Nax. Functional analyses of this cell line revealed that the [Na+]-sensitivity of Nax in neuronal cells was similar to that expressed in glial cells. The cation selectivity sequence of the Nax channel in cations was revealed to be Na+ ≈ Li+ > Rb+ > Cs+ for the first time. Furthermore, we demonstrated that Nax bound to postsynaptic density protein 95 (PSD95 through its PSD95/Disc-large/ZO-1 (PDZ-binding motif at the C-terminus in neurons. The interaction between Nax and PSD95 may be involved in promoting the surface expression of Nax channels because the depletion of endogenous PSD95 resulted in a decrease in Nax at the plasma membrane. These results indicated, for the first time, that Nax functions as a [Na+]-sensitive Na channel in neurons as well as in glial cells.

  17. Effects of Angiotensin Ⅱ on Expression of the Gap Junction Channel Protein Connexin 43 in Neonatal Rat Ventricular Myocytes

    Institute of Scientific and Technical Information of China (English)

    Jun Yang; Wei Wu

    2007-01-01

    To study the effects of angiotensin Ⅱ,as a mediator of cardiac hypertrophy,on expression of connexin 43 (Cx43) in cultured neonatal rat ventricular myocytes and correlation of expression of Cx43 and cardiomyocyte hypertrophy.Methods Cardiomyocytes were isolated from newborn SD rats.Angiotensin Ⅱ was added into the media to induce myocyte hypertrophy.Cultures were exposed to 10 ~6 mol/L angiotensin Ⅱ for 72 h,Cx43 expression was characterized by RT-PCR and Immunofluorescence methods.Results Immunofluorescence analysis revealed decreased Cx43 immunoreactivity in cells treated for 72 h with angiotensin Ⅱ.RT-PCR analysis demonstrated there was an obvious decrease of Cx43 mRNA level in cells exposed to angiotensin Ⅱ for 72 h.The changes of expression of connexin 43 were related to its entrance into S phase of the cell cycle.Cultured neonatal rat cardiomyocytes were exposed for 72 h to increase concentrations of angiotensin Ⅱ ( 1.0 × 10-9 ~ 1.0 × 10-6mol/L),resulting in significantly decreased Cx43 expression.Conclusions Angiotensin Ⅱ leads to a concentration-dependent decrease in Cx43 protein in cultured neonatal rat ventricular myocytes by decreasing Cx43 mRNA synthesis.Signal transduction pathways activated by angiotensin Ⅱ under pathophysiologic conditions of cardiac hypertrophy could initiate remodeling of gap junctions.

  18. [Protein expression and purification].

    Science.gov (United States)

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  19. Elevated extracellular calcium increases expression of bone morphogenetic protein-2 gene via a calcium channel and ERK pathway in human dental pulp cells

    International Nuclear Information System (INIS)

    Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca2+Cao2+ has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Cao2+ signaling in odontogenesis remains unclear. We found that elevated Cao2+ increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca2+ increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca2+ channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca2+, suggesting that the Ca2+ influx from Ca2+ channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca2+-sensing receptors (CaSR) and only respond slightly to other cations such as Sr2+ and spermine, suggesting that dental pulp cells respond to Cao2+ to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Cao2+ among cations.

  20. Analytical Expression for the MIMO Channel Capacity

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yifei; ZHAO Ming; XIAO Limin; WANG Jing

    2006-01-01

    This paper presents analytical expressions for the multiple-input multiple-output (MIMO) channel capacity in frequency-flat Rayleigh fading environments. An exact analytical expression is given for the ergodic capacity for single-input multiple-output (SIMO) channels. The analysis shows that the SIMO channel capacity can be approximated by a Gaussian random variable and that the MIMO channel capacity can be approximated as the sum of multiple SIMO capacities. The SIMO channel results are used to derive approximate closed-form expressions for the MIMO channel ergodic capacity and the complementary cumulative distribution function (CCDF) of the MIMO channel capacity (outage capacity). Simulations show that these theoretical results are good approximations for MIMO systems with an arbitrary number of transmit or receive antennas. Moreover, these analytical expressions are relatively simple which makes them very useful for practical computations.

  1. Membrane channel gene expression in human costal and articular chondrocytes.

    Science.gov (United States)

    Asmar, A; Barrett-Jolley, R; Werner, A; Kelly, R; Stacey, M

    2016-04-01

    Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca(2+) activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  2. Elevated extracellular calcium increases expression of bone morphogenetic protein-2 gene via a calcium channel and ERK pathway in human dental pulp cells

    Energy Technology Data Exchange (ETDEWEB)

    Tada, Hiroyuki [Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Nemoto, Eiji, E-mail: e-nemoto@umin.ac.jp [Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Kanaya, Sousuke; Hamaji, Nozomu; Sato, Hisae; Shimauchi, Hidetoshi [Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan)

    2010-04-16

    Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca{sup 2+}Ca{sub o}{sup 2+} has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca{sub o}{sup 2+} signaling in odontogenesis remains unclear. We found that elevated Ca{sub o}{sup 2+} increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca{sup 2+} increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca{sup 2+} channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca{sup 2+}, suggesting that the Ca{sup 2+} influx from Ca{sup 2+} channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca{sup 2+}-sensing receptors (CaSR) and only respond slightly to other cations such as Sr{sup 2+} and spermine, suggesting that dental pulp cells respond to Ca{sub o}{sup 2+} to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca{sub o}{sup 2+} among cations.

  3. Channel catfish (Ictalurus punctatus Rafinesque, 1818) tetraspanin membrane protein family: Identification, characterization, and expression analysis of CD63 cDNA

    Science.gov (United States)

    CD63, known as lysosome associated membrane protein 3 (LAMP-3), is a member of the tetraspanin integral membrane protein family. This protein plays many important roles in immuno-physiological functions. In this communication, we report the identification, characterization, and expression analysis...

  4. cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

    NARCIS (Netherlands)

    A.B. Vaandrager (Arie); S.M. Lohmann (Suzanne); H.R. de Jonge (Hugo); W.C. Poller; B.C. Tilly (Bernard); A. Smolenski; S. Schneider-Rasp; A.G. Bot (Alice); M.J. Edixhoven (Marcel); B.J. Scholte (Bob); T. Jarchau; U. Walter

    1997-01-01

    textabstractIn order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was ana

  5. Dental enamel cells express functional SOCE channels.

    Science.gov (United States)

    Nurbaeva, Meerim K; Eckstein, Miriam; Concepcion, Axel R; Smith, Charles E; Srikanth, Sonal; Paine, Michael L; Gwack, Yousang; Hubbard, Michael J; Feske, Stefan; Lacruz, Rodrigo S

    2015-10-30

    Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation.

  6. α-actinin2增加SK2通道在HEK293膜蛋白的表达%α-Actinin 2 increases membrane protein expression of SK2 channel in HEK293 cell

    Institute of Scientific and Technical Information of China (English)

    李涛; 陈桂兰; 黄文俊; 毛亮; 李畅; 杨艳; 曾晓荣

    2014-01-01

    目的:证实α-Actinin 2与小电导钙激活钾通道(Small Conductance Ca2+-activated K+Channels, SK)的共同表达增加SK2在HEK293细胞膜上的表达。方法:HEK293细胞用RPMI 1640培养基培养。将HEK293细胞分为两组,对照组细胞转染pIRES-SK2质粒,实验组细胞共转染pIRES-SK2和pcDNA3-α-actinin2质粒。免疫荧光共聚焦显微镜和蛋白印迹技术检测SK2通道蛋白在HEK293细胞膜上的表达。结果:免疫荧光共聚焦显微镜检测到转染pIRES-SK2质粒的HEK293细胞膜上有SK2通道蛋白的表达。蛋白印迹技术显示实验组蛋白条带亮度明显高于对照组。结论:α-Actinin 2能增加SK2在HEK293细胞膜上的表达。%Objective:To confirm whether the protein expression of SK2 channel in HEK293 cell membrane is increased by the co-expression of α-Actinin2. Methods:HEK293 cells were cultured and randomly divided into control group, transfected with plasmid pIRES-SK2,and experimental group,transfected with plasmid pIRES-SK2 plus pcDNA3-α-Actinin2. Immunofluorescence confocal microscopy and western-blotting were used to test the membrane protein expression of SK2 channel in HEK293 cell. Results: SK2 channel protein was detected in the membrane of HEK293 by immunofluorescence confocal microscopy ,and he protein expression level in the experimental group was much higher than that in the control group. Conclusion:α-Actinin2 can increase SK2 channel membrane protein expression in HEK293 cell.

  7. Calreticulin: Roles in Cell-Surface Protein Expression

    Directory of Open Access Journals (Sweden)

    Yue Jiang

    2014-09-01

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

  8. Cloning and expression of a FMRFamide-gated Na+ channel from Helisoma trivolvis and comparison with the native neuronal channel

    Science.gov (United States)

    Jeziorski, Michael C; Green, Kevin A; Sommerville, John; Cottrell, Glen A

    2000-01-01

    We have cloned a cDNA encoding a Phe-Met-Arg-Phe-NH2 (FMRFamide)-gated Na+ channel from nervous tissue of the pond snail Helisoma trivolvis (HtFaNaC) and expressed the channel in Xenopus oocytes. The deduced amino acid sequence of the protein expressed by HtFaNaC is 65 % identical to that of the FMRFamide-gated channel cloned from Helix aspersa (HaFaNaC). HtFaNaC expressed in oocytes was less sensitive to FMRFamide (EC50 = 70 μM) than HaFaNaC (EC50 = 2 μM). The two had a similar selectivity for Na+. The amplitude of the FMRFamide response of HtFaNaC was increased by reducing the extracellular concentration of divalent cations. The conductance of the two channels was similar, but the mean open time of unitary events was shorter for expressed HtFaNaC compared to expressed HaFaNaC. Each channel was susceptible to peptide block by high agonist concentrations. In marked contrast to HaFaNaC and other amiloride-sensitive Na+ channels, amiloride, and the related drugs benzamil and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), enhanced the FMRFamide response in oocytes expressing HtFaNaC cRNA. The potentiating effects of EIPA and benzamil were greater than those of amiloride. Unitary current analysis showed that with such drugs, there was channel blockade as well as an increased probability of channel opening. The similar permeability of the oocyte-expressed HtFaNaC and the Helisoma neuronal channel, and the susceptibility of both to agonist blockade and blockade by divalent cations, suggest that the channels are the same. However, neuronal channels were less susceptible to enhancement by amiloride analogues and in some patches were more sensitive to FMRFamide than expressed HtFaNaC. PMID:10878095

  9. Side-effects of protein kinase inhibitors on ion channels

    Indian Academy of Sciences (India)

    Youn Kyoung Son; Hongzoo Park; Amy L Firth; Won Sun Park

    2013-12-01

    Protein kinases are one of the largest gene families and have regulatory roles in all aspects of eukaryotic cell function. Modulation of protein kinase activity is a desirable therapeutic approach for a number of human diseases associated with aberrant kinase activity, including cancers, arthritis and cardiovascular disorders. Several strategies have been used to develop specific and selective protein kinase modulators, primarily via inhibition of phosphorylation and down-regulation of kinase gene expression. These strategies are effective at regulating intracellular signalling pathways, but are unfortunately associated with several undesirable effects, particularly those that modulate ion channel function. In fact, the side-effects have precluded these inhibitors from being both useful experimental tools and therapeutically viable. This review focuses on the ion channel side-effects of several protein kinase inhibitors and specifically on those modulating K+, Na+ and Ca2+ ion channels. It is hoped that the information provided with a detailed summary in this review will assist the future development of novel specific and selective compounds targeting protein kinases both for experimental tools and for therapeutic approaches.

  10. Inhibition of Voltage-Gated Calcium Channels by RGK Proteins.

    Science.gov (United States)

    Buraei, Zafir; Yang, Jian

    2015-01-01

    Due to their essential biological roles, voltage-gated calcium channels (VGCCs) are regulated by a myriad of molecules and mechanisms. Fifteen years ago, RGK proteins were discovered to bind the VGCC β subunit (Cavβ) and potently inhibit high-voltage activated Ca(2+) channels. RGKs (Rad, Rem, Rem2 and Gem/Kir) are a family of monomeric small GTPases belonging to the superfamily of Ras GTPases. They exert dual inhibitory effects on VGCCs, decreasing surface expression and suppressing surface channels through immobilization of the voltage sensor or reduction of channel open probability. While Cavβ is required for all forms of RGK inhibition, not all inhibition is mediated by the RGK-Cavβ interaction. Some RGK proteins also interact directly with the pore-forming α1 subunit of some types of VGCCs (Cavα1). Importantly, RGK proteins tonically inhibit VGCCs in native cells, regulating cardiac and neural functions. This minireview summarizes the mechanisms, molecular determinants, and physiological impact of RGK inhibition of VGCCs. PMID:25966691

  11. The TRPC2 channel forms protein-protein interactions with Homer and RTP in the rat vomeronasal organ

    Directory of Open Access Journals (Sweden)

    Brann Jessica H

    2010-05-01

    Full Text Available Abstract Background The signal transduction cascade operational in the vomeronasal organ (VNO of the olfactory system detects odorants important for prey localization, mating, and social recognition. While the protein machinery transducing these external cues has been individually well characterized, little attention has been paid to the role of protein-protein interactions among these molecules. Development of an in vitro expression system for the transient receptor potential 2 channel (TRPC2, which establishes the first electrical signal in the pheromone transduction pathway, led to the discovery of two protein partners that couple with the channel in the native VNO. Results Homer family proteins were expressed in both male and female adult VNO, particularly Homer 1b/c and Homer 3. In addition to this family of scaffolding proteins, the chaperones receptor transporting protein 1 (RTP1 and receptor expression enhancing protein 1 (REEP1 were also expressed. RTP1 was localized broadly across the VNO sensory epithelium, goblet cells, and the soft palate. Both Homer and RTP1 formed protein-protein interactions with TRPC2 in native reciprocal pull-down assays and RTP1 increased surface expression of TRPC2 in in vitro assays. The RTP1-dependent TRPC2 surface expression was paralleled with an increase in ATP-stimulated whole-cell current in an in vitro patch-clamp electrophysiological assay. Conclusions TRPC2 expression and channel activity is regulated by chaperone- and scaffolding-associated proteins, which could modulate the transduction of chemosignals. The developed in vitro expression system, as described here, will be advantageous for detailed investigations into TRPC2 channel activity and cell signalling, for a channel protein that was traditionally difficult to physiologically assess.

  12. Newly expressed SUR1-regulated NCCa-ATP channel mediates cerebral edema after ischemic stroke

    OpenAIRE

    Simard, J. Marc; Chen, Mingkui; Tarasov, Kirill V.; Bhatta, Sergei; Ivanova, Svetlana; Melnitchenko, Ludmila; Tsymbalyuk, Natalya; West, G. Alexander; Gerzanich, Volodymyr

    2006-01-01

    Pathological conditions in the central nervous system, including stroke and trauma, are often exacerbated by cerebral edema. We recently identified a nonselective cation channel, the NCCa-ATP channel, in ischemic astrocytes that is regulated by sulfonylurea receptor 1 (SUR1), is opened by depletion of ATP and, when opened, causes cytotoxic edema. Here, we evaluated involvement of this channel in rodent models of stroke. SUR1 protein and mRNA were newly expressed in ischemic neurons, astrocyte...

  13. Massively parallel processor networks with optical express channels

    Science.gov (United States)

    Deri, R.J.; Brooks, E.D. III; Haigh, R.E.; DeGroot, A.J.

    1999-08-24

    An optical method for separating and routing local and express channel data comprises interconnecting the nodes in a network with fiber optic cables. A single fiber optic cable carries both express channel traffic and local channel traffic, e.g., in a massively parallel processor (MPP) network. Express channel traffic is placed on, or filtered from, the fiber optic cable at a light frequency or a color different from that of the local channel traffic. The express channel traffic is thus placed on a light carrier that skips over the local intermediate nodes one-by-one by reflecting off of selective mirrors placed at each local node. The local-channel-traffic light carriers pass through the selective mirrors and are not reflected. A single fiber optic cable can thus be threaded throughout a three-dimensional matrix of nodes with the x,y,z directions of propagation encoded by the color of the respective light carriers for both local and express channel traffic. Thus frequency division multiple access is used to hierarchically separate the local and express channels to eliminate the bucket brigade latencies that would otherwise result if the express traffic had to hop between every local node to reach its ultimate destination. 3 figs.

  14. Correlation of apical fluid-regulating channel proteins with lung function in human COPD lungs.

    Directory of Open Access Journals (Sweden)

    Runzhen Zhao

    Full Text Available Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC, cystic fibrosis transmembrane conductance regulator (CFTR, and aquaporin 5 (AQP5 proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI and II (ATII-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3 was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.

  15. G protein-coupled inwardly rectifying potassium channels in dorsal root ganglion neurons

    Institute of Scientific and Technical Information of China (English)

    Xiao-fei GAO; Hai-lin ZHANG; Zhen-dong YOU; Chang-lin LU; Cheng HE

    2007-01-01

    Aim: G protein-coupled inwardly rectifying potassium channels (GIRK) are important for neuronal signaling and membrane excitability. In the present study, we intend to find whether GIRK channels express functionally in adult rat dorsal root ganglion (DRG) neurons. Methods: We used RT-PCR to detect mRNA for4 subunits of GIRK in the adult DRG. The whole-cell patch clamp recording was used to confirm GIRK channels functionally expressed. Results: The mRNA for the 4 subunits of GIRK were detected in the adult DRG. GTPγS enhanced inwardly rectifying potassium (K+) currents of the DRG neurons, while Ba2+inhibited such currents. Furthermore, the GIRK channels were shown to be coupled to the GABAB receptor, a member of the G protein-coupled receptor family, as baclofen increased the inwardly rectifying K+ currents. Conclusion: GIRK channels are expressed and functionally coupled with GABAB receptors in adult rat DRG neurons.

  16. Functional expression of T-type Ca2+ channels in spinal motoneurons of the adult turtle.

    Directory of Open Access Journals (Sweden)

    Martha Canto-Bustos

    Full Text Available Voltage-gated Ca2+ (CaV channels are transmembrane proteins comprising three subfamilies named CaV1, CaV2 and CaV3. The CaV3 channel subfamily groups the low-voltage activated Ca2+ channels (LVA or T-type a significant role in regulating neuronal excitability. CaV3 channel activity may lead to the generation of complex patterns of action potential firing such as the postinhibitory rebound (PIR. In the adult spinal cord, these channels have been found in dorsal horn interneurons where they control physiological events near the resting potential and participate in determining excitability. In motoneurons, CaV3 channels have been found during development, but their functional expression has not yet been reported in adult animals. Here, we show evidence for the presence of CaV3 channel-mediated PIR in motoneurons of the adult turtle spinal cord. Our results indicate that Ni2+ and NNC55-0396, two antagonists of CaV3 channel activity, inhibited PIR in the adult turtle spinal cord. Molecular biology and biochemical assays revealed the expression of the CaV3.1 channel isotype and its localization in motoneurons. Together, these results provide evidence for the expression of CaV3.1 channels in the spinal cord of adult animals and show also that these channels may contribute to determine the excitability of motoneurons.

  17. Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain.

    Directory of Open Access Journals (Sweden)

    Melissa A Fowler

    Full Text Available The canonical transient receptor potential (TRPC channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+ and G(q/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks. In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS, pyramidal cell layer of the hippocampus (HIP, dentate gyrus (DG, and ventral subiculum (vSUB. TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC, motor cortex (MCx, and somatosensory cortex (SCx. TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.

  18. ThermoTRP channels as modular proteins with allosteric gating.

    Science.gov (United States)

    Latorre, Ramon; Brauchi, Sebastian; Orta, Gerardo; Zaelzer, Cristián; Vargas, Guillermo

    2007-01-01

    Ion channels activate by sensing stimuli such as membrane voltage, ligand binding or temperature and transduce this information into conformational changes that open the channel pore. Thus, a key question in understanding ion channel function is how do the protein domains involved in sensing stimuli (sensors) and opening the pore (gates) communicate. In this regard, transient receptor potential (TRP) channels that confer thermosensation [A. Dhaka, V. Viswanath, A. Patapoutian, TRP ion channels and temperature sensation, Annu. Rev. Neurosci. 29 (2006) 135-161; I.S. Ramsey, M. Delling, D.E. Clapham, An introduction to TRP channels, Annu. Rev. Physiol. 68 (2006) 619-647] (thermoTRP; Q(10)>10) are unique to the extent that they integrate a variety of physical and chemical stimuli. In some cases such as, for example, the vanilloid receptor TRPV1 [M.J. Caterina, M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, D. Julius, The capsaicin receptor: a heat-activated ion channel in the pain pathway, Nature 389 (1997) 816-824] and TRPA1 [G.M. Story, A.M. Peier, A.J. Reeve, S.R. Eid, J. Mosbacher, T.R. Hricik, T.J. Earley, A.C. Hergarden, D.A. Andersson, S.W. Hwang, P. McIntyre, T. Jegla, S. Bevan, A. Patapoutian, ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures, Cell 112 (2003) 819-829; S. Jordt, D. Julius, Molecular basis for species-specific sensitivity to "hot" chilli peppers, Cell 108 (2002) 421-430] the integration of these stimuli elicit pain [M. Tominaga, M.J. Caterina, A.B. Malmberg, T.A. Rosen, H. Gilbert, K. Skinner, B.E. Raumann, A.I. Basbaum, D. Julius, The cloned capsaicin receptor integrates multiple pain-producing stimuli, Neuron 21 (1998) 531-543; M. Bandell, A. Dubin, M. Petrus, A. Orth, J. Mathur, S. Hwang, A. Patapoutian, High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol, Nat. Neurosci. 9 (2006) 466-468; S. Zurborg, B. Yurgionas, JA. Jira, O

  19. Intra-membrane molecular interactions of K+ channel proteins :

    Energy Technology Data Exchange (ETDEWEB)

    Moczydlowski, Edward G.

    2013-07-01

    Ion channel proteins regulate complex patterns of cellular electrical activity and ionic signaling. Certain K+ channels play an important role in immunological biodefense mechanisms of adaptive and innate immunity. Most ion channel proteins are oligomeric complexes with the conductive pore located at the central subunit interface. The long-term activity of many K+ channel proteins is dependent on the concentration of extracellular K+; however, the mechanism is unclear. Thus, this project focused on mechanisms underlying structural stability of tetrameric K+ channels. Using KcsA of Streptomyces lividans as a model K+ channel of known structure, the molecular basis of tetramer stability was investigated by: 1. Bioinformatic analysis of the tetramer interface. 2. Effect of two local anesthetics (lidocaine, tetracaine) on tetramer stability. 3. Molecular simulation of drug docking to the ion conduction pore. The results provide new insights regarding the structural stability of K+ channels and its possible role in cell physiology.

  20. Developmental expression of BK channels in chick cochlear hair cells

    Directory of Open Access Journals (Sweden)

    Tong Mingjie

    2009-12-01

    Full Text Available Abstract Background Cochlear hair cells are high-frequency sensory receptors. At the onset of hearing, hair cells acquire fast, calcium-activated potassium (BK currents, turning immature spiking cells into functional receptors. In non-mammalian vertebrates, the number and kinetics of BK channels are varied systematically along the frequency-axis of the cochlea giving rise to an intrinsic electrical tuning mechanism. The processes that control the appearance and heterogeneity of hair cell BK currents remain unclear. Results Quantitative PCR results showed a non-monotonic increase in BK α subunit expression throughout embryonic development of the chick auditory organ (i.e. basilar papilla. Expression peaked near embryonic day (E 19 with six times the transcript level of E11 sensory epithelia. The steady increase in gene expression from E11 to E19 could not explain the sudden acquisition of currents at E18-19, implicating post-transcriptional mechanisms. Protein expression also preceded function but progressed in a sequence from diffuse cytoplasmic staining at early ages to punctate membrane-bound clusters at E18. Electrophysiology data confirmed a continued refinement of BK trafficking from E18 to E20, indicating a translocation of BK clusters from supranuclear to subnuclear domains over this critical developmental age. Conclusions Gene products encoding BK α subunits are detected up to 8 days before the acquisition of anti-BK clusters and functional BK currents. Therefore, post-transcriptional mechanisms seem to play a key role in the delayed emergence of calcium-sensitive currents. We suggest that regulation of translation and trafficking of functional α subunits, near voltage-gated calcium channels, leads to functional BK currents at the onset of hearing.

  1. Ion channel gene expression predicts survival in glioma patients.

    Science.gov (United States)

    Wang, Rong; Gurguis, Christopher I; Gu, Wanjun; Ko, Eun A; Lim, Inja; Bang, Hyoweon; Zhou, Tong; Ko, Jae-Hong

    2015-08-03

    Ion channels are important regulators in cell proliferation, migration, and apoptosis. The malfunction and/or aberrant expression of ion channels may disrupt these important biological processes and influence cancer progression. In this study, we investigate the expression pattern of ion channel genes in glioma. We designate 18 ion channel genes that are differentially expressed in high-grade glioma as a prognostic molecular signature. This ion channel gene expression based signature predicts glioma outcome in three independent validation cohorts. Interestingly, 16 of these 18 genes were down-regulated in high-grade glioma. This signature is independent of traditional clinical, molecular, and histological factors. Resampling tests indicate that the prognostic power of the signature outperforms random gene sets selected from human genome in all the validation cohorts. More importantly, this signature performs better than the random gene signatures selected from glioma-associated genes in two out of three validation datasets. This study implicates ion channels in brain cancer, thus expanding on knowledge of their roles in other cancers. Individualized profiling of ion channel gene expression serves as a superior and independent prognostic tool for glioma patients.

  2. Lipid ion channels and the role of proteins

    CERN Document Server

    Mosgaard, Lars D

    2013-01-01

    Synthetic lipid membranes in the absence of proteins can display quantized conduction events for ions that are virtually indistinguishable from those of protein channel. By indistinguishable we mean that one cannot decide based on the current trace alone whether conductance events originate from a membrane, which does or does not contain channel proteins. Additional evidence is required to distinguish between the two cases, and it is not always certain that such evidence can be provided. The phenomenological similarities are striking and span a wide range of phenomena: The typical conductances are of equal order and both lifetime distributions and current histograms are similar. One finds conduction bursts, flickering, and multistep-conductance. Lipid channels can be gated by voltage, and can be blocked by drugs. They respond to changes in lateral membrane tension and temperature. Thus, they behave like voltage-gated, temperature-gated and mechano-sensitive protein channels, or like receptors. Lipid channels ...

  3. K-ATP channel expression and pharmacological in vivo and in vitro studies of the K-ATP channel blocker PNU-37883A in rat middle meningeal arteries

    DEFF Research Database (Denmark)

    Ploug, K.B.; Boni, L.J.; Baun, M.;

    2008-01-01

    Background and purpose: Dilatation of cerebral and dural arteries causes a throbbing, migraine-like pain, indicating that these structures are involved in migraine. Clinical trials suggest that adenosine 5'-triphosphate-sensitive K+ (K-ATP) channel opening may cause migraine by dilatating...... intracranial arteries, including the middle meningeal artery (MMA). We studied the K-ATP channel expression profile in rat MMA and examined the potential inhibitory effects of the K-ATP channel blocker PNU-37883A on K-ATP channel opener-induced relaxation of the rat MMA, using the three K-ATP channel openers...... levcromakalim, pinacidil and P-1075. Experimental approach: mRNA and protein expression of K-ATP channel subunits in the rat MMA were studied by quantitative real-time PCR and western blotting, respectively. The in vivo and in vitro effects of the K-ATP channel drugs on rat MMA were studied in the genuine...

  4. Regions of KCNQ K+ Channels Controlling Functional Expression

    Directory of Open Access Journals (Sweden)

    Frank eChoveau

    2012-10-01

    Full Text Available KCNQ1-5 α-subunits assemble to form K+ channels that play critical roles in the function of numerous tissues. The channels are tetramers of subunits containing six transmembrane domains. Each subunit consists of a pore region (S5-pore-S6 and a voltage sensor domain (S1-S4. Despite similar structures, KCNQ2 and KCNQ3 homomers yield small current amplitudes compared to other KCNQ homomers and KCNQ2/3 heteromers. Two major mechanisms have been suggested as governing functional expression. The first involves control of channel trafficking to the plasma membrane by the distal part of the C-terminus, containing two coiled-coiled domains, required for channel trafficking and assembly. The proximal half of the C-terminus is the crucial region for channel modulation by signaling molecules such as calmodulin, which may mediate C- and N-terminal interactions. The N-terminus of KCNQ channels has also been postulated as critical for channel surface expression. The second mechanism suggests networks of interactions between the pore helix and the selectivity filter, and between the pore helix and the S6 domain that govern KCNQ current amplitudes. Here, we summarize the role of these different regions in expression of functional KCNQ channels.

  5. Expression profiles of seven channel catfish antimicrobial peptides in response to Edwardsiella ictaluri infection

    Science.gov (United States)

    Using quantitative PCR technique, the relative transcriptional levels of seven channel catfish antimicrobial peptide (AMP) genes [NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, bactericidal permeability-increasing protein (BPI), cathepsin D, hepcidin, and liver-expressed antimicrobial peptide 2 ...

  6. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    Science.gov (United States)

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  7. Ancient association between cation leak channels and Mid1 proteins is conserved in fungi and animals

    Directory of Open Access Journals (Sweden)

    Alfredo eGhezzi

    2014-03-01

    Full Text Available Neuronal resting potential can tune the excitability of neural networks, affecting downstream behavior. Sodium leak channels (NALCN play a key role in rhythmic behaviors by helping set, or subtly changing neuronal resting potential. The full complexity of these newly described channels is just beginning to be appreciated, however. NALCN channels can associate with numerous subunits in different tissues and can be activated by several different peptides and second messengers. We recently showed that NALCN channels are closely related to fungal calcium channels, which they functionally resemble. Here, we use this relationship to predict a family of NALCN-associated proteins in animals on the basis of homology with the yeast protein Mid1, the subunit of the yeast calcium channel. These proteins all share a cysteine-rich region that is necessary for Mid1 function in yeast. We validate this predicted association by showing that the Mid1 homolog in Drosophila, encoded by the CG33988 gene, is coordinately expressed with NALCN, and that knockdown of either protein creates identical phenotypes in several behaviors associated with NALCN function. The relationship between Mid1 and leak channels has therefore persisted over a billion years of evolution, despite drastic changes to both proteins and the organisms in which they exist.

  8. Noise analysis of ionization kinetics in a protein ion channel

    Science.gov (United States)

    Bezrukov, Sergey M.; Kasianowicz, John J.

    1993-08-01

    We observed excess current noise generated by the reversible ionization of sites in a transmembrane protein ion channel, which is analogous to current fluctuations found recently in solid state microstructure electronic devices. Specifically the current through fully open single channels formed by Staphylococcus aureus α-toxin shows pH dependent fluctuations. We show that noise analysis of the open channel current can be used to evaluate the ionization rate constants, the number of sites participating in the ionization process, and the effect of recharging a single site on the channel conductance.

  9. Calcium-dependent expression of transient receptor potential canonical type 3 channels in patients with chronic kidney disease

    DEFF Research Database (Denmark)

    Liu, Ying; Krueger, Katharina; Hovsepian, Anahit;

    2011-01-01

    It is unknown whether extracellular calcium may regulate the expression of transient receptor potential canonical type 3 (TRPC3) channels in patients with chronic kidney disease. Using quantitative in-cell Western assay we compared the expression of TRPC3 channel protein in monocytes from 20...... patients with chronic kidney disease and 19 age- and sex-matched healthy control subjects. TRPC3 channels were identified by immunoblotting using specific antibodies and TRPC3 protein was further confirmed by mass spectrometry. We observed a significant increase of TRPC3 channel protein expression...... in patients with chronic kidney disease compared to healthy control subjects (normalized expression, 0.42±0.06 vs. 0.19±0.03; p...

  10. Heterologous expression and purification of an active human TRPV3 ion channel

    DEFF Research Database (Denmark)

    Kol, Stefan; Braun, Christian; Thiel, Gerhard;

    2013-01-01

    The transient receptor potential vanilloid 3 (TRPV3) cation channel is widely expressed in human tissues and has been shown to be activated by mild temperatures or chemical ligands. In spite of great progress in the TRP‐channel characterization, very little is known about their structure and...... interactions with other proteins at the atomic level. This is mainly caused by difficulties in obtaining functionally active samples of high homogeneity. Here, we report on the high‐level Escherichia coli expression of the human TRPV3 channel, for which no structural information has been reported to date. We...... retains its current inducing activity, as shown by electrophysiology experiments. The ability to produce the TRPV3 channel heterologously will aid future functional and structural studies. TRPV3 and TRPV3 bind by molecular sieving (1, 2) TRPV3 and TRPV3 bind by blue native page (1, 2, 3)...

  11. Differential effects of ethanol on electrical properties of various potassium channels expressed in oocytes.

    Science.gov (United States)

    Anantharam, V; Bayley, H; Wilson, A; Treistman, S N

    1992-09-01

    The effects of ethanol on a number of electrophysiological parameters were examined in 10 different voltage-gated potassium channels expressed in Xenopus oocytes. None of the channels examined was highly sensitive to ethanol, but there was significant variability among the channels tested at concentrations of ethanol of 200 mM and greater. The response to ethanol was not determined exclusively by membership in a genetic subfamily. In addition, the relative sensitivity among different channels could vary independently for different electrical parameters. For example, current amplitude in DRK1 was insensitive to ethanol, even at concentrations as high as 600 mM, whereas this was one of the more sensitive channels with respect to the kinetics of current inactivation. The opposite situation was true for ShA1. Therefore, ethanol at high concentrations may selectively perturb discrete regions of channel proteins. This is supported by the finding that removal of 318 amino acids from the cytoplasmic carboxyl terminus of DRK1 results in a channel whose current amplitude shows greater sensitivity to ethanol than does DRK1. Thus, the effects of ethanol on the channel may not be limited to interactions at the lipid-protein interface. PMID:1406600

  12. Ether à go-go potassium channel expression in soft tissue sarcoma patients

    Directory of Open Access Journals (Sweden)

    Stühmer Walter

    2006-10-01

    Full Text Available Abstract Background The expression of the human Eag1 potassium channel (Kv10.1 is normally restricted to the adult brain, but it has been detected in both tumour cell lines and primary tumours. Our purpose was to determine the frequency of expression of Eag1 in soft tissue sarcoma and its potential clinical implications. Results We used specific monoclonal antibodies to determine the expression levels of Eag1 in soft tissue sarcomas from 210 patients by immunohistochemistry. Eag1 was expressed in 71% of all tumours, with frequencies ranging from 56% (liposarcoma to 82% (rhabdomyosarcoma. We detected differences in expression levels depending on the histological type, but no association was seen between expression of this protein and sex, age, grade or tumour size. Four cell lines derived from relevant sarcoma histological types (fibrosarcoma and rhabdomyosarcoma were tested for Eag1 expression by real-time RT-PCR. We found all four lines to be positive for Eag1. In these cell lines, blockage of Eag1 by RNA interference led to a decrease in proliferation. Conclusion Eag1 is aberrantly expressed in over 70% sarcomas. In sarcoma cell lines, inhibition of Eag1 expression and/or function leads to reduced proliferation. The high frequency of expression of Eag1 in primary tumours and the restriction of normal expression of the channel to the brain, suggests the application of this protein for diagnostic or therapeutic purposes.

  13. Voltage-gated sodium channel expression and action potential generation in differentiated NG108-15 cells

    OpenAIRE

    Liu Jinxu; Tu Huiyin; Zhang Dongze; Zheng Hong; Li Yu-Long

    2012-01-01

    Abstract Background The generation of action potential is required for stimulus-evoked neurotransmitter release in most neurons. Although various voltage-gated ion channels are involved in action potential production, the initiation of the action potential is mainly mediated by voltage-gated Na+ channels. In the present study, differentiation-induced changes of mRNA and protein expression of Na+ channels, Na+ currents, and cell membrane excitability were investigated in NG108-15 cells. Result...

  14. T-type calcium channel expression in cultured human neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    Xianjie Wen; Shiyuan Xu; Lingling Wang; Hua Liang; Chengxiang Yang; Hanbing Wang; Hongzhen Liu

    2011-01-01

    Human neuroblastoma cells (SH-SY5Y) have similar structures and functions as neural cells and have been frequently used for cell culture studies of neural cell functions. Previous studies have revealed Land N-type calcium channels in SH-SY5Y cells. However, the distribution of the low -voltage activated calcium channel (namely called T-type calcium channel, including Cav3.1, Cav3.2, and Cav3.3) in SH-SY5Y cells remains poorly understood. The present study detected mRNA and protein expression of the T-type calcium channel (Cav3.1, Cav3.2, and Cav3.3) in cultured SH-SY5Y cells using real-time polymerase chain reaction (PCR) and western blot analysis. Results revealed mRNA and protein expression from all three T-type calcium channel subtypes in SH-SY5Y cells. Moreover,Cav3.1 was the predominant T-type calcium channel subtype in SH-SY5Y cells.

  15. Regulation of heartbeat by G protein-coupled ion channels.

    Science.gov (United States)

    Brown, A M

    1990-12-01

    The coupling of ion channels to receptors by G proteins is the subject of this American Physiological Society Walter B. Cannon Memorial "Physiology in Perspective" Lecture. This subject is particularly appropriate because it includes a molecular explanation of a homeostatic mechanism involving the autonomic nervous system and the latter subject preoccupied Dr. Cannon during most of his career. With the use of reconstitution methods, we and others have shown that heterotrimeric guanine nucleotide-binding (G) proteins couple receptors to ion channels by both membrane-delimited, direct pathways and cytoplasmic second messenger pathways. Furthermore, one set of receptors may be coupled to as many as three different sets of ion channels to form networks. Dual G protein pathways lead to the prediction of biphasic ion current responses in cell signaling, and this prediction was confirmed. In sinoatrial pacemaker cells, the pacemaking hyperpolarization-activated inward current (If) is directly regulated by the G proteins Gs and Go, and the two can act simultaneously. This could explain the classical observation that vagal inhibition of heart rate is greater during sympathetic stimulation. Because deactivation of the muscarinic response occurs much faster than the G protein alpha-subunit hydrolyzes guanosine 5'-triphosphate, we looked for accessory cellular factors. A surprising result was that the small monomeric ras G protein blocked the muscarinic pathway. The significance of this observation is unknown, but it appears that small and large G proteins may interact in ion channel signaling pathways.

  16. Importance of NPA motifs in the expression and function of water channel aquaporin-1

    Institute of Scientific and Technical Information of China (English)

    JIANG Yong; MA TongHui

    2007-01-01

    The asparagine-proline-alanine sequences (NPA motifs) are highly conserved in aquaporin water channel family. Crystallographic studies of AQP1 structure demonstrated that the two NPA motifs are in the narrow central constriction of the channel, serving to bind water molecules for selective and efficient water passage. To investigate the importance of the two NPA motifs in the structure, function and biogenesis of aquaporin water channels, we generated AQP1 mutations with NPA1 deletion, NPA2 deletion and NPA1,2 double deletion. The coding sequences of the three mutated cDNAs were subcloned into the mammalian expression vector pcDNA3.1 to form expression plasmids. We established stably transfected CHO cell lines expressing these AQP1 mutants. Immunofluorescence indicated that all the three mutated AQP1 proteins are expressed normally on the plasma membrane of stably transfected CHO cells, suggesting that deletion of NPA motifs does not influence the expression and intracellular processing of AQP1. Functional analysis demonstrated that NPA1 or NPA2 deletion reduced AQP1 water permeability by 49.6% and 46.7%, respectively, while NPA1,2 double deletion had little effect on AQP1 water permeability. These results provide evidence that NPA motifs are important for water per-meation but not essential for the expression, intracellular processing and the basic structure of AQP1 water channel.

  17. Reduced thermal sensitivity and Nav1.8 and TRPV1 channel expression in sensory neurons of aged mice

    OpenAIRE

    Wang, Shuying; Davis, Brian M.; Zwick, Melissa; Waxman, Stephen G.; Albers, Kathryn M.

    2005-01-01

    Sensory neurons in aging mammals undergo changes in anatomy, physiology and gene expression that correlate with reduced sensory perception. In this study we compared young and aged mice to identify proteins that might contribute to this loss of sensation. We first show using behavioral testing that thermal sensitivity in aged male and female mice is reduced. Expression of sodium channel (Nav1.8 and Nav1.9) and transient receptor potential vanilloid (TRPV) channels in DRG and peripheral nerves...

  18. SLOB, a SLOWPOKE channel binding protein, regulates insulin pathway signaling and metabolism in Drosophila.

    Directory of Open Access Journals (Sweden)

    Amanda L Sheldon

    Full Text Available There is ample evidence that ion channel modulation by accessory proteins within a macromolecular complex can regulate channel activity and thereby impact neuronal excitability. However, the downstream consequences of ion channel modulation remain largely undetermined. The Drosophila melanogaster large conductance calcium-activated potassium channel SLOWPOKE (SLO undergoes modulation via its binding partner SLO-binding protein (SLOB. Regulation of SLO by SLOB influences the voltage dependence of SLO activation and modulates synaptic transmission. SLO and SLOB are expressed especially prominently in median neurosecretory cells (mNSCs in the pars intercerebralis (PI region of the brain; these cells also express and secrete Drosophila insulin like peptides (dILPs. Previously, we found that flies lacking SLOB exhibit increased resistance to starvation, and we reasoned that SLOB may regulate aspects of insulin signaling and metabolism. Here we investigate the role of SLOB in metabolism and find that slob null flies exhibit changes in energy storage and insulin pathway signaling. In addition, slob null flies have decreased levels of dilp3 and increased levels of takeout, a gene known to be involved in feeding and metabolism. Targeted expression of SLOB to mNSCs rescues these alterations in gene expression, as well as the metabolic phenotypes. Analysis of fly lines mutant for both slob and slo indicate that the effect of SLOB on metabolism and gene expression is via SLO. We propose that modulation of SLO by SLOB regulates neurotransmission in mNSCs, influencing downstream insulin pathway signaling and metabolism.

  19. Functional Expression of an Arachnid Sodium Channel Reveals Residues Responsible for Tetrodotoxin Resistance in Invertebrate Sodium Channels*

    OpenAIRE

    Du, Yuzhe; Nomura, Yoshiko; Liu, Zhiqi; Huang, Zachary Y.; Dong, Ke

    2009-01-01

    Tetrodotoxin (TTX) is a potent blocker of voltage-gated sodium channels, but not all sodium channels are equally sensitive to inhibition by TTX. The molecular basis of differential TTX sensitivity of mammalian sodium channels has been largely elucidated. In contrast, our knowledge about the sensitivity of invertebrate sodium channels to TTX remains poor, in part because of limited success in functional expression of these channels. In this study, we report the functional characterization in X...

  20. Identification of cyclic nucleotide gated channels using regular expressions

    KAUST Repository

    Zelman, Alice K.

    2013-09-03

    Cyclic nucleotide-gated channels (CNGCs) are nonselective cation channels found in plants, animals, and some bacteria. They have a six-transmembrane/one- pore structure, a cytosolic cyclic nucleotide-binding domain, and a cytosolic calmodulin-binding domain. Despite their functional similarities, the plant CNGC family members appear to have different conserved amino acid motifs within corresponding functional domains than animal and bacterial CNGCs do. Here we describe the development and application of methods employing plant CNGC-specific sequence motifs as diagnostic tools to identify novel candidate channels in different plants. These methods are used to evaluate the validity of annotations of putative orthologs of CNGCs from plant genomes. The methods detail how to employ regular expressions of conserved amino acids in functional domains of annotated CNGCs and together with Web tools such as PHI-BLAST and ScanProsite to identify novel candidate CNGCs in species including Physcomitrella patens. © Springer Science+Business Media New York 2013.

  1. Functional roles of γ2, γ3 and γ4, three new Ca2+ channel subunits, in P/Q-type Ca2+ channel expressed in Xenopus oocytes

    Science.gov (United States)

    Rousset, M; Cens, T; Restituito, S; Barrere, C; Black, J L; McEnery, M W; Charnet, P

    2001-01-01

    Stargazin or γ2, the product of the gene mutated in the stargazer mouse, is a homologue of the γ1 protein, an accessory subunit of the skeletal muscle L-type Ca2+ channel. γ2 is selectively expressed in the brain, and considered to be a putative neuronal Ca2+ channel subunit based mainly on homology to γ1. Two new members of the γ family expressed in the brain have recently been identified: γ3 and γ4. We have co-expressed, in Xenopus oocytes, the human γ2,γ3 and γ4 subunits with the P/Q-type (CaV2.1) Ca2+ channel and different regulatory subunits (α2-δ; β1, β2, β3 or β4). Subcellular distribution of the γ subunits confirmed their membrane localization. Ba2+ currents, recorded using two-electrode voltage clamp, showed that the effects of the γ subunits on the electrophysiological properties of the channel are, most of the time, minor. However, a fraction of the oocytes expressing β subunits displayed an unusual slow-inactivating Ba2+ current. Expression of both β and γ subunits increased the appearance of the slow-inactivating current. Our data support a role for the γ subunit as a brain Ca2+ channel modulatory subunit and suggest that β and γ subunits are involved in a switch between two regulatory modes of the P/Q-type channel inactivation. PMID:11313431

  2. Rv1698 of Mycobacterium tuberculosis represents a new class of channel-forming outer membrane proteins.

    Science.gov (United States)

    Siroy, Axel; Mailaender, Claudia; Harder, Daniel; Koerber, Stephanie; Wolschendorf, Frank; Danilchanka, Olga; Wang, Ying; Heinz, Christian; Niederweis, Michael

    2008-06-27

    Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 +/- 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes. PMID:18434314

  3. Unidirectional photoreceptor-to-Muller glia coupling and unique K+ channel expression in Caiman retina.

    Directory of Open Access Journals (Sweden)

    Astrid Zayas-Santiago

    Full Text Available BACKGROUND: Müller cells, the principal glial cells of the vertebrate retina, are fundamental for the maintenance and function of neuronal cells. In most vertebrates, including humans, Müller cells abundantly express Kir4.1 inwardly rectifying potassium channels responsible for hyperpolarized membrane potential and for various vital functions such as potassium buffering and glutamate clearance; inter-species differences in Kir4.1 expression were, however, observed. Localization and function of potassium channels in Müller cells from the retina of crocodiles remain, hitherto, unknown. METHODS: We studied retinae of the Spectacled caiman (Caiman crocodilus fuscus, endowed with both diurnal and nocturnal vision, by (i immunohistochemistry, (ii whole-cell voltage-clamp, and (iii fluorescent dye tracing to investigate K+ channel distribution and glia-to-neuron communications. RESULTS: Immunohistochemistry revealed that caiman Müller cells, similarly to other vertebrates, express vimentin, GFAP, S100β, and glutamine synthetase. In contrast, Kir4.1 channel protein was not found in Müller cells but was localized in photoreceptor cells. Instead, 2P-domain TASK-1 channels were expressed in Müller cells. Electrophysiological properties of enzymatically dissociated Müller cells without photoreceptors and isolated Müller cells with adhering photoreceptors were significantly different. This suggests ion coupling between Müller cells and photoreceptors in the caiman retina. Sulforhodamine-B injected into cones permeated to adhering Müller cells thus revealing a uni-directional dye coupling. CONCLUSION: Our data indicate that caiman Müller glial cells are unique among vertebrates studied so far by predominantly expressing TASK-1 rather than Kir4.1 K+ channels and by bi-directional ion and uni-directional dye coupling to photoreceptor cells. This coupling may play an important role in specific glia-neuron signaling pathways and in a new type of K

  4. Gas Channels for NH3: Proteins from Hyperthermophiles Complement an Escherichia coli Mutant

    OpenAIRE

    Soupene, Eric; Chu, Tony; Corbin, Rebecca W.; Hunt, Donald F.; Kustu, Sydney

    2002-01-01

    Ammonium transport (Amt) proteins appear to be bidirectional channels for NH3. The amt genes of the hyperthermophiles Aquifex aeolicus and Methanococcus jannaschii complement enteric amtB mutants for growth at 25 nM NH3 at 37°C. To our knowledge, Amt proteins are the first hyperthermophilic membrane transport proteins shown to be active in a mesophilic bacterium. Despite low expression levels, His-tagged Aquifex Amt could be purified by heating and nickel chelate affinity chromatography. It c...

  5. Predictable tuning of protein expression in bacteria

    DEFF Research Database (Denmark)

    Bonde, Mads; Pedersen, Margit; Klausen, Michael Schantz;

    2016-01-01

    We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expressi...

  6. Complement regulatory protein genes in channel catfish and their involvement in disease defense response.

    Science.gov (United States)

    Jiang, Chen; Zhang, Jiaren; Yao, Jun; Liu, Shikai; Li, Yun; Song, Lin; Li, Chao; Wang, Xiaozhu; Liu, Zhanjiang

    2015-11-01

    Complement system is one of the most important defense systems of innate immunity, which plays a crucial role in disease defense responses in channel catfish. However, inappropriate and excessive complement activation could lead to potential damage to the host cells. Therefore the complement system is controlled by a set of complement regulatory proteins to allow normal defensive functions, but prevent hazardous complement activation to host tissues. In this study, we identified nine complement regulatory protein genes from the channel catfish genome. Phylogenetic and syntenic analyses were conducted to determine their orthology relationships, supporting their correct annotation and potential functional inferences. The expression profiles of the complement regulatory protein genes were determined in channel catfish healthy tissues and after infection with the two main bacterial pathogens, Edwardsiella ictaluri and Flavobacterium columnare. The vast majority of complement regulatory protein genes were significantly regulated after bacterial infections, but interestingly were generally up-regulated after E. ictaluri infection while mostly down-regulated after F. columnare infection, suggesting a pathogen-specific pattern of regulation. Collectively, these findings suggested that complement regulatory protein genes may play complex roles in the host immune responses to bacterial pathogens in channel catfish.

  7. Activation of purified calcium channels by stoichiometric protein phosphorylation

    International Nuclear Information System (INIS)

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45Ca2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45Ca2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd2+, Ni2+, and Mg2+. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels

  8. Coevolution of gene expression among interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. Structure and inhibition of the SARS coronavirus envelope protein ion channel.

    Directory of Open Access Journals (Sweden)

    Konstantin Pervushin

    2009-07-01

    Full Text Available The envelope (E protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA, but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV that the transmembrane domain of E protein (ETM forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293 cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA, but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

  10. Ion permeation of AQP6 water channel protein. Single channel recordings after Hg2+ activation.

    Science.gov (United States)

    Hazama, Akihiro; Kozono, David; Guggino, William B; Agre, Peter; Yasui, Masato

    2002-08-01

    Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed in Xenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 microm HgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 microm HgCl2. Reduction of both Na+ and Cl- concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl-. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by 22Na+ influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 +/- 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel. PMID:12034750

  11. Probing Protein Channel Dynamics At The Single Molecule Level.

    Science.gov (United States)

    Lee, M. Ann; Dunn, Robert C.

    1997-03-01

    It would be difficult to overstate the importance played by protein ion channels in cellular function. These macromolecular pores allow the passage of ions across the cellular membrane and play indispensable roles in all aspects of neurophysiology. While the patch-clamp technique continues to provide elegant descriptions of the kinetic processes involved in ion channel gating, the associated conformational changes remain a mystery. We are using the spectroscopic capabilities and single molecule fluorescence sensitivity of near-field scanning optical microscopy (NSOM) to probe these dynamics at the single channel level. Using a newly developed cantilevered NSOM probe capable of probing soft biological samples with single molecule fluorescence sensitivity, we have begun mapping the location of single NMDA receptors in intact rat cortical neurons with <100 nm spatial resolution. We will also present recent results exploring the conformational changes accompanying activation of nuclear pore channels located in the nuclear membrane of Xenopus oocytes. Our recent NSOM and AFM measurements on single nuclear pore complexes reveal large conformational changes taking place upon activation, providing rich, new molecular level details of channel function.

  12. Protein expression strategies for identification of novel target proteins.

    Science.gov (United States)

    Schuster, M; Wasserbauer, E; Einhauer, A; Ortner, C; Jungbauer, A; Hammerschmid, F; Werner, G

    2000-04-01

    Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cDNAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The alpha-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of

  13. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties

    International Nuclear Information System (INIS)

    The small envelope (E) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is a hydrophobic 73 amino acid protein encoded in the internal open reading frame (ORF) of the bicistronic mRNA2. As a first step towards understanding the biological role of E protein during PRRSV replication, E gene expression was blocked in a full-length infectious clone by mutating the ATG translational initiation to GTG, such that the full-length mutant genomic clone was unable to synthesize the E protein. DNA transfection of PRRSV-susceptible cells with the E gene knocked-out genomic clone showed the absence of virus infectivity. P129-ΔE-transfected cells however produced virion particles in the culture supernatant, and these particles contained viral genomic RNA, demonstrating that the E protein is essential for PRRSV infection but dispensable for virion assembly. Electron microscopy suggests that the P129-ΔE virions assembled in the absence of E had a similar appearance to the wild-type particles. Strand-specific RT-PCR demonstrated that the E protein-negative, non-infectious P129-ΔE virus particles were able to enter cells but further steps of replication were interrupted. The entry of PRRSV has been suggested to be via receptor-mediated endocytosis, and lysomotropic basic compounds and known ion-channel blocking agents both inhibited PRRSV replication effectively during the uncoating process. The expression of E protein in Escherichia coli-mediated cell growth arrests and increased the membrane permeability. Cross-linking experiments in cells infected with PRRSV or transfected with E gene showed that the E protein was able to form homo-oligomers. Taken together, our data suggest that the PRRSV E protein is likely an ion-channel protein embedded in the viral envelope and facilitates uncoating of virus and release of the genome in the cytoplasm

  14. Functional expression of KCNQ (Kv7) channels in guinea pig bladder smooth muscle and their contribution to spontaneous activity

    OpenAIRE

    Anderson, U. A.; Carson, C.; Johnston, L; Joshi, S; Gurney, A M; McCloskey, K. D.

    2013-01-01

    Background and Purpose: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. Experimental Approach: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activ...

  15. Expression of calcium-activated chloride channels Ano1 and Ano2 in mouse taste cells.

    Science.gov (United States)

    Cherkashin, Alexander P; Kolesnikova, Alisa S; Tarasov, Michail V; Romanov, Roman A; Rogachevskaja, Olga A; Bystrova, Marina F; Kolesnikov, Stanislav S

    2016-02-01

    Specialized Ca(2+)-dependent ion channels ubiquitously couple intracellular Ca(2+) signals to a change in cell polarization. The existing physiological evidence suggests that Ca(2+)-activated Cl(-) channels (CaCCs) are functional in taste cells. Because Ano1 and Ano2 encode channel proteins that form CaCCs in a variety of cells, we analyzed their expression in mouse taste cells. Transcripts for Ano1 and Ano2 were detected in circumvallate (CV) papillae, and their expression in taste cells was confirmed using immunohistochemistry. When dialyzed with CsCl, taste cells of the type III exhibited no ion currents dependent on cytosolic Ca(2+). Large Ca(2+)-gated currents mediated by TRPM5 were elicited in type II cells by Ca(2+) uncaging. When TRPM5 was inhibited by triphenylphosphine oxide (TPPO), ionomycin stimulated a small but resolvable inward current that was eliminated by anion channel blockers, including T16Ainh-A01 (T16), a specific Ano1 antagonist. This suggests that CaCCs, including Ano1-like channels, are functional in type II cells. In type I cells, CaCCs were prominently active, blockable with the CaCC antagonist CaCCinh-A01 but insensitive to T16. By profiling Ano1 and Ano2 expressions in individual taste cells, we revealed Ano1 transcripts in type II cells only, while Ano2 transcripts were detected in both type I and type II cells. P2Y agonists stimulated Ca(2+)-gated Cl(-) currents in type I cells. Thus, CaCCs, possibly formed by Ano2, serve as effectors downstream of P2Y receptors in type I cells. While the role for TRPM5 in taste transduction is well established, the physiological significance of expression of CaCCs in type II cells remains to be elucidated.

  16. Expression of calcium-activated chloride channels Ano1 and Ano2 in mouse taste cells.

    Science.gov (United States)

    Cherkashin, Alexander P; Kolesnikova, Alisa S; Tarasov, Michail V; Romanov, Roman A; Rogachevskaja, Olga A; Bystrova, Marina F; Kolesnikov, Stanislav S

    2016-02-01

    Specialized Ca(2+)-dependent ion channels ubiquitously couple intracellular Ca(2+) signals to a change in cell polarization. The existing physiological evidence suggests that Ca(2+)-activated Cl(-) channels (CaCCs) are functional in taste cells. Because Ano1 and Ano2 encode channel proteins that form CaCCs in a variety of cells, we analyzed their expression in mouse taste cells. Transcripts for Ano1 and Ano2 were detected in circumvallate (CV) papillae, and their expression in taste cells was confirmed using immunohistochemistry. When dialyzed with CsCl, taste cells of the type III exhibited no ion currents dependent on cytosolic Ca(2+). Large Ca(2+)-gated currents mediated by TRPM5 were elicited in type II cells by Ca(2+) uncaging. When TRPM5 was inhibited by triphenylphosphine oxide (TPPO), ionomycin stimulated a small but resolvable inward current that was eliminated by anion channel blockers, including T16Ainh-A01 (T16), a specific Ano1 antagonist. This suggests that CaCCs, including Ano1-like channels, are functional in type II cells. In type I cells, CaCCs were prominently active, blockable with the CaCC antagonist CaCCinh-A01 but insensitive to T16. By profiling Ano1 and Ano2 expressions in individual taste cells, we revealed Ano1 transcripts in type II cells only, while Ano2 transcripts were detected in both type I and type II cells. P2Y agonists stimulated Ca(2+)-gated Cl(-) currents in type I cells. Thus, CaCCs, possibly formed by Ano2, serve as effectors downstream of P2Y receptors in type I cells. While the role for TRPM5 in taste transduction is well established, the physiological significance of expression of CaCCs in type II cells remains to be elucidated. PMID:26530828

  17. Fluctuation driven active molecular transport in passive channel proteins

    Science.gov (United States)

    Kosztin, Ioan

    2006-03-01

    Living cells interact with their extracellular environment through the cell membrane, which acts as a protective permeability barrier for preserving the internal integrity of the cell. However, cell metabolism requires controlled molecular transport across the cell membrane, a function that is fulfilled by a wide variety of transmembrane proteins, acting as either passive or active transporters. In this talk it is argued that, contrary to the general belief, in active cell membranes passive and spatially asymmetric channel proteins can act as active transporters by consuming energy from nonequilibrium fluctuations fueled by cell metabolism. This assertion is demonstrated in the case of the E. coli aquaglyceroporin GlpF channel protein, whose high resolution crystal structure is manifestly asymmetric. By calculating the glycerol flux through GlpF within the framework of a stochastic model, it is found that, as a result of channel asymmetry, glycerol uptake driven by a concentration gradient is enhanced significantly in the presence of non-equilibrium fluctuations. Furthermore, the enhancement caused by a ratchet-like mechanism is larger for the outward, i.e., from the cytoplasm to the periplasm, flux than for the inward one, suggesting that the same non-equilibrium fluctuations also play an important role in protecting the interior of the cell against poisoning by excess uptake of glycerol. Preliminary data on water and sugar transport through aquaporin and maltoporin channels, respectively, are indicative of the universality of the proposed nonequilibrium-fluctuation-driven active transport mechanism. This work was supported by grants from the Univ. of Missouri Research Board, the Institute for Theoretical Sciences and the Department of Energy (DOE Contract W-7405-ENG-36), and the National Science Foundation (FIBR-0526854).

  18. Expression and function of transient receptor potential channels in the female bovine reproductive tract.

    Science.gov (United States)

    Ghavideldarestani, Maryam; Atkin, Stephen L; Leese, Henry J; Sturmey, Roger G

    2016-07-15

    The epithelium lining the oviduct is critical for early reproductive events, many of which are mediated via intracellular calcium ions. Despite this, little is known about the regulation of calcium homeostasis in the oviductal epithelium. Epithelial transient receptor potential channels (TRPCs) modulate calcium flux in other tissues, and their expression and functional regulation have therefore been examined using the bovine oviduct as a model for the human. The effects of FSH, LH, 17β-estradiol, and progesterone on TRPCs expression and intracellular calcium flux were determined. Transient receptor potential channels 1, 2, 3, 4, and 6 were expressed in the bovine reproductive tract, and their gene expression varied throughout the estrous cycle. In more detailed studies undertaken on TRPC1 and 6, we show that protein expression varied through the estrus cycle; specifically, 17β-estradiol, FSH, and LH individually and in combination upregulated TRPC1 and 6 expression in cultured bovine oviduct epithelial cells although progesterone antagonized these effects. Functional studies showed changes in calcium mobilization in bovine oviduct epithelial cells were dependent on TRPCs. In conclusion, TRPC1, 2, 3, 4, and 6 are present in the epithelium lining the bovine oviduct, and TRPC1 and 6 vary through the estrous cycle suggesting an important role in early reproductive function. PMID:27001231

  19. Differential Liver Protein Expression during Schistosomiasis▿ †

    OpenAIRE

    Harvie, Marina; Jordan, Thomas William; La Flamme, Anne Camille

    2006-01-01

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

  20. CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein.

    Science.gov (United States)

    Palma, Alejandra G; Galizia, Luciano; Kotsias, Basilio A; Marino, Gabriela I

    2016-05-01

    Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases.

  1. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

    KAUST Repository

    Ooi, Amanda Siok Lee

    2016-07-19

    The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

  2. Analysis of correlations between protein complex and protein-protein interaction and mRNA expression

    Institute of Scientific and Technical Information of China (English)

    CAI Lun; XUE Hong; LU Hongchao; ZHAO Yi; ZHU Xiaopeng; BU Dongbo; LING Lunjiang; CHEN Runsheng

    2003-01-01

    Protein-protein interaction is a physical interaction of two proteins in living cells. In budding yeast Saccharomyces cerevisiae, large-scale protein-protein interaction data have been obtained through high-throughput yeast two-hybrid systems (Y2H) and protein complex purification techniques based on mass-spectrometry. Here, we collect 11855 interactions between total 2617 proteins. Through seriate genome-wide mRNA expression data, similarity between two genes could be measured. Protein complex data can also be obtained publicly and can be translated to pair relationship that any two proteins can only exist in the same complex or not. Analysis of protein complex data, protein-protein interaction data and mRNA expression data can elucidate correlations between them. The results show that proteins that have interactions or similar expression patterns have a higher possibility to be in the same protein complex than randomized selected proteins, and proteins which have interactions and similar expression patterns are even more possible to exist in the same protein complex. The work indicates that comprehensive integration and analysis of public large-scale bioinformatical data, such as protein complex data, protein-protein interaction data and mRNA expression data, may help to uncover their relationships and common biological information underlying these data. The strategies described here may help to integrate and analyze other functional genomic and proteomic data, such as gene expression profiling, protein-localization mapping and large-scale phenotypic data, both in yeast and in other organisms.

  3. The voltage-gated sodium channel nav1.8 is expressed in human sperm.

    Directory of Open Access Journals (Sweden)

    Antonio Cejudo-Roman

    Full Text Available The role of Na(+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na(+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 μM, the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca(2+-containing or Ca(2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na(+, [Na(+]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na(+ channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

  4. TRPC1 protein forms only one type of native store-operated channels in HEK293 cells.

    Science.gov (United States)

    Skopin, Anton; Shalygin, Alexey; Vigont, Vladimir; Zimina, Olga; Glushankova, Lyubov; Mozhayeva, Galina N; Kaznacheyeva, Elena

    2013-02-01

    TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I(max) channels, while the properties of I(min) and I(NS) channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current-voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I(max) channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I(max) channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I(max) channels but is not an essential subunit for other store-operated channel types in HEK293 cells.

  5. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

    Directory of Open Access Journals (Sweden)

    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

  6. The first discovered water channel protein, later called aquaporin 1: molecular characteristics, functions and medical implications.

    Science.gov (United States)

    Benga, Gheorghe

    2012-01-01

    After a decade of work on the water permeability of red blood cells (RBC) Benga group in Cluj-Napoca, Romania, discovered in 1985 the first water channel protein in the RBC membrane. The discovery was reported in publications in 1986 and reviewed in subsequent years. The same protein was purified by chance by Agre group in Baltimore, USA, in 1988, who called in 1991 the protein CHIP28 (CHannel forming Integral membrane Protein of 28 kDa), suggesting that it may play a role in linkage of the membrane skeleton to the lipid bilayer. In 1992 the Agre group identified CHIP28's water transport property. One year later CHIP28 was named aquaporin 1, abbreviated as AQP1. In this review the molecular structure-function relationships of AQP1 are presented. In the natural or model membranes AQP1 is in the form of a homotetramer, however, each monomer has an independent water channel (pore). The three-dimensional structure of AQP1 is described, with a detailed description of the channel (pore), the molecular mechanisms of permeation through the channel of water molecules and exclusion of protons. The permeability of the pore to gases (CO(2), NH(3), NO, O(2)) and ions is also mentioned. I have also reviewed the functional roles and medical implications of AQP1 expressed in various organs and cells (microvascular endothelial cells, kidney, central nervous system, eye, lacrimal and salivary glands, respiratory apparatus, gastrointestinal tract, hepatobiliary compartments, female and male reproductive system, inner ear, skin). The role of AQP1 in cell migration and angiogenesis in relation with cancer, the genetics of AQP1 and mutations in human subjects are also mentioned. The role of AQP1 in red blood cells is discussed based on our comparative studies of water permeability in over 30 species. PMID:22705445

  7. Expression and subcellular localization of aquaporin water channels in the polarized hepatocyte cell line, WIF-B

    OpenAIRE

    Marinelli Raúl A; Splinter Patrick L; Tietz Pamela S; Gradilone Sergio A; LaRusso Nicholas F

    2005-01-01

    Abstract Background Recent data suggest that canalicular bile secretion involves selective expression and coordinated regulation of aquaporins (AQPs), a family of water channels proteins. In order to further characterize the role of AQPs in this process, an in vitro cell system with retained polarity and expression of AQPs and relevant solute transporters involved in bile formation is highly desirable. The WIF-B cell line is a highly differentiated and polarized rat hepatoma/human fibroblast ...

  8. Biotechnology Protein Expression and Purification Facility

    Science.gov (United States)

    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.

  9. Pannexin1 channel proteins in the zebrafish retina have shared and unique properties.

    Directory of Open Access Journals (Sweden)

    Sarah Kurtenbach

    Full Text Available In mammals, a single pannexin1 gene (Panx1 is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.

  10. TRP channel-associated factors are a novel protein family that regulates TRPM8 trafficking and activity.

    NARCIS (Netherlands)

    Gkika, D.; Lemonnier, L.; Shapovalov, G.; Gordienko, D.; Poux, C.; Bernardini, M.; Bokhobza, A.; Bidaux, G.; Degerny, C.; Verreman, K.; Guarmit, B.; Benahmed, M.; Launoit, Y. de; Bindels, R.J.M.; Fiorio Pla, A.; Prevarskaya, N.

    2015-01-01

    TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two pr

  11. Src family protein tyrosine kinase regulates the basolateral K channel in the distal convoluted tubule (DCT) by phosphorylation of KCNJ10 protein.

    Science.gov (United States)

    Zhang, Chengbiao; Wang, Lijun; Thomas, Sherin; Wang, Kemeng; Lin, Dao-Hong; Rinehart, Jesse; Wang, Wen-Hui

    2013-09-01

    The loss of function of the basolateral K channels in the distal nephron causes electrolyte imbalance. The aim of this study is to examine the role of Src family protein tyrosine kinase (SFK) in regulating K channels in the basolateral membrane of the mouse initial distal convoluted tubule (DCT1). Single-channel recordings confirmed that the 40-picosiemen (pS) K channel was the only type of K channel in the basolateral membrane of DCT1. The suppression of SFK reversibly inhibited the basolateral 40-pS K channel activity in cell-attached patches and decreased the Ba(2+)-sensitive whole-cell K currents in DCT1. Inhibition of SFK also shifted the K reversal potential from -65 to -43 mV, suggesting a role of SFK in determining the membrane potential in DCT1. Western blot analysis showed that KCNJ10 (Kir4.1), a key component of the basolateral 40-pS K channel in DCT1, was a tyrosine-phosphorylated protein. LC/MS analysis further confirmed that SFK phosphorylated KCNJ10 at Tyr(8) and Tyr(9). The single-channel recording detected the activity of a 19-pS K channel in KCNJ10-transfected HEK293T cells and a 40-pS K channel in the cells transfected with KCNJ10+KCNJ16 (Kir.5.1) that form a heterotetramer in the basolateral membrane of the DCT. Mutation of Tyr(9) did not alter the channel conductance of the homotetramer and heterotetramer. However, it decreased the whole-cell K currents, the probability of finding K channels, and surface expression of KCNJ10 in comparison to WT KCNJ10. We conclude that SFK stimulates the basolateral K channel activity in DCT1, at least partially, by phosphorylating Tyr(9) on KCNJ10. We speculate that the modulation of tyrosine phosphorylation of KCNJ10 should play a role in regulating membrane transport function in DCT1.

  12. Polyester Modification of the Mammalian TRPM8 Channel Protein: Implications for Structure and Function

    Directory of Open Access Journals (Sweden)

    Chike Cao

    2013-07-01

    Full Text Available The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmental cues, such as cold temperatures and chemical compounds, including menthol and icilin. The channel functional activity is regulated by various physical and chemical factors and is likely to be preconditioned by its molecular composition. Our studies indicate that the TRPM8 channel forms a structural-functional complex with the polyester poly-(R-3-hydroxybutyrate (PHB. We identified by mass spectrometry a number of PHB-modified peptides in the N terminus of the TRPM8 protein and in its extracellular S3-S4 linker. Removal of PHB by enzymatic hydrolysis and site-directed mutagenesis of both the serine residues that serve as covalent anchors for PHB and adjacent hydrophobic residues that interact with the methyl groups of the polymer resulted in significant inhibition of TRPM8 channel activity. We conclude that the TRPM8 channel undergoes posttranslational modification by PHB and that this modification is required for its normal function.

  13. Functional expression of an arachnid sodium channel reveals residues responsible for tetrodotoxin resistance in invertebrate sodium channels.

    Science.gov (United States)

    Du, Yuzhe; Nomura, Yoshiko; Liu, Zhiqi; Huang, Zachary Y; Dong, Ke

    2009-12-01

    Tetrodotoxin (TTX) is a potent blocker of voltage-gated sodium channels, but not all sodium channels are equally sensitive to inhibition by TTX. The molecular basis of differential TTX sensitivity of mammalian sodium channels has been largely elucidated. In contrast, our knowledge about the sensitivity of invertebrate sodium channels to TTX remains poor, in part because of limited success in functional expression of these channels. In this study, we report the functional characterization in Xenopus oocytes of the first non-insect, invertebrate voltage-gated sodium channel from the varroa mite (Varroa destructor), an ecto-parasite of the honeybee. This arachnid sodium channel activates and inactivates rapidly with half-maximal activation at -18 mV and half-maximal fast inactivation at -29 mV. Interestingly, this arachnid channel showed surprising TTX resistance. TTX blocked this channel with an IC(50) of 1 microM. Subsequent site-directed mutagenesis revealed two residues, Thr-1674 and Ser-1967, in the pore-forming region of domains III and IV, respectively, which were responsible for the observed resistance to inhibition by TTX. Furthermore, sequence comparison and additional amino acid substitutions suggested that sequence polymorphisms at these two positions could be a widespread mechanism for modulating TTX sensitivity of sodium channels in diverse invertebrates. PMID:19828457

  14. Sodium channel gene expression in mosquitoes, Aedes albopictus (S.)

    Institute of Scientific and Technical Information of China (English)

    NANNAN LIU; QIANG XU; LEE ZHANG

    2006-01-01

    A mosquito strain of Aerdes albopictus,HAmAalG0,from Huntsville,Alabama,USA,showed a normal susceptibility and low tolerance to permethrin and resmethrin (pyrethroid insecticides) compared to a susceptible Ikaken strain,even though these pyrethroid insecticides have been used in the field for a long period of time in Alabama.Recently,we treated HAmAalG0 in the laboratory with permethrin for five generations and detected no significant change in the level of resistance to permethrin in the selected mosquitoes,HAmAalG5,compared with the parental strain HAmAalG0. We then examined the allelic expression at the L-to-F kdr site of the sodium channel gene in the Aedes mosquitoes to address our hypothesis that the L-to-F kdr mutation was not present in HAmAalG0 and HAmAalG5 mosquitoes. We found that every tested individual in Ikaken,HAmAalG0,and HAmAalG5 populations expressed a codon of CTA at the L-to-F kdr site encoding Leu,strongly corresponding to their susceptibility to insecticides.

  15. Expression Pattern of Id Proteins in Medulloblastoma

    OpenAIRE

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

    2013-01-01

    Inhibitor of DNA binding or inhibitor of differentiation (Id) proteins are up regulated in a variety of neoplasms, particularly in association with high-grade, poorly differentiated tumors, while differentiated tissues show little or no Id expression. The four Id genes are members of the helix-loop-helix (HLH) family of transcription factors and act as negative regulators of transcription by binding to and sequestering HLH complexes. We tested the hypothesis that Id proteins are overexpressed...

  16. Evidence for functional diversity between the voltage-gated proton channel Hv1 and its closest related protein HVRP1.

    Directory of Open Access Journals (Sweden)

    Iris H Kim

    Full Text Available The Hv1 channel and voltage-sensitive phosphatases share with voltage-gated sodium, potassium, and calcium channels the ability to detect changes in membrane potential through voltage-sensing domains (VSDs. However, they lack the pore domain typical of these other channels. NaV, KV, and CaV proteins can be found in neurons and muscles, where they play important roles in electrical excitability. In contrast, VSD-containing proteins lacking a pore domain are found in non-excitable cells and are not involved in neuronal signaling. Here, we report the identification of HVRP1, a protein related to the Hv1 channel (from which the name Hv1 Related Protein 1 is derived, which we find to be expressed primarily in the central nervous system, and particularly in the cerebellum. Within the cerebellar tissue, HVRP1 is specifically expressed in granule neurons, as determined by in situ hybridization and immunohistochemistry. Analysis of subcellular distribution via electron microscopy and immunogold labeling reveals that the protein localizes on the post-synaptic side of contacts between glutamatergic mossy fibers and the granule cells. We also find that, despite the similarities in amino acid sequence and structural organization between Hv1 and HVRP1, the two proteins have distinct functional properties. The high conservation of HVRP1 in vertebrates and its cellular and subcellular localizations suggest an important function in the nervous system.

  17. The role of water channel proteins and nitric oxide signaling in rice seed germination

    Institute of Scientific and Technical Information of China (English)

    Hong-Yan Liu; Xin Yu; Da-Yong Cui; Mei-Hao Sun; Wei-Ning Sun; Zhang-Cheng Tang; Sang-Soo Kwak; Wei-Ai Su

    2007-01-01

    Previous studies have demonstrated the possible role of several aquaporins in seed germination. But systematic investigation of the role of aquaporin family members in this process is lacking. Here, the developmental regulation of plasma membrane intrinsic protein (PIP) expression throughout germination and post-germination processes in rice embryos was analyzed. The expression patterns of the PIPs suggest these aquaporins play different roles in seed germination and seedling growth. Partial silencing of the water channel genes, OsPIP1; 1 and OsPIP1;3, reduced seed germination while over-expression of OsPIPl;3 promoted seed germination under water-stress conditions. Moreover, spatial expression analysis indicates that OsPIP1;3 is expressed predominantly in embryo during seed germination. Our data also revealed that the nitric oxide (NO) donors, sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO), promoted seed germination; furthermore, the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibited germination and reduced the stimulative effects of SNP and GSNO on rice germination. Exogenous NO stimulated the transcription of OsPIP1;1, OsPIP1;2, OsPIP1;3 and OsPIP2;8 in germinating seeds. These results suggest that water channels play an important role in seed germination, acting, at least partly, in response to the NO signaling pathway.

  18. A ligand channel through the G protein coupled receptor opsin.

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    Peter W Hildebrand

    Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  20. Calcium binding protein-mediated regulation of voltage-gated calcium channels linked to human diseases

    Institute of Scientific and Technical Information of China (English)

    Nasrin NFJATBAKHSH; Zhong-ping FENG

    2011-01-01

    Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases.

  1. Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes

    NARCIS (Netherlands)

    Money, T. T.; King, R. G.; Wong, M.H.; Stevenson, J. L.; Kalionis, B.; Erwich, J. J. H. M.; Huisman, Marcel; Timmer, A.; Hiden, U.; Desoye, G.; Gude, N. M.

    2007-01-01

    Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular ch

  2. Expression of stretch-activated two-pore potassium channels in human myometrium in pregnancy and labor.

    Directory of Open Access Journals (Sweden)

    Iain L O Buxton

    Full Text Available BACKGROUND: We tested the hypothesis that the stretch-activated, four-transmembrane domain, two pore potassium channels (K2P, TREK-1 and TRAAK are gestationally-regulated in human myometrium and contribute to uterine relaxation during pregnancy until labor. METHODOLOGY: We determined the gene and protein expression of K2P channels in non-pregnant, pregnant term and preterm laboring myometrium. We employed both molecular biological and functional studies of K2P channels in myometrial samples taken from women undergoing cesarean delivery of a fetus. PRINCIPAL FINDINGS: TREK-1, but not TREK-2, channels are expressed in human myometrium and significantly up-regulated during pregnancy. Down-regulation of TREK-1 message was seen by Q-PCR in laboring tissues consistent with a role for TREK-1 in maintaining uterine quiescence prior to labor. The TRAAK channel was unregulated in the same women. Blockade of stretch-activated channels with a channel non-specific tarantula toxin (GsMTx-4 or the more specific TREK-1 antagonist L-methionine ethyl ester altered contractile frequency in a dose-dependent manner in pregnant myometrium. Arachidonic acid treatment lowered contractile tension an effect blocked by fluphenazine. Functional studies are consistent with a role for TREK-1 in uterine quiescence. CONCLUSIONS: We provide evidence supporting a role for TREK-1 in contributing to uterine quiescence during gestation and hypothesize that dysregulation of this mechanism may underlie certain cases of spontaneous pre-term birth.

  3. Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

    Science.gov (United States)

    Hristov, Kiril L; Chen, Muyan; Afeli, Serge A Y; Cheng, Qiuping; Rovner, Eric S; Petkov, Georgi V

    2012-06-01

    The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

  4. Interactions between β-catenin and the HSlo potassium channel regulates HSlo surface expression.

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    Shumin Bian

    Full Text Available BACKGROUND: The large conductance calcium-activated potassium channel alpha-subunit (Slo is widely distributed throughout the body and plays an important role in a number of diseases. Prior work has shown that Slo, through its S10 region, interacts with β-catenin, a key component of the cytoskeleton framework and the Wnt signaling pathway. However, the physiological significance of this interaction was not clear. METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of proteomic and cell biology tools we show the existence of additional multiple binding sites in Slo, and explore in detail β-catenin interactions with the S10 region. We demonstrate that deletion of this region reduces Slo surface expression in HEK cells, which indicates that interaction with beta-catenin is important for Slo surface expression. This is confirmed by reduced expression of Slo in HEK cells and chicken (Gallus gallus domesticus leghorn white hair cells treated with siRNA to β-catenin. HSlo reciprocally co-immunoprecipitates with β-catenin, indicating a stable binding between these two proteins, with the S10 deletion mutant having reduced binding with β-catenin. We also observed that mutations of the two putative GSK phosphorylation sites within the S10 region affect both the surface expression of Slo and the channel's voltage and calcium sensitivities. Interestingly, expression of exogenous Slo in HEK cells inhibits β-catenin-dependent canonical Wnt signaling. CONCLUSIONS AND SIGNIFICANCE: These studies identify for the first time a central role for β-catenin in mediating Slo surface expression. Additionally we show that Slo overexpression can lead to downregulation of Wnt signaling.

  5. Mechanosensitive channels of Escherichia coli: the MscL gene, protein, and activities

    Science.gov (United States)

    Sukharev, S. I.; Blount, P.; Martinac, B.; Kung, C.

    1997-01-01

    Although mechanosensory responses are ubiquitous and diverse, the molecular bases of mechanosensation in most cases remain mysterious MscL, a mechanosensitive channel of large conductance of Escherichia coli and its bacterial homologues are the first and currently only channel molecules shown to directly sense mechanical stretch of the membrane. In response to the tension conveyed via the lipid bilayer, MscL increases its open probability by several orders of magnitude. In the present review we describe the identification, cloning, and first sets of biophysical and structural data on this simplest mechanosensory molecule. We discovered a 2.5-ns mechanosensitive conductance in giant E. coli spheroplasts. Using chromatographies to enrich the target and patch clamp to assay the channel activity in liposome-reconstituted fractions, we identified the MscL protein and cloned the mscL gene. MscL comprises 136 amino acid residues (15 kDa), with two highly hydrophobic regions, and resides in the inner membrane of the bacterium. PhoA-fusion experiments indicate that the protein spans the membrane twice with both termini in the cytoplasm. Spectroscopic techniques show that it is highly helical. Expression of MscL tandems and covalent cross-linking suggest that the active channel complex is a homo-hexamer. We have identified several residues, which when deleted or substituted, affect channel kinetics or mechanosensitivity. Although unique when discovered, highly conserved MscL homologues in both gram-negative and gram-positive bacteria have been found, suggesting their ubiquitous importance among bacteria.

  6. Functional study of the effect of phosphatase inhibitors on KCNQ4 channels expressed in Xenopus oocytes

    Institute of Scientific and Technical Information of China (English)

    Tzu-rong SU; Cay-huyen CHEN; Shih-jen HUANG; Chun-yi LEE; Mao-chang SU; Gwan-hong CHEN; Shuan-yow LI; Jiann-jou YANG; Min-jon LIN

    2009-01-01

    Aim: KCNQ4 channels play an important part in adjusting the function of cochlear outer hair cells. The aim of this study was to investigate the effects of ser/thr phosphatase inhibitors on human KCNQ4 channels expressed in Xenopus laevis oocytes. Methods: Synthetic cRNA encoding human KCNQ4 channels was injected into Xenopus oocytes. We used a two-electrode voltage clamp to measure the ion currents in the oocytes. Results: Wild-type KCNQ4 expressed in Xenopus oocytes showed the typical properties of slow activation kinetics and low threshold activation. The outward K~+ current was almost completely blocked by a KCNQ4 blocker, linopirdine (0.25 mmol/L). BIMI (a PKC inhibitor) prevented the effects of PMA (a PKC activator) on the KCNQ4 current, indicating that PKC may be involved in the regulation of KCNQ4 expressed in the Xenopus oocyte system. Treatment with the ser/thr phosphatase inhibitors, cyclosporine (2 μmoVL), calyculin A (2 μmol/L) or okadaic acid (1 μmol/L), caused a significant positive shift in V_(1/2) and a decrease in the conductance of KCNQ4 chan-nels. The V_(1/2) was shifted from-14.6±0.5 to-6.4±0.4 mV by cyclosporine, -18.8±0.5 to-9.2±0.4 mV by calyculin A, and-14.1±0.5 to -0.7±0.6 mV by okadaic acid. Moreover, the effects of these phosphatase inhibitors (okadaic acid or calyculin A) on the induction of a positive shift of V_(1/2) were augmented by further addition of PMA. Conclusion: These results indicate that ser/thr phosphatase inhibitors can induce a shift to more positive potentials of the activation curve of the KCNQ4 current. It is highly likely that the phosphatase functions to balance the phosphorylated state of substrate protein and thus has an important role in the regulation of human KCNQ4 channels expressed in Xenopus oocytes.

  7. Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants.

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    Toru Kobayashi

    Full Text Available Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+ (GIRK, Kir3 channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.

  8. Aberrant expression of ether à go-go potassium channel in colorectal cancer patients and cell lines

    Institute of Scientific and Technical Information of China (English)

    Xiang-Wu Ding; Juan-Juan Yan; Ping An; Peng Lü; He-Sheng Luo

    2007-01-01

    AIM: To study the expression of ether à go-go (Eag1) potassium channel in colorectal cancer and the relation ship between their expression and clinico-pathological features.METHODS: The expression levels of Eag1 protein were determined in 76 cancer tissues with paired noncancerous matched tissues as well as 9 colorectal adenoma tissues by immunohistochemistry. Eag1 mRNA expression was detected in 13 colorectal cancer tissues with paired non-cancerous matched tissues and 4 colorectal adenoma tissues as well as two colorectal cancer cell lines (LoVo and HT-29) by reverse transcription PCR.RESULTS: The frequency of positive expression of Eag1 protein was 76.3% (58/76) and Eag1 mRNA was 76.9% (10/13) in colorectal cancer tissue. Expression level of Eag1 protein was dependent on the tumor size,lymphatic node metastasis, other organ metastases and Dukes' stage (P < 0.05), while not dependent on age,sex, site and degree of differentiation. Eag1 protein and mRNA were negative in normal colorectal tissue, and absolutely negative in colorectal adenomas except that one case was positively stained for Eag1 protein.CONCLUSION: Eag1 protein and mRNA are aberrantly expressed in colorectal cancer and occasionally expressed in colorectal adenoma. The high frequency of expression of Eag1 in tumors and the restriction of normal expression to the brain suggest the potential of this protein for diagnostic, prognostic and therapeutic purposes.

  9. IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels.

    Science.gov (United States)

    Harmar, Anthony J; Hills, Rebecca A; Rosser, Edward M; Jones, Martin; Buneman, O Peter; Dunbar, Donald R; Greenhill, Stuart D; Hale, Valerie A; Sharman, Joanna L; Bonner, Tom I; Catterall, William A; Davenport, Anthony P; Delagrange, Philippe; Dollery, Colin T; Foord, Steven M; Gutman, George A; Laudet, Vincent; Neubig, Richard R; Ohlstein, Eliot H; Olsen, Richard W; Peters, John; Pin, Jean-Philippe; Ruffolo, Robert R; Searls, David B; Wright, Mathew W; Spedding, Michael

    2009-01-01

    The IUPHAR database (IUPHAR-DB) integrates peer-reviewed pharmacological, chemical, genetic, functional and anatomical information on the 354 nonsensory G protein-coupled receptors (GPCRs), 71 ligand-gated ion channel subunits and 141 voltage-gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes represent the targets of approximately one-third of currently approved drugs and are a major focus of drug discovery and development programs in the pharmaceutical industry. IUPHAR-DB provides a comprehensive description of the genes and their functions, with information on protein structure and interactions, ligands, expression patterns, signaling mechanisms, functional assays and biologically important receptor variants (e.g. single nucleotide polymorphisms and splice variants). In addition, the phenotypes resulting from altered gene expression (e.g. in genetically altered animals or in human genetic disorders) are described. The content of the database is peer reviewed by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR); the data are provided through manual curation of the primary literature by a network of over 60 subcommittees of NC-IUPHAR. Links to other bioinformatics resources, such as NCBI, Uniprot, HGNC and the rat and mouse genome databases are provided. IUPHAR-DB is freely available at http://www.iuphar-db.org. PMID:18948278

  10. Brain-derived neurotrophic factor modulation of Kv1.3 channel is disregulated by adaptor proteins Grb10 and nShc

    Directory of Open Access Journals (Sweden)

    Marks David R

    2009-01-01

    Full Text Available Abstract Background Neurotrophins are important regulators of growth and regeneration, and acutely, they can modulate the activity of voltage-gated ion channels. Previously we have shown that acute brain-derived neurotrophic factor (BDNF activation of neurotrophin receptor tyrosine kinase B (TrkB suppresses the Shaker voltage-gated potassium channel (Kv1.3 via phosphorylation of multiple tyrosine residues in the N and C terminal aspects of the channel protein. It is not known how adaptor proteins, which lack catalytic activity, but interact with members of the neurotrophic signaling pathway, might scaffold with ion channels or modulate channel activity. Results We report the co-localization of two adaptor proteins, neuronal Src homology and collagen (nShc and growth factor receptor-binding protein 10 (Grb10, with Kv1.3 channel as demonstrated through immunocytochemical approaches in the olfactory bulb (OB neural lamina. To further explore the specificity and functional ramification of adaptor/channel co-localization, we performed immunoprecipitation and Western analysis of channel, kinase, and adaptor transfected human embryonic kidney 293 cells (HEK 293. nShc formed a direct protein-protein interaction with Kv1.3 that was independent of BDNF-induced phosphorylation of Kv1.3, whereas Grb10 did not complex with Kv1.3 in HEK 293 cells. Both adaptors, however, co-immunoprecipitated with Kv1.3 in native OB. Grb10 was interestingly able to decrease the total expression of Kv1.3, particularly at the membrane surface, and subsequently eliminated the BDNF-induced phosphorylation of Kv1.3. To examine the possibility that the Src homology 2 (SH2 domains of Grb10 were directly binding to basally phosphorylated tyrosines in Kv1.3, we utilized point mutations to substitute multiple tyrosine residues with phenylalanine. Removal of the tyrosines 111–113 and 449 prevented Grb10 from decreasing Kv1.3 expression. In the absence of either adaptor protein

  11. Regulation of Mutant p53 Protein Expression

    OpenAIRE

    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 e...

  12. Electrochemical evaluation of chemical selectivity of glutamate receptor ion channel proteins with a multi-channel sensor.

    Science.gov (United States)

    Sugawara, M; Hirano, A; Rehák, M; Nakanishi, J; Kawai, K; Sato, H; Umezawa, Y

    1997-01-01

    A new method for evaluating chemical selectivity of agonists towards receptor ion channel proteins is proposed by using glutamate receptor (GluR) ion channel proteins and their agonists N-methyl-D-aspartic acid (NMDA), L-glutamate, and (2S, 3R, 4S) isomer of 2-(carboxycyclopropyl)glycine (L-CCG-IV). Integrated multi-channel currents, corresponding to the sum of total amount of ions passed through the multiple open channels, were used as a measure of agonists' selectivity to recognize ion channel proteins and induce channel currents. GluRs isolated from rat synaptic plasma membranes were incorporated into planar bilayer lipid membranes (BLMs) formed by the folding method. The empirical factors that affect the selectivity were demonstrated: (i) the number of GluRs incorporated into BLMs varied from one membrane to another; (ii) each BLM contained different subtypes of GluRs (NMDA and/or non-NMDA subtypes); and (iii) the magnitude of multi-channel responses induced by L-glutamate at negative applied potentials was larger than at positive potentials, while those by NMDA and L-CCG-IV were linearly related to applied potentials. The chemical selectivity among NMDA, L-glutamate and L-CCG-IV for NMDA subtype of GluRs was determined with each single BLM in which only NMDA subtype of GluRs was designed to be active by inhibiting the non-NMDA subtypes using a specific antagonist DNQX. The order of selectivity among the relevant agonists for the NMDA receptor subtype was found to be L-CCG-IV > L-glutamate > NMDA, which is consistent with the order of binding affinity of these agonists towards the same NMDA subtypes. The potential use of this approach for evaluating chemical selectivity towards non-NMDA receptor subtypes of GluRs and other receptor ion channel proteins is discussed.

  13. Chloride channel protein 2 prevents glutamate-induced apoptosis in retinal ganglion cells

    Science.gov (United States)

    Bi, Miao-Miao; Hong, Sen; Ma, Ling-Jun; Zhou, Hong-Yan; Lu, Jia; Zhao, Jing; Zheng, Ya-Juan

    2016-01-01

    Objective(s): The purpose of this study was to investigate the role of chloride channel protein 2 (ClC-2) in glutamate-induced apoptosis in the retinal ganglion cell line (RGC-5). Materials and Methods: RGC-5 cells were treated with 1 mM glutamate for 24 hr. The expression of ClC-2, Bax, and Bcl-2 was detected by western blot analysis. Cell survival and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Caspase-3 and -9 activities were determined by a colorimetric assay. The roles of ClC-2 in glutamate-induced apoptosis were examined by using ClC-2 complementary deoxyribonucleic acid (cDNA) and small inference ribonucleic acid (RNA) transfection technology. Results: Overexpression of ClC-2 in RGC-5 cells significantly decreased glutamate-induced apoptosis and increased cell viability, whereas silencing of ClC-2 with short hairpin (sh) RNA produced opposite effects. ClC-2 overexpression increased the expression of Bcl-2, decreased the expression of Bax, and decreased caspase-3 and -9 activation in RGC-5 cells treated with glutamate, but silencing of ClC-2 produced opposite effects. Conclusion: Our data suggest that ClC-2 chloride channels might play a protective role in glutamate-induced apoptosis in retinal ganglion cells via the mitochondria-dependent apoptosis pathway. PMID:27635193

  14. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales.

    Science.gov (United States)

    Margres, Mark J; Wray, Kenneth P; Seavy, Margaret; McGivern, James J; Herrera, Nathanael D; Rokyta, Darin R

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our results suggest that various constraints on high-expression proteins reduce the availability of beneficial expression variants relative to low-expression

  15. Ion channel gene expression in lung adenocarcinoma: potential role in prognosis and diagnosis.

    Science.gov (United States)

    Ko, Jae-Hong; Gu, Wanjun; Lim, Inja; Bang, Hyoweon; Ko, Eun A; Zhou, Tong

    2014-01-01

    Ion channels are known to regulate cancer processes at all stages. The roles of ion channels in cancer pathology are extremely diverse. We systematically analyzed the expression patterns of ion channel genes in lung adenocarcinoma. First, we compared the expression of ion channel genes between normal and tumor tissues in patients with lung adenocarcinoma. Thirty-seven ion channel genes were identified as being differentially expressed between the two groups. Next, we investigated the prognostic power of ion channel genes in lung adenocarcinoma. We assigned a risk score to each lung adenocarcinoma patient based on the expression of the differentially expressed ion channel genes. We demonstrated that the risk score effectively predicted overall survival and recurrence-free survival in lung adenocarcinoma. We also found that the risk scores for ever-smokers were higher than those for never-smokers. Multivariate analysis indicated that the risk score was a significant prognostic factor for survival, which is independent of patient age, gender, stage, smoking history, Myc level, and EGFR/KRAS/ALK gene mutation status. Finally, we investigated the difference in ion channel gene expression between the two major subtypes of non-small cell lung cancer: adenocarcinoma and squamous-cell carcinoma. Thirty ion channel genes were identified as being differentially expressed between the two groups. We suggest that ion channel gene expression can be used to improve the subtype classification in non-small cell lung cancer at the molecular level. The findings in this study have been validated in several independent lung cancer cohorts.

  16. Comparative identification of Ca2+ channel expression in INS-1 and rat pancreatic β cells

    Institute of Scientific and Technical Information of China (English)

    Fei Li; Zong-Ming Zhang

    2009-01-01

    AIM: To identify and compare the profile of Ca2+ channel subunit expression in INS-1 and rat pancreatic β cells. METHODS: The rat insulin-secreting INS-1 cell line was cultured in RPMI-1640 with Wistar rats employed as islet donors. Ca2+ channel subunit expression in INS-1 and isolated rat β cells were examined by reverse transcription polymerase chain reaction (RT-PCR). Absolute real-time quantitative PCR was performed in a Bio-Rad iQ5 Gradient Real Time PCR system and the data analyzed using an iQ5 system to identify the expression level of the Ca2+ channel subunits. RESULTS: In INS-1 cells, the L-type Ca2+ channel 1C subunit had the highest expression level and the TPRM2 subunit had the second highest expression. In rat β cells, the TPRC4β subunit expression was dominant and the expression of the L-type 1C subunit exceeded the 1D subunit expression about two-fold. This result agreed with other studies, confirming the important role of the L-type 1C subunit in insulinsecreting cells, and suggested that non-voltageoperated Ca2+ channels may have an important role in biphasic insulin secretion. CONCLUSION: Twelve major Ca2+ channel subunit types were identified in INS-1 and rat β cells and significant differences were observed in the expression of certain subunits between these cells.

  17. Protein kinase C is involved in regulation of Ca2+ channels in plasmalemma of Nitella syncarpa.

    Science.gov (United States)

    Zherelova, O M

    1989-01-01

    Ca2+ current recordings have been made on Nitella syncarpa cells using the intracellular perfusion and the voltage-clamp technique. TPA (12-O-tetradecanoylphorbol-13-acetate), a substance capable of activating protein kinase C from plasmalemma of Nitella cells, modulates voltage-dependent Ca2+ channels. Polymixin B, inhibitor of protein kinase C, blocks the Nitella plasmalemma Ca2+ channels; the rate of channel blockage depends on the concentration and exposure time of the substance. PMID:2536617

  18. Sensory neuron-specific sodium channel SNS is abnormally expressed in the brains of mice with experimental allergic encephalomyelitis and humans with multiple sclerosis

    Science.gov (United States)

    Black, Joel A.; Dib-Hajj, Sulayman; Baker, David; Newcombe, Jia; Cuzner, M. Louise; Waxman, Stephen G.

    2000-10-01

    Clinical abnormalities in multiple sclerosis (MS) have classically been considered to be caused by demyelination and/or axonal degeneration; the possibility of molecular changes in neurons, such as the deployment of abnormal repertoires of ion channels that would alter neuronal electrogenic properties, has not been considered. Sensory Neuron-Specific sodium channel SNS displays a depolarized voltage dependence, slower activation and inactivation kinetics, and more rapid recovery from inactivation than classical "fast" sodium channels. SNS is selectively expressed in spinal sensory and trigeminal ganglion neurons within the peripheral nervous system and is not expressed within the normal brain. Here we show that sodium channel SNS mRNA and protein, which are not present within the cerebellum of control mice, are expressed within cerebellar Purkinje cells in a mouse model of MS, chronic relapsing experimental allergic encephalomyelitis. We also demonstrate SNS mRNA and protein expression within Purkinje cells from tissue obtained postmortem from patients with MS, but not in control subjects with no neurological disease. These results demonstrate a change in sodium channel expression in neurons within the brain in an animal model of MS and in humans with MS and suggest that abnormal patterns of neuronal ion channel expression may contribute to clinical abnormalities such as ataxia in these disorders.

  19. Ca2+ channels as integrators of G protein-mediated signaling in neurons.

    Science.gov (United States)

    Strock, Jesse; Diversé-Pierluissi, María A

    2004-11-01

    The observations from Dunlap and Fischbach that transmitter-mediated shortening of the duration of action potentials could be caused by a decrease in calcium conductance led to numerous studies of the mechanisms of modulation of voltage-dependent calcium channels. Calcium channels are well known targets for inhibition by receptor-G protein pathways, and multiple forms of inhibition have been described. Inhibition of Ca(2+) channels can be mediated by G protein betagamma-subunits or by kinases, such as protein kinase C and tyrosine kinases. In the last few years, it has been shown that integration of G protein signaling can take place at the level of the calcium channel by regulation of the interaction of the channel pore-forming subunit with different cellular proteins.

  20. Effect of the protein expressions of gap junction Cx40 by using Ibutilide to selective act on Kv1.5 potassium channel on atrial fibrillation patients%特异性阻断Kv1.5通道对心房颤动患者缝隙连接蛋白40表达的影响

    Institute of Scientific and Technical Information of China (English)

    张珂; 石开虎; 徐盛松; 曹炜; 宣海洋; 沙纪名

    2013-01-01

    Objective To evaluate the expression of atrial muscle gap junction Cx40 in rheumatic heart disease with chronic atrial fibrillation by using the new class M antiarrhythmic drug ibutilide specific block ultra-rapid delayed rectifier potassium channels( Kvl. 5 ), to discuss possibility of the Kvl. 5 potassium channels to be one of downstream signal transduction pathways of the atrial muscle gap junction protein, and to reveal the possible mechanism of atrial fibrillation( AF ). Methods The experiment specimens were taken from right atrial appendage tissues of rheumatic heart disease patients who accepted the valve replacement surgeries, and these specimens were divided into 2 groups according to the heart rate before surgeries. The sinus rhythm group of rheumatic heart disease [ sinus rhythm ( SR ) group ], and the atrial fibrillation group of rheumatic heart disease heart disease( AF group ). Enzyme was used to dissociate atrial myocytes, atrial myocytes was cultured and Western blot was used to detect the protein expressions of Cx40 in two groups before and after they were selective acted on by the selective Kvl. 5 potassium channel blocker-ibutilide. Results After using the Class M antiarrhythmic agents to selective act on Kvl. 5 potassium channel in rheumatic heart disease with chronic atrial fibrillation, the expression of Cx40 was significantly higher in AF group ( P <0. 01 ). The protein expression of Kvl. 5 was not significantly changed in SR group. Conclusion In the group of rheumatic heart disease with atrial fibrillation, the expression of Cx40 was significantly increased after using the specific inhibitor of Kvl. 5 potassium channel blocker in AF group, proving that the open Kvl. 5 potassium channels may affect the expression of connexin Cx40, which further reveals that electrical remodeling may lead to the diversification of structural remodeling in AF.%目的 利用伊布利特特异性阻断超速延迟整流性钾通道 (Kv1.5)

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

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Grunnet, M; Angelo, K;

    2002-01-01

    will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells...... and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual......-function vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels....

  2. Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells

    OpenAIRE

    ISHIKAWA, TORU; Marunaka, Yoshinori; Rotin, Daniela

    1998-01-01

    The epithelial Na+ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby ca...

  3. Over Expression of Voltage Dependent Anion Channel 2 (VDAC2 in Muscles of Electrically Stunned Chickens

    Directory of Open Access Journals (Sweden)

    Norshahida Abu Samah, Azura Amid, and Faridah Yusof

    2011-12-01

    Full Text Available Water bath stunning is a common practice in commercial slaughterhouses. Such treatment is economic and in line with animal welfare practice. However, the conditions applied for the stunning process may vary from a slaughterhouse to another slaughterhouse. Such a loose regulation on the stunning procedure has opened up doors for food adulteration such as over dose stunning. In this study, a simple and reliable approach using proteomics have been developed to study the effect of different currents and voltages in stunning on the protein expression of the chickens. Protein profiles of the chickens were constructed in order to detect any differences in protein expression and modifications. The different voltage studied were 10 V, 40 V and 70 V while the values for current studied were 0.25 A, 0.5 A, and 0.75 A. After the proteomics analyses using 2D Platinum ImageMaster 6.0 and Matrix-assisted laser desorption ionization- time of flight (MALDI TOF spectrometry identification, Voltage dependent anion channel 2 (VDAC2 was identified to be over expressed in the muscle sample of over stunned chicken. The over expression of VDAC2 was confirmed at the transcriptional level of RNA expression. Real Time PCR showed that all over stunned samples contained higher mRNA expression level for VDAC2 genes. The mRNA level of VDAC2 was up-regulated by 59.87 fold change when normalized with housekeeping gene. In conclusion, VDAC2 could serve as potential biomarkers for identification of electrically stimulated chickens. The existence of these biomarkers will help to monitor the slaughtering and stunning process in the future. It will revolutionize the food authentication field and give a new breathe to the meat industry.ABSTRAK: Kaedah "waterbath stunning" merupakan amalan biasa di pusat-pusat penyembelihan. Kaedah ini adalah ekonomik dan selari dengan amalan kebajikan haiwan. Walaubagaimanapun, syarat-syarat yang digunakan untuk proses kejutan tersebut mungkin

  4. 人类胞内氯离子通道蛋白3在原核细胞及真核细胞内的表达%The expression of human intracellular chloride channel protein 3 in eukaryotic and prokaryotic cells

    Institute of Scientific and Technical Information of China (English)

    李春雨; 潘林鑫; 刘晓颖; 范礼斌

    2014-01-01

    目的研究人类胞内氯离子通道蛋白3(CLIC3)在真核细胞中的定位和表达,及其GST融合蛋白在原核细胞中的表达。方法以含人CLIC3的全长cDNA序列的质粒为模板,PCR扩增CLIC3片段,构建真核表达载体pcDNA3.1-CLIC3-FLAG,检测其定位及表达;构建原核表达载体pGEX-5X-3-CLIC3,转化到大肠杆菌 BL21菌株,IPTG诱导融合蛋白GST-CLIC3表达。结果细胞免疫荧光结果表明 CLIC3在COS7细胞质和细胞核中均有分布;Western blot结果显示CLIC3在HEK-293T细胞中能有效表达;考马斯亮蓝染色结果表明融合蛋白GST-CLIC3在BL21菌株中能有效表达。结论人类的CLIC3蛋白COS7、HEK-293T及大肠杆菌BL21菌株均能有效表达,为进一步了解CLIC3的功能奠定了一定的基础。%Objective To investigate the expression and localization of the human chloride channel protein 3 (CLIC3) in eukaryotic cells, and the expression of GST fusion protein in prokaryotic cells. Methods Plasmids containing full length of human CLIC3 cDNA was used as PCR template to construct the prokaryotic and eukaryotic expression vectors. The pcDNA3. 1-CLIC3-FLAG was transfected into COS7 and HEK-293T cells respectively to detect the localization and expression of CLIC3. The pGEX-5X-3-CLIC3 was transformed into E. coli BL21 to inves-tigate the expression of fusion protein GST-CLIC3 . Results The immunofluorescence results indicated that CLIC3 was distributed in both cytoplasm and nucleus of COS7 cells;Western blot showed CLIC3 could be effectively ex-pressed in HEK-293T cells;Coomassie blue staining proved GST-CLIC3 could be expressed in E. coli BL21. Con-clusion Human CLIC3 protein can be expressed effectively both in eukaryotic and prokaryotic cells, which is im-portant for further research on the function of human CLIC3 .

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

    OpenAIRE

    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 wi...

  6. Functional reconstitution and characterization of AqpZ, the E. coli water channel protein.

    Science.gov (United States)

    Borgnia, M J; Kozono, D; Calamita, G; Maloney, P C; Agre, P

    1999-09-01

    Understanding the selectivity of aquaporin water channels will require structural and functional studies of wild-type and modified proteins; however, expression systems have not previously yielded aquaporins in the necessary milligram quantities. Here we report expression of a histidine-tagged form of Escherichia coli aquaporin-Z (AqpZ) in its homologous expression system. 10-His-AqpZ is solubilized and purified to near homogeneity in a single step with a final yield of approximately 2.5 mg/l of culture. The histidine tag is removed by trypsin, yielding the native protein with the addition of three N-terminal residues, as confirmed by microsequencing. Sucrose gradient sedimentation analysis showed that the native, solubilized AqpZ protein is a trypsin-resistant tetramer. Unlike other known aquaporins, AqpZ tetramers are not readily dissociated by 1% SDS at neutral pH. Hydrophilic reducing agents have a limited effect on the stability of the tetramer in 1% SDS, whereas incubations for more than 24 hours, pH values below 5.6, or exposure to the hydrophobic reducing agent ethanedithiol cause dissociation into monomers. Cys20, but not Cys9, is necessary for the stability of the AqpZ tetramer in SDS. Upon reconstitution into proteoliposomes, AqpZ displays very high osmotic water permeability (pf > or = 10 x 10(-14) cm3 s-1 subunit-1) and low Arrhenius activation energy (Ea = 3.7 kcal/mol), similar to mammalian aquaporin-1 (AQP1). No permeation by glycerol, urea or sorbitol was detected. Expression of native and modified AqpZ in milligram quantities has permitted biophysical characterization of this remarkably stable aquaporin tetramer, which is being utilized for high-resolution structural studies. PMID:10518952

  7. Dengue virus M protein C-terminal peptide (DVM-C) forms ion channels.

    Science.gov (United States)

    Premkumar, A; Horan, C R; Gage, P W

    2005-03-01

    A chemically synthesized peptide consisting of the C-terminus of the M protein of the Dengue virus type 1 strain Singapore S275/90 (DVM-C) produced ion channel activity in artificial lipid bilayers. The channels had a variable conductance and were more permeable to sodium and potassium ions than to chloride ions and more permeable to chloride ions than to calcium ions. Hexamethylene amiloride (100 microM) and amantadine (10 microM), blocked channels formed by DVM-C. Ion channels may play an important role in the life cycle of many viruses and drugs that block these channels may prove to be useful antiviral agents.

  8. Direct protein-protein interactions and substrate channeling between cellular retinoic acid binding proteins and CYP26B1.

    Science.gov (United States)

    Nelson, Cara H; Peng, Chi-Chi; Lutz, Justin D; Yeung, Catherine K; Zelter, Alex; Isoherranen, Nina

    2016-08-01

    Cellular retinoic acid binding proteins (CRABPs) bind all-trans-retinoic acid (atRA) tightly. This study aimed to determine whether atRA is channeled directly to cytochrome P450 (CYP) CYP26B1 by CRABPs, and whether CRABPs interact directly with CYP26B1. atRA bound to CRABPs (holo-CRABP) was efficiently metabolized by CYP26B1. Isotope dilution experiments showed that delivery of atRA to CYP26B1 in solution was similar with or without CRABP. Holo-CRABPs had higher affinity for CYP26B1 than free atRA, but both apo-CRABPs inhibited the formation of 4-OH-RA by CYP26B1. Similar protein-protein interactions between soluble binding proteins and CYPs may be important for other lipophilic CYP substrates.

  9. Genome-wide identification of Hsp70 genes in channel catfish and their regulated expression after bacterial infection.

    Science.gov (United States)

    Song, Lin; Li, Chao; Xie, Yangjie; Liu, Shikai; Zhang, Jiaren; Yao, Jun; Jiang, Chen; Li, Yun; Liu, Zhanjiang

    2016-02-01

    Heat shock proteins 70/110 (Hsp70/110) are a family of conserved ubiquitously expressed heat shock proteins which are produced by cells in response to exposure to stressful conditions. Besides the chaperone and housekeeping functions, they are also known to be involved in immune response during infection. In this study, we identified 16 Hsp70/110 geness in channel catfish (Ictalurus punctatus) through in silico analysis using RNA-Seq and genome databases. Among them 12 members of Hsp70 (Hspa) family and 4 members of Hsp110 (Hsph) family were identified. Phylogenetic and syntenic analyses provided strong evidence in supporting the orthologies of these HSPs. In addition, we also determined the expression patterns of Hsp70/110 genes after Flavobacterium columnare and Edwardsiella ictaluri infections by meta-analyses, for the first time in channel catfish. Ten out of sixteen genes were significantly up/down-regulated after bacterial challenges. Specifically, nine genes were found significantly expressed in gill after F. columnare infection. Two genes were found significantly expressed in intestine after E. ictaluri infection. Pathogen-specific pattern and tissue-specific pattern were found in the two infections. The significantly regulated expressions of catfish Hsp70 genes after bacterial infections suggested their involvement in immune response in catfish. PMID:26693666

  10. Caenorhabditis elegans paraoxonase-like proteins control the functional expression of DEG/ENaC mechanosensory proteins

    Science.gov (United States)

    Chen, Yushu; Bharill, Shashank; Altun, Zeynep; O’Hagan, Robert; Coblitz, Brian; Isacoff, Ehud Y.; Chalfie, Martin

    2016-01-01

    Caenorhabditis elegans senses gentle touch via a mechanotransduction channel formed from the DEG/ENaC proteins MEC-4 and MEC-10. An additional protein, the paraoxonase-like protein MEC-6, is essential for transduction, and previous work suggested that MEC-6 was part of the transduction complex. We found that MEC-6 and a similar protein, POML-1, reside primarily in the endoplasmic reticulum and do not colocalize with MEC-4 on the plasma membrane in vivo. As with MEC-6, POML-1 is needed for touch sensitivity, the neurodegeneration caused by the mec-4(d) mutation, and the expression and distribution of MEC-4 in vivo. Both proteins are likely needed for the proper folding or assembly of MEC-4 channels in vivo as measured by FRET. MEC-6 detectably increases the rate of MEC-4 accumulation on the Xenopus oocyte plasma membrane. These results suggest that MEC-6 and POML-1 interact with MEC-4 to facilitate expression and localization of MEC-4 on the cell surface. Thus MEC-6 and POML-1 act more like chaperones for MEC-4 than channel components. PMID:26941331

  11. Functional expression of mammalian receptors and membrane channels in different cells.

    Science.gov (United States)

    Eifler, Nora; Duckely, Myriam; Sumanovski, Lazar T; Egan, Terrance M; Oksche, Alexander; Konopka, James B; Lüthi, Anita; Engel, Andreas; Werten, Paul J L

    2007-08-01

    In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.

  12. The Sarcoglycan complex is expressed in the cerebrovascular system and is specifically regulated by astroglial Cx30 channels

    Directory of Open Access Journals (Sweden)

    Anne-Cécile eBoulay

    2015-02-01

    Full Text Available Astrocytes, the most prominent glial cell type in the brain, send specialized processes called endfeet, around blood vessels and express a large molecular repertoire regulating the cerebrovascular system physiology. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction protein Connexin 43 and 30 (Cx43 and Cx30 allowing in particular for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. In this study, we addressed the specific role of Cx30 at the gliovascular interface. Using an inactivation mouse model for Cx30 (Cx30Δ/Δ, we showed that absence of Cx30 does not affect blood-brain barrier (BBB organization and permeability. However, it results in the cerebrovascular fraction, in a strong upregulation of Sgcg encoding γ-Sarcoglycan (SG, a member of the Dystrophin-associated protein complex (DAPC connecting cytoskeleton and the extracellular matrix. The same molecular event occurs in Cx30T5M/T5M mutated mice, where Cx30 channels are closed, demonstrating that Sgcg regulation relied on Cx30 channel functions. We further characterized the expression of other Sarcoglycan complex (SGC molecules in the cerebrovascular system and showed the presence of α-, β-, δ-, γ-, ε- and ζ- SG, as well as Sarcospan. Their expression was however not modified in Cx30Δ/Δ. These results suggest that a full SGC might be present in the cerebrovascular system, and that expression of one of its member, γ-Sarcoglycan, depends on Cx30 channels. As described in skeletal muscles, the SGC may contribute to membrane stabilization and signal transduction in the cerebrovascular system, which may therefore be regulated by Cx30 channel-mediated functions.

  13. Ovarian cancer: Ion channel and aquaporin expression as novel targets of clinical potential.

    Science.gov (United States)

    Frede, Julia; Fraser, Scott P; Oskay-Özcelik, Gülten; Hong, Yeosun; Ioana Braicu, E; Sehouli, Jalid; Gabra, Hani; Djamgoz, Mustafa B A

    2013-07-01

    Ovarian cancer is associated with limited overall survival, due to problems in early detection and therapy. Membrane ion channels have been proposed to play a significant, concerted role in the cancer process, from initial proliferation to metastasis, and promise to be early, functional biomarkers. We review the evidence for ion channel and aquaporin expression and functioning in human ovarian cancer cells and tissues. In vitro, K(+) channels, mainly voltage-gated, including Ca(2+)-activated channels, have been found to control the cell cycle, as in other cancers. Voltage-gated, volume-regulated and intracellular Cl(-) channels have been detected in vitro and in vivo and shown to be involved in proliferation, adhesion and invasion. Evidence for 'transient receptor potential', voltage-gated sodium and calcium channels, which have been shown to contribute to pathogenesis of other carcinomas, is also emerging in ovarian cancer. Aquaporins may be involved in cell growth, migration and formation of ascites via increased water permeability of micro-vessels. It is concluded that functional expression of ion channels and their regulation by steroid hormones and growth factors are an integral part of ovarian cancer development and progression. Furthermore, ion channels may be involved in multidrug resistance, commonly associated with treatment of ovarian cancer. We propose that ion channel studies can facilitate our understanding of the pathobiology of ovarian cancer and, ultimately, can serve as viable novel targets for its clinical management. PMID:23683551

  14. Expression of Transient Receptor Potential Vanilloid (TRPV Channels in Different Passages of Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    Richard Barrett-Jolley

    2012-04-01

    Full Text Available Ion channels play important roles in chondrocyte mechanotransduction. The transient receptor potential vanilloid (TRPV subfamily of ion channels consists of six members. TRPV1-4 are temperature sensitive calcium-permeable, relatively non-selective cation channels whereas TRPV5 and TRPV6 show high selectivity for calcium over other cations. In this study we investigated the effect of time in culture and passage number on the expression of TRPV4, TRPV5 and TRPV6 in articular chondrocytes isolated from equine metacarpophalangeal joints. Polyclonal antibodies raised against TRPV4, TRPV5 and TRPV6 were used to compare the expression of these channels in lysates from first expansion chondrocytes (P0 and cells from passages 1–3 (P1, P2 and P3 by western blotting. TRPV4, TRPV5 and TRPV6 were expressed in all passages examined. Immunohistochemistry and immunofluorescence confirmed the presence of these channels in sections of formalin fixed articular cartilage and monolayer cultures of methanol fixed P2 chondrocytes. TRPV5 and TRPV6 were upregulated with time and passage in culture suggesting that a shift in the phenotype of the cells in monolayer culture alters the expression of these channels. In conclusion, several TRPV channels are likely to be involved in calcium signaling and homeostasis in chondrocytes.

  15. Investigation of TRPC channel-modulating progestins and proteins

    OpenAIRE

    Miehe, Susanne

    2008-01-01

    In the first part of this study, we have identified the two steroid hormones progesterone and norgestimate as novel TRPC channel blockers. Both substances blocked TRPC-mediated Ca2+ influx with micromolar activities in fluorometric measurements. TRPC channel inhibition did not seem to be a general steroid effect since another progestin, the norgestimate metabolite levonorgestrel, was not effective. Norgestimate was 4- to 5-fold more active on the TRPC3/6/7 subfamily compared to TRPC4/5, where...

  16. A Unified Channel Charges Expression for Analytic MOSFET Modeling

    OpenAIRE

    Hugues Murray; Patrick Martin

    2012-01-01

    Based on a 1D Poissons equation resolution, we present an analytic model of inversion charges allowing calculation of the drain current and transconductance in the Metal Oxide Semiconductor Field Effect Transistor. The drain current and transconductance are described by analytical functions including mobility corrections and short channel effects (CLM, DIBL). The comparison with the Pao-Sah integral shows excellent accuracy of the model in all inversion modes from strong to weak inversion in ...

  17. Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

    Directory of Open Access Journals (Sweden)

    Piotr Koprowski

    Full Text Available Bacterial mechano-sensitive (MS channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

  18. Phycodnavirus potassium ion channel proteins question the virus molecular piracy hypothesis.

    Directory of Open Access Journals (Sweden)

    Kay Hamacher

    Full Text Available Phycodnaviruses are large dsDNA, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. Among the viral gene products are the smallest proteins known to form functional K(+ channels. To determine if these viral K(+ channels are the product of molecular piracy from their hosts, we compared the sequences of the K(+ channel pore modules from seven phycodnaviruses to the K(+ channels from Chlorella variabilis and Ectocarpus siliculosus, whose genomes have recently been sequenced. C. variabilis is the host for two of the viruses PBCV-1 and NY-2A and E. siliculosus is the host for the virus EsV-1. Systematic phylogenetic analyses consistently indicate that the viral K(+ channels are not related to any lineage of the host channel homologs and that they are more closely related to each other than to their host homologs. A consensus sequence of the viral channels resembles a protein of unknown function from a proteobacterium. However, the bacterial protein lacks the consensus motif of all K(+ channels and it does not form a functional channel in yeast, suggesting that the viral channels did not come from a proteobacterium. Collectively, our results indicate that the viruses did not acquire their K(+ channel-encoding genes from their current algal hosts by gene transfer; thus alternative explanations are required. One possibility is that the viral genes arose from ancient organisms, which served as their hosts before the viruses developed their current host specificity. Alternatively the viral proteins could be the origin of K(+ channels in algae and perhaps even all cellular organisms.

  19. Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein.

    Directory of Open Access Journals (Sweden)

    Guowei Wei

    Full Text Available Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses.

  20. Protein expression in response to folate stress in Escherichia coli.

    OpenAIRE

    Huang, E Y; Mohler, A M; Rohlman, C E

    1997-01-01

    Interruption of folate metabolism by trimethoprim results in the elevated expression of folate stress proteins in Escherichia coli. E. coli grown in culture medium supplemented with the folate-dependent metabolites glycine, methionine, and the purine nucleoside inosine shows reduced expression of folate stress proteins. The folate stress proteins include the universal stress protein, the ferric uptake regulatory repressor, and possibly, lipoamide dehydrogenase, the L protein component of the ...

  1. The KCNQ5 potassium channel from mouse: a broadly expressed M-current like potassium channel modulated by zinc, pH, and volume changes

    DEFF Research Database (Denmark)

    Jensen, Henrik Sindal; Callø, Kirstine; Jespersen, Thomas;

    2005-01-01

    The KCNQ proteins compose a sub-group of the voltage-activated potassium channel family. The family consists of five members (KCNQ1 to 5--also named Kv7.1 to Kv7.5) encoded by single genes, which all give rise to proteins forming slowly activating potassium-selective ion channels. The physiological...

  2. Co- and post-translational translocation through the protein-conducting channel : analogous mechanisms at work?

    NARCIS (Netherlands)

    Mitra, Kakoli; Frank, Joachim; Driessen, Arnold

    2006-01-01

    Many proteins are translocated across, or integrated into, membranes. Both functions are fulfilled by the 'translocon/translocase', which contains a membrane-embedded proteinconducting channel (PCC) and associated soluble factors that drive translocation and insertion reactions using nucleotide trip

  3. Regionally specific expression of high-voltage-activated calcium channels in thalamic nuclei of epileptic and non-epileptic rats.

    Science.gov (United States)

    Kanyshkova, Tatyana; Ehling, Petra; Cerina, Manuela; Meuth, Patrick; Zobeiri, Mehrnoush; Meuth, Sven G; Pape, Hans-Christian; Budde, Thomas

    2014-07-01

    The polygenic origin of generalized absence epilepsy results in dysfunction of ion channels that allows the switch from physiological asynchronous to pathophysiological highly synchronous network activity. Evidence from rat and mouse models of absence epilepsy indicates that altered Ca(2+) channel activity contributes to cellular and network alterations that lead to seizure activity. Under physiological circumstances, high voltage-activated (HVA) Ca(2+) channels are important in determining the thalamic firing profile. Here, we investigated a possible contribution of HVA channels to the epileptic phenotype using a rodent genetic model of absence epilepsy. In this study, HVA Ca(2+) currents were recorded from neurons of three different thalamic nuclei that are involved in both sensory signal transmission and rhythmic-synchronized activity during epileptic spike-and-wave discharges (SWD), namely the dorsal part of the lateral geniculate nucleus (dLGN), the ventrobasal thalamic complex (VB) and the reticular thalamic nucleus (NRT) of epileptic Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) and non-epileptic August Copenhagen Irish (ACI) rats. HVA Ca(2+) current densities in dLGN neurons were significantly increased in epileptic rats compared with non-epileptic controls while other thalamic regions revealed no differences between the strains. Application of specific channel blockers revealed that the increased current was carried by L-type Ca(2+) channels. Electrophysiological evidence of increased L-type current correlated with up-regulated mRNA and protein expression of a particular L-type channel, namely Cav1.3, in dLGN of epileptic rats. No significant changes were found for other HVA Ca(2+) channels. Moreover, pharmacological inactivation of L-type Ca(2+) channels results in altered firing profiles of thalamocortical relay (TC) neurons from non-epileptic rather than from epileptic rats. While HVA Ca(2+) channels influence tonic and burst firing in ACI and WAG

  4. A Unified Channel Charges Expression for Analytic MOSFET Modeling

    Directory of Open Access Journals (Sweden)

    Hugues Murray

    2012-01-01

    Full Text Available Based on a 1D Poissons equation resolution, we present an analytic model of inversion charges allowing calculation of the drain current and transconductance in the Metal Oxide Semiconductor Field Effect Transistor. The drain current and transconductance are described by analytical functions including mobility corrections and short channel effects (CLM, DIBL. The comparison with the Pao-Sah integral shows excellent accuracy of the model in all inversion modes from strong to weak inversion in submicronics MOSFET. All calculations are encoded with a simple C program and give instantaneous results that provide an efficient tool for microelectronics users.

  5. Glial cell-expressed mechanosensitive channel TRPV4 mediates infrasound-induced neuronal impairment.

    Science.gov (United States)

    Shi, Ming; Du, Fang; Liu, Yang; Li, Li; Cai, Jing; Zhang, Guo-Feng; Xu, Xiao-Fei; Lin, Tian; Cheng, Hao-Ran; Liu, Xue-Dong; Xiong, Li-Ze; Zhao, Gang

    2013-11-01

    Vibroacoustic disease, a progressive and systemic disease, mainly involving the central nervous system, is caused by excessive exposure to low-frequency but high-intensity noise generated by various heavy transportations and machineries. Infrasound is a type of low-frequency noise. Our previous studies demonstrated that infrasound at a certain intensity caused neuronal injury in rats but the underlying mechanism(s) is still largely unknown. Here, we showed that glial cell-expressed TRPV4, a Ca(2+)-permeable mechanosensitive channel, mediated infrasound-induced neuronal injury. Among different frequencies and intensities, infrasound at 16 Hz and 130 dB impaired rat learning and memory abilities most severely after 7-14 days exposure, a time during which a prominent loss of hippocampal CA1 neurons was evident. Infrasound also induced significant astrocytic and microglial activation in hippocampal regions following 1- to 7-day exposure, prior to neuronal apoptosis. Moreover, pharmacological inhibition of glial activation in vivo protected against neuronal apoptosis. In vitro, activated glial cell-released proinflammatory cytokines IL-1β and TNF-α were found to be key factors for this neuronal apoptosis. Importantly, infrasound induced an increase in the expression level of TRPV4 both in vivo and in vitro. Knockdown of TRPV4 expression by siRNA or pharmacological inhibition of TRPV4 in cultured glial cells decreased the levels of IL-1β and TNF-α, attenuated neuronal apoptosis, and reduced TRPV4-mediated Ca(2+) influx and NF-κB nuclear translocation. Finally, using various antagonists we revealed that calmodulin and protein kinase C signaling pathways were involved in TRPV4-triggered NF-κB activation. Thus, our results provide the first evidence that glial cell-expressed TRPV4 is a potential key factor responsible for infrasound-induced neuronal impairment. PMID:24002225

  6. Expression of BK Ca channels and the modulatory beta-subunits in the rat and porcine trigeminal ganglion

    DEFF Research Database (Denmark)

    Johansson, Helle Wulf; Hay-Schmidt, Anders; Poulsen, Asser Nyander;

    2009-01-01

    Large conductance calcium-activated potassium (BK(Ca)) channels contribute to electrical impulses, proper signal transmission of information and regulation of neurotransmitter release. Migraine has been proposed to be a trigeminovascular disease involving the sensory trigeminal pathways and the c......Large conductance calcium-activated potassium (BK(Ca)) channels contribute to electrical impulses, proper signal transmission of information and regulation of neurotransmitter release. Migraine has been proposed to be a trigeminovascular disease involving the sensory trigeminal pathways...... and the cerebral arteries. We hypothesize that BK(Ca) channel alpha- and beta-subunits are present in the rat and porcine trigeminal ganglion (TG) thus enabling a role in migraine. BK(Ca) channel mRNA was detected using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. BK(Ca...... revealed beta2- and beta 4-subunit proteins in rat and porcine TG. The present study showed BK(Ca) channel expression in rat and porcine TG. The main modulatory beta-subunits detected in TG of both species were beta2- and beta 4-subunits....

  7. Distribution of Water Channel Protein RWC3 and Its Regulation by GA and Sucrose in Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    SUNMei-Hao; ZHANGMin-Hua; LIUHong-Yan; LILe-Gong; YUXin; SUWei-Ai; TANGZhang-Cheng

    2004-01-01

    Water channel proteins facilitate water flux across cell membranes and play important roles in plant growth and development. By GUS histochemical assay in RWC3 promoter-GUS transgenic rice (Oryza sativa L. cv. Shenxiangjin 4), one of the members of water channel proteins in rice, RWC3, was found to distribute widely in variety of organs, from vegetative and reproductive organs. Further studies showed that gibberellin (GA) enhanced the GUS activity in the transgenic calli, suspension cells and leaves, whereas ancymidol (anc), an inhibitor of GA synthesis, reduced the GUS activity. Sucrose was found to inhibit the effects induced by addition of GA, suggesting a possible cross-talk between GA and sucrose signaling on regulation of the RWC3 gene expression.

  8. Fluorometric functional assay for ion channel proteins in lipid nanovesicle membranes

    Energy Technology Data Exchange (ETDEWEB)

    Patti, J T [Department of Bioengineering, University of California, Los Angeles (United States); Montemagno, C D [College of Engineering, University of Cincinnati, Cincinnati (United States)

    2007-08-15

    Voltage-gated membrane proteins function as biomolecular transistors, making them attractive components for biologically based nanodevices. A functional assay for purified channel proteins is described and demonstrated with sodium selective, voltage-gated NaChBac ion channels. Purified NaChBac proteins were incorporated into a nanovesicle system utilizing oxonol VI, a fluorescent indicator of trans-membrane voltage. The ionophore valinomycin was used to trigger a change in membrane potential, allowing the observation of sodium permeability using a fluorometer. This method is suitable for concurrently testing a large population of purified proteins prior to incorporation in nanodevices.

  9. Fluorometric functional assay for ion channel proteins in lipid nanovesicle membranes

    Science.gov (United States)

    Patti, J. T.; Montemagno, C. D.

    2007-08-01

    Voltage-gated membrane proteins function as biomolecular transistors, making them attractive components for biologically based nanodevices. A functional assay for purified channel proteins is described and demonstrated with sodium selective, voltage-gated NaChBac ion channels. Purified NaChBac proteins were incorporated into a nanovesicle system utilizing oxonol VI, a fluorescent indicator of trans-membrane voltage. The ionophore valinomycin was used to trigger a change in membrane potential, allowing the observation of sodium permeability using a fluorometer. This method is suitable for concurrently testing a large population of purified proteins prior to incorporation in nanodevices.

  10. Expression and Fuactional Role of HERG1, K+ Channels in Leukemic Cells and Leukemic Stem Cells

    Institute of Scientific and Technical Information of China (English)

    LI Huiyu; LIU Liqiong; GUO Tiannan; ZHANG Jiahua; LI Xiaoqing; DU Wen; LIU Wei; CHEN Xiangjun; HUANG Shi'ang

    2007-01-01

    In order to investigate the expression and functional role of HERG1 K+ channels in leukemic cells and leukemic stem cells (LSCs), RT-PCR was used to detect the HERG1 K+ channels expression in leukemic cells and LSCs. The functional role of HERG1 K+ channels in leukemic cell proliferation was measured by MTT assay, and cell cycle and apoptosis were analyzed by flow cytometry. The results showed that herg mRNA was expressed in CD34+/CD38-, CD123+ LSCs but not in circulating CD34+ cells. Herg mRNA was also up-regulated in leukemia cell lines K562 and HL60 as well as almost all the primary leukemic cells while not in normal peripheral blood mononuclear cells (PBMNCs) and the expression of herg mRNA was not associated with the clinical and cytogenetic features of leukemia. In addition, leukemic cell proliferation was dramatically inhibited by HERG K+ channel special inhibitor E-4031. Moreover, E-4031 suppressed the cell growth by inducing a specific block at the G1/S transition phase of the cell cycle but had no effect on apoptosis in leukemic cells. The results suggested that HERG1 K+ channels could regulate leukemic cells proliferation and were necessary for leukemic cells to proceed with the cell cycle. HERG1 K+ channels may also have oncogenic potential and may be a biomarker for diagnosis of leukemia and a novel potential pharmacological target for leukemia therapy.

  11. Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

    Institute of Scientific and Technical Information of China (English)

    GAO Lei; LI Xia; GUO Zheng; ZHU MingZhu; LI YanHui; RAO ShaoQi

    2007-01-01

    GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interaction data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automatically selects the most appropriate functional classes as specific as possible during the learning process, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to "biology process" by three measures particularly designed for functional classes organized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.

  12. Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interac-tion data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automati-cally selects the most appropriate functional classes as specific as possible during the learning proc-ess, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to “biology process” by three measures particularly designed for functional classes organ-ized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.

  13. The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel

    Science.gov (United States)

    Iftinca, Mircea; Flynn, Robyn; Basso, Lilian; Melo, Helvira; Aboushousha, Reem; Taylor, Lauren

    2016-01-01

    Background Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund’s Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. Results We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund’s Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Conclusions Our work identified Hsc70 and its ATPase activity as a central

  14. Expression of inwardly rectifying potassium channels (GIRKs and beta-adrenergic regulation of breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Cakir Yavuz

    2004-12-01

    Full Text Available Abstract Background Previous research has indicated that at various organ sites there is a subset of adenocarcinomas that is regulated by beta-adrenergic and arachidonic acid-mediated signal transduction pathways. We wished to determine if this regulation exists in breast adenocarcinomas. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1 has been shown in tissue samples from approximately 40% of primary human breast cancers. Previously, GIRK channels have been associated with beta-adrenergic signaling. Methods Breast cancer cell lines were screened for GIRK channels by RT-PCR. Cell cultures of breast cancer cells were treated with beta-adrenergic agonists and antagonists, and changes in gene expression were determined by both relative competitive and real time PCR. Potassium flux was determined by flow cytometry and cell signaling was determined by western blotting. Results Breast cancer cell lines MCF-7, MDA-MB-361 MDA-MB 453, and ZR-75-1 expressed mRNA for the GIRK1 channel, while MDA-MB-468 and MDA-MB-435S did not. GIRK4 was expressed in all six breast cancer cell lines, and GIRK2 was expressed in all but ZR-75-1 and MDA-MB-435. Exposure of MDA-MB-453 cells for 6 days to the beta-blocker propranolol (1 μM increased the GIRK1 mRNA levels and decreased beta2-adrenergic mRNA levels, while treatment for 30 minutes daily for 7 days had no effect. Exposure to a beta-adrenergic agonist and antagonist for 24 hours had no effect on gene expression. The beta adrenergic agonist, formoterol hemifumarate, led to increases in K+ flux into MDA-MB-453 cells, and this increase was inhibited by the GIRK channel inhibitor clozapine. The tobacco carcinogen 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK, a high affinity agonist for beta-adrenergic receptors stimulated activation of Erk 1/2 in MDA-MB-453 cells. Conclusions Our data suggests β-adrenergic receptors and GIRK channels may play a role in breast cancer.

  15. The ABC protein turned chloride channel whose failure causes cystic fibrosis

    Science.gov (United States)

    Gadsby, David C.; Vergani, Paola; Csanády, László

    2006-03-01

    CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

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

    Science.gov (United States)

    Mayfield, Stephen P.

    2010-03-16

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, Stephen P

    2015-01-13

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

  18. Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in severe asthma

    OpenAIRE

    McGarvey, Lorcan P.; Butler, Claire A; Stokesberry, Susan; Polley, Liam; McQuaid, Stephen; Abdullah, Hani'ah; Ashraf, Sadaf; McGahon, Mary K; Curtis, Tim M; Arron, Joe; Choy, David; Warke, Tim J.; Bradding, Peter; Ennis, Madeleine; Zholos, Alexander

    2014-01-01

    BACKGROUND: The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of ...

  19. Potassium Channel Ether à go-go1 Is Aberrantly Expressed in Human Liposarcoma and Promotes Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Jin Wu

    2014-01-01

    Full Text Available The ether à go-go1 (Eag1 channel is overexpressed in a variety of cancers. However, the expression and function of Eag1 in liposarcoma are poorly understood. In the present study, the mRNA expression of Eag1 in different adipose tissue samples was examined by real-time PCR. Then, the protein expression of Eag1 in 131 different adipose tissues from 109 patients was detected by immunohistochemistry. Next, the associations between Eag1 expression and clinicopathological features of liposarcoma were analyzed. In addition, the effects of Eag1 on liposarcoma cell proliferation and cycle were evaluated by CCK-8, colony formation, xenograft mouse model, and flow cytometry, respectively. Finally, the activation of p38 mitogen-activated protein kinase (MAPK was detected by Western blot analysis to explain the detailed mechanisms of oncogenic potential of Eag1 in liposarcoma. It was found that Eag1 was aberrantly expressed in over 67% liposarcomas, with a higher frequency than in lipoma, hyperplasia, inflammation, and normal adipose tissues. However, Eag1 expression was not correlated with clinicopathological features of liposarcoma. Eag1 inhibitor imipramine or Eag1-shRNA significantly suppressed the proliferation of liposarcoma cells in vitro and in vivo, accompanying with accumulation of cells in the G1 phase. These results suggest that Eag1 plays an important role in regulating the proliferation and cell cycle of liposarcoma cells and might be a potential therapeutic target for liposarcoma.

  20. Estrogens and human papilloma virus oncogenes regulate human ether-à-go-go-1 potassium channel expression.

    Science.gov (United States)

    Díaz, Lorenza; Ceja-Ochoa, Irais; Restrepo-Angulo, Iván; Larrea, Fernando; Avila-Chávez, Euclides; García-Becerra, Rocío; Borja-Cacho, Elizabeth; Barrera, David; Ahumada, Elías; Gariglio, Patricio; Alvarez-Rios, Elizabeth; Ocadiz-Delgado, Rodolfo; Garcia-Villa, Enrique; Hernández-Gallegos, Elizabeth; Camacho-Arroyo, Ignacio; Morales, Angélica; Ordaz-Rosado, David; García-Latorre, Ethel; Escamilla, Juan; Sánchez-Peña, Luz Carmen; Saqui-Salces, Milena; Gamboa-Dominguez, Armando; Vera, Eunice; Uribe-Ramírez, Marisela; Murbartián, Janet; Ortiz, Cindy Sharon; Rivera-Guevara, Claudia; De Vizcaya-Ruiz, Andrea; Camacho, Javier

    2009-04-15

    Ether-à-go-go-1 (Eag1) potassium channels are potential tools for detection and therapy of numerous cancers. Here, we show human Eag1 (hEag1) regulation by cancer-associated factors. We studied hEag1 gene expression and its regulation by estradiol, antiestrogens, and human papillomavirus (HPV) oncogenes (E6/E7). Primary cultures from normal placentas and cervical cancer tissues; tumor cell lines from cervix, choriocarcinoma, keratinocytes, and lung; and normal cell lines from vascular endothelium, keratinocytes, and lung were used. Reverse transcription-PCR (RT-PCR) experiments and Southern blot analysis showed Eag1 expression in all of the cancer cell types, normal trophoblasts, and vascular endothelium, in contrast to normal keratinocytes and lung cells. Estradiol and antiestrogens regulated Eag1 in a cell type-dependent manner. Real-time RT-PCR experiments in HeLa cells showed that Eag1 estrogenic regulation was strongly associated with the expression of estrogen receptor-alpha. Eag1 protein was detected by monoclonal antibodies in normal placenta and placental blood vessels. Patch-clamp recordings in normal trophoblasts treated with estradiol exhibited potassium currents resembling Eag1 channel activity. Eag1 gene expression in keratinocytes depended either on cellular immortalization or the presence of HPV oncogenes. Eag1 protein was found in keratinocytes transfected with E6/E7 HPV oncogenes. Cell proliferation of E6/E7 keratinocytes was decreased by Eag1 antibodies inhibiting channel activity and by the nonspecific Eag1 inhibitors imipramine and astemizole; the latter also increased apoptosis. Our results propose novel oncogenic mechanisms of estrogen/antiestrogen use and HPV infection. We also suggest Eag1 as an early indicator of cell proliferation leading to malignancies and a therapeutic target at early stages of cellular hyperproliferation. PMID:19351862

  1. Effects of sodium metabisulfite on the expression of BK(Ca), K(ATP), and L-Ca(2+) channels in rat aortas in vivo and in vitro.

    Science.gov (United States)

    Zhang, Quanxi; Bai, Yunlong; Tian, Jingjing; Lei, Xiaodong; Li, Mei; Yang, Zhenhua; Meng, Ziqiang

    2015-03-01

    Sodium metabisulfite (SMB) is most commonly used as the preservative in many food preparations and drugs. So far, few studies about its negative effects were reported. The purpose of this study was to investigate the effect of SMB on the expression of big-conductance Ca(2+)-activated K(+) (BKCa), ATP-sensitive K(+) (KATP), and L-type calcium (L-Ca(2+)) channels in rat aorta in vivo and in vitro. The results showed that the mRNA and protein levels of the BKCa channel subunits α and β1 of aorta in rats were increased by SMB in vivo and in vitro. Similarly, the expression of the KATP channel subunits Kir6.1, Kir6.2, and SUR2B were increased by SMB. However, SMB at the highest concentration significantly decreased the expression of the L-Ca(2+) channel subunits Cav1.2 and Cav1.3. These results suggest that SMB can activate BKCa and KATP channels by increasing the expression of α, β1, and Kir6.1, Kir6.2, SUR2B respectively, while also inhibit L-Ca(2+) channels by decreasing the expression of Cav1.2 and Cav1.3 of aorta in rats. The molecular mechanism of SMB-induced vasorelaxant effect might be related to the expression changes of BKCa, KATP, and L-Ca(2+) channels subunits. Further work is needed to determine the relative contribution of each channel in SMB-mediated vasorelaxant effect.

  2. Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in gastrointestinal system

    Institute of Scientific and Technical Information of China (English)

    Shun-Ying Jin; Yan-Li Liu; Li-Na Xu; Yong Jiang; Ying Wang; Bao-Xue Yang; Hong Yang; Tong-Hui Ma

    2006-01-01

    AIM: To clone and characterize the porcine aquaporins (AQPs) in the gastrointestinal system.METHODS: A PCR-based cloning strategy and RACE were used to clone full-length AQP coding sequence from reversely transcribed pig liver cDNA. Stopped-flow light scattering and a YFP-based fluorescence method were used to measure the osmotic water permeability of erythrocytes and the stably transfected CHO cells.RT-PCR, Northern blot, and immunohistochemistry were used to determine the gastrointestinal expression and localization of cloned AQPs. Protein expression in transfected cells and red blood cells was analyzed by Western blot.RESULTS: An 813 bp cDNA encoding a 271 amino acid porcine aquaporin (designated pAQP1) was cloned from liver mRNA (pAQP1 has a 93% identity with human AQP1 and contains two NPA motifs conserved in AQP family, one consensus sequence for N-linked glycosylation, and one mercury-sensitive site at cysteine 191). RT-PCR analysis revealed extensive expression of pAQP1 mRNA in porcine digestive glands and gut.Northern blot showed a single 3.0 kb transcript in selected digestive organs, pAQP1 protein was localized at central lacteals of the small intestine, microvessles of salivary glands, as well as epithelium of intrahepatic bile ducts by immunoperoxydase. High osmotic water permeability that is inhibitable by HgCl2 was detected in porcine erythrocytes and CHO cells stably transfected with pAQP1 cDNA. Tmmunoblot analysis of porcine erythrocytes and pAQP-transfected CHO cells revealed an unglycosylated 28 ku band and larger glycosylated proteins.CONCLUSION: pAQP1 is the first porcine aquaporin that can be molecularly identified so far. The broad distribution of pAQP1 in epithelium and endothelium of porcine digestive organs may suggest an important role of channel-mediated water transport in fluid secretion/absorption as well as in digestive function and pathophysiology of the gastrointestinal system.

  3. Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.

    Science.gov (United States)

    Eckford, Paul D W; Li, Canhui; Bear, Christine E

    2015-03-09

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

  4. Protein kinase A modulation of CaV1.4 calcium channels.

    Science.gov (United States)

    Sang, Lingjie; Dick, Ivy E; Yue, David T

    2016-01-01

    The regulation of L-type Ca(2+) channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca(2+) channels, relatively little is known about the closely related CaV1.4 L-type Ca(2+) channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca(2+)-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca(2+)-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family. PMID:27456671

  5. Protein kinase A modulation of CaV1.4 calcium channels

    Science.gov (United States)

    Sang, Lingjie; Dick, Ivy E.; Yue, David T.

    2016-07-01

    The regulation of L-type Ca2+ channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca2+ channels, relatively little is known about the closely related CaV1.4 L-type Ca2+ channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca2+-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca2+-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family.

  6. In vivo and in vitro CYP1B mRNA expression in channel catfish.

    Science.gov (United States)

    Willett, Kristine L; Ganesan, Shobana; Patel, Monali; Metzger, Christine; Quiniou, Sylvie; Waldbieser, Geoff; Scheffler, Brian

    2006-07-01

    Our goal was to study the induction of CYP1B mRNA expression in channel catfish (Ictalurus punctatus). CYP1B belongs to the cytochrome P450 superfamily of genes, is involved in the oxidation of endogenous and exogenous compounds, and could potentially be a useful biomarker in fish for exposure to AhR ligands. The full-length catfish CYP1B cDNA is 2417 nt to the polyA tail and encodes a putative protein of 536 amino acids. It has 67% amino acid similarity to carp and zebrafish CYP1B and 68% similarity to carp CYP1B2. Male channel catfish were collected from three Mississippi Delta sites: Lake Roebuck, Itta Bena; Bee Lake, Thornton; and Sunflower River, Indianola. Total RNA was isolated from wild-caught catfish gill, blood, gonad and liver tissues. Quantitative real-time reverse transcriptase PCR was used to determine relative induction of CYP1B in wild catfish compared to laboratory control and BaP-exposed catfish (20mg/kg i.p. after 4 days). BaP exposure significantly induced CYP1B message in blood, gonad, and liver of laboratory catfish. In these same tissues of wild catfish from sites with relatively low sediment contaminants, CYP1B message was not statistically increased relative to laboratory control catfish. CYP1B transcript abundance was higher in gills compared to other tissues in both laboratory and wild catfish. When primary cultured gill cells were treated with increasing concentrations of BaP, TCDD, and PCBs 77, 126 and 169, CYP1B mRNA was induced more than 10-fold while PCB153 and 4,4'DDT did not cause significant CYP1B induction. Our results suggest that catfish CYP1B is induced by the classic AhR ligands. PMID:16697458

  7. Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D;

    2001-01-01

    The distribution of voltage-dependent calcium channels in kidney pre- and postglomerular resistance vessels was determined at the molecular and functional levels. Reverse transcription-polymerase chain reaction analysis of microdissected rat preglomerular vessels and cultured smooth muscle cells...... showed coexpression of mRNAs for T-type subunits (Ca(V)3.1, Ca(V)3.2) and for an L-type subunit (Ca(V)1.2). The same expression pattern was observed in juxtamedullary efferent arterioles and outer medullary vasa recta. No calcium channel messages were detected in cortical efferent arterioles. Ca(V)1.......2 protein was demonstrated by immunochemical labeling of rat preglomerular vasculature and juxtamedullary efferent arterioles and vasa recta. Cortical efferent arterioles were not immunopositive. Recordings of intracellular calcium concentration with digital fluorescence imaging microscopy showed a...

  8. E Protein Prokaryotic Expression of Avian Infectious Bronchitis Virus

    Institute of Scientific and Technical Information of China (English)

    WEI Ping; ZHANG Fang; MING Xiaobo; ZENG Xiangwei; ZHU Yuqing; WANG Lin

    2008-01-01

    The small envelope protein (E) gene of avian infectious bronchitis virus (IBV) M41 strain was cloned,and then it was subeloned into prokaryotic expressing vector pGEX-6P-1.The recombinant plasmid was transformed into E.coli.BL21 and induced by IPTG.SDS-PAGE result showed that when objective protein fused with GST (about 20 ku), the relative molecular mass of fusion protein was 38 ku.It indicated that objective protein was about 12.4 ku.The result showed that E protein was expressed successfully, it was useful to the subsequent E protein research.

  9. Piezo proteins are pore-forming subunits of mechanically activated channels.

    Science.gov (United States)

    Coste, Bertrand; Xiao, Bailong; Santos, Jose S; Syeda, Ruhma; Grandl, Jörg; Spencer, Kathryn S; Kim, Sung Eun; Schmidt, Manuela; Mathur, Jayanti; Dubin, Adrienne E; Montal, Mauricio; Patapoutian, Ardem

    2012-03-01

    Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

  10. Cloning and expression of special F protein from human liver

    Institute of Scientific and Technical Information of China (English)

    Shu-Ye Liu; Xin-Da Yu; Chun-Juan Song; Wei Lu; Jian-Dong Zhang; Xin-Rong Shi; Ying Duan; Ju Zhang

    2007-01-01

    AIM:To clone human liver special F protein and to express it in a prokaryotic system.METHODS:Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this,cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein's cDNA was subcloned into the expression vector pET-15b and transformed into E coli BL21 (DEB) pLyss. Isopropy-β-D-thiogalactoside (IPTG) was then used to induce expression of the target protein.RESULTS:The cDNA clone of human liver special F protein (1134bp) was successfully produced,with the cDNA sequence being published in Gene-bank:DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted.CONCLUSION:F protein expresses cDNA clone in a proKaryotic system,which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein.

  11. Boost protein expression through co-expression of LEA-like peptide in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Shinya Ikeno

    Full Text Available The boost protein expression has been done successfully by simple co-expression with a late embryogenesis abundant (LEA-like peptide in Escherichia coli. Frequently, overexpression of a recombinant protein fails to provide an adequate yield. In the study, we developed a simple and efficient system for overexpressing transgenic proteins in bacteria by co-expression with an LEA-like peptide. The design of this peptide was based on part of the primary structure of an LEA protein that is known hydrophilic protein to suppress aggregation of other protein molecules. In our system, the expression of the target protein was increased remarkably by co-expression with an LEA-like peptide consisting of only 11 amino acid residues. This could provide a practical method for producing recombinant proteins efficiently.

  12. Data presenting a modified bacterial expression vector for expressing and purifying Nus solubility-tagged proteins.

    Science.gov (United States)

    Gupta, Nidhi; Wu, Heng; Terman, Jonathan R

    2016-09-01

    Bacteria are the predominant source for producing recombinant proteins but while many exogenous proteins are expressed, only a fraction of those are soluble. We have found that a new actin regulatory enzyme Mical is poorly soluble when expressed in bacteria but the use of a Nus fusion protein tag greatly increases its solubility. However, available vectors containing a Nus tag have been engineered in a way that hinders the separation of target proteins from the Nus tag during protein purification. We have now used recombinant DNA approaches to overcome these issues and reengineer a Nus solubility tag-containing bacterial expression vector. The data herein present a modified bacterial expression vector useful for expressing proteins fused to the Nus solubility tag and separating such target proteins from the Nus tag during protein purification. PMID:27547802

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

    NARCIS (Netherlands)

    Busso, D.; Peleg, Y.; Folkers, G.E.; Celie, P.H.N.

    2011-01-01

    Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for opti

  14. Expression in Pichia pastoris and characterization of APETx2, a specific inhibitor of acid sensing ion channel 3.

    Science.gov (United States)

    Anangi, Raveendra; Chen, Chih-Cheng; Lin, Yi-Wen; Cheng, Yuan-Ren; Cheng, Chun-Ho; Chen, Yi-Chun; Chu, Yuan-Ping; Chuang, Woei-Jer

    2010-12-01

    Acid sensing ion channels (ASICs) are family of proteins predominantly present in the central and peripheral nervous system. They are known to play important roles in the pathophysiology of pain and ischemic stroke. APETx2 is a potent and selective inhibitor of ASIC3-containing channels and was isolated from sea anemone Anthopleura elegantissima. To facilitate the study on the molecular determinants of ASIC3-ligand interactions, we expressed recombinant APETx2 in the Pichia pastoris (P. pastoris) expression system and purified it to homogeneity. Recombinant APETx2 produced in P. pastoris inhibited the acid-evoked ASIC3 current with the IC(50) value of 37.3 nM. The potency of recombinant toxin is similar to that of native APETx2. The sequential assignment and structure analysis of APETx2 were obtained by 2D and 3D (15)N-edited NMR spectra. Our NMR data suggests that APETx2 produced in P. pastoris retained its native fold. The results presented here provide the first direct evidence that highly disulfide bonded peptide inhibitor of ASIC3, APETx2, can be expressed in P. pastoris with correct fold and high yield. We also showed that the R17A mutant exhibited a decrease in activity, suggesting the feasibility of the use of this expression system to study the interactions between APETx2 and ASIC3. These evidences may serve as the basis for understanding the selectivity and activity of APETx2. PMID:20813121

  15. Expression and subcellular localization of aquaporin water channels in the polarized hepatocyte cell line, WIF-B

    Directory of Open Access Journals (Sweden)

    Marinelli Raúl A

    2005-08-01

    Full Text Available Abstract Background Recent data suggest that canalicular bile secretion involves selective expression and coordinated regulation of aquaporins (AQPs, a family of water channels proteins. In order to further characterize the role of AQPs in this process, an in vitro cell system with retained polarity and expression of AQPs and relevant solute transporters involved in bile formation is highly desirable. The WIF-B cell line is a highly differentiated and polarized rat hepatoma/human fibroblast hybrid, which forms abundant bile canalicular structures. This cell line has been reported to be a good in vitro model for studying hepatocyte polarity. Results Using RT-PCR, immunoblotting and confocal immunofluorescence, we showed that WIF-B cells express the aquaporin water channels that facilitate the osmotically driven water movements in the liver, i.e. AQP8, AQP9, and AQP0; as well as the key solute transporters involved in the generation of canalicular osmotic gradients, i.e., the bile salt export pump Bsep, the organic anion transporter Mrp2 and the chloride bicarbonate exchanger AE2. The subcellular localization of the AQPs and the solute transporters in WIF-B cells was similar to that in freshly isolated rat hepatocytes and in intact liver. Immunofluorescent costaining studies showed intracellular colocalization of AQP8 and AE2, suggesting the possibility that these transporters are expressed in the same population of pericanalicular vesicles. Conclusion The hepatocyte cell line WIF-B retains the expression and subcellular localization of aquaporin water channels as well as key solute transporters for canalicular bile secretion. Thus, these cells can work as a valuable tool for regulatory and mechanistic studies of the biology of bile formation.

  16. Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes

    DEFF Research Database (Denmark)

    Lundbæk, Jens August; Collingwood, S.A.; Ingolfsson, H.I.;

    2010-01-01

    Membrane protein function is regulated by the host lipid bilayer composition. This regulation may depend on specific chemical interactions between proteins and individual molecules in the bilayer, as well as on non-specific interactions between proteins and the bilayer behaving as a physical enti...... use of gramicidin channels as molecular force probes for studying this mechanism, with a unique ability to discriminate between consequences of changes in monolayer curvature and bilayer elastic moduli....

  17. Suprachiasmatic nucleus function and circadian entrainment are modulated by G protein-coupled inwardly rectifying (GIRK) channels

    Science.gov (United States)

    Hablitz, L M; Molzof, H E; Paul, J R; Johnson, R L; Gamble, K L

    2014-01-01

    Abstract G protein signalling within the central circadian oscillator, the suprachiasmatic nucleus (SCN), is essential for conveying time-of-day information. We sought to determine whether G protein-coupled inwardly rectifying potassium channels (GIRKs) modulate SCN physiology and circadian behaviour. We show that GIRK current and GIRK2 protein expression are greater during the day. Pharmacological inhibition of GIRKs and genetic loss of GIRK2 depolarized the day-time resting membrane potential of SCN neurons compared to controls. Behaviourally, GIRK2 knockout (KO) mice failed to shorten free running period in response to wheel access in constant darkness and entrained more rapidly to a 6 h advance of a 12 h:12 h light–dark (LD) cycle than wild-type (WT) littermate controls. We next examined whether these effects were due to disrupted signalling of neuropeptide Y (NPY), which is known to mediate non-photic phase shifts, attenuate photic phase shifts and activate GIRKs. Indeed, GIRK2 KO SCN slices had significantly fewer silent cells in response to NPY, likely contributing to the absence of NPY-induced phase advances of PER2::LUC rhythms in organotypic SCN cultures from GIRK2 KO mice. Finally, GIRK channel activation is sufficient to cause a non-photic-like phase advance of PER2::LUC rhythms on a Per2Luc+/− background. These results suggest that rhythmic regulation of GIRK2 protein and channel function in the SCN contributes to day-time resting membrane potential, providing a mechanism for the fine tuning responses to non-photic and photic stimuli. Further investigation could provide insight into disorders with circadian disruption comorbidities such as epilepsy and addiction, in which GIRK channels have been implicated. PMID:25217379

  18. SPAK and OSR1 Sensitive Cell Membrane Protein Abundance and Activity of KCNQ1/E1 K+ Channels

    Directory of Open Access Journals (Sweden)

    Bernat Elvira

    2015-11-01

    Full Text Available Background/Aims: KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase and OSR1 (oxidative stress-responsive kinase 1, which are under control of WNK (with-no-K[Lys] kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. Methods: cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1. Results: KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. Conclusions: SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity.

  19. Expression of Helicobacter pylori Hsp60 protein and its immunogenicity

    Institute of Scientific and Technical Information of China (English)

    Yang Bai; Liang-Ren Li; Ji-De Wang; Ye Chen; Jian-Feng Jin; Zhao-Shan Zhang; Dian-Yuan Zhou; Ya-Li Zhang

    2003-01-01

    AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity.METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+), which was transformed into BL21 (DE3) E. coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal experiments.RESULTS: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2 % of the total bacterial protein,and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 itself.CONCLUSION: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.

  20. Signatures of protein structure in the cooperative gating of mechanosensitive ion channels

    CERN Document Server

    Kahraman, Osman; Haselwandter, Christoph A

    2016-01-01

    Membrane proteins deform the surrounding lipid bilayer, which can lead to membrane-mediated interactions between neighboring proteins. Using the mechanosensitive channel of large conductance (MscL) as a model system, we demonstrate how the observed differences in protein structure can affect membrane-mediated interactions and cooperativity among membrane proteins. We find that distinct oligomeric states of MscL lead to distinct gateway states for the clustering of MscL, and predict signatures of MscL structure and spatial organization in the cooperative gating of MscL. Our modeling approach establishes a quantitative relation between the observed shapes and cooperative function of membrane~proteins.

  1. Polyunsaturated fatty acids are potent openers of human M-channels expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Liin, Sara I; Karlsson, Urban; Bentzen, Bo Hjorth;

    2016-01-01

    threshold current to evoke action potentials in dorsal root ganglion neurons. The polyunsaturated fatty acids docosahexaenoic acid, α-linolenic acid, and eicosapentaenoic acid facilitated opening of the human M-channel, comprised of the heteromeric human KV 7.2/3 channel expressed in Xenopus oocytes, by...... shifting the conductance-versus-voltage curve towards more negative voltages (by -7.4 to -11.3 mV by 70 μM). Uncharged docosahexaenoic acid methyl ester and monounsaturated oleic acid did not facilitate opening of the human KV 7.2/3 channel. CONCLUSIONS: These findings suggest that circulating...... polyunsaturated fatty acids, with a minimum requirement of multiple double bonds and a charged carboxyl group, dampen excitability by opening neuronal M-channels. Collectively, our data bring light to the molecular targets of polyunsaturated fatty acids and thus a possible mechanism by which polyunsaturated fatty...

  2. Differential expression of genes encoding subthreshold-operating voltage-gated K+ channels in brain.

    Science.gov (United States)

    Saganich, M J; Machado, E; Rudy, B

    2001-07-01

    The members of the three subfamilies (eag, erg, and elk) of the ether-a-go-go (EAG) family of potassium channel pore-forming subunits express currents that, like the M-current (I(M)), could have considerable influence on the subthreshold properties of neuronal membranes, and hence the control of excitability. A nonradioactive in situ hybridization (NR-ISH) study of the distribution of the transcripts encoding the eight known EAG family subunits in rat brain was performed to identify neuronal populations in which the physiological roles of EAG channels could be studied. These distributions were compared with those of the mRNAs encoding the components of the classical M-current (Kcnq2 and Kcnq3). NR-ISH was combined with immunohistochemistry to specific neuronal markers to help identify expressing neurons. The results show that each EAG subunit has a specific pattern of expression in rat brain. EAG and Kcnq transcripts are prominent in several types of excitatory neurons in the cortex and hippocampus; however, only one of these channel components (erg1) was consistently expressed in inhibitory interneurons in these areas. Some neuronal populations express more than one product of the same subfamily, suggesting that the subunits may form heteromeric channels in these neurons. Many neurons expressed multiple EAG family and Kcnq transcripts, such as CA1 pyramidal neurons, which contained Kcnq2, Kcnq3, eag1, erg1, erg3, elk2, and elk3. This indicates that the subthreshold current in many neurons may be complex, containing different components mediated by a number of channels with distinct properties and neuromodulatory responses. PMID:11425889

  3. Improved technique for studying ion channels expressed in Xenopus oocytes, including fast superfusion.

    OpenAIRE

    Costa, A.C.; Patrick, J W; Dani, J. A.

    1994-01-01

    The study of whole-cell currents from ion channels expressed in Xenopus oocytes with conventional two-electrode voltage clamp has two major limitations. First, the large diameter and spherical geometry of oocytes prevent extremely fast solution changes. Second, the internal medium is not controlled, which limits the experimental versatility of the oocyte expression system. For example, because the internal medium is not controlled, endogenous calcium-activated chloride conductances can contam...

  4. Design Methodology of the Structure of Postal Express Mail Networks of Aviation Channels in China

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    On the basis of the postal area center office system, the continuous space approximation is used to study the structure of postal express mail networks of aviation channels in China. Tradeoffs among sorting, handling, transportation, administrative and facilities costs are examined. The optimizing design methodology proposed in this paper can be used to analyze and design the postal express mail network. The objective is to minimize the total system cost.

  5. Expression of eag1 channel associated with the aggressive clinicopathological features and subtype of breast cancer

    OpenAIRE

    Liu, Guang-Xu; Yu, Yun-Cui; He, Xiang-ping; Ren, Sheng-Nan; Fang, Xue-Dong; Liu, Fen; He, Yan

    2015-01-01

    Backgrounds: Expression of eag1 channel (Eag1) is associated with cell malignant transformation, tumor cell metastasis and poor prognosis of the patient. This study aimed at examining whether expression of the Eag1 associated with aggressive clinicopathological feature and the molecular subtype of breast cancer. Materials and Methods: 109 patients who received breast cancer operation during January 2009 to December 2010 in Chinese-Japanese Friendship Hospital of Jilin University were recruite...

  6. Confirmation of human protein interaction data by human expression data

    OpenAIRE

    Talwar Priti; Rahnenführer Jörg; Hahn Andreas; Lengauer Thomas

    2005-01-01

    Abstract Background With microarray technology the expression of thousands of genes can be measured simultaneously. It is well known that the expression levels of genes of interacting proteins are correlated significantly more strongly in Saccharomyces cerevisiae than those of proteins that are not interacting. The objective of this work is to investigate whether this observation extends to the human genome. Results We investigated the quantitative relationship between expression levels of ge...

  7. Action of the pyrethroid insecticide cypermethrin on rat brain IIa sodium channels expressed in xenopus oocytes.

    Science.gov (United States)

    Smith, T J; Soderlund, D M

    1998-12-01

    Pyrethroid insecticides bind to a unique site on voltage-dependent sodium channels and prolong sodium currents, leading to repetitive bursts of action potentials or use-dependent nerve block. To further characterize the site and mode of action of pyrethroids on sodium channels, we injected synthetic mRNA encoding the rat brain IIa sodium channel alpha subunit, either alone or in combination with synthetic mRNA encoding the rat sodium channel beta1 subunit, into oocytes of the frog Xenopus laevis and assessed the actions of the pyrethroid insecticide [1R,cis,alphaS]-cypermethrin on expressed sodium currents by two-electrode voltage clamp. In oocytes expressing only the rat brain IIa alpha subunit, cypermethrin produced a slowly-decaying sodium tail current following a depolarizing pulse. In parallel experiments using oocytes expressing the rat brain IIa alpha subunit in combination with the rat beta1 subunit, cypermethrin produced qualitatively similar tail currents following a depolarizing pulse and also induced a sustained component of the sodium current measured during a step depolarization of the oocyte membrane. The voltage dependence of activation and steady-state inactivation of the cypermethrin-dependent sustained current were identical to those of the peak transient sodium current measured in the absence of cypermethrin. Concentration-response curves obtained using normalized tail current amplitude as an index of the extent of sodium channel modification by cypermethrin revealed that coexpression of the rat brain IIa alpha subunit with the rat beta1 subunit increased the apparent affinity of the sodium channel binding site for cypermethrin by more than 20-fold. These results confirm that the pyrethroid binding site is intrinsic to the sodium channel alpha subunit and demonstrate that coexpression of the rat brain IIa alpha subunit with the rat beta1 subunit alters the apparent affinity of this site for pyrethroids.

  8. Plant Antifreeze Proteins and Their Expression Regulatory Mechanism

    Institute of Scientific and Technical Information of China (English)

    Lin Yuan-zhen; Lin Shan-zhi; Zhang Zhi-yi; Zhang Wei; Liu Wen-feng

    2005-01-01

    Low temperature is one of the major limiting environmental factors which constitutes the growth, development,productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identification and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.

  9. Myometrial relaxation of mice via expression of two pore domain acid sensitive K+ (TASK-2) channels

    Science.gov (United States)

    Kyeong, Kyu-Sang; Hong, Seung Hwa; Cho, Woong; Myung, Sun Chul; Lee, Moo Yeol; You, Ra Young; Kim, Chan Hyung; Kwon, So Yeon; Suzuki, Hikaru; Park, Yeon Jin; Jeong, Eun-Hwan; Kim, Hak Soon; Kim, Heon; Lim, Seung Woon; Xu, Wen-Xie; Lee, Sang Jin

    2016-01-01

    Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K+ channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K+ current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K+ channels (TASK-2). NIOK in the presence of K+ channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery. PMID:27610042

  10. Myometrial relaxation of mice via expression of two pore domain acid sensitive K(+) (TASK-2) channels.

    Science.gov (United States)

    Kyeong, Kyu-Sang; Hong, Seung Hwa; Kim, Young Chul; Cho, Woong; Myung, Sun Chul; Lee, Moo Yeol; You, Ra Young; Kim, Chan Hyung; Kwon, So Yeon; Suzuki, Hikaru; Park, Yeon Jin; Jeong, Eun-Hwan; Kim, Hak Soon; Kim, Heon; Lim, Seung Woon; Xu, Wen-Xie; Lee, Sang Jin; Ji, Il Woon

    2016-09-01

    Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K(+) channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K(+) current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K(+) channels (TASK-2). NIOK in the presence of K(+) channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery. PMID:27610042

  11. Sexual dimorphism and oestrogen regulation of KCNE3 expression modulates the functional properties of KCNQ1 K channels.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2012-02-01

    The KCNQ1 potassium channel associates with various KCNE ancillary subunits that drastically affect channel gating and pharmacology. Co-assembly with KCNE3 produces a current with nearly instantaneous activation, some time-dependent activation at very positive potentials, a linear current-voltage relationship and a 10-fold higher sensitivity to chromanol 293B. KCNQ1:KCNE3 channels are expressed in colonic crypts and mediate basolateral K(+) recycling required for Cl(-) secretion. We have previously reported the female-specific anti-secretory effects of oestrogen via KCNQ1:KCNE3 channel inhibition in colonic crypts. This study was designed to determine whether sex and oestrogen regulate the expression and function of KCNQ1 and KCNE3 in rat distal colon. Colonic crypts were isolated from Sprague-Dawley rats and used for whole-cell patch-clamp and to extract total RNA and protein. Sheets of epithelium were used for short-circuit current recordings. KCNE1 and KCNE3 mRNA and protein abundance were significantly higher in male than female crypts. No expression of KCNE2 was found and no difference was observed in KCNQ1 expression between male and female (at oestrus) colonic crypts. Male crypts showed a 2.2-fold higher level of association of KCNQ1 and KCNE3 compared to female cells. In female colonic crypts, KCNQ1 and KCNE3 protein expression fluctuated throughout the oestrous cycle and 17beta-oestradiol (E2 10 nM) produced a rapid (<15 min) dissociation of KCNQ1 and KCNE3 in female crypts only. Whole-cell K(+) currents showed a linear current-voltage relationship in male crypts, while K(+) currents in colonic crypts isolated from females displayed voltage-dependent outward rectification. Currents in isolated male crypts and epithelial sheets were 10-fold more sensitive to specific KCNQ1 inhibitors, such as chromanol 293B and HMR-1556, than in female. The effect of E2 on K(+) currents mediated by KCNQ1 with or without different beta-subunits was assayed from current

  12. Transient calnexin interaction confers long-term stability on folded K+ channel protein in the ER.

    Science.gov (United States)

    Khanna, Rajesh; Lee, Eun Jeon; Papazian, Diane M

    2004-06-15

    We recently showed that an unglycosylated form of the Shaker potassium channel protein is retained in the endoplasmic reticulum (ER) and degraded by proteasomes in mammalian cells despite apparently normal folding and assembly. These results suggest that channel proteins with a native structure can be substrates for ER-associated degradation. We have now tested this hypothesis using the wild-type Shaker protein. Wild-type Shaker is degraded by cytoplasmic proteasomes when it is trapped in the ER and prevented from interacting with calnexin. Neither condition alone is sufficient to destabilize the protein. Proteasomal degradation of the wild-type protein is abolished when ER mannosidase I trimming of the core glycan is inhibited. Our results indicate that transient interaction with calnexin provides long-term protection from ER-associated degradation. PMID:15161937

  13. Expression, Purification and Antibacterial Activity of NK-Lysin Mature Peptides from the Channel Catfish (Ictalurus punctatus

    Directory of Open Access Journals (Sweden)

    Shurui Cai

    2016-08-01

    Full Text Available Antimicrobial peptides (AMPs are small peptides and play important roles in host innate immune response against microbial invasion. Aquatic animals secrete different kinds of antimicrobial peptides which have antimicrobial activity towards microorganisms. NK-lysins, mature peptides produced by cytotoxic T lymphocytes and natural killer cells, are comprised of 74–78 amino acid residues, demonstrating broad-spectrum antimicrobial activity against bacteria, fungi, protozoa, and parasites. In this study, three distinct NK-lysin mature peptide (mNKLs, transcripts (76 amino acid residues cloned from the channel catfish (Ictalurus punctatus head kidney were ligated into plasmid vector pET-32a(+ to express the mNKLs fusion protein. The fusion protein was successfully expressed in E. coli Rosetta (DE3 under optimized conditions. After purification by affinity column chromatography, the fusion protein was successfully cleaved by enterokinase and released the peptide mNKLs. Tricine-SDS-PAGE results showed that mNKLs (approximately 8.6 kDa were successfully expressed. The purified peptide mNKLs exhibited antibacterial activity against Staphylococcus aureus and E. coli.

  14. Protein and cell patterning in closed polymer channels by photoimmobilizing proteins on photografted poly(ethylene glycol) diacrylate

    DEFF Research Database (Denmark)

    Larsen, Esben Kjær Unmack; Mikkelsen, Morten Bo Lindholm; Larsen, Niels Bent

    2014-01-01

    Definable surface chemistry is essential for many applications of microfluidic polymer systems. However, small cross-section channels with a high surface to volume ratio enhance passive adsorption of molecules that depletes active molecules in solution and contaminates the channel surface. Here, we...... to greatly improve cell adhesion compared to unexposed areas. This method opens for easy surface modification of closed microfluidic systems through combining a low protein binding PEG-based coating with spatially defined protein patterns of interest....... present a one-step photochemical process to coat the inner surfaces of closed microfluidic channels with a nanometer thick layer of poly(ethylene glycol) (PEG), well known to strongly reduce non-specific adsorption, using only commercially available reagents in an aqueous environment. The coating consists...

  15. Heroin-Induces Differential Protein Expression by Normal Human Astrocytes (NHA

    Directory of Open Access Journals (Sweden)

    Jessica L. Reynolds

    2006-01-01

    Full Text Available Heroin use is postulated to act as a cofactor in the neuropathogenesis of human immunodeficiency virus (HIV-1 infection. Astrocytes, integral components of the CNS, are reported to be susceptible to HIV-1 infection. Upon activation, astrocytes release a number of immunoregulatory products or modulate the expression of a number of proteins that foster the immunopathogenesis of HIV-1 infection. However, the role of heroin on HIV-1 infectivity and the expression of the proteome of normal human astrocytes (NHA have not been elucidated. We hypothesize that heroin modulates the expression of a number of proteins by NHA that foster the neuoropathogenesis of HIV-1 infection. We utilized LTR amplification and the p24 antigen assay to quantitate the effect of heroin on HIV-1 infectivity while difference gel electrophoresis (DIGE combined with protein identification through high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS to analyze the effects of heroin on the proteomic profile of NHA. Results demonstrate that heroin potentiates HIV-1 replication in NHA. Furthermore, heroin significantly increased protein expression levels for protein kinase C (PKC, reticulocalbin 1 precursor, reticulocalbin 1, tyrosine 3-monooxgenase/tryptophan 5-monooxgenase activation protein, chloride intracellular channel 1, cathepsin D preproprotein, galectin 1 and myosin light chain alkali. Heroin also significantly decreased protein expression for proliferating cell nuclear antigen, proteasome beta 6 subunit, tropomyosin 3, laminin receptor 1, tubulin alpha 6, vimentin, EF hand domain family member D2, Tumor protein D54 (hD54, ATP synthase, H+ transporting, mitochondrial F1 complex and ribosomal protein S14. Identification of unique, heroin-induced proteins may help to develop novel markers for diagnostic, preventative and therapeutic targeting in heroin using subjects.

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

    Directory of Open Access Journals (Sweden)

    Kędzierska K

    2014-09-01

    Full Text Available Karolina Kędzierska,1 Katarzyna Sporniak-Tutak,2 Krzysztof Sindrewicz,2 Joanna Bober,3 Leszek Domański,1 Mirosław Parafiniuk,4 Elżbieta Urasińska,5 Andrzej Ciechanowicz,6 Maciej Domański,1 Tomasz Smektała,2 Marek Masiuk,5 Wiesław Skrzypczak,6 Małgorzata Ożgo,6 Joanna Kabat-Koperska,1 Kazimierz Ciechanowski1 1Department of Nephrology, Transplantology, and Internal Medicine, 2Department of Dental Surgery, 3Department of Medical Chemistry, 4Department of Forensic Medicine, 5Department of Pathomorphology, Pomeranian Medical University, 6Department of Physiology, Cytobiology, and Proteomics, West Pomeranian University of Technology, Szczecin, Poland Abstract: The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents' toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins' synthesis. Very slight differences

  17. The low PLC-δ1 expression in cystic fibrosis bronchial epithelial cells induces upregulation of TRPV6 channel activity.

    Science.gov (United States)

    Vachel, Laura; Norez, Caroline; Jayle, Christophe; Becq, Frédéric; Vandebrouck, Clarisse

    2015-01-01

    Increase of Ca(2+) influx in Cystic Fibrosis (CF) cells has been reported to be related to Transient Receptor Potential Canonical (TRPC6) channel, which is implicated in a functional coupling with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Several members of the Transient Receptor Potential Vanilloid (TRPV) channels family have already been described as emerging target for respiratory diseases. Two specific isoforms, TRPV5 and TRPV6 are of particular interest in the context of CF Ca(2+) homeostasis as they are highly selective toward Ca(2+) and constitutively activated. Thus, we investigated the involvement of these channels in Ca(2+) influx in CF and non-CF human bronchial epithelial cell lines. 16HBE14o-, CFBE41o- cell lines, primary human airway epithelial cells (hAEC) and freshly isolated human airway epithelial cells from CF and non-CF individuals were used. We showed that both channels are expressed in CF and non-CF cells and constitutive Ca(2+) influx was significantly higher (85%) in cells from CF individuals compared to cells from non-CF ones. Using the selective inhibitor of TRPV6 channel SOR-C27 and a siRNA strategy, our results revealed that TRPV6 was mostly involved in the increase of Ca(2+) influx. TRPV6 channel is negatively regulated by the PLC-PIP2 pathway. We measured the Ca(2+) influx in the presence of the non-specific PLC inhibitor, U73122, in non-CF human bronchial epithelial cells. Ca(2+) influx was increased by 33% with U73122 and this increase was largely reduced in the presence of SOR-C27. PLC inhibition in CF cells by U73122 had no effect on Ca(2+) influx. These results showed that PLC-PIP2 pathway is dysregulated in CF cells and leads to the increase of TRPV6 activity. The regulation of TRPV6 by PLC-PIP2 pathway implicates the specific PLC isoform, PLC-δ1. Immunoblot experiments revealed that expression of PLC-δ1 was decreased by 70% in CF cells. TRPV6 activity was normalized but not the level of expression of PLC-δ1

  18. Loss of ATP-Sensitive Potassium Channel Surface Expression in Heart Failure Underlies Dysregulation of Action Potential Duration and Myocardial Vulnerability to Injury.

    Science.gov (United States)

    Gao, Zhan; Sierra, Ana; Zhu, Zhiyong; Koganti, Siva Rama Krishna; Subbotina, Ekaterina; Maheshwari, Ankit; Anderson, Mark E; Zingman, Leonid V; Hodgson-Zingman, Denice M

    2016-01-01

    The search for new approaches to treatment and prevention of heart failure is a major challenge in medicine. The adenosine triphosphate-sensitive potassium (KATP) channel has been long associated with the ability to preserve myocardial function and viability under stress. High surface expression of membrane KATP channels ensures a rapid energy-sparing reduction in action potential duration (APD) in response to metabolic challenges, while cellular signaling that reduces surface KATP channel expression blunts APD shortening, thus sacrificing energetic efficiency in exchange for greater cellular calcium entry and increased contractile force. In healthy hearts, calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the Kir6.2 KATP channel subunit initiating a cascade responsible for KATP channel endocytosis. Here, activation of CaMKII in a transaortic banding (TAB) model of heart failure is coupled with a 35-40% reduction in surface expression of KATP channels compared to hearts from sham-operated mice. Linkage between KATP channel expression and CaMKII is verified in isolated cardiomyocytes in which activation of CaMKII results in downregulation of KATP channel current. Accordingly, shortening of monophasic APD is slowed in response to hypoxia or heart rate acceleration in failing compared to non-failing hearts, a phenomenon previously shown to result in significant increases in oxygen consumption. Even in the absence of coronary artery disease, failing myocardium can be further injured by ischemia due to a mismatch between metabolic supply and demand. Ischemia-reperfusion injury, following ischemic preconditioning, is diminished in hearts with CaMKII inhibition compared to wild-type hearts and this advantage is largely eliminated when myocardial KATP channel expression is absent, supporting that the myocardial protective benefit of CaMKII inhibition in heart failure may be substantially mediated by KATP channels. Recognition of Ca

  19. Loss of ATP-Sensitive Potassium Channel Surface Expression in Heart Failure Underlies Dysregulation of Action Potential Duration and Myocardial Vulnerability to Injury.

    Directory of Open Access Journals (Sweden)

    Zhan Gao

    Full Text Available The search for new approaches to treatment and prevention of heart failure is a major challenge in medicine. The adenosine triphosphate-sensitive potassium (KATP channel has been long associated with the ability to preserve myocardial function and viability under stress. High surface expression of membrane KATP channels ensures a rapid energy-sparing reduction in action potential duration (APD in response to metabolic challenges, while cellular signaling that reduces surface KATP channel expression blunts APD shortening, thus sacrificing energetic efficiency in exchange for greater cellular calcium entry and increased contractile force. In healthy hearts, calcium/calmodulin-dependent protein kinase II (CaMKII phosphorylates the Kir6.2 KATP channel subunit initiating a cascade responsible for KATP channel endocytosis. Here, activation of CaMKII in a transaortic banding (TAB model of heart failure is coupled with a 35-40% reduction in surface expression of KATP channels compared to hearts from sham-operated mice. Linkage between KATP channel expression and CaMKII is verified in isolated cardiomyocytes in which activation of CaMKII results in downregulation of KATP channel current. Accordingly, shortening of monophasic APD is slowed in response to hypoxia or heart rate acceleration in failing compared to non-failing hearts, a phenomenon previously shown to result in significant increases in oxygen consumption. Even in the absence of coronary artery disease, failing myocardium can be further injured by ischemia due to a mismatch between metabolic supply and demand. Ischemia-reperfusion injury, following ischemic preconditioning, is diminished in hearts with CaMKII inhibition compared to wild-type hearts and this advantage is largely eliminated when myocardial KATP channel expression is absent, supporting that the myocardial protective benefit of CaMKII inhibition in heart failure may be substantially mediated by KATP channels. Recognition of Ca

  20. Major cancer protein amplifies global gene expression

    Science.gov (United States)

    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

  1. Expression of Recombinant Proteins in Microalgae

    OpenAIRE

    Mayfield, Stephen P.; Franklin, Scott E.

    2003-01-01

    Our initial objectives in this project were three-fold. I. Express functional antibodies in the chloroplast of Chlamydomonas reinhardtii II. Express functional antibodies in the nucleocytoplasmic compartment of C. reinhardtii and III. Define optimal parameters for the large scale culture of antibody producing strains of C. reinhardtii.

  2. Insulin influenced expression of myelin proteins in diabetic peripheral neuropathy.

    Science.gov (United States)

    Rachana, Kuruvanthe S; Manu, Mallahalli S; Advirao, Gopal M

    2016-08-26

    Diabetic peripheral neuropathy (DPN) is one of the downstream complications of diabetes. This complication is caused by the deficiency of insulin action and subsequent hyperglycemia, but the details of their pathogenesis remain unclear. Hence, it is of critical importance to understand how such hormonal variation affects the expression of myelin proteins such as myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in the peripheral nerve. An earlier report from our lab has demonstrated the expression of insulin receptors (IR) in Schwann cells (SCs) of sciatic nerve. To assess the neurotrophic role of insulin in diabetic neuropathy, we studied the expression of these myelin proteins under control, DPN and insulin treated DPN subjects at developmental stages. Further, the expression of these myelin proteins was correlated with the expression of insulin receptor. Expression of myelin proteins was significantly reduced in the diabetic model compared to normal, and upregulated in insulin treated diabetic rats. Similarly, an in vitro study was also carried out in SCs grown at high glucose and insulin treated conditions. The expression pattern of myelin proteins in SCs was comparable to that of in vivo samples. In addition, quantitative study of myelin genes by real time PCR has also showed the significant expression pattern change in the insulin treated and non-treated DPN subjects. Taken together, these results corroborate the critical importance of insulin as a neurotrophic factor in demyelinized neurons in diabetic neuropathy.

  3. Temporal protein expression pattern in intracellular signalling cascade during T-cell activation: A computational study

    Indian Academy of Sciences (India)

    Piyali Ganguli; Saikat Chowdhury; Rupa Bhowmick; Ram Rup Sarkar

    2015-10-01

    Various T-cell co-receptor molecules and calcium channel CRAC play a pivotal role in the maintenance of cell’s functional responses by regulating the production of effector molecules (mostly cytokines) that aids in immune clearance and also maintaining the cell in a functionally active state. Any defect in these co-receptor signalling pathways may lead to an altered expression pattern of the effector molecules. To study the propagation of such defects with time and their effect on the intracellular protein expression patterns, a comprehensive and largest pathway map of T-cell activation network is reconstructed manually. The entire pathway reactions are then translated using logical equations and simulated using the published time series microarray expression data as inputs. After validating the model, the effect of in silico knock down of co-receptor molecules on the expression patterns of their downstream proteins is studied and simultaneously the changes in the phenotypic behaviours of the T-cell population are predicted, which shows significant variations among the proteins expression and the signalling routes through which the response is propagated in the cytoplasm. This integrative computational approach serves as a valuable technique to study the changes in protein expression patterns and helps to predict variations in the cellular behaviour.

  4. Temporal protein expression pattern in intracellular signalling cascade during T-cell activation: a computational study.

    Science.gov (United States)

    Ganguli, Piyali; Chowdhury, Saikat; Bhowmick, Rupa; Sarkar, Ram Rup

    2015-10-01

    Various T-cell co-receptor molecules and calcium channel CRAC play a pivotal role in the maintenance of cell's functional responses by regulating the production of effector molecules (mostly cytokines) that aids in immune clearance and also maintaining the cell in a functionally active state. Any defect in these co-receptor signalling pathways may lead to an altered expression pattern of the effector molecules. To study the propagation of such defects with time and their effect on the intracellular protein expression patterns, a comprehensive and largest pathway map of T-cell activation network is reconstructed manually. The entire pathway reactions are then translated using logical equations and simulated using the published time series microarray expression data as inputs. After validating the model, the effect of in silico knock down of co-receptor molecules on the expression patterns of their downstream proteins is studied and simultaneously the changes in the phenotypic behaviours of the T-cell population are predicted, which shows significant variations among the proteins expression and the signalling routes through which the response is propagated in the cytoplasm. This integrative computational approach serves as a valuable technique to study the changes in protein expression patterns and helps to predict variations in the cellular behaviour. PMID:26564978

  5. Clinical significance of PHPT1 protein expression in lung cancer

    Institute of Scientific and Technical Information of China (English)

    XU An-jian; XIA Xiang-hou; DU Song-tao; GU Jun-chao

    2010-01-01

    Background in our previous studies, we found the expression of 14-kD phosphohistidine phosphatase (PHPT1) was associated with lung cancer cells migration and invasion, and PHPT1 mRNA expression level in lung cancer tissues clinically correlated with lymph node metastasis. in the present study, we aimed to further investigate the expression of PHPT1 protein in lung cancer.Methods Expression of PHPT1 protein in tissue samples from 146 lung cancers and 30 normal tissues adjacent to lung cancers was assessed using immunohistochemical method. Fisher's exact test was used to analyze expression patterns of PHPT1 protein in these tissue types. Meanwhile, we studied the correlation between expression of PHPT1 protein and clinicopathological features in lung cancer.Results Significantly higher expression levels of PHPT1 protein were found in lung cancer samples (53.42%) than in normal tissues adjacent to lung cancer (23.33%) (P=0.003). Fisher's exact test showed that lung cancer stage positively correlated with expression of PHPT1 protein (P=0.02), and lung cancer samples with lymph node metastasis showed higher PHPT1 protein expression (P=0.016) than the samples without lymph node metastasis.Conclusions The results of this study agree with findings from our previous study of PHPT1 mRNA expression in lung cancer tissues, and strongly suggest that PHPT1 protein is closely associated with the carcinogenesis and metastasis of lung cancer. Thus, therapy targeting PHPT1 (inhibition or silencing) could be potentially benefited for lung cancer patients.

  6. Constitutive and Inducible Green Fluorescent Protein Expression in Bartonella henselae

    OpenAIRE

    Lee, Anthea K.; Falkow, Stanley

    1998-01-01

    The green fluorescent protein (GFP) gene was expressed on a plasmid in B. henselae, and GFP-expressing bacteria were visualized by fluorescence microscopy. HEp-2 cells infected with GFP-expressing bacteria were separated from uninfected cells with a fluorescence activated cell sorter. Promoter fusions of B. henselae chromosomal DNA to gfp were examined by flow cytometry, and a B. henselae groEL promoter fusion which induced expression at 37°C was isolated.

  7. Differential expression of BK channel isoforms and beta-subunits in rat neuro-vascular tissues

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Johansson, Helle Wulf; Hay-Schmidt, Anders;

    2009-01-01

    We investigated the expression of splice variants and beta-subunits of the BK channel (big conductance Ca(2+)-activated K(+) channel, Slo1, MaxiK, K(Ca)1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An alpha-subunit splice variant X1(+24) was found expressed...... (RT-PCR) in nervous tissue only where also the SS4(+81) variant was dominating with little expression of the short form SS4(0). SS4(+81) was present in some cerebral vessels too. The SS2(+174) variant (STREX) was found in both blood vessels and in nervous tissue. In situ hybridization data supported...

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

    Science.gov (United States)

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

    2009-02-01

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

  9. Sequence and functional expression in Xenopus oocytes of a human insulinoma and islet potassium channel

    International Nuclear Information System (INIS)

    Regulation of insulin secretion involves the coordinated control of ion channels in the β-cell membrane. The authors have isolated and characterized cDNA and genomic clones encoding a voltage-dependent K+ channel isoform expressed in human islets and in a human insulinoma. This K+ channel isoform, designated hPCN1, with a deduced amino acid sequence of 613 residues is related to the Shaker family of Drosophila K+ channels. hPCN1 is homologous to two other human K+ channel isoforms. They have isolated, hPCN2 and hPCN3, with 55% and 65% amino acid sequence identity, respectively. The electrophysiological characteristics of hPCN1 were determined after microinjuection of synthetic RNA into Xenopus oocytes. Two-microelectrode voltage-clamp recordings of oocytes injected with hPCN1 RNA revealed a voltage-dependent outward K+ current that inactivated slowly with time. Outward currents were inhibited by 4-aminopyridine with a Ki less that 0.01 mM and were relatively insensitive to tetraethylammonium ion or Ba2+. A delayed rectifier K+ channel such as hPCN1 could restore the resting membrane potential of β cells after depolarization and thereby contribute to the regulation of insulin secretion

  10. Dexamethasone increases aquaporin-2 protein expression in ex vivo inner medullary collecting duct suspensions.

    Science.gov (United States)

    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

  11. Chemometrics of differentially expressed proteins from colorectal cancer patients

    Institute of Scientific and Technical Information of China (English)

    Lay-Chin Yeoh; Saravanan Dharmaraj; Boon-Hui Gooi; Manjit Singh; Lay-Harn Gam

    2011-01-01

    AIM: To evaluate the usefulness of differentially expressed proteins from colorectal cancer (CRC) tissues for differentiating cancer and normal tissues. METHODS: A Proteomic approach was used to identify the differentially expressed proteins between CRC and normal tissues. The proteins were extracted using Tris buffer and thiourea lysis buffer (TLB) for extraction of aqueous soluble and membrane-associated proteins, respectively. Chemometrics, namely principal component analysis (PCA) and linear discriminant analysis (LDA), were used to assess the usefulness of these proteins for identifying the cancerous state of tissues. RESULTS: Differentially expressed proteins identified were 37 aqueous soluble proteins in Tris extracts and 24 membrane-associated proteins in TLB extracts. Based on the protein spots intensity on 2D-gel images, PCA by applying an eigenvalue > 1 was successfully used to reduce the number of principal components (PCs) into 12 and seven PCs for Tris and TLB extracts, respectively, and subsequently six PCs, respectively from both the extracts were used for LDA. The LDA classification for Tris extract showed 82.7% of original samples were correctly classified, whereas 82.7% were correctly classified for the cross-validated samples. The LDA for TLB extract showed that 78.8% of original samples and 71.2% of the cross-validated samples were correctly classified. CONCLUSION: The classification of CRC tissues by PCA and LDA provided a promising distinction between normal and cancer types. These methods can possibly be used for identification of potential biomarkers among the differentially expressed proteins identified.

  12. Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's β-Amyloid Protein

    Directory of Open Access Journals (Sweden)

    Masahiro Kawahara

    2011-01-01

    Full Text Available Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer's β-amyloid protein (AβP play crucial roles in the pathogenesis of Alzheimer's disease (AD. Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”, and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.

  13. HERG K+ channels expression in gastric cancers and analysis of its regulation in tumor cell proliferation and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Qing Lü; Huiyu Li; Xiaoming Lu; Guobin Wang

    2009-01-01

    Objective: To investigate the expression of herg 1 gene in tumor tissues from gastric carcinomas and gastric carcinoma cell lines, and study the relationship between HERG K+ channel expressions and tumor cell proliferation and apoptosis. Methods: RT-PCR and PCR assays were used to detect the expression of herg 1 gene in 64 gastric carcinomas and the gastric cancer cell line SGC-7901. Blocking the HERG K+ channels was used to evaluate their effects on tumor cell proliferation and apoptosis. Results:The statistically significant expression of herg 1 gene was detected in all the gastric cancers and SGC-7901 cells, but not in normal tissues. The HERG K+ channel blocker, E-4031, increased the cell population in G0/G1 (P<0.05) and the number of apoptotic tumor cells(P<0.05). Conclusion: HERG K+ channels were expressed in all gastric carcinomas tested and these channels appear to modulate tumor cell proliferation and apoptosis.

  14. Expression of a Carrot Antifreeze Protein Gene in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Ma Xinyu; Shen Xin; Lu Cunfu

    2003-01-01

    The recombinant expression vectorpET43. lb-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol. L-1 IPTG (isopropyl-β-D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein.The analysis of product solubility revealed that pET43. 1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h.

  15. Regulator of G-protein signalling and GoLoco proteins suppress TRPC4 channel function via acting at Gαi/o.

    Science.gov (United States)

    Jeon, Jae-Pyo; Thakur, Dhananjay P; Tian, Jin-Bin; So, Insuk; Zhu, Michael X

    2016-05-15

    Transient receptor potential canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions. Previously, TRPC4 was shown to be activated by the Gi/o subgroup of heterotrimeric G-proteins involving Gαi/o, rather than Gβγ, subunits. Because the lifetime and availability of Gα-GTP are regulated by regulators of G-protein signalling (RGS) and Gαi/o-Loco (GoLoco) domain-containing proteins via their GTPase-activating protein (GAP) and guanine-nucleotide-dissociation inhibitor (GDI) functions respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch-clamp recordings, we show that both RGS and GoLoco proteins [RGS4, RGS6, RGS12, RGS14, LGN or activator of G-protein signalling 3 (AGS3)] suppress receptor-mediated TRPC4 activation without causing detectable basal current or altering surface expression of the channel protein. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco protein AGS3. In addition, RGS, but not GoLoco, proteins accelerate desensitization of receptor-activation evoked TRPC4 currents. The inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS12 and RGS14, which contain both RGS and GoLoco domains. Our data support the notion that the Gα, but not Gβγ, arm of the Gi/o signalling is involved in TRPC4 activation and unveil new roles for RGS and GoLoco domain proteins in fine-tuning TRPC4 activities. The versatile and diverse functions of RGS and GoLoco proteins in regulating G-protein signalling may underlie the complexity of receptor-operated TRPC4 activation in various cell types under different conditions. PMID:26987813

  16. Functional Expression of Voltage-Gated Sodium Channels Navl.5 in Human Breast Caner Cell Line MDA-MB-231

    Institute of Scientific and Technical Information of China (English)

    Rui GAO; Jing WANG; Yi SHEN; Ming LEI; Zehua WANG

    2009-01-01

    Voltage-gated sodium channels (VGSCs) are known to be involved in the initiation and progression of many malignancies,and the different subtypes of VGSCs play important roles in the metastasis cascade of many tumors.This study investigated the functional expression of Nav 1.5 and its effect on invasion behavior of human breast cancer cell line MDA-MB-231.The mRNA and pro-tein expression of Navl.5 was detected by real time PCR,Western Blot and immunofluorescence.The effects of Navl.5 on cell proliferation,migration and invasion were respectively assessed by MTT and Transwell.The effects of Nav1.5 on the secretion of matrix metalloproteases (MMPs) by MDA-MB-231 were analyzed by RT-PCR.The over-expressed Navl.5 was present on the membrane of MDA-MB-231 cells.The invasion ability in vitro and the MMP-9 mRNA expression were respec-tively decreased to (47.82±0.53)% and (43.97±0.64)% (P<0.05) respectively in MDA-MB-231 cells treated with VGSCs specific inhibitor tetrodotoxin (TTX) by blocking Navl.5 activity.It was con-eluded that Nav1.5 functional expression potentiated the invasive behavior of human breast cancer cell line MDA-MB-231 by increasing the secretion of MMP-9.

  17. Engineering of an E. coli outer membrane protein FhuA with increased channel diameter

    Directory of Open Access Journals (Sweden)

    Dworeck Tamara

    2011-08-01

    Full Text Available Abstract Background Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. Results The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%. Conclusion In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

  18. Communication of emotions in vocal expression and music performance: different channels, same code?

    Science.gov (United States)

    Juslin, Patrik N; Laukka, Petri

    2003-09-01

    Many authors have speculated about a close relationship between vocal expression of emotions and musical expression of emotions. but evidence bearing on this relationship has unfortunately been lacking. This review of 104 studies of vocal expression and 41 studies of music performance reveals similarities between the 2 channels concerning (a) the accuracy with which discrete emotions were communicated to listeners and (b) the emotion-specific patterns of acoustic cues used to communicate each emotion. The patterns are generally consistent with K. R. Scherer's (1986) theoretical predictions. The results can explain why music is perceived as expressive of emotion, and they are consistent with an evolutionary perspective on vocal expression of emotions. Discussion focuses on theoretical accounts and directions for future research. PMID:12956543

  19. Expression data on liver metabolic pathway genes and proteins

    OpenAIRE

    Mooli Raja Gopal Reddy; Chodisetti Pavan Kumar; Malleswarapu Mahesh; Manchiryala Sravan Kumar; Jeyakumar, Shanmugam M

    2016-01-01

    Here, we present the expression data on various metabolic pathways of liver with special emphasize on lipid and carbohydrate metabolism and long chain polyunsaturated fatty acid (PUFA) synthesis, both at gene and protein levels. The data were obtained to understand the effect of vitamin A deficiency on the expression status (both gene and protein levels) of some of the key factors involved in lipogenesis, fatty acid oxidation, triglyceride secretion, long chain PUFA, resolvin D1 synthesis, gl...

  20. Expression pattern and function of alternative splice variants of glutamate-gated chloride channel in the housefly Musca domestica.

    Science.gov (United States)

    Kita, Tomo; Ozoe, Fumiyo; Ozoe, Yoshihisa

    2014-02-01

    Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. cDNAs encoding two alternative splice variants (MdGluClB and C) of the GluCl subunit were cloned from the housefly Musca domestica. The expression patterns of three variants, including the previously reported MdGluClA, differed among the body parts (head, thorax, abdomen, and leg) of the adult housefly and among developmental stages (embryo, larva, pupa, and adult). The MdGluClA and B transcripts were abundant in the central nervous system of the adult, whereas the MdGluClC transcript was expressed in the central nervous system and as the predominant variant in the peripheral tissues. The sensitivities to the agonist glutamate and the allosteric activator ivermectin B1a did not differ between channels containing MdGluCl variants when they were singly or co-expressed in Xenopus oocytes. By contrast, MdGluClA and B channels were more sensitive to the channel blockers fipronil and picrotoxinin than was MdGluClC channels. Heteromeric channels containing different subunit variants were more sensitive to picrotoxinin than were homomeric channels. Heteromeric channels were more sensitive to fipronil than were homomeric MdGluClC channels but not than homomeric MdGluClA and B channels. These results suggest that functionally indistinguishable but pharmacologically distinct GluCls are expressed in a spatially and temporally distinct manner in the housefly.

  1. Differential regulation of human Eag1 channel expression by serum and epidermal growth factor in lung and breast cancer cells

    Directory of Open Access Journals (Sweden)

    Acuña-Macías I

    2015-10-01

    Full Text Available Isabel Acuña-Macías,1 Eunice Vera,1 Alma Yolanda Vázquez-Sánchez,1 María Eugenia Mendoza-Garrido,2 Javier Camacho1 1Department of Pharmacology, 2Department of Physiology, Biophysics and Neurosciences, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico City, Mexico Abstract: Oncogenic ether à-go-go-1 (Eag1 potassium channels are overexpressed in most primary human solid tumors. Low oxygen and nutrient/growth factor concentrations play critical roles in tumorigenesis. However, the mechanisms by which tumor cells survive and proliferate under growth factor-depleted conditions remain elusive. Here, we investigated whether serum-deprived conditions and epidermal growth factor (EGF regulate Eag1 expression in human lung and breast cancer cells. The human cancer cell lines A549 and MCF-7 (from the lungs and breast, respectively were obtained from the American Type Culture Collection and cultured following the manufacturer’s recommendations. Eag1 gene and protein expression were studied by real-time PCR and immunocytochemistry, respectively. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay, and ERK1/2 phosphorylation was investigated by Western blot. Serum-deprived conditions increased Eag1 mRNA and protein expression in both cell lines. This Eag1 upregulation was prevented by EGF and the ERK1/2 inhibitor U0126 in only lung cancer cells; vascular endothelial growth factor did not prevent Eag1 upregulation. Our results suggest that Eag1 may act as a survival and mitogenic factor under low-serum and nutrient conditions and may be a clinical target during the early stages of tumor development. Keywords: lung cancer, serum deprivation, ether à-go-go, potassium channels, EGF, epidermal growth factor, ERK 1/2

  2. A Calcium-Dependent Protein Kinase Interactswith and Activates A Calcium Channel toRequlate Pollen Tube Growth

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    ABSTRACT Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neu-rons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code anddecode Ca2+ signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32,controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca2+accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activationof CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity.Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, simi-lar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading tomore severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in whichcalcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca2+ oscilla-tions in the polar growth of pollen tubes.

  3. Description and control of dissociation channels in gas-phase protein complexes

    Science.gov (United States)

    Thachuk, Mark; Fegan, Sarah K.; Raheem, Nigare

    2016-08-01

    Using molecular dynamics simulations of a coarse-grained model of the charged apo-hemoglobin protein complex, this work expands upon our initial report [S. K. Fegan and M. Thachuk, J. Am. Soc. Mass Spectrom. 25, 722-728 (2014)] about control of dissociation channels in the gas phase using specially designed charge tags. Employing a charge hopping algorithm and a range of temperatures, a variety of dissociation channels are found for activated gas-phase protein complexes. At low temperatures, a single monomer unfolds and becomes charge enriched. At higher temperatures, two additional channels open: (i) two monomers unfold and charge enrich and (ii) two monomers compete for unfolding with one eventually dominating and the other reattaching to the complex. At even higher temperatures, other more complex dissociation channels open with three or more monomers competing for unfolding. A model charge tag with five sites is specially designed to either attract or exclude charges. By attaching this tag to the N-terminus of specific monomers, the unfolding of those monomers can be decidedly enhanced or suppressed. In other words, using charge tags to direct the motion of charges in a protein complex provides a mechanism for controlling dissociation. This technique could be used in mass spectrometry experiments to direct forces at specific attachment points in a protein complex, and hence increase the diversity of product channels available for quantitative analysis. In turn, this could provide insight into the function of the protein complex in its native biological environment. From a dynamics perspective, this system provides an interesting example of cooperative behaviour involving motions with differing time scales.

  4. Screening and analysis of Chinese herbs with liver channel tropism acting on human liver cancer cell differential expressed protein tyrosine kinase%归肝经中药作用于人肝癌细胞差异表达 蛋白酪氨酸激酶的筛选与分析

    Institute of Scientific and Technical Information of China (English)

    田雪飞; 黄晓蒂; 周青; 王志琪; 田莎; 陈园

    2015-01-01

    PTKs差异表达在化瘀消癥药对组有17种,其中9种RTKs〔受体酪氨酸激酶样孤儿受体(ROR2)、干细胞因子受体(SCFR)、间变性淋巴瘤激酶(ALK)、血小板源性生长因子受体-β(PDGFR-β)、胰岛素样生长因子-IR(IGF-IR)、ErbB2、ErbB3、EphB1、EphA2〕,1种nrPTKs〔fps/fes相关酪氨酸激酶(FER)〕,7种PTKs,包括3种RTKs(M-CSFR、FGFR2-α、EphA3)和4种nrPTKs〔乙酸激酶1(ACK1)、布鲁顿酪氨酸蛋白激酶(Btk)、非受体酪氨酸激酶ABL1(ABL1)、BMX〕;在攻毒散结药对组有7种,包括5种RTKs(M-CSFR、FGFR1、ROR2、EphB1、ErbB2)和2种nrPTKs〔缺乏C端调节与N端烷基化位点的Src相关激酶(SRMS)、FER〕.结论 具有攻毒散结作用的蜈蚣和全蝎药对比具有化瘀消癥作用的三棱和莪术药有更好的抗肝癌作用,其机制可能是通过调节PTKs信号通路.归肝经活血化瘀药对三棱和莪术可能存在独立于PTKs信号系统外的抗肝癌效应途径.%Objective To screen the Chinese drugs with liver channel tropism acting on the characteristic differential expressed protein of human liver cancer cell protein tyrosine kinase (PTKs) system by protein chip technology, and to analyze the difference in different Chinese drugs with liver channel tropism in relation to PTKs regulation. Methods Forty BALB/C nude mice were chosen; a piece of subcutaneous tumor mass was implanted into the left lobe of liver parenchyma to reduplicate the orthotopically implanted tumor models. After modeling for 10 days, the tumorigenicity was confirmed, and all the nude mice models were divided into four groups; different Chinese medicine extracts were injected intra-peritoneally into corresponding treatment groups respectively, and the methods of treatment in the 4 groups were as follows: In liver channel tropism drug pair of Huayu Xiaozhen group, rhizoma sparganii and curcuma zedoary with dosage containing crude drug 4.5 g·kg-1·d-1 was given, in liver channel tropism drug pair of Gongdu

  5. Molecular Characterization of LRB7 Gene and a Water Channel Protein TIP2 in Chorispora bungeana

    Science.gov (United States)

    Liang, Zhaoxu; Di, Cuixia; Fang, Weikuan; Wu, Kaichao; Chen, Maoshan; He, Shanshan; Zeng, Yuan; Jing, Yan; Liang, Jun; Tan, Fang; Li, Song; Chen, Tuo; Liu, Guangxiu

    2016-01-01

    Background. Water channel proteins, also called aquaporins, are integral membrane proteins from major intrinsic protein (MIP) family and involved in several pathways including not only water transport but also cell signaling, reproduction, and photosynthesis. The full cDNA and protein sequences of aquaporin in Chorispora bungeana Fisch. & C.A. Mey (C. bungeana) are still unknown. Results. In this study, PCR and rapid amplification of cDNA ends approaches were used to clone the full cDNA of LRB7 (GenBank accession number: EU636988) of C. bungeana. Sequence analysis indicated that it was 1235 bp, which had two introns and encoded a protein of 250 amino acids. Structure analysis revealed that the protein had two conserved NPA motifs, one of which is MIP signature sequence (SGxHxNPAVT), six membrane helix regions, and additional membrane-embedded domains. Phylogenetic analysis suggested that the protein was from TIP2 subgroup. Surprisingly, semiquantitative RT-PCR experiment and western blot analysis showed that LRB7 and TIP2 were only detectable in roots, unlike Arabidopsis and Raphanus. Connecting with our previous studies, LRB7 was supported to associate with chilling-tolerance in C. bungeana. Conclusion. This is the first time to characterize the full sequences of LRB7 gene and water channel protein in C. bungeana. Our findings contribute to understanding the water transports in plants under low temperatures.

  6. Molecular Characterization of LRB7 Gene and a Water Channel Protein TIP2 in Chorispora bungeana

    Directory of Open Access Journals (Sweden)

    Ming Li

    2016-01-01

    Full Text Available Background. Water channel proteins, also called aquaporins, are integral membrane proteins from major intrinsic protein (MIP family and involved in several pathways including not only water transport but also cell signaling, reproduction, and photosynthesis. The full cDNA and protein sequences of aquaporin in Chorispora bungeana Fisch. & C.A. Mey (C. bungeana are still unknown. Results. In this study, PCR and rapid amplification of cDNA ends approaches were used to clone the full cDNA of LRB7 (GenBank accession number: EU636988 of C. bungeana. Sequence analysis indicated that it was 1235 bp, which had two introns and encoded a protein of 250 amino acids. Structure analysis revealed that the protein had two conserved NPA motifs, one of which is MIP signature sequence (SGxHxNPAVT, six membrane helix regions, and additional membrane-embedded domains. Phylogenetic analysis suggested that the protein was from TIP2 subgroup. Surprisingly, semiquantitative RT-PCR experiment and western blot analysis showed that LRB7 and TIP2 were only detectable in roots, unlike Arabidopsis and Raphanus. Connecting with our previous studies, LRB7 was supported to associate with chilling-tolerance in C. bungeana. Conclusion. This is the first time to characterize the full sequences of LRB7 gene and water channel protein in C. bungeana. Our findings contribute to understanding the water transports in plants under low temperatures.

  7. The G. L. Brown Prize Lecture. Hypoxic regulation of ion channel function and expression.

    Science.gov (United States)

    Peers, Chris

    2002-07-01

    Acute hypoxia regulates the activity of specific ion channels in a rapid and reversible manner. Such effects underlie appropriate cellular responses to hypoxia which are designed to initiate cardiorespiratory reflexes and contribute importantly to other tissue responses, all of which are designed to improve tissue O2 supply. These responses include excitation of chemoreceptors as well as pulmonary vasoconstriction and systemic vasodilatation. However, such responses may also contribute to the adverse responses to hypoxia, such as excitotoxicity in the central nervous system. Whilst numerous ion channel types are known to be modulated by acute hypoxia, the nature of the O2 sensor in most tissues remains to be identified. Prolonged (chronic) hypoxia regulates functional expression of ion channels, and so remodels excitability of various cell types. Whilst this may contribute to adaptive responses such as high-altitude acclimatization, such altered channel expression may also contribute to the onset of pathological disorders, including Alzheimer's disease. Indeed, evidence is emerging that production of pathological peptides associated with Alzheimer's disease is increased during prolonged hypoxia. Such effects may account for the known increased incidence of this disease in patients who have previously endured hypoxic episodes, such as congestive heart failure and stroke. Identification of the mechanisms coupling hypoxia to the increased production of these peptides is likely to be of therapeutic benefit. PMID:12392105

  8. The G. L. Brown Prize Lecture. Hypoxic regulation of ion channel function and expression.

    Science.gov (United States)

    Peers, Chris

    2002-07-01

    Acute hypoxia regulates the activity of specific ion channels in a rapid and reversible manner. Such effects underlie appropriate cellular responses to hypoxia which are designed to initiate cardiorespiratory reflexes and contribute importantly to other tissue responses, all of which are designed to improve tissue O2 supply. These responses include excitation of chemoreceptors as well as pulmonary vasoconstriction and systemic vasodilatation. However, such responses may also contribute to the adverse responses to hypoxia, such as excitotoxicity in the central nervous system. Whilst numerous ion channel types are known to be modulated by acute hypoxia, the nature of the O2 sensor in most tissues remains to be identified. Prolonged (chronic) hypoxia regulates functional expression of ion channels, and so remodels excitability of various cell types. Whilst this may contribute to adaptive responses such as high-altitude acclimatization, such altered channel expression may also contribute to the onset of pathological disorders, including Alzheimer's disease. Indeed, evidence is emerging that production of pathological peptides associated with Alzheimer's disease is increased during prolonged hypoxia. Such effects may account for the known increased incidence of this disease in patients who have previously endured hypoxic episodes, such as congestive heart failure and stroke. Identification of the mechanisms coupling hypoxia to the increased production of these peptides is likely to be of therapeutic benefit.

  9. Expression of ATP sensitive K+ channel subunit Kir6.1 in rat kidney

    Directory of Open Access Journals (Sweden)

    M Zhou

    2009-06-01

    Full Text Available ATP-sensitive K+ (KATP channels in kidney are considered to play roles in regulating membrane potential during the change in intracellular ATP concentration. They are composed of channel subunits (Kir6.1, Kir6.2, which are members of the inwardly rectifying K+ channel family, and sulphonylurea receptors (SUR1, SUR2A and SUR2B, which belong to the ATP-binding cassette superfamily. In the present study, we have investigated the expression and localization of Kir6.1 in rat kidney with Western blot analysis, immunohistochemistry, in situ hybridization histochemistry, and immunoelectron microscopy. Western blot analysis showed that Kir6.1 was expressed in the mitochondria and microsome fractions of rat kidney and very weakly in the membrane fractions. Immunohistochemistry revealed that Kir6.1 was widely distributed in renal tubular epithelial cells, glomerular mesangial cells, and smooth muscles of blood vessels. In immunoelectron microscopy, Kir6.1 is mainly localized in the mitochondria, endoplasmic reticulum (ER, and very weakly in cell membranes. Thus, Kir6.1 is contained in the kidney and may be a candidate of mitochondrial KATP channels.

  10. Expression and activity of acid-sensing ion channels in the mouse anterior pituitary.

    Directory of Open Access Journals (Sweden)

    Jianyang Du

    Full Text Available Acid sensing ion channels (ASICs are proton-gated cation channels that are expressed in the nervous system and play an important role in fear learning and memory. The function of ASICs in the pituitary, an endocrine gland that contributes to emotions, is unknown. We sought to investigate which ASIC subunits were present in the pituitary and found mRNA expression for all ASIC isoforms, including ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4. We also observed acid-evoked ASIC-like currents in isolated anterior pituitary cells that were absent in mice lacking ASIC1a. The biophysical properties and the responses to PcTx1, amiloride, Ca2+ and Zn2+ suggested that ASIC currents were mediated predominantly by heteromultimeric channels that contained ASIC1a and ASIC2a or ASIC2b. ASIC currents were also sensitive to FMRFamide (Phe-Met-Arg-Phe amide, suggesting that FMRFamide-like compounds might endogenously regulate pituitary ASICs. To determine whether ASICs might regulate pituitary cell function, we applied low pH and found that it increased the intracellular Ca2+ concentration. These data suggest that ASIC channels are present and functionally active in anterior pituitary cells and may therefore influence their function.

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  14. Genome-wide screens for expressed hypothetical proteins

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Durhuus, Jon Ambæk; Rasmussen, Lene Juel

    2012-01-01

    A hypothetical protein (HP) is defined as a protein that is predicted to be expressed from an open reading frame, but for which there is no experimental evidence of translation. HPs constitute a substantial fraction of proteomes of human as well as of other organisms. With the general belief that...

  15. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  16. Expression of the transient receptor potential channels TRPV1, TRPA1 and TRPM8 in mouse trigeminal primary afferent neurons innervating the dura

    Directory of Open Access Journals (Sweden)

    Huang Dongyue

    2012-09-01

    Full Text Available Abstract Background Migraine and other headache disorders affect a large percentage of the population and cause debilitating pain. Activation and sensitization of the trigeminal primary afferent neurons innervating the dura and cerebral vessels is a crucial step in the “headache circuit”. Many dural afferent neurons respond to algesic and inflammatory agents. Given the clear role of the transient receptor potential (TRP family of channels in both sensing chemical stimulants and mediating inflammatory pain, we investigated the expression of TRP channels in dural afferent neurons. Methods We used two fluorescent tracers to retrogradely label dural afferent neurons in adult mice and quantified the abundance of peptidergic and non-peptidergic neuron populations using calcitonin gene-related peptide immunoreactivity (CGRP-ir and isolectin B4 (IB4 binding as markers, respectively. Using immunohistochemistry, we compared the expression of TRPV1 and TRPA1 channels in dural afferent neurons with the expression in total trigeminal ganglion (TG neurons. To examine the distribution of TRPM8 channels, we labeled dural afferent neurons in mice expressing farnesylated enhanced green fluorescent protein (EGFPf from a TRPM8 locus. We used nearest-neighbor measurement to predict the spatial association between dural afferent neurons and neurons expressing TRPA1 or TRPM8 channels in the TG. Results and conclusions We report that the size of dural afferent neurons is significantly larger than that of total TG neurons and facial skin afferents. Approximately 40% of dural afferent neurons exhibit IB4 binding. Surprisingly, the percentage of dural afferent neurons containing CGRP-ir is significantly lower than those of total TG neurons and facial skin afferents. Both TRPV1 and TRPA1 channels are expressed in dural afferent neurons. Furthermore, nearest-neighbor measurement indicates that TRPA1-expressing neurons are clustered around a subset of dural afferent

  17. Formation of individual protein channels in lipid bilayers suspended in nanopores.

    Science.gov (United States)

    Studer, André; Han, Xiaojun; Winkler, Fritz K; Tiefenauer, Louis X

    2009-10-15

    Free-standing lipid bilayers are formed in regularly arranged nanopores of 200, 400 and 800 nm in a 300 nm thin hydrophobic silicon nitride membrane separating two fluid compartments. The extraordinary stability of the lipid bilayers allows us to monitor channel formation of the model peptide melittin and alpha-hemolysin from Staphylococcus aureus using electrochemical impedance spectroscopy and chronoamperometry. We observed that melittin channel formation is voltage-dependent and transient, whereas transmembrane heptameric alpha-hemolysin channels in nano-BLMs persist for hours. The onset of alpha-hemolysin-mediated conduction depends on the applied protein concentration and strongly on the diameter of the nanopores. Heptameric channel formation from adsorbed alpha-hemolysin monomers needs more time in bilayers suspended in 200 nm pores compared to bilayers in pores of 400 and 800 nm diameters. Diffusion of sodium ions across alpha-hemolysin channels present in a sufficiently high number in the bilayers was quantitatively and specifically determined using ion selective electrodes. The results demonstrate that relatively small variations of nano-dimensions have a tremendous effect on observable dynamic biomolecular processes. Such nanopore chips are potentially useful as supports for stable lipid bilayers to establish functional assays of membrane proteins needed in basic research and drug discovery.

  18. Expression of Potassium Channels in Uterine Smooth Muscle Cells from Patients with Adenomyosis

    Directory of Open Access Journals (Sweden)

    Jing-Hua Shi

    2016-01-01

    Full Text Available Background: Adenomyosis (AM has impaired contraction. This study aimed to explore the expression of potassium channels related to contraction in myometrial smooth muscle cells (MSMCs of AM. Methods: Uterine tissue samples from 22 patients (cases with histologically confirmed AM and 12 (controls with cervical intraepithelial neoplasia were collected for both immunohistochemistry and real-time polymerase chain reaction to detect the expression of large conductance calcium- and voltage-sensitive K + channel (BKCa-α/β subunits, voltage-gated potassium channel (Kv 4.2, and Kv4.3. Student′s t-test was used to compare the expression. Results: The BKCa-α/β subunits, Kv4.2, and Kv4.3 were located in smooth muscle cells, glandular epithelium, and stromal cells. However, BKCa-β subunit expression in endometrial glands of the controls was weak, and Kv4.3 was almost undetectable in the controls. The expression of BKCa-α messenger RNA (mRNA (0.62 ± 0.19-fold decrease, P < 0.05 and Kv4.3 mRNA (0.67 ± 0.20-fold decrease, P < 0.05 decreased significantly in the MSMCs of the control group compared with the AM group. However, there were no significant differences in BKCa-β subunit mRNA or Kv4.2 mRNA. Conclusions: The BKCa-α mRNA and the Kv4.3 mRNA are expressed significantly higher in AM than those in the control group, that might cause the abnormal uterus smooth muscle contractility, change the microcirculation of uterus to accumulate the inflammatory factors, impair the endometrium further, and aggravate the pain.

  19. Expression of Potassium Channels in Uterine Smooth Muscle Cells from Patients with Adenomyosis

    Institute of Scientific and Technical Information of China (English)

    Jing-Hua Shi; Li Jin; Jin-Hua Leng; Jing-He Lang

    2016-01-01

    Background:Adenomyosis (AM) has impaired contraction.This study aimed to explore the expression of potassium channels related to contraction in myometrial smooth muscle cells (MSMCs) of AM.Methods:Uterine tissue samples from 22 patients (cases) with histologically confirmed AM and 12 (controls) with cervical intraepithelial neoplasia were collected for both immunohistochemistry and real-time polymerase chain reaction to detect the expression of large conductance calcium-and voltage-sensitive K+ channel (BKCa)-α/β subunits,voltage-gated potassium channel (Kv) 4.2,and Kv4.3.Student's t-test was used to compare the expression.Results:The BKCa-α/β subunits,Kv4.2,and Kv4.3 were located in smooth muscle cells,glandular epithelium,and stromal cells.However,BKCa-β subunit expression in endometrial glands of the controls was weak,and Kv4.3 was almost undetectable in the controls.The expression of BKCa-α messenger RNA (mRNA) (0.62 ± 0.19-fold decrease,P < 0.05) and Kv4.3 mRNA (0.67 ± 0.20-fold decrease,P < 0.05) decreased significantly in the M SMCs of the control group compared with the AM group.However,there were no significant differences in BKCa-β subunit mRNA or Kv4.2 mRNA.Conclusions:The BKCa-α mRNA and the Kv4.3 mRNA are expressed significantly higher in AM than those in the control group,that might cause the abnormal uterus smooth muscle contractility,change the microcirculation of uterus to accumulate the inflammatory factors,impair the endometrium further,and aggravate the pain.

  20. Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating

    DEFF Research Database (Denmark)

    Bildl, Wolfgang; Strassmaier, Tim; Thurm, Henrike;

    2004-01-01

    Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and...

  1. The proteome response to amyloid protein expression in vivo.

    Directory of Open Access Journals (Sweden)

    Ricardo A Gomes

    Full Text Available Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE. We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

  2. The Influence of Polychlorinated Biphenyls Contamination on Soil Protein Expression

    OpenAIRE

    Xi Zhang; Feng Li; Tingting Liu; Cheng Peng; Dechao Duan; Chen Xu; Shenhai Zhu; Jiyan Shi

    2013-01-01

    Polychlorinated biphenyls (PCBs) are typical representative of chlorinated organic pollutants. Given the toxicity of PCBs, there is an urgent need to select an appropriate indicator to monitor their biological effects on soil ecosystems. For this purpose, we investigated the impacts of PCBs on soil protein and the potential of using protein as a biological indicator to assess soil contamination due to PCBs. This study demonstrated that soil protein concentration and expression were negatively...

  3. Recombinant Brucella abortus gene expressing immunogenic protein

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-06-11

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

  4. Oxaliplatin-induced cold hypersensitivity is due to remodelling of ion channel expression in nociceptors.

    Science.gov (United States)

    Descoeur, Juliette; Pereira, Vanessa; Pizzoccaro, Anne; Francois, Amaury; Ling, Bing; Maffre, Violette; Couette, Brigitte; Busserolles, Jérôme; Courteix, Christine; Noel, Jacques; Lazdunski, Michel; Eschalier, Alain; Authier, Nicolas; Bourinet, Emmanuel

    2011-05-01

    Cold hypersensitivity is the hallmark of oxaliplatin-induced neuropathy, which develops in nearly all patients under this chemotherapy. To date, pain management strategies have failed to alleviate these symptoms, hence development of adapted analgesics is needed. Here, we report that oxaliplatin exaggerates cold perception in mice as well as in patients. These symptoms are mediated by primary afferent sensory neurons expressing the thermoreceptor TRPM8. Mechanistically, oxaliplatin promotes over-excitability by drastically lowering the expression of distinct potassium channels (TREK1, TRAAK) and by increasing the expression of pro-excitatory channels such as the hyperpolarization-activated channels (HCNs). These findings are corroborated by the analysis of TREK1-TRAAK null mice and use of the specific HCN inhibitor ivabradine, which abolishes the oxaliplatin-induced cold hypersensibility. These results suggest that oxaliplatin exacerbates cold perception by modulating the transcription of distinct ionic conductances that together shape sensory neuron responses to cold. The translational and clinical implication of these findings would be that ivabradine may represent a tailored treatment for oxaliplatin-induced neuropathy. PMID:21438154

  5. Gene expressions of TRP channels in glioblastoma multiforme and relation with survival.

    Science.gov (United States)

    Alptekin, M; Eroglu, S; Tutar, E; Sencan, S; Geyik, M A; Ulasli, M; Demiryurek, A T; Camci, C

    2015-12-01

    Glioblastoma multiforme (GBM) is one of the most lethal forms of cancer in humans, with a median survival of 10 to 12 months. Glioblastoma is highly malignant since the cells are supported by a great number of blood vessels. Although new treatments have been developed by increasing knowledge of molecular nature of the disease, surgical operation remains the standard of care. The TRP (transient receptor potential) superfamily consists of cation-selective channels that have roles in sensory physiology such as thermo- and osmosensation and in several complex diseases such as cancer, cardiovascular, and neuronal diseases. The aim of this study was to investigate the expression levels of TRP channel genes in patients with glioblastoma multiforme and to evaluate the relationship between TRP gene expressions and survival of the patients. Thirty-three patients diagnosed with glioblastoma were enrolled to the study. The expression levels of 21 TRP genes were quantified by using qRT-PCR with dynamic array 48 × 48 chip (BioMark HD System, Fluidigm, South San Francisco, CA, USA). TRPC1, TRPC6, TRPM2, TRPM3, TRPM7, TRPM8, TRPV1, and TRPV2 were found significantly higher in glioblastoma patients. Moreover, there was a significant relationship between the overexpression of TRP genes and the survival of the patients. These results demonstrate for the first time that TRP channels contribute to the progression and survival of the glioblastoma patients. PMID:26088448

  6. Mapping the expression of soluble olfactory proteins in the honeybee.

    Science.gov (United States)

    Dani, Francesca Romana; Iovinella, Immacolata; Felicioli, Antonio; Niccolini, Alberto; Calvello, Maria Antonietta; Carucci, Maria Giovanna; Qiao, Huili; Pieraccini, Giuseppe; Turillazzi, Stefano; Moneti, Gloriano; Pelosi, Paolo

    2010-04-01

    Chemical communication in insects is mediated by soluble binding proteins, belonging to two large families, Odorant-binding Proteins (OBPs) and Chemosensory Proteins (CSPs). Recently, evidence has been provided that OBPs are involved in recognition of chemical stimuli. We therefore decided to investigate the expression of OBPs and CSPs in the honeybee at the protein level, using a proteomic approach. Our results are in agreement with previous reports of expression at the RNA level and show that 12 of the 21 OBPs predicted in the genome of the honeybee Apis mellifera and 2 of the 6 CSPs are present in the foragers' antennae, while the larvae express only three OBPs and a single CSP. MALDI mass spectrometry on crude antennal extracts and MALDI profiling on sections of antennae demonstrated that these techniques can be applied to investigate individual differences in the expression of abundant proteins, such as OBPs and CSPs, as well as to detect the presence of proteins in different regions of the antenna. Finally, as part of a project aimed at the characterization of all OBPs and CSPs of the honeybee, we expressed 5 OBPs and 4 CSPs in a bacterial system and measured their affinity to a number of ligands. Clear differences in their binding spectra have been observed between OBPs, as well as CSPs. PMID:20155982

  7. Expression of genes encoding multi-transmembrane proteins in specific primate taste cell populations.

    Directory of Open Access Journals (Sweden)

    Bryan D Moyer

    Full Text Available BACKGROUND: Using fungiform (FG and circumvallate (CV taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed. METHODOLOGY/PRINCIPAL FINDINGS: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive, sour cells (PKD2L1-positive, as well as other taste cell populations. Transmembrane protein 44 (TMEM44, a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1, a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1, a calcium-binding transmembrane protein; and anoctamin 7 (ANO7, a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B, a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release. CONCLUSIONS/SIGNIFICANCE: Identification of genes encoding multi-transmembrane domain proteins

  8. Regulation of epithelial sodium channel a-subunit expression by adenosine receptor A2a in alveolar epithelial cells

    Institute of Scientific and Technical Information of China (English)

    DENG Wang; WANG Dao-xin; ZHANG Wei; LI Chang-yi

    2011-01-01

    Background The amiloride-sensitive epithelial sodium channel a-subunit (a-ENaC) is an important factor for alveolar fluid clearance during acute lung injury. The relationship between adenosine receptor A2a (A2aAR) expressed in alveolar epithelial cells and aα-ENaC is poorly understood. We targeted the A2aAR in this study to investigate its role in the expression of αa-ENaC and in acute lung injury.Methods A549 cells were incubated with different concentrations of A2aAR agonist CGS-21680 and with 100 μmol/L CGS-21680 for various times. Rats were treated with lipopolysaccharide (LPS) after CGS-21680 was injected. Animals were sacrificed and tissue was harvested for evaluation of lung injury by analysis of the lung wet-to-dry weight ratio, lung permeability and myeloperoxidase activity. RT-PCR and Western blotting were used to determine the mRNA and protein expression levels of α-ENaC in A549 cells and alveolar type II epithelial cells.Results Both mRNA and protein levels of α-ENaC were markedly higher from 4 hours to 24 hours after exposure to 100μmol/L CGS-21680. There were significant changes from 0.1 umol/L to 100 μmol/L CGS-21680, with a positive correlation between increased concentrations of CGS-21680 and expression of α-ENaC. Treatment with CGS-21680during LPS induced lung injury protected the lung and promoted α-ENaC expression in the alveolar epithelial cells.Conclusion Activation of A2aAR has a protective effect during the lung injury, which may be beneficial to the prognosis of acute lung injury.

  9. Differential Protein Expression in Congenital and Acquired Cholesteatomas.

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    Seung-Ho Shin

    Full Text Available 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

  10. The DEG/ENaC cation channel protein UNC-8 drives activity-dependent synapse removal in remodeling GABAergic neurons

    Science.gov (United States)

    Miller-Fleming, Tyne W; Petersen, Sarah C; Manning, Laura; Matthewman, Cristina; Gornet, Megan; Beers, Allison; Hori, Sayaka; Mitani, Shohei; Bianchi, Laura; Richmond, Janet; Miller, David M

    2016-01-01

    Genetic programming and neural activity drive synaptic remodeling in developing neural circuits, but the molecular components that link these pathways are poorly understood. Here we show that the C. elegans Degenerin/Epithelial Sodium Channel (DEG/ENaC) protein, UNC-8, is transcriptionally controlled to function as a trigger in an activity-dependent mechanism that removes synapses in remodeling GABAergic neurons. UNC-8 cation channel activity promotes disassembly of presynaptic domains in DD type GABA neurons, but not in VD class GABA neurons where unc-8 expression is blocked by the COUP/TF transcription factor, UNC-55. We propose that the depolarizing effect of UNC-8-dependent sodium import elevates intracellular calcium in a positive feedback loop involving the voltage-gated calcium channel UNC-2 and the calcium-activated phosphatase TAX-6/calcineurin to initiate a caspase-dependent mechanism that disassembles the presynaptic apparatus. Thus, UNC-8 serves as a link between genetic and activity-dependent pathways that function together to promote the elimination of GABA synapses in remodeling neurons. DOI: http://dx.doi.org/10.7554/eLife.14599.001 PMID:27403890

  11. Identification of Differentially Expressed Serum Proteins in Infectious Purpura Fulminans

    Directory of Open Access Journals (Sweden)

    Ting He

    2014-01-01

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

  12. Similar expression patterns of bestrophin-4 and cGMP dependent Ca2+-activated chloride channel activity in the vasculature

    DEFF Research Database (Denmark)

    Bouzinova, Elena V.; Larsen, Per; Matchkov, Vladimir;

    2008-01-01

    (abstract by Matchkov et. al) that siRNA mediated downregulation of bestrophin-4 is associated with the disappearance of a recently demonstrated2 cGMP-dependent Ca2+-activated Cl- current in vascular smooth muscle cells (SMCs). Here we study the distribution of bestrophin-4-and cGMP dependent Cl- channel...... expressed epitope) Western blot detected a ~65 kDa band in cell lysates from rat mesenteric small arteries and aorta, which was not seen in pulmonary arteries and when preincubated with the immunizing peptide. The distribution of bestrophin-4 mRNA and protein has a pattern similar to the cGMP-dependent Cl......- current in SMCs of different origins. Immunohistochemistry identified bestrophin-4 both in endothelial and SMCs of the vascular tree in the brain, heart, kidney and mesentery, but not in the lungs. We suggest that bestrophin-4 is important for the cGMP dependent, Ca2+ activated Cl- conductance in many...

  13. l-Lactate mediates neuroprotection against ischaemia by increasing TREK1 channel expression in rat hippocampal astrocytes in vitro.

    Science.gov (United States)

    Banerjee, Aditi; Ghatak, Swagata; Sikdar, Sujit Kumar

    2016-07-01

    Brain ischaemia is a highly debilitating condition where shortage of oxygen and glucose leads to profuse cell death. Lactate is a neuroprotective metabolite whose concentrations increase up to 15-30 mmol/L during ischaemia and TREK1 is a neuroprotective potassium channel which is upregulated during ischaemia. The aim of this study was to investigate the effect of l-lactate on TREK1 expression and to evaluate the role of l-lactate-TREK1 interaction in conferring neuroprotection in ischaemia-prone hippocampus. We show that 15-30 mmol/L l-lactate increases functional TREK1 protein expression by 1.5-3-fold in hippocampal astrocytes using immunostaining and electrophysiology. Studies with transcription blocker actinomycin-D and quantitative PCR indicate that the increase in TREK1 expression is due to enhanced TREK1 mRNA transcription. We further report that l-lactate-mediated increase in TREK1 expression is via protein kinase A (PKA)-dependent pathway. This is the first report of an ischaemic metabolite affecting functional expression of an ion channel. Our studies in an in vitro model of ischaemia using oxygen glucose deprivation show that 30 mmol/L l-lactate fails to reduce cell death in rat hippocampal slices treated with TREK1 blockers, PKA inhibitors and gliotoxin. The above effects were specific to l-lactate as pyruvate failed to increase TREK1 expression and reduce cell death. l-Lactate-induced TREK1 upregulation is a novel finding of physiological significance as TREK1 channels contribute to neuroprotection by enhancing potassium buffering and glutamate clearance capacity of astrocytes. We propose that l-lactate promotes neuronal survival in hippocampus by increasing TREK1 channel expression via PKA pathway in astrocytes during ischaemia. Insufficient blood supply to the brain leads to cerebral ischaemia and increase in extracellular lactate concentrations. We incubated hippocampal astrocytes in lactate and observed increase in TREK1 channel expression via

  14. A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion.

    Science.gov (United States)

    Grefen, Christopher; Karnik, Rucha; Larson, Emily; Lefoulon, Cécile; Wang, Yizhou; Waghmare, Sakharam; Zhang, Ben; Hills, Adrian; Blatt, Michael R

    2015-01-01

    Growth in plants depends on ion transport for osmotic solute uptake and secretory membrane trafficking to deliver material for wall remodelling and cell expansion. The coordination of these processes lies at the heart of the question, unresolved for more than a century, of how plants regulate cell volume and turgor. Here we report that the SNARE protein SYP121 (SYR1/PEN1), which mediates vesicle fusion at the Arabidopsis plasma membrane, binds the voltage sensor domains (VSDs) of K(+) channels to confer a voltage dependence on secretory traffic in parallel with K(+) uptake. VSD binding enhances secretion in vivo subject to voltage, and mutations affecting VSD conformation alter binding and secretion in parallel with channel gating, net K(+) concentration, osmotic content and growth. These results demonstrate a new and unexpected mechanism for secretory control, in which a subset of plant SNAREs commandeer K(+) channel VSDs to coordinate membrane trafficking with K(+) uptake for growth.

  15. Construction, Expression and Purification of SUMO1-GST Fusion Protein

    Institute of Scientific and Technical Information of China (English)

    QIAO Xiao-fang; FANG Xue-dong; LIU Jun

    2011-01-01

    Sumoylation is an important protein modification discovered recently. SUMO(small ubiquitin-related modifier) pathway regulates the protein stability and transcriptional activity with a 12-kDa small molecular protein,SUMO, ligated to the target protein. The purification of SUMO proteins is a key step to reveal their function. The purpose of this study was to construct the recombinant SUMO1 gene cloned to a pGEX-4T-1 vector to express and purify the SUMO1-GST fusion protein in Escherichia coli. First, the full length DNA sequence of SUMO1 gene was amplified by PCR and was ligated to pMD18-T vector. Then the SUMO1 gene was subcloned to pGEX-4T-1 prokaryotic expression vector between BamHI and XhoI sites, and transformed in Escherichia coli DH5a cells. The right colonies were identified by restrictive enzyme digestion and sequencing. The correct rebombinant plasmid of pGEX-4T-1-SUMO1 was transformed in Escherichia coli BL21 cells and then induced by IPTG(isopropyl-β-D-lthiogalacto-pyranoside) to express the SUMO1-GST fusion protein. The highly purified SUMOl-GST(glutathione S-transferase) fusion protein was obtained by affinity chromatography. Finally, the properties of SUMO1-GST fusion protein were confirmed by Coomassie brilliant blue strain and Western blot analysis. The recombinant plasmid of pGEX-4T-1-SUMO 1 was successfully constructed, and SUMO1-GST fusion proteins were successfully expressed.

  16. The role of water channel proteins in facilitating recovery of leaf hydraulic conductance from water stress in Populus trichocarpa.

    Directory of Open Access Journals (Sweden)

    Joan Laur

    Full Text Available Gas exchange is constrained by the whole-plant hydraulic conductance (Kplant. Leaves account for an important fraction of Kplant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (Kleaf decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs. Here we assessed changes in the hydraulic conductance of Populus trichocarpa leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, Kleaf recovered only 2 hours after plants were rewatered. Recovery of Kleaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in Kleaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in Kleaf.

  17. Expression of genes encoding extracellular matrix proteins: a macroarray study.

    Science.gov (United States)

    Futyma, Konrad; Miotła, Paweł; Różyńska, Krystyna; Zdunek, Małgorzata; Semczuk, Andrzej; Rechberger, Tomasz; Wojcierowski, Jacek

    2014-12-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs.

  18. Expression of SKP2 Protein in Lung Carcinoma

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jun; YANG Chun-lu; ZHANG Huan; DING Wei-zhong; LIU Zhi-ping; LIU Jing-yi

    2008-01-01

    Objective:To study the expressive characteristics of SKP2 protein in lung carcinoma and its implication for prognosis.Methods:The expression of SKP2 protein was detected in 89 non small cell lung carcinoma,13 small cell lung carcinoma,10 lung benign lesion tissues by Tissue Chip and Immunohistochemistry technology.Results:The positive rate of SKP2 protein staining was(23.52±13.57)% in non small cell lung carcinoma and (53.85+12.26)% in small cell lung carcinoma,which were significantly higher than(2.91±1.27)% in lung benign lesion tissues.It was highest in small cell lung carcinoma and lowest in lung benign lesion tissues,with a significant difference between them(P=0.000).The expressive level of SKP2 protein in lung carcinoma tissues was closely related to cell differentiation,lymph node metastasis and pathological types,but not to age,sex,smoking history,tumor site and size,and TNM staging.The survival analysis revealed that the 5-year survival rate of lung carcinoma patients was lower in SKP2 protein positive expression group than that in negative expression group(P1=0.003/0.002;r=-0.275,P2=0.005).Conclusion:The positive expression of SKP2 protein is higher in lung carcinoma than in lung benign lesion tissues.in particular,much higher in small cell lung carcinoma.In lung carcinoma,its expressive level was closely related to cell differentiation,lymph node metastasis and pathological types.Moreover,it may be an independent factor to prognosis of patients with lung carcinoma.

  19. EXPRESSION AND SIGNIFICANCE OF ERK PROTEIN IN HUMAN BREAST CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    张秀梅; 李柏林; 宋敏; 宋继谒

    2004-01-01

    Objective: To investigate the expression of ERK and p-ERK protein in human breast cancer and their corresponding tissue, to assess the significance of ERK signal pathway in tumorigenesis and progression of breast carcinoma. Methods: 40 breast cancer cases were used in S-P immunohistochemistry technique and Western Blot study. Results: The expression of ERK1, ERK2, and p- ERK protein levels increased remarkably in breast cancer tissues in comparison to normal tissues (P<0.01). The expression was upregulated by 1.32-, 1.53-and 4.27-fold, respectively. The overexpressions of ERK1, ERK2, and p- ERK proteins were obviously correlated with clinical stage of breast cancer. Protein levels of ERK and p-ERK were higher in stage III patients than in stage I and stage II patients (P<0.05). These proteins were strongly related with axillary lymph node metastasis of breast cancer, but not correlated with histopathological type and status of ER and PR of breast cancer. Expression of ERK1, and ERK2, protein showed a positive linear correlation. Conclusion: ERK signal transduction pathway is a key factor during human breast tumorigenesis and breast cancer progression.

  20. Expression of Extracellular Superoxide Dismutase Protein in Diabetes

    Directory of Open Access Journals (Sweden)

    Chul Han Kim

    2013-09-01

    Full Text Available Background Diabetes is characterized by chronic hyperglycemia, which can increase reactiveoxygen species (ROS production by the mitochondrial electron transport chain. The formationof ROS induces oxidative stress and activates oxidative damage-inducing genes in cells. Noresearch has been published on oxidative damage-related extracellular superoxide dismutase(EC-SOD protein levels in human diabetic skin. We investigated the expression of EC-SOD indiabetic skin compared with normal skin tissue in vivo.Methods The expression of EC-SOD protein was evaluated by western blotting in 6 diabeticskin tissue samples and 6 normal skin samples. Immunohistochemical staining was also carriedout to confirm the EC-SOD expression level in the 6 diabetic skin tissue samples.Results The western blotting showed significantly lower EC-SOD protein expression in thediabetic skin tissue than in the normal tissue. Immunohistochemical examination of EC-SODprotein expression supported the western blotting analysis.Conclusions Diabetic skin tissues express a relatively small amount of EC-SOD protein andmay not be protected against oxidative stress. We believe that EC-SOD is related to the alteredmetabolic state in diabetic skin, which elevates ROS production.

  1. Altered expression of renal bumetanide-sensitive sodium-pota-ssium-2 chloride cotransporter and Cl- channel -K2 gene in angiotensin Ⅱ-infused hypertensive rats

    Institute of Scientific and Technical Information of China (English)

    YE Tao; LIU Zhi-quan; SUN Chao-feng; ZHENG Yong; MA Ai-qun; FANG Yuan

    2005-01-01

    Background Little information is available regarding the effect of angiotensin Ⅱ (Ang Ⅱ) on the bumetanide-sensitive sodium-potassium-2 chloride cotransporter (NKCC2), the thiazide-sensitive sodium-chloride cotransporter (NCC), and the Cl- channel (CLC)-K2 at both mRNA and protein expression level in Ang Ⅱ-induced hypertensive rats. This study was conducted to investigate the influence of Ang Ⅱ with chronic subpressor infusion on nephron-specific gene expression of NKCC2, NCC and CLC-K2. Results Ang Ⅱ significantly increased blood pressure and up-regulated NKCC2 mRNA and protein expression in the kidney. Expression of CLC-K2 mRNA in the kidney increased 1.6 fold (P<0.05).There were no changes in NCC mRNA or protein expression in AngII-treated rats versus control. Conclusions Chronic subpressor Ang Ⅱ infusion can significantly alter NKCC2 and CLC-K2 mRNA expression in the kidney, and protein abundance of NKCC2 in kidney is positively regulated by Ang Ⅱ. These effects may contribute to enhanced renal Na+ and Cl- reabsorption in response to Ang Ⅱ.

  2. Differential effects of two-pore channel protein 1 and 2 silencing in MDA-MB-468 breast cancer cells.

    Science.gov (United States)

    Jahidin, Aisyah H; Stewart, Teneale A; Thompson, Erik W; Roberts-Thomson, Sarah J; Monteith, Gregory R

    2016-09-01

    Two-pore channel proteins, TPC1 and TPC2, are calcium permeable ion channels found localized to the membranes of endolysosomal calcium stores. There is increasing interest in the role of TPC-mediated intracellular signaling in various pathologies; however their role in breast cancer has not been extensively evaluated. TPC1 and TPC2 mRNA was present in all non-tumorigenic and tumorigenic breast cell lines assessed. Silencing of TPC2 but not TPC1 attenuated epidermal growth factor-induced vimentin expression in MDA-MB-468 breast cancer cells. This effect was not due to a general inhibition of epithelial to mesenchymal transition (EMT) as TPC2 silencing had no effect on epidermal growth factor (EGF)-induced changes on E-cadherin expression. TPC1 and TPC2 were also shown to differentially regulate cyclopiazonic acid (CPA)-mediated changes in cytosolic free Ca(2+). These findings indicate potential differential regulation of signaling processes by TPC1 and TPC2 in breast cancer cells. PMID:27353380

  3. Proteomics of protein expression profiling in tissues with different radiosensitivity

    International Nuclear Information System (INIS)

    The purpose of this study was to identify Radiosensitivity of proteins in tissues with different radiosensitivity. C3H/HeJ mice were exposed to 10 Gy. The mice were sacrifiud 8 hrs after radiation. Their spleen and liver tissues were collected and analyzed histologically for apoptosis. The expressions of radiosusceptibility protein were analyzed by 2-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The peak of apoptosis levels were 35.3 ± 1.7% in spleen and 0.6 ± 0.2% in liver at 8 hrs after radiation. Liver, radioresistant tissues, showed that the levels of ROS metabolism related to proteins such as cytochromm c, glutathione S transferase, NADH dehydrogenase, riken cDNA and peroxiredoxin VI increased after radiation. The expression of cytochrome c increased significantly in spleen and liver tissues after radiation. In spleen, radiosensitivity tissue, the identified proteins showed a significantly quantitative alteration following radiation. It was categorized as signal transduction, apoptosis, cytokine, Ca signal related protein, stress-related protein, cytoskeletal regulation, ROS metabolism, and others. Differences of radiation-induced apoptosis by tissues specifted were coupled with the induction of related radiosensitivity and radioresistant proteins. The result suggests that apoptosis relate protein and redox proteins play important roles in this radiosusceptibility

  4. Raf-1 kinase inhibitory protein expression in thyroid carcinomas.

    Science.gov (United States)

    Kim, Hyun-Soo; Kim, Gou Young; Lim, Sung-Jig; Kim, Youn Wha

    2010-12-01

    Raf-1 kinase inhibitory protein (RKIP) has been implicated in several fundamental signal transduction pathways that control cellular growth, differentiation, apoptosis and migration. RKIP is reduced in a variety of human carcinomas, but RKIP expression in thyroid carcinomas has not been analyzed at the protein level. In this study, we examined the immunohistochemical expression of RKIP in various subtypes of thyroid carcinoma. Immunostaining for RKIP was performed on 104 cases of primary thyroid carcinoma (40 papillary, 29 follicular, 11 medullary, 11 poorly differentiated, and 13 anaplastic carcinomas) and 26 cases of nodal metastatic tumor (17 papillary, 4 medullary, and 5 anaplastic carcinomas). Normal thyroid tissue and all cases of follicular, papillary, and medullary carcinomas showed uniform, strong cytoplasmic immunoreactivity for RKIP. With the exception of one case, poorly differentiated carcinomas also revealed strong RKIP expression. In contrast, RKIP expression was completely absent in all anaplastic carcinomas. The transition zone from the differentiated carcinoma component (strong RKIP expression) to the anaplastic carcinoma component (no RKIP expression) demonstrated a completely opposite pattern of RKIP immunoreactivity. This reduction of RKIP expression in anaplastic carcinoma was statistically significant (P carcinomas showed uniform, strong cytoplasmic RKIP immunoreactivity, in contrast, in metastatic anaplastic carcinomas, RKIP expression was completely absent. RKIP expression is significantly reduced in anaplastic thyroid carcinoma as compared to other subtypes of thyroid carcinoma. Further studies are necessary to elucidate the precise mechanism of RKIP action in anaplastic thyroid carcinoma.

  5. Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25

    Directory of Open Access Journals (Sweden)

    Medler Kathryn F

    2006-03-01

    Full Text Available Abstract Background Taste receptor cells are responsible for transducing chemical stimuli from the environment and relaying information to the nervous system. Bitter, sweet and umami stimuli utilize G-protein coupled receptors which activate the phospholipase C (PLC signaling pathway in Type II taste cells. However, it is not known how these cells communicate with the nervous system. Previous studies have shown that the subset of taste cells that expresses the T2R bitter receptors lack voltage-gated Ca2+ channels, which are normally required for synaptic transmission at conventional synapses. Here we use two lines of transgenic mice expressing green fluorescent protein (GFP from two taste-specific promoters to examine Ca2+ signaling in subsets of Type II cells: T1R3-GFP mice were used to identify sweet- and umami-sensitive taste cells, while TRPM5-GFP mice were used to identify all cells that utilize the PLC signaling pathway for transduction. Voltage-gated Ca2+ currents were assessed with Ca2+ imaging and whole cell recording, while immunocytochemistry was used to detect expression of SNAP-25, a presynaptic SNARE protein that is associated with conventional synapses in taste cells. Results Depolarization with high K+ resulted in an increase in intracellular Ca2+ in a small subset of non-GFP labeled cells of both transgenic mouse lines. In contrast, no depolarization-evoked Ca2+ responses were observed in GFP-expressing taste cells of either genotype, but GFP-labeled cells responded to the PLC activator m-3M3FBS, suggesting that these cells were viable. Whole cell recording indicated that the GFP-labeled cells of both genotypes had small voltage-dependent Na+ and K+ currents, but no evidence of Ca2+ currents. A subset of non-GFP labeled taste cells exhibited large voltage-dependent Na+ and K+ currents and a high threshold voltage-gated Ca2+ current. Immunocytochemistry indicated that SNAP-25 was expressed in a separate population of taste cells

  6. Protein Co-Expression Network Analysis (ProCoNA)

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. Expression of mRNA coding voltage - gated sodium channel α-subunit in spontaneously epileptic rat

    Institute of Scientific and Technical Information of China (English)

    DUWa; CAIJi-Qun

    2004-01-01

    OBJECTIVE Subtypes Ⅰ,Ⅱ and Ⅲ of sodium channel α- subunit mRNA were analyzed in adult rat brain of spontaneously epileptic rats, and investigated the relationship between sodium channel expression and epilepsy. METHODS Tissue samples were microdissected from occipital neocortex, CA1 and CA3 hippocampus areas and dentate gyms, observe

  8. Cardiac Expression of Skeletal Muscle Sodium Channels Increases Longitudinal Conduction Velocity in the Canine One Week Myocardial Infarction

    Science.gov (United States)

    Coronel, Ruben; Lau, David H; Sosunov, Eugene A; Janse, Michiel J; Danilo, Peter; Anyukhovsky, Evgeny P; Wilms-Schopman, Francien JG; Opthof, Tobias; Shlapakova, Iryna N; Ozgen, Nazira; Prestia, Kevin; Kryukova, Yelena; Cohen, Ira S.; Robinson, Richard B; Rosen, Michael R

    2013-01-01

    Background Skeletal muscle sodium channel (Nav1.4) expression in border zone myocardium increases action potential upstroke velocity in depolarized isolated tissue. Because resting membrane potential in the 1 week canine infarct is reduced, we hypothesized that conduction velocity (CV) is greater in Nav1.4 dogs compared to control dogs. Objective To measure CV in the infarct border zone border in dogs with and without Nav1.4 expression. Methods Adenovirus was injected in the infarct border zone in 34 dogs. The adenovirus incorporated the Nav1.4- and a green fluorescent protein (GFP) gene (Nav1.4 group, n=16) or only GFP (n=18). After 1 week, upstroke velocity and CV were measured by sequential microelectrode recordings at 4 and 7 mM [K+] in superfused epicardial slabs. High density in vivo epicardial activation mapping was performed in a subgroup (8 Nav1.4, 6 GFP) at 3–4 locations in the border zone. Microscopy and antibody staining confirmed GFP or Nav1.4 expression. Results Infarct sizes were similar between groups (30.6+/−3 % of LV mass, mean+/−SEM). Longitudinal CV was greater in Nav1.4- than in GFP- sites (58.5+/−1.8 vs 53.3+/−1.2 cm/s, 20 and 15 sites, respectively, p<0.05). Transverse CV was not different between the groups. In tissue slabs dV/dtmax was higher and CV was greater in Nav1.4 than in control at 7 mM [K+] (P<0.05). Immunohistochemical Nav1.4 staining was seen at the longitudinal ends of the myocytes. Conclusion Nav1.4 channels in myocardium surviving 1 week infarction increases longitudinal but not transverse CV, consistent with the increased dV/dtmax and with the cellular localization of Nav1.4. PMID:20385252

  9. Expression and Purification of SARS Coronavirus Membrane Protein

    Institute of Scientific and Technical Information of China (English)

    戴五星; 雷明军; 吴少庭; 陈智浩; 梁靓; 潘晖榕; 秦莉; 高士同; 袁仕善; 张仁利

    2004-01-01

    To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The re combinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropylβ-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0. 992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.

  10. Expression of Structural Protein E2 of Hepatitis C Virus

    Institute of Scientific and Technical Information of China (English)

    GUO Tai-lin; YE Lin-bo; MAO Can-quan

    2005-01-01

    The E2 glycoprotein is one of the structural components of the hepatitis C virus (HCV) virion. It elicits production of neutralising antibodies against the virus, and is involved in viral morphogenesis. The protein is considered as a major candidate for anti- HCV vaccine. Despitethis, little is known about this protein. Previous studies have focused on the and functional analysis of the glycosylated forms. This report describes expression of the E2 (recE2) in different forms and in different expression systems in Escherichia coli cells and in mammalian cells in order to obtain enough protein efficiently in vitro, in addition we also analysed the usage of rare codons in the genes of E2 and CORE. All results have shown that great efforts should be made to improve the expression efficiency of E2 in bacteria or mammalian cells.

  11. High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension.

    Science.gov (United States)

    Subedi, Ganesh P; Johnson, Roy W; Moniz, Heather A; Moremen, Kelley W; Barb, Adam

    2015-01-01

    The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield. PMID:26779721

  12. Commitment of Satellite Cells Expressing the Calcium Channel α2δ1 Subunit to the Muscle Lineage

    Directory of Open Access Journals (Sweden)

    Tammy Tamayo

    2012-01-01

    Full Text Available Satellite cells can maintain or repair muscle because they possess stem cell properties, making them a valuable option for cell therapy. However, cell transplants into skeletal muscle of patients with muscular dystrophy are limited by donor cell attachment, migration, and survival in the host tissue. Cells used for therapy are selected based on specific markers present in the plasma membrane. Although many markers have been identified, there is a need to find a marker that is expressed at different states in satellite cells, activated, quiescent, or differentiated cell. Furthermore, the marker has to be present in human tissue. Recently we reported that the plasma membrane α2δ1 protein is involved in cell attachment and migration in myoblasts. The α2δ1 subunit forms a part of the L-type voltage-dependent calcium channel in adult skeletal muscle. We found that the α2δ1 subunit is expressed in the majority of newly isolated satellite cells and that it appears earlier than the α1 subunits and at higher levels than the β or γ subunits. We also found that those cells that expressed α2δ1 would differentiate into muscle cells. This evidence indicates that the α2δ1 may be used as a marker of satellite cells that will differentiate into muscle.

  13. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    Science.gov (United States)

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. PMID:27284048

  14. A novel regulatory mechanism for whey acidic protein gene expression.

    OpenAIRE

    Chen, L.H.; Bissell, M J

    1989-01-01

    When primary mouse mammary epithelial cells (PMME) are cultured on a basement membrane type matrix, they undergo extensive morphogenesis leading to the formation of 3-dimensional alveoli-like spherical structures surrounding a closed lumen. We show for the first time that cells cultured on basement membrane-type matrix express high levels of whey acidic protein (WAP) mRNA and secrete the protein into the lumen. The expression of WAP appears to be dependent upon the formation of the alveoli-li...

  15. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Directory of Open Access Journals (Sweden)

    Jacob L Lilly

    Full Text Available 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.

  16. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Science.gov (United States)

    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.

  17. Expression of surface hydrophobic proteins by Candida albicans in vivo.

    OpenAIRE

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

    1995-01-01

    Candida albicans modulates cell surface hydrophobicity during growth and morphogenesis in vitro. To determine if surface hydrophobicity is expressed during pathogenesis, we generated a polyclonal antiserum against yeast hydrophobic proteins. The antiserum was then used for indirect immunofluorescence analysis of tissues from mice colonized and chronically infected with C. albicans. Results demonstrated that yeast hydrophobic proteins are exposed on fungal cells present in host tissues. The po...

  18. Coordination of murine parotid secretory protein and salivary amylase expression.

    OpenAIRE

    Poulsen, K; Jakobsen, B K; Mikkelsen, B M; Harmark, K; Nielsen, J T; Hjorth, J P

    1986-01-01

    PSP, parotid secretory protein, and salivary amylase are the major secretory proteins of mouse parotid gland where they appear in a constant ratio. Here we describe the isolation of the PSP gene and show through expression analysis on this and the salivary amylase gene that the two genes are transcribed in a coordinate fashion in adult animals, whereas the activation profiles are different during postnatal development. An explanation is put forward that involves activation of the genes at dif...

  19. Differential protein expression in perfusates from metastasized rat livers

    OpenAIRE

    Zhang, Yang; Li, Menglin; Wei, Lilong; Zhu, Lisi; Hu, Siqi; Wu, Shuzhen; Ma, Sucan; Gao, Youhe

    2013-01-01

    Background Liver perfusates exhibit theoretical advantages regarding the discovery of disease biomarkers because they contain proteins that readily enter the blood-stream, and perfusion preserves the disease state in its natural context. The purpose of the study is to explore the value of liver perfusate proteome in the biomarker discovery of liver diseases. Results In this study, 86 differentially expressed proteins were identified in perfusates from isolated rat livers metastasized by Walke...

  20. Molecular characterization and functional expression of the Apis mellifera voltage-dependent Ca2+ channels.

    Science.gov (United States)

    Cens, Thierry; Rousset, Matthieu; Collet, Claude; Charreton, Mercedes; Garnery, Lionel; Le Conte, Yves; Chahine, Mohamed; Sandoz, Jean-Christophe; Charnet, Pierre

    2015-03-01

    Voltage-gated Ca(2+) channels allow the influx of Ca(2+) ions from the extracellular space upon membrane depolarization and thus serve as a transducer between membrane potential and cellular events initiated by Ca(2+) transients. Most insects are predicted to possess three genes encoding Cavα, the main subunit of Ca(2+) channels, and several genes encoding the two auxiliary subunits, Cavβ and Cavα2δ; however very few of these genes have been cloned so far. Here, we cloned three full-length cDNAs encoding the three Cavα subunits (AmelCav1a, AmelCav2a and AmelCav3a), a cDNA encoding a novel variant of the Cavβ subunit (AmelCavβc), and three full-length cDNAs encoding three Cavα2δ subunits (AmelCavα2δ1 to 3) of the honeybee Apis mellifera. We identified several alternative or mutually exclusive exons in the sequence of the AmelCav2 and AmelCav3 genes. Moreover, we detected a stretch of glutamine residues in the C-terminus of the AmelCav1 subunit that is reminiscent of the motif found in the human Cav2.1 subunit of patients with Spinocerebellar Ataxia type 6. All these subunits contain structural domains that have been identified as functionally important in their mammalian homologues. For the first time, we could express three insect Cavα subunits in Xenopus oocytes and we show that AmelCav1a, 2a and 3a form Ca(2+) channels with distinctive properties. Notably, the co-expression of AmelCav1a or AmelCav2a with AmelCavβc and AmCavα2δ1 produces High Voltage-Activated Ca(2+) channels. On the other hand, expression of AmelCav3a alone leads to Low Voltage-Activated Ca(2+) channels. PMID:25602183

  1. RNA Binding Proteins that Control Human Papillomavirus Gene Expression.

    OpenAIRE

    Naoko Kajitani; Stefan Schwartz

    2015-01-01

    The human papillomavirus (HPV) life cycle is strictly linked to the differentiation program of the infected mucosal epithelial cell. In the basal and lower levels of the epithelium, early genes coding for pro-mitotic proteins and viral replication factors are expressed, while terminal cell differentiation is required for activation of late gene expression and production of viral particles at the very top of the epithelium. Such productive infections are normally cleared within 18–24 months. I...

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

    International Nuclear Information System (INIS)

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

  3. Recombinant protein expression by targeting pre-selected chromosomal loci

    Directory of Open Access Journals (Sweden)

    Krömer Wolfgang

    2009-12-01

    Full Text Available Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs were targeted by Flp recombinase mediated cassette exchange (RMCE. The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context

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

    Directory of Open Access Journals (Sweden)

    Meligonis G

    2005-01-01

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

  5. Angiotensin-2-mediated Ca2+ signaling in the retinal pigment epithelium: role of angiotensin-receptor-associated-protein and TRPV2 channel.

    Directory of Open Access Journals (Sweden)

    Rene Barro-Soria

    Full Text Available Angiotensin II (AngII receptor (ATR is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap, and transient-receptor-potential channel-V2 (TRPV2. AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE cells by AngII results in biphasic increases in intracellular free Ca(2+inhibited by losartan. Xestospongin C (xest C and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca(2+response. RPE cells from Atrap(-/- mice showed smaller AngII-evoked Ca(2+peak (by 22% and loss of sustained Ca(2+elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD at 15 µM stimulates intracellular Ca(2+-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor reduced the cannabidiol-induced Ca(2+-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca(2+transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca(2+transients in the RPE by releasing Ca(2+from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca(2+elevation.

  6. Controlling for gene expression changes in transcription factor protein networks.

    Science.gov (United States)

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

    2014-06-01

    The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein-protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions.

  7. Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium.

    Science.gov (United States)

    Monaghan, Kevin; McNaughten, Jennifer; McGahon, Mary K; Kelly, Catriona; Kyle, Daniel; Yong, Phaik Har; McGeown, J Graham; Curtis, Tim M

    2015-01-01

    Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25 mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months' streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the

  8. Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium.

    Directory of Open Access Journals (Sweden)

    Kevin Monaghan

    Full Text Available Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs. Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA, which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25 mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months' streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes

  9. VELOCITY DISTRIBUTION IN TRAPEZOID-SECTION OPEN CHANNEL FLOW WITH A NEW REYNOLDS-STRESS EXPRESSION

    Institute of Scientific and Technical Information of China (English)

    Ma Zheng

    2003-01-01

    By considering that the coherent structure is the main cause of the Reynolds stress, a new Reynolds stress expression was given. On this basis the velocity distribution in the trapezoid-section open channel flow was worked out with the pseudo-spectral method. The results were compared with experimental data and the influence of the ratio of length to width of the cross-section and the lateral inclination on the velocity distribution was analyzed. This model can be used the large flux in rivers and open channes.

  10. Aberrant LRP16 protein expression in primary neuroendocrine lung tumors

    OpenAIRE

    Shao, Yun; Li, Xiaoying; Lu, Yali; Liu, Lin; Zhao, Po

    2015-01-01

    Background: The Leukemia related protein 16 gene (LRP16) localized on chromosome 11q12.1, is an important estrogen-responsive gene and a crucial regulator for NF-kB activation. LRP16 is frequently expressed in human cancers; however, the LRP16 gene remains unexplored in lung neuroendocrine tumors. The aim of this study was to investigate the role of LRP16 expression in primary lung neuroendocrine tumors. Methods: lung neuroendocrine tumors were analyzed for LRP16 gene expression by two-step n...

  11. TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

    Science.gov (United States)

    Carvacho, Ingrid; Ardestani, Goli; Lee, Hoi Chang; McGarvey, Kaitlyn; Fissore, Rafael A.; Lykke-Hartmann, Karin

    2016-01-01

    The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes (germinal vesicle stage), matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development. PMID:27681336

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

    Science.gov (United States)

    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…

  13. Protein expression on Cr resistant microorganism using electrophoresis method

    Directory of Open Access Journals (Sweden)

    SAJIDAN

    2009-01-01

    Full Text Available Fatmawati U, Suranto, Sajidan. 2009. Protein expression on Cr resistant microorganism using electrophoresis method. Nusantara Bioscience 1: 31-37. Hexavalent chromium (Cr(VI is known as toxic heavy metals, so the need is reduced to Cr(III is much less toxicity. Pseudomonas aeruginosa, Pseudomonas putida, Klebsiella pneumoniae, Pantoea sp. and Saccharomyces cerevisiae are resistant Cr(VI microorganism and have ability to reduce Cr(VI. The aim of this research is to know ability of microorganism to reduce Cr(VI and to know protein band pattern between Cr(VI resistant microorganism and non resistant microorganism which inoculated on LB broth. SDS-PAGE was used to indentify protein expression. While, Cr(VI concentration was identified by 1.5 diphenylcarbazide method. The quantitative data was analyzed by two factorial ANOVA that continued with DMRT at 1% level test. The qualitative data i.e. protein expression analyzed by relative mobility (Rf. The results showed that the ability of microorganisms to reduce Cr(VI at initial concentration of 0.5 ppm, 1 ppm, 5 ppm and 10 ppm may vary, the average percentage of the ability of each microorganism in reducing Cr(VI is P. putida (65% > S. cerevisiae (64.45% >. P. aeruginosa (60.73% > Pantoea sp. (50.22% > K. pneumoniae (47.82% > without microorganisms (34.25%. The adding microorganisms have significantly influenced toward reduction of Cr(VI. The SDS-PAGE shows that protein expression between resistant and not resistant microorganisms are no different, but resistant microorganisms have more protein (protein band is thicker.

  14. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    Directory of Open Access Journals (Sweden)

    Nishizaki Takashi

    2006-07-01

    Full Text Available Abstract Background Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. Methods The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. Results The Bcl-2 protein expression was found to be decreased in 105 (42% cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089 associated with a worse disease free survival (DFS, while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. Conclusion The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer.

  15. ASIC3, an acid-sensing ion channel, is expressed in metaboreceptive sensory neurons

    Directory of Open Access Journals (Sweden)

    Fierro Leonardo

    2005-11-01

    Full Text Available Abstract Background ASIC3, the most sensitive of the acid-sensing ion channels, depolarizes certain rat sensory neurons when lactic acid appears in the extracellular medium. Two functions have been proposed for it: 1 ASIC3 might trigger ischemic pain in heart and muscle; 2 it might contribute to some forms of touch mechanosensation. Here, we used immunocytochemistry, retrograde labelling, and electrophysiology to ask whether the distribution of ASIC3 in rat sensory neurons is consistent with either of these hypotheses. Results Less than half (40% of dorsal root ganglion sensory neurons react with anti-ASIC3, and the population is heterogeneous. They vary widely in cell diameter and express different growth factor receptors: 68% express TrkA, the receptor for nerve growth factor, and 25% express TrkC, the NT3 growth factor receptor. Consistent with a role in muscle nociception, small ( Conclusion Our data indicates that: 1 ASIC3 is expressed in a restricted population of nociceptors and probably in some non-nociceptors; 2 co-expression of ASIC3 and CGRP, and the absence of P2X3, are distinguishing properties of a class of sensory neurons, some of which innervate blood vessels. We suggest that these latter afferents may be muscle metaboreceptors, neurons that sense the metabolic state of muscle and can trigger pain when there is insufficient oxygen.

  16. STIM1 and STIM2 proteins differently regulate endogenous store-operated channels in HEK293 cells.

    Science.gov (United States)

    Shalygin, Alexey; Skopin, Anton; Kalinina, Vera; Zimina, Olga; Glushankova, Lyuba; Mozhayeva, Galina N; Kaznacheyeva, Elena

    2015-02-20

    The endoplasmic reticulum calcium sensors stromal interaction molecules 1 and 2 (STIM1 and STIM2) are key modulators of store-operated calcium entry. Both these sensors play a major role in physiological functions in normal tissue and in pathology, but available data on native STIM2-regulated plasma membrane channels are scarce. Only a few studies have recorded STIM2-induced CRAC (calcium release-activated calcium) currents. On the other hand, many cell types display store-operated currents different from CRAC. The STIM1 protein regulates not only CRAC but also transient receptor potential canonical (TRPC) channels, but it has remained unclear whether STIM2 is capable of regulating store-operated non-CRAC channels. Here we present for the first time experimental evidence for the existence of endogenous non-CRAC STIM2-regulated channels. As shown in single-channel patch clamp experiments on HEK293 cells, selective activation of native STIM2 proteins or STIM2 overexpression results in store-operated activation of Imin channels, whereas STIM1 activation blocks this process. Changes in the ratio between active STIM2 and STIM1 proteins can switch the regulation of Imin channels between store-operated and store-independent modes. We have previously characterized electrophysiological properties of different Ca(2+) influx channels coexisting in HEK293 cells. The results of this study show that STIM1 and STIM2 differ in the ability to activate these store-operated channels; Imin channels are regulated by STIM2, TRPC3-containing INS channels are induced by STIM1, and TRPC1-composed Imax channels are activated by both STIM1 and STIM2. These new data about cross-talk between STIM1 and STIM2 and their different roles in store-operated channel activation are indicative of an additional level in the regulation of store-operated calcium entry pathways.

  17. THE CLINICAL EXPRESSION OF HEREDITARY PROTEIN-C AND PROTEIN-S DEFICIENCY - A RELATION TO CLINICAL THROMBOTIC RISK-FACTORS AND TO LEVELS OF PROTEIN-C AND PROTEIN-S

    NARCIS (Netherlands)

    HENKENS, CMA; VANDERMEER, J; HILLEGE, JL; BOM, VJJ; HALIE, MR; van der Schaaf, W

    1993-01-01

    We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were

  18. Breast Cancer Resistance Protein Expression and 5-Fluorouracil Resistance

    Institute of Scientific and Technical Information of China (English)

    JIAN-HUI YUAN; ZHI-XIONG ZHUANG; JIN-QUAN CHENG; LONG-YUAN JIANG; WEI-DONG JI; LIANG-FENG GUO; JIAN-JUN LIU; XING-YUN XU; JING-SONG HE; XIAN-MING WANG

    2008-01-01

    To filtrate breast cancer resistance protein (BCRP)-mediated resistant agents and to investigate clinical relationship between BCRP expression and drug resistance. Methods MTT assay was performed to filtrate BCRP-mediated resistant agents with BCRP expression cell model and to detect chemosensitivity of breast cancer tissue specimens to these agents. A high performance liquid chromatography (HPLC) assay was established, and was used to measure the relative dose of intracellular retention resistant agents. RT-PCR and immununohistochemistry (IHC) were employed to investigate the BCRP expression in breast cancer tissue specimens. Results MTT assay showed that the expression of BCRP increased with the increasing resistance of 5-fluorouracil (5-Fu) (P=0.8124, P<0.01). Condusion Resistance to 5-Fu can be mediated by BCRP. Clinical chemotherapy for breast cancer patients can be optimized based on BCRP-positive expression.

  19. Expression and localization of inwardly rectifying potassium channel Kit2.1 in glia cells of native bovine retina

    Institute of Scientific and Technical Information of China (English)

    PAN Ai-hua; LUO Xue-gang

    2005-01-01

    Objective The purpose of this study is to identify the molecular basis of the contacting -neuron membrane K+ conductance in glia cells of native bovine retina. Methods RT-PCR, Northern blot and Western blot analyses were used to detect the expression of the inwardly rectifying K+ (Kir) channel subunits Kir2.1 in native bovine RPE and neural retina. The distribution of Kir2.1 protein was determined in frozen sections of bovine retina-RPEchoroid by indirect immunofluorescence analysis. Results RT-PCR analysis reveals Kir2.1 transcript in both RPE and neural retina. In Northern blots, Kir2.1 probe hybridizes to an appropriately sized-transcript in neural retina but not in RPE. In Western blots, Kir2.1 antibody recognizes a major monomer of about 60 kDa in neural retina but not in RPE. Immunofluorescence reveals that Kir2.1 immunostaining is expressed at many parts of Muller cells, especially in the membrane domains of Muller cells that contact retinal neurons, i. e. , along the two stem processes,over the soma, and in the side branches extending into the synaptic layers. No immunostaining is seen in RPE. Doubling staining shows that Kir2.1 proteins and glutamine synthetase proteins which are a marker of Muller cell co-localized well. Conclusions These results reveal that Kir2.1 is localized in the Muller cells, no Kir2.1 in RPE. These data suggests that Kir2.1 may be involved in the transport of K+ in the bovine neural retina.

  20. Expression and motor functional roles of voltage-dependent type 7 K(+) channels in the human taenia coli.

    Science.gov (United States)

    Adduci, Alice; Martire, Maria; Taglialatela, Maurizio; Arena, Vincenzo; Rizzo, Gianluca; Coco, Claudio; Currò, Diego

    2013-12-01

    Voltage-dependent type 7 K(+) (KV7 or KCNQ) channels modulate the excitability of neurons and muscle cells. The aims of the present study were to investigate the motor effects of KV7 channel modulators and the expression of KV7 channels in the human taenia coli. The effects of KV7 channel modulators on the muscle tone of human taenia coli strips were investigated under nonadrenergic non-nitrergic conditions by organ bath studies. Gene expression and tissue localisation of channels were studied by real-time PCR and immunohistochemistry, respectively. Under basal conditions, the KV7 channel blocker XE-991 induced concentration-dependent contractions, with mean EC50 and Emax of 18.7 μM and 30.5% respectively of the maximal bethanechol-induced contraction, respectively. The KV7 channel activators retigabine and flupirtine concentration-dependently relaxed the taenia coli, with mean EC50s of 19.2 μM and 29.9 μM, respectively. Retigabine also relaxed bethanechol-precontracted strips, with maximal relaxations of 79.2% of the bethanecol-induced precontraction. The motor effects induced by the KV7 channel modulators were not affected by tetrodotoxin or ω-conotoxin GVIA. XE-991 greatly reduced retigabine- and flupirtine-induced relaxations. Transcripts encoded by all KCNQ genes were detected in the taenia coli, with KCNQ4 showing the highest expression levels. KV7.4 channels were clearly visualised by immunohistochemistry in colonic epithelium, circular muscle layer and taenia coli. KV7 channels appear to contribute to the resting muscle tone of the human taenia coli. In addition, KV7 channel activators significantly relax the taenia coli. Thus, they could be useful therapeutic relaxant agents for colonic motor disorders. PMID:24120659

  1. Mechanical stimulation increases proliferation, differentiation and protein expression in culture

    DEFF Research Database (Denmark)

    Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

    2007-01-01

    the cells are able to to regulate Myo-D and myogenin is poorly understood. In the present work, we investigate the role of mechanical loading, through specific receptors to intracellular matrix proteins such as laminin and fibronectin, in both Myo-D and myogenin expression in C(2)C(12) cells. We propose...... recepotors plays a role in myoblast differentiation and fusion....

  2. Proteomics of protein expression profiling in tissues with different radiosensitivity

    International Nuclear Information System (INIS)

    Ionizing radiation activates multiple signaling pathways, resulting in diverse stress responses including apoptosis, cell cycle arrest, and gene induction. Liver tissue is known to be rather resistant to radiation while a spleen tissue is highly radiosentitive. Our purpose was to compare radioresponse in liver and spleen following exposure to radiation to further investigate the differentially protein expression profile in radiosensitive and radioresistant tissues

  3. Heterologous expression of membrane proteins: choosing the appropriate host.

    Directory of Open Access Journals (Sweden)

    Florent Bernaudat

    Full Text Available 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.

  4. Optimization of translation profiles enhances protein expression and solubility.

    Directory of Open Access Journals (Sweden)

    Anne-Katrin Hess

    Full Text Available mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  5. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins.

    Science.gov (United States)

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-04-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

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

    Energy Technology Data Exchange (ETDEWEB)

    Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

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

    International Nuclear Information System (INIS)

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1∧E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1∧E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1∧E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1∧E4, these kinases

  8. Cooperative regulation by G proteins and Na+ of neuronal GIRK2 K+ channels

    Science.gov (United States)

    Wang, Weiwei; Touhara, Kouki K; Weir, Keiko; Bean, Bruce P; MacKinnon, Roderick

    2016-01-01

    G protein gated inward rectifier K+ (GIRK) channels open and thereby silence cellular electrical activity when inhibitory G protein coupled receptors (GPCRs) are stimulated. Here we describe an assay to measure neuronal GIRK2 activity as a function of membrane-anchored G protein concentration. Using this assay we show that four Gβγ subunits bind cooperatively to open GIRK2, and that intracellular Na+ – which enters neurons during action potentials – further amplifies opening mostly by increasing Gβγ affinity. A Na+ amplification function is characterized and used to estimate the concentration of Gβγ subunits that appear in the membrane of mouse dopamine neurons when GABAB receptors are stimulated. We conclude that GIRK2, through its dual responsiveness to Gβγ and Na+, mediates a form of neuronal inhibition that is amplifiable in the setting of excess electrical activity. DOI: http://dx.doi.org/10.7554/eLife.15751.001 PMID:27074662

  9. L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

    Science.gov (United States)

    Maddala, Rupalatha; Nagendran, Tharkika; de Ridder, Gustaaf G; Schey, Kevin L; Rao, Ponugoti Vasantha

    2013-01-01

    Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial

  10. L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

    Directory of Open Access Journals (Sweden)

    Rupalatha Maddala

    Full Text Available Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations

  11. Aplysia synapse associated protein (APSAP): identification, characterization, and selective interactions with Shaker-type potassium channels

    OpenAIRE

    Reissner, Kathryn J.; Boyle, Heather D.; Ye, Xiaojing; Carew, Thomas J.

    2007-01-01

    The vertebrate post-synaptic density (PSD) is a region of high molecular complexity in which dynamic protein interactions modulate receptor localization and synaptic function. Members of the membrane-associated guanylate kinase (MAGUK) family of proteins represent a major structural and functional component of the vertebrate PSD. In order to investigate the expression and significance of orthologous PSD components associated with the Aplysia sensory neuron-motor neuron synapse, we have cloned...

  12. Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

    Science.gov (United States)

    Sawatsky, Bevan; Bente, Dennis A; Czub, Markus; von Messling, Veronika

    2016-05-01

    The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle. PMID:26813519

  13. Expression and localization of X11 family proteins in neurons.

    Science.gov (United States)

    Motodate, Rika; Saito, Yuhki; Hata, Saori; Suzuki, Toshiharu

    2016-09-01

    The X11/Mint family of proteins comprises X11/X11α/Mint1, X11L/X11β/Mint2, and X11L2/X11γ/Mint3. Each of these molecules is an adaptor protein that contains a phosphotyrosine interaction/binding (PI/PTB) and two PDZ domains in its carboxy-terminal region. X11/Mint family members associate with a broad spectrum of membrane proteins, including Alzheimer's β-amyloid precursor protein (APP), alcadeins, and low density lipoprotein receptor proteins, as well as various cytoplasmic proteins including Arf, kalirin-7, and Munc18. In particular, X11 and X11L are thought to play various roles in the regulation of neural functions in brain. Nevertheless, the protein levels and respective localization of individual family members remain controversial. We analyzed the protein levels of X11 and X11L in the corresponding single- and double-knockout mice. X11 and X11L did not exhibit obvious changes of their protein levels when the other was absent, especially in cerebrum in which they were widely co-expressed. In cerebellum, X11 and X11L localized in characteristic patterns in various types of neurons, and X11 protein level increased without an obvious ectopic localization in X11L-knockout mice. Interestingly, only X11L protein existed specifically in brain, whereas, contrary to the accepted view, X11 protein was detected at the highest levels in brain but was also strongly detected in pancreas, testis, and paranephros. Together, our results indicate that both X11 and X11L exert largely in brain neurons, but X11 may also function in peripheral tissues. PMID:27268412

  14. Expression patterns, mutation detection and RNA interference of Rhopalosiphum padi voltage-gated sodium channel genes

    Science.gov (United States)

    Zuo, Yayun; Peng, Xiong; Wang, Kang; Lin, Fangfei; Li, Yuting; Chen, Maohua

    2016-07-01

    The voltage-gated sodium channel (VGSC) is the target of sodium-channel-blocking insecticides. Traditionally, animals were thought to have only one VGSC gene comprising a α-subunit with four homologous domains (DI–DIV). The present study showed that Rhopalosiphum padi, an economically important crop pest, owned a unique heterodimeric VGSC (H1 and H2 subunits) encoded by two genes (Rpvgsc1 and Rpvgsc2), which is unusual in insects and other animals. The open reading frame (ORF) of Rpvgsc1 consisted 1150 amino acids, and the ORF of Rpvgsc2 had 957 amino acids. Rpvgsc1 showed 64.1% amino acid identity to DI–DII of Drosophila melanogaster VGSC and Rpvgsc2 showed 64.0% amino acid identity to DIII–DIV of D. melanogaster VGSC. A M918L mutation previously reported in pyrethroids-resistant strains of other insects was found in the IIS4-S6 region of R. padi field sample. The two R. padi VGSC genes were expressed at all developmental stages and showed similar expression patterns after treatment with beta-cypermethrin. Knockdown of Rpvgsc1 or Rpvgsc2 caused significant reduction in mortality rate of R. padi after exposure to beta-cypermethrin. These findings suggest that the two R. padi VGSC genes are both functional genes.

  15. Expression patterns, mutation detection and RNA interference of Rhopalosiphum padi voltage-gated sodium channel genes

    Science.gov (United States)

    Zuo, Yayun; Peng, Xiong; Wang, Kang; Lin, Fangfei; Li, Yuting; Chen, Maohua

    2016-07-01

    The voltage-gated sodium channel (VGSC) is the target of sodium-channel-blocking insecticides. Traditionally, animals were thought to have only one VGSC gene comprising a α-subunit with four homologous domains (DI-DIV). The present study showed that Rhopalosiphum padi, an economically important crop pest, owned a unique heterodimeric VGSC (H1 and H2 subunits) encoded by two genes (Rpvgsc1 and Rpvgsc2), which is unusual in insects and other animals. The open reading frame (ORF) of Rpvgsc1 consisted 1150 amino acids, and the ORF of Rpvgsc2 had 957 amino acids. Rpvgsc1 showed 64.1% amino acid identity to DI-DII of Drosophila melanogaster VGSC and Rpvgsc2 showed 64.0% amino acid identity to DIII-DIV of D. melanogaster VGSC. A M918L mutation previously reported in pyrethroids-resistant strains of other insects was found in the IIS4-S6 region of R. padi field sample. The two R. padi VGSC genes were expressed at all developmental stages and showed similar expression patterns after treatment with beta-cypermethrin. Knockdown of Rpvgsc1 or Rpvgsc2 caused significant reduction in mortality rate of R. padi after exposure to beta-cypermethrin. These findings suggest that the two R. padi VGSC genes are both functional genes.

  16. Selective expression of KCNS3 potassium channel α-subunit in parvalbumin-containing GABA neurons in the human prefrontal cortex.

    Directory of Open Access Journals (Sweden)

    Danko Georgiev

    Full Text Available The cognitive deficits of schizophrenia appear to be associated with altered cortical GABA neurotransmission in the subsets of inhibitory neurons that express either parvalbumin (PV or somatostatin (SST. Identification of molecular mechanisms that operate selectively in these neurons is essential for developing targeted therapeutic strategies that do not influence other cell types. Consequently, we sought to identify, in the human cortex, gene products that are expressed selectively by PV and/or SST neurons, and that might contribute to their distinctive functional properties. Based on previously reported expression patterns in the cortex of mice and humans, we selected four genes: KCNS3, LHX6, KCNAB1, and PPP1R2, encoding K(+ channel Kv9.3 modulatory α-subunit, LIM homeobox protein 6, K(+ channel Kvβ1 subunit, and protein phosphatase 1 regulatory subunit 2, respectively, and examined their colocalization with PV or SST mRNAs in the human prefrontal cortex using dual-label in situ hybridization with (35S- and digoxigenin-labeled antisense riboprobes. KCNS3 mRNA was detected in almost all PV neurons, but not in SST neurons, and PV mRNA was detected in >90% of KCNS3 mRNA-expressing neurons. LHX6 mRNA was detected in almost all PV and >90% of SST neurons, while among all LHX6 mRNA-expressing neurons 50% expressed PV mRNA and >44% expressed SST mRNA. KCNAB1 and PPP1R2 mRNAs were detected in much larger populations of cortical neurons than PV or SST neurons. These findings indicate that KCNS3 is a selective marker of PV neurons, whereas LHX6 is expressed by both PV and SST neurons. KCNS3 and LHX6 might be useful for characterizing cell-type specific molecular alterations of cortical GABA neurotransmission and for the development of novel treatments targeting PV and/or SST neurons in schizophrenia.

  17. Selective expression of KCNS3 potassium channel α-subunit in parvalbumin-containing GABA neurons in the human prefrontal cortex.

    Science.gov (United States)

    Georgiev, Danko; González-Burgos, Guillermo; Kikuchi, Mitsuru; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

    2012-01-01

    The cognitive deficits of schizophrenia appear to be associated with altered cortical GABA neurotransmission in the subsets of inhibitory neurons that express either parvalbumin (PV) or somatostatin (SST). Identification of molecular mechanisms that operate selectively in these neurons is essential for developing targeted therapeutic strategies that do not influence other cell types. Consequently, we sought to identify, in the human cortex, gene products that are expressed selectively by PV and/or SST neurons, and that might contribute to their distinctive functional properties. Based on previously reported expression patterns in the cortex of mice and humans, we selected four genes: KCNS3, LHX6, KCNAB1, and PPP1R2, encoding K(+) channel Kv9.3 modulatory α-subunit, LIM homeobox protein 6, K(+) channel Kvβ1 subunit, and protein phosphatase 1 regulatory subunit 2, respectively, and examined their colocalization with PV or SST mRNAs in the human prefrontal cortex using dual-label in situ hybridization with (35)S- and digoxigenin-labeled antisense riboprobes. KCNS3 mRNA was detected in almost all PV neurons, but not in SST neurons, and PV mRNA was detected in >90% of KCNS3 mRNA-expressing neurons. LHX6 mRNA was detected in almost all PV and >90% of SST neurons, while among all LHX6 mRNA-expressing neurons 50% expressed PV mRNA and >44% expressed SST mRNA. KCNAB1 and PPP1R2 mRNAs were detected in much larger populations of cortical neurons than PV or SST neurons. These findings indicate that KCNS3 is a selective marker of PV neurons, whereas LHX6 is expressed by both PV and SST neurons. KCNS3 and LHX6 might be useful for characterizing cell-type specific molecular alterations of cortical GABA neurotransmission and for the development of novel treatments targeting PV and/or SST neurons in schizophrenia.

  18. Role of protein sulfation in vasodilation induced by minoxidil sulfate, a K+ channel opener

    Energy Technology Data Exchange (ETDEWEB)

    Meisheri, K.D.; Oleynek, J.J.; Puddington, L. (Cardiovascular Diseases Research, Upjohn Laboratories, Upjohn Company, Kalamazoo, MI (United States))

    1991-09-01

    Evidence from contractile, radioisotope ion flux and electrophysiological studies suggest that minoxidil sulfate (MNXS) acts as a K+ channel opener in vascular smooth muscle. This study was designed to examine possible biochemical mechanisms by which MNXS exerts such an effect. Experiments performed in the isolated rabbit mesenteric artery (RMA) showed that MNXS, 5 microM, but not the parent compound minoxidil, was a potent vasodilator. Whereas the relaxant effects of an another K+ channel opener vasodilator, BRL-34915 (cromakalim), were removed by washing with physiological saline solution, the effects of MNXS persisted after repeated washout attempts. Furthermore, after an initial exposure of segments of intact RMA to (35S) MNXS, greater than 30% of the radiolabel was retained 2 hr after removal of the drug. In contrast, retention of radiolabel was not detected with either (3H)MNXS (label on the piperidine ring of MNXS) or (3H)minoxidil (each less than 3% after a 2-hr washout). These data suggested that the sulfate moiety from MNXS was closely associated with the vascular tissue. To determine if proteins were the acceptors of sulfate from MNXS, intact RMAs were incubated with (35S)MNXS, and then 35S-labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by fluorography. Preferential labeling of a 116 kD protein was detected by 2 and 5 min of treatment. A 43 kD protein (resembling actin) also showed significant labeling. A similar profile of 35S-labeled proteins was observed in (35S) MNXS-treated A7r5 rat aortic smooth muscle cells, suggesting that the majority of proteins labeled by (35S)MNXS in intact RMA were components of smooth muscle cells.

  19. Online multi-channel microfluidic chip-mass spectrometry and its application for quantifying noncovalent protein-protein interactions.

    Science.gov (United States)

    Liu, Wu; Chen, Qiushui; Lin, Xuexia; Lin, Jin-Ming

    2015-03-01

    To establish an automatic and online microfluidic chip-mass spectrometry (chip-MS) system, a device was designed and fabricated for microsampling by a hybrid capillary. The movement of the capillary was programmed by a computer to aspirate samples from different microfluidic channels in the form of microdroplets (typically tens of nanoliters in volume), which were separated by air plugs. The droplets were then directly analyzed by MS via paper spray ionization without any pretreatment. The feasibility and performance were demonstrated by a concentration gradient experiment. Furthermore, after eliminating the effect of nonuniform response factors by an internal standard method, determination of the association constant within a noncovalent protein-protein complex was successfully accomplished with the MS-based titration indicating the versatility and the potential of this novel platform for widespread applications. PMID:25597452

  20. Differential gene expression patterns and colocalization of ATP-gated P2X6/P2X4 ion channels during rat small intestine ontogeny.

    Science.gov (United States)

    Padilla, Karla; Gonzalez-Mendoza, David; Berumen, Laura C; Escobar, Jesica E; Miledi, Ricardo; García-Alcocer, Guadulupe

    2016-07-01

    Gene coding for ATP-gated receptor ion channels (P2X1-7) has been associated with the developmental process in various tissues; among these ion channel subtypes, P2X6 acts as a physiological regulator of P2X4 receptor functions when the two receptors form heteroreceptors. The P2X4 receptor is involved in pain sensation, the inflammatory process, and body homeostasis by means of Mg(2+) absorption through the intestine. The small intestine is responsible for the absorption and digestion of nutrients; throughout its development, several gene expressions are induced that are related to nutrients received, metabolism, and other intestine functions. Previous work has shown a differential P2X4 and P2X6 protein distribution in the small intestine of newborn and adult rats; however, it is not well-known at what age the change in the relationship between the gene and protein expression occurs and whether or not these receptors are colocalized. In this work, we evaluate P2X4 and P2X6 gene expression patterns by qPCR from embryonic (E18, P0, P7, P17, P30) to adult age in rat gut, as well as P2X6/P2X4 colocalization using qRT-PCR and confocal immunofluorescence in proximal and distal small intestine sections. The results showed that P2X6 and P2X4 gene expression levels of both receptors decreased at the embryonic-perinatal transition, whereas from ages P17 to P30 (suckling-weaning transition) both receptors increased their gene expression levels. Furthermore, P2X4 and P2X6 proteins were expressed in a different way during rat small intestine development, showing a higher colocalization coefficient at age P30 in both intestine regions. Those results suggest that purinergic receptors may play a role in intestinal maturation, which is associated with age and intestinal region.

  1. AB095. Increased expression of TMEM16A/Ano1 chloride channel associated with diabetic erectile dysfunction

    Science.gov (United States)

    Ruan, Yajun; Chen, Yingwei; Li, Mingchao; Wang, Tao; Yang, Jun; Rao, Ke; Wang, Shaogang; Yang, Weimin; Liu, Jihong; Ye, Zhangqun

    2016-01-01

    Objective To investigate the presence, location and functional role of TMEM16A/anotamin-1 (Ano1) calcium-activated chloride channel (CaCC) in the penile of rats with diabetic erectile dysfunction. Methods Eight-week-old male Sprague-Dawley (SD) rats were administrated streptozotocin (diabetic) or citrate buffer (control) randomly. Erectile function was measured by cavernous nerve electrostimulation at 12th week after diabetes was induced. The effect of Ano1 specific inhibitor—T16Ainh-A01 on intracavernous pressure (ICP) was evaluated. Then the penile tissues were harvested for molecular exploration. Real-time PCR and Western Blotting were used to assess the expression of Ano1 in penile tissues. Immunofluorescent labelling of penile tissue allowed localization of Ano1. Cavernous smooth muscle cell (CSMC) was cultured in high glucose medium. The change of Ano1 was measured using Western Blotting. The proliferation of CSMC was evaluated by cell counting kit-8 (CCK-8). Results Erectile function was impaired in diabetic rats. The expression of Ano1 was increased in rats with diabetic erectile dysfunction at mRNA and protein levels. Immunofluorescent labelling revealed the presence of Ano1 mainly in cavernous smooth muscle cells. The inhibition of Ano1 increased the ICP of DED rats. High glucose in vitro enhanced the proliferation of CSMC and the expression level of Ano1. Conclusions Ano1 is expressed in rat penile tissue and is increased with diabetes mellitus. The inhibition of Ano1 increased the ICP of DED rats. The alerted Ano1 may be associated with diabetic erectile dysfunction. It is a potential therapy target for ED in the future.

  2. Functional Interaction of the SNARE Protein NtSyp121 in Ca2+ Channel Gating,Ca2+ Transients and ABA Signalling of Stomatal Guard Cells

    Institute of Scientific and Technical Information of China (English)

    Sergei Sokolovski; Adrian Hills; Robert A.Gay; Michael R.Blatt

    2008-01-01

    There is now growing evidence that membrane vesicle trafficking proteins,especially of the superfamily of SNAREs,are critical for cellular signalling in plants.Work from this laboratory first demonstrated that a soluble,inhibitory (dominant-negative) fragment of the SNARE NtSyp121 blocked K+ and Cl- channel responses to the stress-related hormone abscisic acid (ABA),but left open a question about functional impacts on signal intermediates,especially on Ca2+-mediated signalling events.Here,we report one mode of action for the SNARE mediated directly through alterations in Caz+ channel gating and its consequent effects on cytosolic-free [Ca2+] ([Ca2+]i) elevation.We find that expressing the same inhibitory fragment of NtSyp121 blocks ABA-evoked stomatal closure,but only partially suppresses stomatal closure in the presence of the NO donor,SNAP,which promotes [Ca2+]i elevation independently of the plasma membrane Ca2+ channels.Consistent with these observations,Ca2+ channel gating at the plasma membrane is altered by the SNARE fragment in a manner effective in reducing the potential for triggering a rise in [Ca2+]i,and we show directly that its expression in vivo leads to a pronounced suppression of evoked [Ca2+]i transients.These observations offer primary evidence for the functional coupling of the SNARE with Ca2+ channels at the plant cell plasma membrane and,because [Ca2+]i plays a key role in the control of K+ and Cl- channel currents in guard cells,they underscore an important mechanism for SNARE integration with ion channel regulation during stomatal closure.

  3. Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits

    OpenAIRE

    Tong, XiaoYong; Porter, Lisa M.; Liu, GongXin; Dhar-Chowdhury, Piyali; Srivastava, Shekhar; Pountney, David J.; Yoshida, Hidetada; Artman, Michael; Fishman, Glenn I.; Yu, Cindy; Iyer, Ramesh; Morley, Gregory E.; Gutstein, David E.; Coetzee, William A.

    2006-01-01

    Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits. Am J Physiol Heart Circ Physiol 291: H543–H551, 2006. First published February 24, 2006; doi:10.1152/ajpheart.00051.2006.—Cardiac ATP-sensitive K+ (KATP) channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice that express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression ...

  4. Survivin and related proteins in canine mammary tumors: immunohistochemical expression.

    Science.gov (United States)

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

    2015-03-01

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

  5. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    Science.gov (United States)

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  6. Novel MGF-based expressions for the average bit error probability of binary signalling over generalized fading channels

    KAUST Repository

    Yilmaz, Ferkan

    2014-04-01

    The main idea in the moment generating function (MGF) approach is to alternatively express the conditional bit error probability (BEP) in a desired exponential form so that possibly multi-fold performance averaging is readily converted into a computationally efficient single-fold averaging - sometimes into a closed-form - by means of using the MGF of the signal-to-noise ratio. However, as presented in [1] and specifically indicated in [2] and also to the best of our knowledge, there does not exist an MGF-based approach in the literature to represent Wojnar\\'s generic BEP expression in a desired exponential form. This paper presents novel MGF-based expressions for calculating the average BEP of binary signalling over generalized fading channels, specifically by expressing Wojnar\\'s generic BEP expression in a desirable exponential form. We also propose MGF-based expressions to explore the amount of dispersion in the BEP for binary signalling over generalized fading channels.

  7. A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies.

    Science.gov (United States)

    Grefen, Christopher; Donald, Naomi; Hashimoto, Kenji; Kudla, Jörg; Schumacher, Karin; Blatt, Michael R

    2010-10-01

    Fluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently-tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway-compatable vectors with fluorescent tags incorporating the ubiqutin-10 gene promoter (P(UBQ10) ) of Arabidopsis that gives prolonged expression of the fluorescently-tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicle-trafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K(+) channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co-cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P(UBQ10) promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S-driven construct, expression under the P(UBQ10) promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant

  8. Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns.

    Directory of Open Access Journals (Sweden)

    Guang-Jian Jiang

    Full Text Available It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.

  9. Expression of hepatitis B virus X protein in transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Jun Xiong; Yi-Ping Hu; Yu-Cheng Yao; Xiao-Yuan Zi; Jian-Xiu Li; Xin-Min Wang; Xu-Ting Ye; Shu-Min Zhao; Yong-Bi Yan; Hong-Yu Yu

    2003-01-01

    AIM: To establish a mice model harboring hepatitis B virusx gene (adr subtype) for studying the function of hepatitis Bvirus X protein, a transactivator of viral and cellular promoter/enhancer elements.METHODS: Expression vector pcDNA3-HBx, containing CMVpromoter and hepatitis B virus x gene open reading fragment,was constructed by recombination DNA technique. Hela cellswere cultured in DMEM and transfected with pcDNA3-HBxor control pcDNA3 plasmids using FuGENE6 TransfectionReagent. Expression of pcDNA3-HBx vectors in thetransfected Hela cells was confirmed by Western blotting.After restriction endonudease digestion, the coding elementswere microinjected into male pronuclei of mice zygotes. Thepups were evaluated by multiplex polymerase chain reaction(PCR) at genomic DNA level. The x gene transgenic micefounders were confirmed at protein level by Western blotting,immunohistochemistry and immunogold transmissionelectron microscopy.RESULTS: Expression vector pcDNA3-HBxwas constructedby recombination DNA technique and identified right byrestriction endonuclease digestion and DNA directsequencing. With Western blotting, hepatitis X protein wasdetected in Hela cells transfected with pcDNA3-HBxplasmids,suggesting pcDNA3-HBxplasmids could express in eukaryoticcells. Following microinjection of coding sequence ofpcDNA3-HBx, the embryos were transferred to oviducts ofpsedopregnant females. Four pups were born and survived.Two of them were verified to have the HBxgene integratedin their genomic DNA by multiplex PCR assay, and namedC57-TgN(HBx)SMMU1 and C57-TgN(HBx)SMMU3respectively. They expressed 17KD X protein in liver tissueby Western blotting assay. With the immunohistochemistry,X protein was detected mainly in hepatocytes cytoplasm oftransgenic mice, which was furthermore confirmed byimmunogold transmission electon microscopy.CONCLUSION: We have constructed the expression vectorpcDNA3-HBxthat can be used to study the function of HBxgene in eukaryotic cellsin vitro. We

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

    Energy Technology Data Exchange (ETDEWEB)

    Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    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. Heat Shock Protein 90 (Hsp90 Expression and Breast Cancer

    Directory of Open Access Journals (Sweden)

    Christos A. Papadimitriou

    2012-09-01

    Full Text Available Hsp90 is an abundant protein in mammalian cells. It forms several discrete complexes, each containing distinct groups of co-chaperones that assist protein folding and refolding during stress, protein transport and degradation. It interacts with a variety of proteins that play key roles in breast neoplasia including estrogen receptors, tumor suppressor p53 protein, angiogenesis transcription factor HIF-1alpha, antiapoptotic kinase Akt, Raf-1 MAP kinase and a variety of receptor tyrosine kinases of the erbB family. Elevated Hsp90 expression has been documented in breast ductal carcinomas contributing to the proliferative activity of breast cancer cells; whilst a significantly decreased Hsp90 expression has been shown in infiltrative lobular carcinomas and lobular neoplasia. Hsp90 overexpression has been proposed as a component of a mechanism through which breast cancer cells become resistant to various stress stimuli. Therefore, pharmacological inhibition of HSPs can provide therapeutic opportunities in the field of cancer treatment. 17-allylamino,17-demethoxygeldanamycin is the first Hsp90 inhibitor that has clinically been investigated in phase II trial, yielding promising results in patients with HER2-overexpressing metastatic breast cancer, whilst other Hsp90 inhibitors (retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, etc. are currently under evaluation.

  12. Neuroprotective effects of a mitochondrial K+-ATP channel opener (diazoxide) are mediated by Bcl-2 expression upregulation

    Institute of Scientific and Technical Information of China (English)

    Majid Katebi; Mansooreh Soleimani; Mehdi Mehdizadeh

    2011-01-01

    Mitochondrial K+-ATP (mito-KATP) channels play an important role in cellular function and survival following ischemic stress. The present results revealed that intervention with diazoxide, a mito-KATP channel opener, led to an increase in Bcl-2 expression in the cerebral cortex of rats subjected to cerebral ischemia reperfusion injury. In addition, the intervention also led to clear improvements in neuronal mitochondrial morphology and consciousness post-injury. Glibenclamide, a mito-KATP channel blocker, exhibited the converse effects. Both diazoxide and glibenclamide exerted dose-dependent effects (in particular, at 18 mg/kg diazoxide and 25 mg/kg glibenclamide). These findings suggest that diazoxide exerts a neuroprotective effect on cerebral ischemia reperfusion injury by opening mito-KATP channels and upregulating Bcl-2 expression.

  13. Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: potential role in regulation of a nonselective cation channel

    Science.gov (United States)

    Yamaguchi, T.; Ye, C.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D(3) (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in Ca

  14. Regulation of voltage-gated sodium channel expression in cancer:hormones, growth factors and auto-regulation

    OpenAIRE

    Fraser, Scott P.; Ozerlat-Gunduz, Iley; Brackenbury, William J; Fitzgerald, Elizabeth M.; Campbell, Thomas M.; Coombes, R. Charles; Djamgoz, Mustafa B. A.

    2014-01-01

    Although ion channels are increasingly being discovered in cancer cells in vitro and in vivo, and shown to contribute to different aspects and stages of the cancer process, much less is known about the mechanisms controlling their expression. Here, we focus on voltage-gated Na(+) channels (VGSCs) which are upregulated in many types of carcinomas where their activity potentiates cell behaviours integral to the metastatic cascade. Regulation of VGSCs occurs at a hierarchy of levels from transcr...

  15. Dynamics of energy distribution in three channel alpha helix protein based on Davydov’s ansatz

    Energy Technology Data Exchange (ETDEWEB)

    Ahmad, Faozan; Alatas, Husin [Theoretical Physics Division, Department of Physics, Faculty of Mathematics and Sciences Bogor Agricultural University, Bogor, Indonesia, 16680 faozan@ipb.ac.id (Indonesia)

    2015-04-16

    An important aspect of many biological processes at molecular level is the transfer and storage mechanism of bioenergy released in the reaction of the hydrolysis of Adenosinetriphosphate (ATP) by biomacromolecule especially protein. Model of Soliton Davydov is a new break-through that could describe that mechanism. Here we have reformulated quantum mechanical the Davydov theory, using least action principle. Dynamical aspect of the model is analyzed by numerical calculation. We found two dynamical cases: the traveling and pinning soliton that we suggest they are related to the energy transfer and storage mechanism in the protein. Traveling and pinning soliton can be controlled by strength of coupling. In 3- channel approach, we found the breather phenomena in which its frequency is determined by interchannel coupling parameter.

  16. Disposable bioreactors for inoculum production and protein expression.

    Science.gov (United States)

    Eibl, Regine; Löffelholz, Christian; Eibl, Dieter

    2014-01-01

    Disposable bioreactors have been increasingly implemented over the past ten years. This relates to both R & D and commercial manufacture, in particular, in animal cell-based processes. Among the numerous disposable bioreactors which are available today, wave-mixed bag bioreactors and stirred bioreactors are predominant. Whereas wave-mixed bag bioreactors represent the system of choice for inoculum production, stirred systems are often preferred for protein expression. For this reason, the authors present protocols instructing the reader how to use the wave-mixed BIOSTAT CultiBag RM 20 L for inoculum production and the stirred UniVessel SU 2 L for recombinant protein production at benchtop scale. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing the human placental secreted alkaline phosphatase (SEAP).

  17. Recombinant Dragline Silk-Like Proteins-Expression and Purification.

    Science.gov (United States)

    Gaines, William A; Marcotte, William R

    2011-03-01

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

  18. Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria*

    Science.gov (United States)

    Curran, Jerry; Musa, Hassan; Kline, Crystal F.; Makara, Michael A.; Little, Sean C.; Higgins, John D.; Hund, Thomas J.; Band, Hamid; Mohler, Peter J.

    2015-01-01

    Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca2+ channels (CaV3.1, CaV3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Cav3.1 and CaV3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca2+ current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both CaV3.1 and CaV3.2 in co-immunoprecipitation experiments. T-type Ca2+ channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify CaV3.1 and CaV3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction. PMID:25825486

  19. Protein tyrosine phosphatases expression during development of mouse superior colliculus

    OpenAIRE

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

    2009-01-01

    Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior col...

  20. Expression of P16 protein and Bcl-2 protein in malignant eyelid tumors

    Institute of Scientific and Technical Information of China (English)

    牛膺筠; 周占宇; 刘夫玲; 王红云

    2002-01-01

    Objective To investigate the relationship between P16 gene (the tumor suppressor gene) and the bcl-2 gene (the apoptosis inhibitor gene) and the incidence and development of malignant eyelid tumors. Methods The streptavidin-biotin-peroxidase complex immunohistochemistry method was used to study the expression of P16 gene and the bcl-2 gene in 96 cases of malignant eyelid tumors. Results Among the 96 cases, there were 40 basal cell carcinomas (BCCs), 33 squamous carcinomas and 23 sebaceous carcinoma, with P16 protein positive (nuclear staining) rates 70%, 54.6% and 56.5%, respectively. The P16 positive rate was negatively correlated with the degree of tumor histological differentiation, and the rate difference between the high differentiated carcinomas was significant (P<0.05). Positive Bcl-2 protein expression was detected in the cytoplasm. All 40 BCC cases were Bcl-2 positive, and nearly all of the tumor cells showed positive cytoplasmic expression, while in the 33 squamous cell carcinoma cases only one showed positive focal reaction, and the staining in the other 32 cases was relatively faint. None of the 23 sebaceous carcinomas expressed Bcl-2. Conclusions The expression of the P16 protein was related to the occurrence and degree of differentiation of malignant eyelid tumors. The overexpression of the Bcl-2 protein suggests that suppression of apoptosis might play a role in the tumorigenesis of BCC.

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

    OpenAIRE

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

    1995-01-01

    We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal lu...

  2. Expression of DNA-dependent protein kinase in human granulocytes

    Institute of Scientific and Technical Information of China (English)

    Annahita SALLMYR; Anna MILLER; Aida GABDOULKHAKOVA; Valentina SAFRONOVA; Gunnel HENRIKSSON; Anders BREDBERG

    2004-01-01

    Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNAPK in P MN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration.In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.

  3. Differential expression of ribosomal proteins in myelodysplastic syndromes.

    Science.gov (United States)

    Rinker, Elizabeth B; Dueber, Julie C; Qualtieri, Julianne; Tedesco, Jason; Erdogan, Begum; Bosompem, Amma; Kim, Annette S

    2016-02-01

    Aberrations of ribosomal biogenesis have been implicated in several congenital bone marrow failure syndromes, such as Diamond-Blackfan anaemia, Shwachman-Diamond syndrome and Dyskeratosis Congenita. Recent studies have identified haploinsufficiency of RPS14 in the acquired bone marrow disease isolated 5q minus syndrome, a subtype of myelodysplastic syndromes (MDS). However, the expression of various proteins comprising the ribosomal subunits and other proteins enzymatically involved in the synthesis of the ribosome has not been explored in non-5q minus MDS. Furthermore, differences in the effects of these expression alterations among myeloid, erythroid and megakaryocyte lineages have not been well elucidated. We examined the expression of several proteins related to ribosomal biogenesis in bone marrow biopsy specimens from patients with MDS (5q minus patients excluded) and controls with no known myeloid disease. Specifically, we found that there is overexpression of RPS24, DKC1 and SBDS in MDS. This overexpression is in contrast to the haploinsufficiency identified in the congenital bone marrow failure syndromes and in acquired 5q minus MDS. Potential mechanisms for these differences and aetiology for these findings in MDS are discussed.

  4. Bassoon specifically controls presynaptic P/Q-type Ca(2+) channels via RIM-binding protein.

    Science.gov (United States)

    Davydova, Daria; Marini, Claudia; King, Claire; Klueva, Julia; Bischof, Ferdinand; Romorini, Stefano; Montenegro-Venegas, Carolina; Heine, Martin; Schneider, Romy; Schröder, Markus S; Altrock, Wilko D; Henneberger, Christian; Rusakov, Dmitri A; Gundelfinger, Eckart D; Fejtova, Anna

    2014-04-01

    Voltage-dependent Ca(2+) channels (CaVs) represent the principal source of Ca(2+) ions that trigger evoked neurotransmitter release from presynaptic boutons. Ca(2+) influx is mediated mainly via CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels, which differ in their properties. Their relative contribution to synaptic transmission changes during development and tunes neurotransmission during synaptic plasticity. The mechanism of differential recruitment of CaV2.1 and CaV2.2 to release sites is largely unknown. Here, we show that the presynaptic scaffolding protein Bassoon localizes specifically CaV2.1 to active zones via molecular interaction with the RIM-binding proteins (RBPs). A genetic deletion of Bassoon or an acute interference with Bassoon-RBP interaction reduces synaptic abundance of CaV2.1, weakens P/Q-type Ca(2+) current-driven synaptic transmission, and results in higher relative contribution of neurotransmission dependent on CaV2.2. These data establish Bassoon as a major regulator of the molecular composition of the presynaptic neurotransmitter release sites. PMID:24698275

  5. Dramatic nano-fluidic properties of carbon nanotube membranes as a platform for protein channel mimetics

    Science.gov (United States)

    Hinds, Bruce

    2013-03-01

    Carbon nanotubes have three key attributes that make them of great interest for novel membrane applications: 1) atomically flat graphite surface allows for ideal fluid slip boundary conditions and extremely fast flow rates 2) the cutting process to open CNTs inherently places functional chemistry at CNT core entrance for chemical selectivity and 3) CNT are electrically conductive allowing for electrochemical reactions and application of electric fields gradients at CNT tips. Pressure driven flux of a variety of solvents (H2O, hexane, decane ethanol, methanol) are 4-5 orders of magnitude higher than conventional Newtonian flow [Nature 2005, 438, 44] due to atomically flat graphite planes inducing nearly ideal slip conditions. However this is eliminated with selective chemical functionalization [ACS Nano 2011 5(5) 3867-3877] needed to give chemical selectivity. These unique properties allow us to explore the hypothesis of producing ``Gatekeeper'' membranes that mimic natural protein channels to actively pump through rapid nm-scale channels. With anionic tip functionality strong electroosmotic flow is induced by unimpeded cation flow with similar 10,000 fold enhancements [Nature Nano 2012 7(2) 133-39]. With enhanced power efficiency, carbon nanotube membranes were employed as the active element of a switchable transdermal drug delivery device that can facilitate more effective treatments of drug abuse and addiction. Recently methods to deposit Pt monolayers on CNT surface have been developed making for highly efficient catalytic platforms. Discussed are other applications of CNT protein channel mimetics, for large area robust engineering platforms, including water purification, flow battery energy storage, and biochemical/biomass separations. DOE EPSCoR (DE-FG02-07ER46375) and DARPA, W911NF-09-1-0267

  6. Interaction of Human Chloride Intracellular Channel Protein 1 (CLIC1) with Lipid Bilayers: A Fluorescence Study.

    Science.gov (United States)

    Hare, Joanna E; Goodchild, Sophia C; Breit, Samuel N; Curmi, Paul M G; Brown, Louise J

    2016-07-12

    Chloride intracellular channel protein 1 (CLIC1) is very unusual as it adopts a soluble glutathione S-transferase-like canonical fold but can also autoinsert into lipid bilayers to form an ion channel. The conversion between these forms involves a large, but reversible, structural rearrangement of the CLIC1 module. The only identified environmental triggers controlling the metamorphic transition of CLIC1 are pH and oxidation. Until now, there have been no high-resolution structural data available for the CLIC1 integral membrane state, and consequently, a limited understanding of how CLIC1 unfolds and refolds across the bilayer to form a membrane protein with ion channel activity exists. Here we show that fluorescence spectroscopy can be used to establish the interaction and position of CLIC1 in a lipid bilayer. Our method employs a fluorescence energy transfer (FRET) approach between CLIC1 and a dansyl-labeled lipid analogue to probe the CLIC1-lipid interface. Under oxidizing conditions, a strong FRET signal between the single tryptophan residue of CLIC1 (Trp35) and the dansyl-lipid analogue was detected. When considering the proportion of CLIC1 interacting with the lipid bilayer, as estimated by fluorescence quenching experiments, the FRET distance between Trp35 and the dansyl moiety on the membrane surface was determined to be ∼15 Å. This FRET-detected interaction provides direct structural evidence that CLIC1 associates with membranes. The results presented support the current model of an oxidation-driven interaction of CLIC1 with lipid bilayers and also propose a membrane anchoring role for Trp35. PMID:27299171

  7. Improved functional expression of recombinant human ether-a-go-go (hERG K+ channels by cultivation at reduced temperature

    Directory of Open Access Journals (Sweden)

    Hamilton Bruce

    2007-12-01

    Full Text Available Abstract Background HERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. There is a strong interest in the pharmaceutical industry to develop high quality medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. Cultivation of cells at lower temperature has been used to improve the folding and membrane localization of trafficking defective hERG mutant proteins. The objective of this study was to investigate the effect of lower temperature maintenance on wild type hERG expression and assay performance. Results Wild type hERG was stably expressed in CHO-K1 cells, with the majority of channel protein being located in the cytoplasm, but relatively little on the cell surface. Expression at both locations was increased several-fold by cultivation at lower growth temperatures. Intracellular hERG protein levels were highest at 27°C and this correlated with maximal 3H-dofetilide binding activity. In contrast, the expression of functionally active cell surface-associated hERG measured by patch clamp electrophysiology was optimal at 30°C. The majority of the cytoplasmic hERG protein was associated with the membranes of cytoplasmic vesicles, which markedly increased in quantity and size at lower temperatures or in the presence of the Ca2+-ATPase inhibitor, thapsigargin. Incubation with the endocytic trafficking blocker, nocodazole, led to an increase in hERG activity at 37°C, but not at 30°C. Conclusion Our results are consistent with the concept that maintenance of cells at reduced temperature can be used to boost the functional expression of difficult-to-express membrane proteins and improve the quality of assays for medium to high-throughput compound screening. In addition, these results shed some light on the trafficking of hERG protein under these growth conditions.

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

    Science.gov (United States)

    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.

  9. Sperm protein 17 is expressed in the sperm fibrous sheath

    Directory of Open Access Journals (Sweden)

    Albani Elena

    2009-07-01

    Full Text Available Abstract Background Sperm protein 17 (Sp17 is a highly conserved mammalian protein characterized in rabbit, mouse, monkey, baboon, macaque, human testis and spermatozoa. mRNA encoding Sp17 has been detected in a range of murine and human somatic tissues. It was also recognized in two myeloma cell lines and in neoplastic cells from patients with multiple myeloma and ovarian carcinoma. These data all indicate that Sp17 is widely distributed in humans, expressed not only in germinal cells and in a variety of somatic tissues, but also in neoplastic cells of unrelated origin. Methods Sp17 expression was analyzed by immunocytochemistry and transmission electron microscopy on spermatozoa. Results Here, we demonstrate the ultrastructural localization of human Sp17 throughout the spermatozoa flagellar fibrous sheath, and its presence in spermatozoa during in vitro states from their ejaculation to the oocyte fertilization. Conclusion These findings suggest a possible role of Sp17 in regulating sperm maturation, capacitation, acrosomal reaction and interactions with the oocyte zona pellucida during the fertilization process. Further, the high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP-binding motif within this region, suggest that Sp17 might play a regulatory role in a protein kinase A-independent AKAP complex in both germinal and somatic cells.

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

    Science.gov (United States)

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

    2010-06-01

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

  11. Expression of TRPV1 channels after nerve injury provides an essential delivery tool for neuropathic pain attenuation.

    Directory of Open Access Journals (Sweden)

    Hossain Md Zakir

    Full Text Available Increased expression of the transient receptor potential vanilloid 1 (TRPV1 channels, following nerve injury, may facilitate the entry of QX-314 into nociceptive neurons in order to achieve effective and selective pain relief. In this study we hypothesized that the level of QX-314/capsaicin (QX-CAP--induced blockade of nocifensive behavior could be used as an indirect in-vivo measurement of functional expression of TRPV1 channels. We used the QX-CAP combination to monitor the functional expression of TRPV1 in regenerated neurons after inferior alveolar nerve (IAN transection in rats. We evaluated the effect of this combination on pain threshold at different time points after IAN transection by analyzing the escape thresholds to mechanical stimulation of lateral mental skin. At 2 weeks after IAN transection, there was no QX-CAP mediated block of mechanical hyperalgesia, implying that there was no functional expression of TRPV1 channels. These results were confirmed immunohistochemically by staining of regenerated trigeminal ganglion (TG neurons. This suggests that TRPV1 channel expression is an essential necessity for the QX-CAP mediated blockade. Furthermore, we show that 3 and 4 weeks after IAN transection, application of QX-CAP produced a gradual increase in escape threshold, which paralleled the increased levels of TRPV1 channels that were detected in regenerated TG neurons. Immunohistochemical analysis also revealed that non-myelinated neurons regenerated slowly compared to myelinated neurons following IAN transection. We also show that TRPV1 expression shifted towards myelinated neurons. Our findings suggest that nerve injury modulates the TRPV1 expression pattern in regenerated neurons and that the effectiveness of QX-CAP induced blockade depends on the availability of functional TRPV1 receptors in regenerated neurons. The results of this study also suggest that the QX-CAP based approach can be used as a new behavioral tool to detect

  12. TGF-β1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-β1/Smad pathway

    International Nuclear Information System (INIS)

    Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts plays a critical role in the development of liver fibrosis, since myofibroblasts are the key cells responsible for excessive deposition of ECM proteins. Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel with protein serine/threonine kinase activity, has been demonstrated to function in the proliferation of activated HSCs. Here, we investigated the functional role of TRPM7 in collagen deposition in activated HSC-T6 cells (a rat hepatic stellate cell line). TRPM7 mRNA and protein were measured by Real-time PCR and Western blot in TGF-β1-activated HSC-T6 cells in vitro. Results demonstrated that TRPM7 protein was dramatically increased in fibrotic human livers. Stimulation of HSC-T6 cells with TGF-β1 increased TRPM7 mRNA and protein level in a time-dependent manner. Nevertheless, TGF-β1-elicited upregulation of TRPM7 in HSC-T6 cells was abrogated by SB431542 (TGF-β1 receptor blocker) or SIS3 (inhibitor of Smad3 phosphorylation). Additionally, blockade of TRPM7 channels with non-specific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7 attenuated TGF-β1-induced expression of myofibroblast markers, as measured by the induction of α-SMA and Col1α1. Silencing TRPM7 also increased the ratio of MMPs/TIMPs by increasing MMP-13 expression and decreasing TIMP-1 and TIMP-2 levels. Strikingly, phosphorylation of p-Smad2 and p-Smad3, associated with collagen production, was decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TGF-β1 elevates TRPM7 expression in HSCs via Smad3-dependant mechanisms, which in turn contributes Smad protein phosphorylation, and subsequently increases fibrous collagen expression. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis. - Highlights: • Upregulation of TRPM7 protein in human fibrotic livers • Upregulation of TRPM7 by TGF-β1 elicited Smad signaling in HSC-T6 cells

  13. TGF-β1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-β1/Smad pathway

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Ling, E-mail: fangling_1984@126.com [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China); The First Affiliated Hospital of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Huang, Cheng; Meng, Xiaoming; Wu, Baoming; Ma, Taotao; Liu, Xuejiao; Zhu, Qian [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China); Zhan, Shuxiang [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China); The First Affiliated Hospital of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Li, Jun, E-mail: lj@ahmu.edu.cn [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China)

    2014-10-15

    Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts plays a critical role in the development of liver fibrosis, since myofibroblasts are the key cells responsible for excessive deposition of ECM proteins. Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel with protein serine/threonine kinase activity, has been demonstrated to function in the proliferation of activated HSCs. Here, we investigated the functional role of TRPM7 in collagen deposition in activated HSC-T6 cells (a rat hepatic stellate cell line). TRPM7 mRNA and protein were measured by Real-time PCR and Western blot in TGF-β1-activated HSC-T6 cells in vitro. Results demonstrated that TRPM7 protein was dramatically increased in fibrotic human livers. Stimulation of HSC-T6 cells with TGF-β1 increased TRPM7 mRNA and protein level in a time-dependent manner. Nevertheless, TGF-β1-elicited upregulation of TRPM7 in HSC-T6 cells was abrogated by SB431542 (TGF-β1 receptor blocker) or SIS3 (inhibitor of Smad3 phosphorylation). Additionally, blockade of TRPM7 channels with non-specific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7 attenuated TGF-β1-induced expression of myofibroblast markers, as measured by the induction of α-SMA and Col1α1. Silencing TRPM7 also increased the ratio of MMPs/TIMPs by increasing MMP-13 expression and decreasing TIMP-1 and TIMP-2 levels. Strikingly, phosphorylation of p-Smad2 and p-Smad3, associated with collagen production, was decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TGF-β1 elevates TRPM7 expression in HSCs via Smad3-dependant mechanisms, which in turn contributes Smad protein phosphorylation, and subsequently increases fibrous collagen expression. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis. - Highlights: • Upregulation of TRPM7 protein in human fibrotic livers • Upregulation of TRPM7 by TGF-β1 elicited Smad signaling in HSC-T6 cells

  14. Yellow fluorescent protein-based assay to measure GABA(A channel activation and allosteric modulation in CHO-K1 cells.

    Directory of Open Access Journals (Sweden)

    Teres Johansson

    Full Text Available The γ-aminobutyric acid A (GABA(A ion channels are important drug targets for treatment of neurological and psychiatric disorders. Finding GABA(A channel subtype selective allosteric modulators could lead to new improved treatments. However, the progress in this area has been obstructed by the challenging task of developing functional assays to support screening efforts and the generation of cells expressing functional GABA(A ion channels with the desired subtype composition. To address these challenges, we developed a yellow fluorescent protein (YFP-based assay to be able to study allosteric modulation of the GABA(A ion channel using cryopreserved, transiently transfected, assay-ready cells. We show for the first time how the MaxCyte STX electroporation instrument can be used to generate CHO-K1 cells expressing functional GABA(A α2β3γ2 along with a halide sensing YFP-H148Q/I152L (YFP-GABA(A2 cells. As a basis for a cell-based assay capable of detecting allosteric modulators, experiments with antagonist, ion channel blocker and modulators were used to verify GABA(A subunit composition and functionality. We found that the I(- concentration used in the YFP assay affected both basal quench of YFP and potency of GABA. For the first time the assay was used to study modulation of GABA with 7 known modulators where statistical analysis showed that the assay can distinguish modulatory pEC50 differences of 0.15. In conclusion, the YFP assay proved to be a robust, reproducible and inexpensive assay. These data provide evidence that the assay is suitable for high throughput screening (HTS and could be used to discover novel modulators acting on GABA(A ion channels.

  15. Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

    Directory of Open Access Journals (Sweden)

    Schlarman Maggie S

    2012-03-01

    Full Text Available Abstract Background Plasmodium falciparum malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development. Methods The P. falciparum gene, PF11_0394, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR and green fluorescent protein (GFP-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined. Results The PF11_0394 protein has orthologs in other Plasmodium species and Apicomplexans, but none outside of the group Apicomplexa. PF11_0394 transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites. Conclusions PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in P. falciparum biology, it could be another candidate for a new vaccine.

  16. Strain differences in pH-sensitive K+ channel-expressing cells in chemosensory and nonchemosensory brain stem nuclei

    OpenAIRE

    Martino, Paul F.; Olesiak, S.; Batuuka, D.; Riley, D; Neumueller, S.; Forster, H. V.; Hodges, M. R.

    2014-01-01

    The ventilatory CO2 chemoreflex is inherently low in inbred Brown Norway (BN) rats compared with other strains, including inbred Dahl salt-sensitive (SS) rats. Since the brain stem expression of various pH-sensitive ion channels may be determinants of the CO2 chemoreflex, we tested the hypothesis that there would be fewer pH-sensitive K+ channel-expressing cells in BN relative to SS rats within brain stem sites associated with respiratory chemoreception, such as the nucleus tractus solitarius...

  17. Expression and Function of CLC and Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in Renal Epithelial Tubule Cells: Pathophysiological Implications

    OpenAIRE

    Alain Vandewalle

    2007-01-01

    Cl- channels play important roles in the regulation of a variety of functions, includingelectrical excitability, cell volume regulation, transepithelial transport and acidification ofcellular organelles. They are expressed in plasma membranes or reside in intracellularorganelles. A large number of Cl- channels with different functions have been identified.Some of them are highly expressed in the kidney. They include members of the CLC Clchannelfamily: ClC-K1 (or ClC-Ka), ClC-K2 (or ClC-Kb) an...

  18. An electronic channel switching-based aptasensor for ultrasensitive protein detection

    Energy Technology Data Exchange (ETDEWEB)

    Li Hongbo; Wang Cui [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Wu Zaisheng, E-mail: wuzaisheng@163.com [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Lu Limin; Qiu Liping; Zhou Hui; Shen Guoli [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Yu Ruqin, E-mail: rqyu@hnu.edu.cn [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China)

    2013-01-03

    Highlights: Black-Right-Pointing-Pointer Target IgE is successfully designed to serve as a barrier to separate enzyme from its substrate. Black-Right-Pointing-Pointer This sensing platform of electronic channel switching-based aptasensor can be simply manipulated. Black-Right-Pointing-Pointer The stable hairpin structure of anti-IgE aptamer is utilized to detect target IgE. Black-Right-Pointing-Pointer The sensor is ultrasensitive sensitivity, excellent selectivity and small volume of sample. Black-Right-Pointing-Pointer It is a powerful platform to be further expanded to detect more kinds of proteins and even cells. - Abstract: Due to the ubiquity and essential of the proteins in all living organisms, the identification and quantification of disease-specific proteins are particularly important. Because the conformational change of aptamer upon its target or probe/target/probe sandwich often is the primary prerequisite for the design of an electrochemical aptameric assay system, it is extremely difficult to construct the electrochemical aptasensor for protein assay because the corresponding aptamers cannot often meet the requirement. To circumvent the obstacles mentioned, an electronic channel switching-based (ECS) aptasensor for ultrasensitive protein detection is developed. The essential achievement made is that an innovative sensing concept is proposed: the hairpin structure of aptamer is designed to pull electroactive species toward electrode surface and makes the surface-immobilized IgE serve as a barrier that separates enzyme from its substrate. It seemingly ensures that the ECS aptasensor exhibits most excellent assay features, such as, a detection limit of 4.44 Multiplication-Sign 10{sup -6} {mu}g mL{sup -1} (22.7 fM, 220 zmol in 10-{mu}L sample) (demonstrating a 5 orders of magnitude improvement in detection sensitivity compared with classical electronic aptasensors) and dynamic response range from 4.44 Multiplication-Sign 10{sup -6} to 4.44 Multiplication

  19. Hyperpolarization-activated cation and T-type calcium ion channel expression in porcine and human renal pacemaker tissues.

    Science.gov (United States)

    Hurtado, Romulo; Smith, Carl S

    2016-05-01

    Renal pacemaker activity triggers peristaltic upper urinary tract contractions that propel waste from the kidney to the bladder, a process prone to congenital defects that are the leading cause of pediatric kidney failure. Recently, studies have discovered that hyperpolarization-activated cation (HCN) and T-type calcium (TTC) channel conductances underlie murine renal pacemaker activity, setting the origin and frequency and coordinating upper urinary tract peristalsis. Here, we determined whether this ion channel expression is conserved in the porcine and human urinary tracts, which share a distinct multicalyceal anatomy with multiple pacemaker sites. Double chromagenic immunohistochemistry revealed that HCN isoform 3 is highly expressed at the porcine minor calyces, the renal pacemaker tissues, whereas the kidney and urinary tract smooth muscle lacked this HCN expression. Immunofluorescent staining demonstrated that HCN(+) cells are integrated within the porcine calyx smooth muscle, and that they co-express TTC channel isoform Cav3.2. In humans, the anatomic structure of the minor calyx pacemaker was assayed via hematoxylin and eosin analyses, and enabled the visualization of the calyx smooth muscle surrounding adjacent papillae. Strikingly, immunofluorescence revealed that HCN3(+) /Cav3.2(+) cells are also localized to the human minor calyx smooth muscle. Collectively, these data have elucidated a conserved molecular signature of HCN and TTC channel expression in porcine and human calyx pacemaker tissues. These findings provide evidence for the mechanisms that can drive renal pacemaker activity in the multi-calyceal urinary tract, and potential causes of obstructive uropathies. PMID:26805464

  20. Expression of livin protein in lung cancer and its relation with the expression of pro-caspase3 protein

    Directory of Open Access Journals (Sweden)

    Hongru LI

    2008-10-01

    Full Text Available Background and objective Livin is a novel inhibitor of apoptosis protein (IAP, recent studies showed it overexpresses in a variety of carcinomas including lung cancer and contributes much to the cancerous development. The objective of this study is to explore the expression of livin in tissues of lung cancer and its relationshipwith histological types, chemotherapy, Lymph node metastasis and to study its correlation with the expression of pro-caspase3 as well. Methods Expressions of Livin and caspase3 were detected by Western blot assay in lung cancer tissues as well as in controls. Results Livin was expressed in 15 of 27 lung cancer, significantly more than those in lung para-cancerous (1/5 or benign disease lung tissues (2/12 (P 0.05. Conclusion Livin are differently expressed in different histological types of lung cancer; High levels of livin expression do not relate to chemotherapy, lymph node metastasis (P >0.05. The levels of livin tends to be positively associated with those of accordingly pro-caspase3, it is presumed that livin could bind pro-caspase3 and suppress its activation.

  1. A molecular clock regulates angiopoietin-like protein 2 expression.

    Science.gov (United States)

    Kadomatsu, Tsuyoshi; Uragami, Shota; Akashi, Makoto; Tsuchiya, Yoshiki; Nakajima, Hiroo; Nakashima, Yukiko; Endo, Motoyoshi; Miyata, Keishi; Terada, Kazutoyo; Todo, Takeshi; Node, Koichi; Oike, Yuichi

    2013-01-01

    Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

  2. A molecular clock regulates angiopoietin-like protein 2 expression.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Kadomatsu

    Full Text Available Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2 contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

  3. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

    Directory of Open Access Journals (Sweden)

    Cui Zhu

    2016-08-01

    Full Text Available Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11 have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes, goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

  4. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

    Science.gov (United States)

    Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

    2016-01-01

    Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines. PMID:27589719

  5. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

    Science.gov (United States)

    Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

    2016-08-29

    Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

  6. Calcitriol inhibits Ether-a go-go potassium channel expression and cell proliferation in human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Becerra, Rocio [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Diaz, Lorenza, E-mail: lorenzadiaz@gmail.com [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Camacho, Javier [Department of Pharmacology, Centro de Investigacion y de Estudios Avanzados, Instituto Politecnico Nacional, Av. Instituto Politecnico Nacional 2508, San Pedro Zacatenco 07360, Mexico, D.F. (Mexico); Barrera, David; Ordaz-Rosado, David; Morales, Angelica [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Ortiz, Cindy Sharon [Department of Pathology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Avila, Euclides [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Bargallo, Enrique [Department of Breast Tumors, Instituto Nacional de Cancerologia, Av. San Fernando No. 22, Tlalpan 14080, Mexico, D.F. (Mexico); Arrecillas, Myrna [Department of Pathology, Instituto Nacional de Cancerologia, Av. San Fernando No. 22, Tlalpan 14080, Mexico, D.F. (Mexico); Halhali, Ali; Larrea, Fernando [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico)

    2010-02-01

    Antiproliferative actions of calcitriol have been shown to occur in many cell types; however, little is known regarding the molecular basis of this process in breast carcinoma. Ether-a-go-go (Eag1) potassium channels promote oncogenesis and are implicated in breast cancer cell proliferation. Since calcitriol displays antineoplastic effects while Eag1 promotes tumorigenesis, and both factors antagonically regulate cell cycle progression, we investigated a possible regulatory effect of calcitriol upon Eag1 as a mean to uncover new molecular events involved in the antiproliferative activity of this hormone in human breast tumor-derived cells. RT real-time PCR and immunocytochemistry showed that calcitriol suppressed Eag1 expression by a vitamin D receptor (VDR)-dependent mechanism. This effect was accompanied by inhibition of cell proliferation, which was potentiated by astemizole, a nonspecific Eag1 inhibitor. Immunohistochemistry and Western blot demonstrated that Eag1 and VDR abundance was higher in invasive-ductal carcinoma than in fibroadenoma, and immunoreactivity of both proteins was located in ductal epithelial cells. Our results provide evidence of a novel mechanism involved in the antiproliferative effects of calcitriol and highlight VDR as a cancer therapeutic target for breast cancer treatment and prevention.

  7. Calcitriol inhibits Ether-a go-go potassium channel expression and cell proliferation in human breast cancer cells

    International Nuclear Information System (INIS)

    Antiproliferative actions of calcitriol have been shown to occur in many cell types; however, little is known regarding the molecular basis of this process in breast carcinoma. Ether-a-go-go (Eag1) potassium channels promote oncogenesis and are implicated in breast cancer cell proliferation. Since calcitriol displays antineoplastic effects while Eag1 promotes tumorigenesis, and both factors antagonically regulate cell cycle progression, we investigated a possible regulatory effect of calcitriol upon Eag1 as a mean to uncover new molecular events involved in the antiproliferative activity of this hormone in human breast tumor-derived cells. RT real-time PCR and immunocytochemistry showed that calcitriol suppressed Eag1 expression by a vitamin D receptor (VDR)-dependent mechanism. This effect was accompanied by inhibition of cell proliferation, which was potentiated by astemizole, a nonspecific Eag1 inhibitor. Immunohistochemistry and Western blot demonstrated that Eag1 and VDR abundance was higher in invasive-ductal carcinoma than in fibroadenoma, and immunoreactivity of both proteins was located in ductal epithelial cells. Our results provide evidence of a novel mechanism involved in the antiproliferative effects of calcitriol and highlight VDR as a cancer therapeutic target for breast cancer treatment and prevention.

  8. Calcitriol inhibits Ether-à go-go potassium channel expression and cell proliferation in human breast cancer cells.

    Science.gov (United States)

    García-Becerra, Rocío; Díaz, Lorenza; Camacho, Javier; Barrera, David; Ordaz-Rosado, David; Morales, Angélica; Ortiz, Cindy Sharon; Avila, Euclides; Bargallo, Enrique; Arrecillas, Myrna; Halhali, Ali; Larrea, Fernando

    2010-02-01

    Antiproliferative actions of calcitriol have been shown to occur in many cell types; however, little is known regarding the molecular basis of this process in breast carcinoma. Ether-à-go-go (Eag1) potassium channels promote oncogenesis and are implicated in breast cancer cell proliferation. Since calcitriol displays antineoplastic effects while Eag1 promotes tumorigenesis, and both factors antagonically regulate cell cycle progression, we investigated a possible regulatory effect of calcitriol upon Eag1 as a mean to uncover new molecular events involved in the antiproliferative activity of this hormone in human breast tumor-derived cells. RT real-time PCR and immunocytochemistry showed that calcitriol suppressed Eag1 expression by a vitamin D receptor (VDR)-dependent mechanism. This effect was accompanied by inhibition of cell proliferation, which was potentiated by astemizole, a nonspecific Eag1 inhibitor. Immunohistochemistry and Western blot demonstrated that Eag1 and VDR abundance was higher in invasive-ductal carcinoma than in fibroadenoma, and immunoreactivity of both proteins was located in ductal epithelial cells. Our results provide evidence of a novel mechanism involved in the antiproliferative effects of calcitriol and highlight VDR as a cancer therapeutic target for breast cancer treatment and prevention. PMID:19932096

  9. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  10. Heat shock protein 70-hom gene polymorphism and protein expression in multiple sclerosis.

    Science.gov (United States)

    Boiocchi, C; Monti, M C; Osera, C; Mallucci, G; Pistono, C; Ferraro, O E; Nosari, G; Romani, A; Cuccia, M; Govoni, S; Pascale, A; Montomoli, C; Bergamaschi, R

    2016-09-15

    Immune-mediated and neurodegenerative mechanisms are involved in multiple sclerosis (MS). Growing evidences highlight the role of HSP70 genes in the susceptibility of some neurological diseases. In this explorative study we analyzed a polymorphism (i.e. HSP70-hom rs2227956) of the gene HSPA1L, which encodes for the protein hsp70-hom. We sequenced the polymorphism by polymerase chain reaction (PCR), in 191 MS patients and 365 healthy controls. The hsp70-hom protein expression was quantified by western blotting. We reported a strong association between rs2227956 polymorphism and MS risk, which is independent from the association with HSP70-2 rs1061581, and a significant link between hsp70-hom protein expression and MS severity. PMID:27609295

  11. Protein tyrosine phosphatases expression during development of mouse superior colliculus.

    Science.gov (United States)

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

    2009-12-01

    Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis. PMID:19727691

  12. Simvastatin enhances bone morphogenetic protein receptor type II expression

    International Nuclear Information System (INIS)

    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

  13. Dynamic expression pattern of kinesin accessory protein in Drosophila

    Indian Academy of Sciences (India)

    Ritu Sarpal; Krishanu Ray

    2002-09-01

    We have identified the Drosophila homologue of the non-motor accessory subunit of kinesin-II motor complex. It is homologous to the SpKAP115 of the sea urchin, KAP3A and KAP3B of the mouse, and SMAP protein in humans. In situ hybridization using a DmKAP specific cRNA probe has revealed a dynamic pattern of expression in the developing nervous system. The staining first appears in a subset of cells in the embryonic central nervous system at stage 13 and continues till the first instar larva stage. At the third instar larva stage the staining gets restricted to a few cells in the optic lobe and in the ventral ganglion region. It has also stained a subset of sensory neurons from late stage 13 and till the first instar larva stage. The DmKAP expression pattern in the nervous system corresponds well with that of Klp64D and Klp68D as reported earlier. In addition, we have found that the DmKAP gene is constitutively expressed in the germline cells and in follicle cells during oogenesis. These cells are also stained using an antibody to KLP68D protein, but mRNA in situ hybridization using KLP64D specific probe has not stained these cells. Together these results proved a basis for further analysis of tissue specific function of DmKAP in future.

  14. A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

    OpenAIRE

    AYDIN, Halil; Azimi, Farshad C.; Jonathan D Cook; 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. cerevisi...

  15. Enhanced cell-free protein expression by fusion with immunoglobulin Cκ domain

    OpenAIRE

    Palmer, Elizabeth; Liu, Hong; Khan, Farid; Taussig, Michael J; He, Mingyue

    2006-01-01

    While cell-free systems are increasingly used for protein expression in structural and functional studies, several proteins are difficult to express or expressed only at low levels in cell-free lysates. Here, we report that fusion of the human immunoglobulin κ light chain constant domain (Cκ) at the C terminus of four representative proteins dramatically improved their production in the Escherichia coli S30 system, suggesting that enhancement of cell-free protein expression by Cκ fusion will ...

  16. Ryanodine receptors, a family of intracellular calcium ion channels, are expressed throughout early vertebrate development

    Directory of Open Access Journals (Sweden)

    Wu Houdini HT

    2011-12-01

    Full Text Available Abstract Background Calcium signals ([Ca2+]i direct many aspects of embryo development but their regulation is not well characterised. Ryanodine receptors (RyRs are a family of intracellular Ca2+ release channels that control the flux of Ca2+ from internal stores into the cytosol. RyRs are primarily known for their role in excitation-contraction coupling in adult striated muscle and ryr gene mutations are implicated in several human diseases. Current evidence suggests that RyRs do not have a major role to play prior to organogenesis but regulate tissue differentiation. Findings The sequences of the five zebrafish ryr genes were confirmed, their evolutionary relationship established and the primary sequences compared to other vertebrates, including humans. RyRs are differentially expressed in slow (ryr1a, fast (ryr3 and both types (ryr1b of developing skeletal muscle. There are two ryr2 genes (ryr2a and ryr2b which are expressed exclusively in developing CNS and cardiac tissue, respectively. In addition, ryr3 and ryr2a mRNA is detectable in the initial stages of development, prior to embryonic axis formation. Conclusions Our work reveals that zebrafish ryr genes are differentially expressed throughout the developing embryo from cleavage onwards. The data suggests that RyR-regulated Ca2+ signals are associated with several aspects of embryonic development, from organogenesis through to the differentiation of the musculoskeletal, cardiovascular and nervous system. These studies will facilitate further work to explore the developmental function of RyRs in each of these tissue types.

  17. The protein kinase KIS impacts gene expression during development and fear conditioning in adult mice.

    Directory of Open Access Journals (Sweden)

    Valérie Manceau

    Full Text Available The brain-enriched protein kinase KIS (product of the gene UHMK1 has been shown to phosphorylate the human splicing factor SF1 in vitro. This phosphorylation in turn favors the formation of a U2AF(65-SF1-RNA complex which occurs at the 3' end of introns at an early stage of spliceosome assembly. Here, we analyzed the effects of KIS knockout on mouse SF1 phosphorylation, physiology, adult behavior, and gene expression in the neonate brain. We found SF1 isoforms are differently expressed in KIS-ko mouse brains and fibroblasts. Re-expression of KIS in fibroblasts restores a wild type distribution of SF1 isoforms, confirming the link between KIS and SF1. Microarray analysis of transcripts in the neonate brain revealed a subtle down-regulation of brain specific genes including cys-loop ligand-gated ion channels and metabolic enzymes. Q-PCR analyses confirmed these defects and point to an increase of pre-mRNA over mRNA ratios, likely due to changes in splicing efficiency. While performing similarly in prepulse inhibition and most other behavioral tests, KIS-ko mice differ in spontaneous activity and contextual fear conditioning. This difference suggests that disregulation of gene expression due to KIS inactivation affects specific brain functions.

  18. Expression-dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Rivera-Acevedo, Ricardo E; Pless, Stephan Alexander; Schwarz, Stephan K W;

    2013-01-01

    Transient receptor potential vanilloid subfamily member 1 channels are polymodal sensors of noxious stimuli and integral players in thermosensation, inflammation and pain signaling. It has been shown previously that under prolonged stimulation, these channels show dynamic pore dilation, providing...

  19. High extracellular potassium ion concentration attenuates the blockade action of ketanserin on Kvl.3 channels expressed in xenopus oocytes

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Background Ketanserin (KT), a selective serotonin (5-HT) 2-receptor antagonist, reduces peripheral blood pressure by blocking the activation of peripheral 5-HT receptors. In this study electrophysiological method was used to investigate the effect of KT and potassium ion on Kv1.3 potassium channels and explore the role of blocker KT in the alteration of channel kinetics contributing to the potassium ion imbalances. Methods Kvl.3 channels were expressed in xenopus oocytes, and currents were measured using the two-microelectrode voltage-clamp technique. Results KCI made a left shift of activation and an inactivation curve of Kv1.3 current and accelerated the activation and inactivation time constant. High extracellular [K+] attenuated the blockade effect of KT on Kv1.3 channels. In the presence of KT and KCI the activation and inactivation time constants were not influenced significantly no matter what was administered first. KT did not significantly inhibit Kv1.3 current induced by tetraethylammonium (TEA). Conclusions KT is a weak blocker of Kv1.3 channels at different concentrations of extracellular potassium and binds to the intracellular side of the channel pore. The inhibitor KT of ion channels is not fully effective in clinical use because of high [K+]o and other electrolyte disorders.

  20. Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Gavillet, Bruno; van Bemmelen, Miguel X;

    2006-01-01

    In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull...

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Eileen Marie Hanna

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  4. Potassium Channel Interacting Protein 2 (KChIP2) is not a transcriptional regulator of cardiac electrical remodeling.

    Science.gov (United States)

    Winther, Sine V; Tuomainen, Tomi; Borup, Rehannah; Tavi, Pasi; Antoons, Gudrun; Thomsen, Morten B

    2016-01-01

    The heart-failure relevant Potassium Channel Interacting Protein 2 (KChIP2) augments CaV1.2 and KV4.3. KChIP3 represses CaV1.2 transcription in cardiomyocytes via interaction with regulatory DNA elements. Hence, we tested nuclear presence of KChIP2 and if KChIP2 translocates into the nucleus in a Ca(2+) dependent manner. Cardiac biopsies from human heart-failure patients and healthy donor controls showed that nuclear KChIP2 abundance was significantly increased in heart failure; however, this was secondary to a large variation of total KChIP2 content. Administration of ouabain did not increase KChIP2 content in nuclear protein fractions in anesthetized mice. KChIP2 was expressed in cell lines, and Ca(2+) ionophores were applied in a concentration- and time-dependent manner. The cell lines had KChIP2-immunoreactive protein in the nucleus in the absence of treatments to modulate intracellular Ca(2+) concentration. Neither increasing nor decreasing intracellular Ca(2+) concentrations caused translocation of KChIP2. Microarray analysis did not identify relief of transcriptional repression in murine KChIP2(-/-) heart samples. We conclude that although there is a baseline presence of KChIP2 in the nucleus both in vivo and in vitro, KChIP2 does not directly regulate transcriptional activity. Moreover, the nuclear transport of KChIP2 is not dependent on Ca(2+). Thus, KChIP2 does not function as a conventional transcription factor in the heart. PMID:27349185

  5. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    Science.gov (United States)

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  6. Expression and detection of the FMDV VP1 transgene and expressed structural protein in Arabidopsis thaliana

    OpenAIRE

    Pan, Li; Zhang, Yongguang; Wang, Yonglu; Lv, Jianliang; Zhou, Peng; Zhang, Zhongwang

    2011-01-01

    To explore the feasibility of developing a new type of plantderived foot-and-mouth disease virus (FMDV) oral vaccine, the plant seed-specific expression vector p7SBin438/VP1 carrying the VP1 gene of the FMDV strain O/China/99 was constructed and transformed into Agrobacterium tumefaciens strain GV3101. This strain was used for transformation of Arabidopsis thaliana via the floral-dip method. The kanamycin-resistant transgenic plants were selected, and the VP1 gene and protein expressions were...

  7. Immunolocalization and expression of small-conductance calcium-activated potassium channels in human myometrium

    DEFF Research Database (Denmark)

    Rosenbaum, Sofia T; Svalø, Julie; Nielsen, Karsten;

    2012-01-01

    Small-conductance calcium-activated potassium (SK3) channels have been detected in human myometrium and we have previously shown a functional role of SK channels in human myometrium in vitro. The aims of this study were to identify the precise localization of SK3 channels and to quantify SK3 mRNA...

  8. The voltage-gated potassium channel subunit, Kv1.3, is expressed in epithelia

    DEFF Research Database (Denmark)

    Grunnet, Morten; Rasmussen, Hanne B; Hay-Schmidt, Anders;

    2003-01-01

    The Shaker-type voltage-gated potassium channel, Kv1.3, is believed to be restricted in distribution to lymphocytes and neurons. In lymphocytes, this channel has gained intense attention since it has been proven that inhibition of Kv1.3 channels compromise T lymphocyte activation. To investigate...

  9. NRAMP, TNF, TLR5, and Hepcidin Expression in Resistant and Susceptible Families of Channel Catfish Following Challenge With E. ictaluri

    Science.gov (United States)

    Real-time PCR was used to measure gene expression of Nramp, TNF, TLR5, and Hepcidin, in spleen and liver tissue from two families of channel catfish, one resistant and one susceptible to ESC, following challenge with Edwardsiella ictaluri. There were no significant differences in relative copy numbe...

  10. Expressed protein ligation for metalloprotein design and engineering.

    Science.gov (United States)

    Clark, Kevin M; van der Donk, Wilfred A; Lu, Yi

    2009-01-01

    Metalloproteins contain highly specialized metal-binding sites that are designed to accept specific metal ions to maintain correct function. Although many of the sites have been modified with success, the relative paucity of functional group availability within proteinogenic amino acids can sometimes leave open questions about specific functions of the metal binding ligands. Attaining a more thorough analysis of individual amino acid function within metalloproteins has been realized using expressed protein ligation (EPL). Here we describe our recent efforts using EPL to incorporate nonproteinogenic cysteine and methionine analogues into the type 1 copper site found in Pseudomonas aeruginosa azurin.

  11. Plant lipid transfer proteins : Evolution, expression and function

    OpenAIRE

    Edstam, Monika

    2013-01-01

    The plant non-specific lipid transfer proteins (nsLTPs) are known for the ability to transfer different lipids in vitro, but their in vivo functions have not yet been elucidated. They seem to play a role in the defense against biotic and abiotic stresses; the gene expression of nsLTPs is often upregulated when exposed to stresses. Further, two different nsLTPs have been shown to affect the lipid composition of the plant cuticle, a structure acting as a protective barrier. However, more eviden...

  12. Environmental Impact of Genetically Modified Maize Expressing Cry1 Proteins

    DEFF Research Database (Denmark)

    Bartsch, Detlef; Devos, Yann; Hails, Rosie;

    2010-01-01

    For more than a decade, genes of Bacillus thuringiensis (‘Bt’) that encode lepidopteran-specific protein toxins (Cry1Ab and Cry1F) have been engineered into maize for protection against lepidopteran pests. An extensive body of research data and environmental risk assessments (ERA) has been...... assembled on the potential environmental impact of Cry1 expressing maize. The available literature so far suggests only minor environmental effects. The majority of laboratory studies and all the field studies reviewed did not reveal any unexpected adverse or long-term effectson the environment. Negative...

  13. Analysis of cDNA sequence, protein structure and expression of parotid secretory protein in pig

    Institute of Scientific and Technical Information of China (English)

    YIN Haifang; FAN Baoliang; ZHAO Zhihui; LIU Zhaoliang; FEI Jing; LI Ning

    2003-01-01

    Parotid secretory protein (PSP) secreted abundantly in saliva, whose function is related with the anti-bacterial effect. The PSP cDNA has been isolated from pig parotid glands by 3′ and 5′ rapid amplification of cDNA end (RACE),based on the conserved signal peptide region among the known mammalian PSP. Theresult of homologous comparison shows that pig PSP and human PSP shares the high identity at the level of the primary, secondary and tertiary protein structure. A search for functionally significant protein motifs revealed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu- X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is important to its function. RT-PCR, Dot blot and Northern blot analysis demonstrated that PSP was strongly expressed in parotid glands, but not in other tissues.

  14. Development of transgenic chickens expressing enhanced green fluorescent protein

    International Nuclear Information System (INIS)

    In this work we demonstrated the successful production of transgenic chickens expressing the enhanced green fluorescence protein (EGFP) gene. Replication-defective recombinant retroviruses produced from vesicular stomatitis virus G glycoprotein pseudotyped retrovirus vector system were injected beneath the blastoderm of non-incubated chicken embryos (stage X). From 129 injected eggs, 13 chicks hatched after 21 days of incubation. All hatched chicks were found to express vector-encoded EGFP gene, which was under the control of the Rous sarcoma virus promoter and boosted post-transcriptionally by woodchuck hepatitis virus post-transcriptional regulatory element sequence. Green fluorescent signals, indicative of the EGFP gene expression, were detected in various body parts, including head, limb, eye, toe, and several internal organs. Genomic incorporation of the transgene was also proven by Southern blot assay. Our results show the exceptional versatile effectiveness of the EGFP gene as a marker in the gene expression-related studies which therefore would be very helpful in establishing a useful transgenic chicken model system for studies on embryo development and for efficient production of transgenic chickens as bioreactors

  15. Sperm protein 17 is expressed in human nervous system tumours

    Directory of Open Access Journals (Sweden)

    Frezza Eldo E

    2006-01-01

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

  16. Sperm protein 17 is expressed in human nervous system tumours

    International Nuclear Information System (INIS)

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

  17. Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Pei-Hong Jiang; Yoshiharu Motoo; Stéphane Garcia; Juan Lucio Iovanna; Marie-Josèphe Pébusque; Norio Sawabu

    2006-01-01

    AIM: Overexpression of tumor protein p53-induced nuclear protein 1 (TP53INP1) induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1and p53 expression in gastric cancer.METHODS: TP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed.RESULTS: TP53INP1 was expressed in 98% (139/142cases) of non-cancerous gastric tissues and was downexpressed in 64% (91/142 cases) of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased (43.9%) in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma (81.6%).Cancers invading the submucosa or deeper showed lower positively (59.1%) compared with mucosal cancers (85.2%). Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion (54.3%vs 82.0% without lymphatic invasion) and node-positive patients (31.3% vs 68.3% in node-negative patients).P53 was expressed in 68 (47.9%) patients of gastric cancer, whereas it was absent in normal gastric tissues.A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells: the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP1-negative portions. Finally, when survival data were analyzed,loss of TP53INP1 expression had a significant effect in predicting a poor prognosis (P= 0.0006).CONCLUSION: TP53INP1-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1is related to the apoptosis of gastric cancer cells. The decreased expression of the TP53INP1 protein may

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

    OpenAIRE

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

    2010-01-01

    Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear if this is due ...

  19. ABA Signaling in Guard Cells Entails a Dynamic Protein-Protein Interaction Relay from the PYL-RCAR Family Receptors to Ion Channels

    Institute of Scientific and Technical Information of China (English)

    Sung Chul Lee; Chae Woo Lim; Wenzhi Lan; Kai He; Sheng Luan

    2013-01-01

    Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells.We previously reported that SLACl,an outward anion channel required for stomatal closure,was regulated via reversible protein phosphorylation events involving ABA signaling components,including protein phosphatase 2C members and a SnRK2-type kinase (OST1).In this study,we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors,to the PP2C-SnRK2 phosphatase-kinase pairs,to the ion channel SLACl.The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase,releasing active SnRK2 kinase to phosphorylate,and activate the SLACl channel,leading to reduced guard cell turgor and stomatal closure.Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway.These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners.The SLACl channel activity was used as an endpoint readout for the strength of the signaling pathway,depending on the presence of different combinations of signaling components.Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yamakoshi, Takako [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Makino, Teruhiko, E-mail: tmakino@med.u-toyama.ac.jp [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Ur Rehman, Mati; Yoshihisa, Yoko [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Sugimori, Michiya [Department of Integrative Neuroscience, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Shimizu, Tadamichi, E-mail: shimizut@med.u-toyama.ac.jp [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan)

    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.

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

    International Nuclear Information System (INIS)

    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

  2. Growing functional modules from a seed protein via integration of protein interaction and gene expression data

    Directory of Open Access Journals (Sweden)

    Dimitrakopoulou Konstantina

    2007-10-01

    Full Text Available Abstract Background Nowadays modern biology aims at unravelling the strands of complex biological structures such as the protein-protein interaction (PPI networks. A key concept in the organization of PPI networks is the existence of dense subnetworks (functional modules in them. In recent approaches clustering algorithms were applied at these networks and the resulting subnetworks were evaluated by estimating the coverage of well-established protein complexes they contained. However, most of these algorithms elaborate on an unweighted graph structure which in turn fails to elevate those interactions that would contribute to the construction of biologically more valid and coherent functional modules. Results In the current study, we present a method that corroborates the integration of protein interaction and microarray data via the discovery of biologically valid functional modules. Initially the gene expression information is overlaid as weights onto the PPI network and the enriched PPI graph allows us to exploit its topological aspects, while simultaneously highlights enhanced functional association in specific pairs of proteins. Then we present an algorithm that unveils the functional modules of the weighted graph by expanding a kernel protein set, which originates from a given 'seed' protein used as starting-point. Conclusion The integrated data and the concept of our approach provide reliable functional modules. We give proofs based on yeast data that our method manages to give accurate results in terms both of structural coherency, as well as functional consistency.

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

    Science.gov (United States)

    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.

  4. A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders.

    Science.gov (United States)

    Yang, Sheng; Pyati, Prashant; Fitches, Elaine; Gatehouse, John A

    2014-04-01

    Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the "carrier" snowdrop lectin (GNA) to confer oral toxicity. The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th - 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide. PMID:24486516

  5. Decreased levels of canonical transient receptor potential channel 3 protein in the rat cerebral cortex after chronic treatment with lithium or valproate.

    Science.gov (United States)

    Zaeri, Sasan; Farjadian, Shirin; Emamghoreishi, Masoumeh

    2015-01-01

    Lithium and valproate modulate disturbances in intracellular calcium homeostasis implicated in the pathophysiology of bipolar disorder, but the molecular mechanisms are not fully understood. Two subtypes of transient receptor potential (TRP) channel family, i.e. TRPC3 and TRPM2, are potential candidates involved in calcium signaling and implicated in the pathophysiology of bipolar disorder. This study was designed to investigate whether mood stabilizers such as lithium and valproate affect the expression of TRPC3 and TRPM2. Rats were treated with intraperitoneal injections of lithium (2 mEq/kg b.i.d.) or valproate (300 mg/kg b.i.d.) acutely (for 24 h) or chronically (for 4 weeks). The changes in mRNA and protein levels of TRPC3 and TRPM2 were measured with real-time polymerase chain reaction and western blotting. The chronic administration of lithium and valproate significantly reduced levels of TRPC3 by 19.7% and 19.3%, respectively. No change was detected in the mRNA level of this channel. Neither acute nor chronic treatment with lithium or valproate had any effect on TRPM2 levels. The results suggest that downregulation of the TRPC3 channel is an important shared mechanism by which lithium and valproate can modulate calcium disturbances, whereas the TRPM2 channel does not appear to be affected by mood stabilizers, at least under non stressed conditions. PMID:26752988

  6. Expresión de canales de potasio voltaje dependientes en ovocitos de Xenopus laevis (Amphibia Voltage gated potassium channels expressed in Xenopus laevis(AMPHIBIA oocytes

    Directory of Open Access Journals (Sweden)

    Clavijo Carlos

    2003-06-01

    Full Text Available La expresión en sistemas heterólogos ha sido una herramienta ampliamente utilizada enlos últimos años para el estudio funcional y estructural de proteínas. Para la carac-terización de las propiedades biofísicas de canales, bombas y transportadores engeneral su expresión en ovocitos de Xenopus laevis, ha sido fundamental. Este estudioreporta la expresión de dos canales de potasio voltaje dependientes, Kv1.1y Shakerenovocitos de X. laevisusando un protocolo ajustado a las condiciones de latitud y altitudde Bogotá para la extracción, aislamiento, cultivo y microinyección de éstas células.Heterologous expression has been an important tool for structural and functionalcharacterization of proteins. The study of biophysical properties of ion channels,pumps and transporters has been possible thanks to their expression in Xenopuslaevisoocytes. Here we report the expression of two voltage gated channels, Kv1.1and Shaker, in X. laevisoocytes using a method for oocyte extraction, isolation, cul-ture, and microinjection adapted to the latitude and altitude conditions of Bogotá,Colombia.

  7. Characterization of protein expression levels with label-free detected reverse phase protein arrays.

    Science.gov (United States)

    Guo, Xuexue; Deng, Yihong; Zhu, Chenggang; Cai, Junlong; Zhu, Xiangdong; Landry, James P; Zheng, Fengyun; Cheng, Xunjia; Fei, Yiyan

    2016-09-15

    In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies. PMID:27372609

  8. Cell-Free Expression of Protein Kinase A for Rapid Activity Assays

    OpenAIRE

    Leippe, Donna M.; Kate Qin Zhao; Kevin Hsiao; Slater, Michael R.

    2010-01-01

    Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag® fusion protein. The cell-free protein synthesis systems provide quick access t...

  9. Expression and phosphorylation of neurofilament protein in different neuronal tissues

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The neurofilament proteins (NFPs) from different neuronal tissues including Alzheimer and Huntington disease gray matter, rat brain gray, white matter and spinal cord were separated biochemically into two major fractions. A systematic investigation on the distribution, expression and phosphorylation of NFPs in those fractions was undertaken in the present study. It was found that only non-phosphorylated NF-H and NF-M, but not NF-L subunit were detected in Alzheimer brain gray matter high speed supernatant, whereas all neurofilament subunits including non-phosphorylated and phosphorylated were measured in high speed pellet fraction of the same tissue. The hyperphosphorylation of NF-H and NF-M in Alzheimer brain was shown by phosphorylation dependent monoclonal antibodies SMI31 and SMI34. This hyperphosphorylation was confirmed by non-phosphorylation dependent antibody SMI32 with dephosphosphorylation of the samples. Furthermore, an increased amount of NF-H, NH-M and NF-L, detected by SMI33 and NR4 respectively, was also observed in Alzheimer samples, in which the elevation in NF-L was significant. A significantly different immunoblot patterns in distribution, expression and phosphorylation were determined in various position of the neural system and alternative fractions. To our best knowledge, this is the first data shown definite abnormality of NFPs in Alzheimer disease. The information obtained in the present study will be extremely valuable in further study of the proteins both in physiological and pathological conditions.

  10. Neurotoxocarosis alters myelin protein gene transcription and expression.

    Science.gov (United States)

    Heuer, Lea; Beyerbach, Martin; Lühder, Fred; Beineke, Andreas; Strube, Christina

    2015-06-01

    Neurotoxocarosis is an infection of the central nervous system caused by migrating larvae of the common dog and cat roundworms (Toxocara canis and Toxocara cati), which are zoonotic agents. As these parasites are prevalent worldwide and neuropathological and molecular investigations on neurotoxocarosis are scare, this study aims to characterise nerve fibre demyelination associated with neurotoxocarosis on a molecular level. Transcription of eight myelin-associated genes (Cnp, Mag, Mbp, Mog, Mrf-1, Nogo-A, Plp1, Olig2) was determined in the mouse model during six time points of the chronic phase of infection using qRT-PCR. Expression of selected proteins was analysed by Western blotting or immunohistochemistry. Additionally, demyelination and neuronal damage were investigated histologically. Significant differences (p ≤ 0.05) between transcription rates of T. canis-infected and uninfected control mice were detected for all analysed genes while T. cati affected five of eight investigated genes. Interestingly, 2', 3 ´-cyclic nucleotide 3'-phosphodiesterase (Cnp) and myelin oligodendrocyte glycoprotein (Mog) were upregulated in both T. canis- and T. cati-infected mice preceding demyelination. Later, CNPase expression was additionally enhanced. As expected, myelin basic protein (Mbp) was downregulated in cerebra and cerebella of T. canis-infected mice when severe demyelination was present 120 days post infectionem (dpi). The transcriptional pattern observed in the present study appears to reflect direct traumatic and hypoxic effects of larval migration as well as secondary processes including host immune reactions, demyelination and attempts to remyelinate damaged areas.

  11. T-type Calcium Channel Regulation of Neural Tube Closure and EphrinA/EPHA Expression

    Directory of Open Access Journals (Sweden)

    Sarah Abdul-Wajid

    2015-10-01

    Full Text Available A major class of human birth defects arise from aberrations during neural tube closure (NTC. We report on a NTC signaling pathway requiring T-type calcium channels (TTCCs that is conserved between primitive chordates (Ciona and Xenopus. With loss of TTCCs, there is a failure to seal the anterior neural folds. Accompanying loss of TTCCs is an upregulation of EphrinA effectors. Ephrin signaling is known to be important in NTC, and ephrins can affect both cell adhesion and repulsion. In Ciona, ephrinA-d expression is downregulated at the end of neurulation, whereas, with loss of TTCC, ephrinA-d remains elevated. Accordingly, overexpression of ephrinA-d phenocopied TTCC loss of function, while overexpression of a dominant-negative Ephrin receptor was able to rescue NTC in a Ciona TTCC mutant. We hypothesize that signaling through TTCCs is necessary for proper anterior NTC through downregulation of ephrins, and possibly elimination of a repulsive signal.

  12. The design of a three-channel nuclear spectrometry tool for express logging

    International Nuclear Information System (INIS)

    This paper describes a three-channel nuclear spectrometry logging tool, which is designed to meet the needs of platform express logging. The application of complicated programmable logical device (CPLD) expands the tool's function and minimizes its scale. This tool can be used to analyse pulse height for natural gamma spectrometry, long-space and short-space detectors of Litho-density. Compensated neutron log (CNL) and other conventional analog and pulse logging signals can also be acquired. Manchester's code is used in data transmission. Half-duplex mode with 20 kb/s data rate can be selected when it is connected to systems which are compatible with Atlas 3508 telemetry cartridge; or full-duplex mode with 20 kb/s down-link data rate and 93.75 kb/s (or 41.66 kb/s) up-link data rate can be selected when it is connected to systems which are compatible with Atlas WTS telemetry system

  13. L—type calcium channel blockers inhibit the development but not the expression of sensitization to morphine in mice

    Institute of Scientific and Technical Information of China (English)

    ZhanQ; ZhenJW

    2002-01-01

    The relationship between opioid actions and L-type calcium channel blockers has been well documented.However,there is no report relevant to L-type calcium channel blockers and morphinesensitization,which is suggested to be an analog of behaviors that are the characteristics of drug addiction.Here the effects of three L-type calcium channel blockers,nimodipine,nifedipine and verapamil,on morphine-induced locomotor activity,the development and the expression of sensitization to morphine were studied systematically.The results showed that both nimodipine and verapamil attenuated,while nifedipine had only a tendency to decrease morphine-induced locomotor activity.All the three drugs inhibited the development of sensitization to morphine.However,none of them showed any effects on the expression of morphine sensitization.These results indicate that blocking L-tpye calcium channel attenuates the locomotor stimulating effects of morphine and inhibits the development but not the expression of morphine-sensitization.

  14. Development of supported biomimetic membranes for insertion of aquaporin protein water channels for novel water filtration applications

    DEFF Research Database (Denmark)

    Hansen, Jesper Søndergaard

    in a horizontal chamber design. Chapter 4 characterizes reconstitution and folding of E. coli Aquaporin–Z (AqpZ) and the spinach plasma integral protein 2;1 (SoPIP2;1) aquaporins into model membranes. A central part of this chapter is the development of a method for formation of giant protein vesicles (≥10 μm......Aquaporins represent a class of membrane protein channels found in all living organisms that selectively transport water molecules across biological membranes. The work presented in this thesis was motivated by the conceptual idea of incorporating aquaporin water channels into biomimetic membranes...... to develop novel water separation technologies. To accomplish this, it is necessary to construct an efficient platform to handle biomimetic membranes. Moreover, general methods are required to reliable and controllable reconstitute membrane proteins into artificially made model membranes...

  15. Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol.

    Directory of Open Access Journals (Sweden)

    Stella M Valenzuela

    Full Text Available The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer.

  16. Activation of Mitochondrial Uncoupling Protein 4 and ATP-Sensitive Potassium Channel Cumulatively Decreases Superoxide Production in Insect Mitochondria.

    Science.gov (United States)

    Slocińska, Malgorzata; Rosinski, Grzegorz; Jarmuszkiewicz, Wieslawa

    2016-01-01

    It has been evidenced that mitochondrial uncoupling protein 4 (UCP4) and ATP-regulated potassium channel (mKATP channel) of insect Gromphadorhina coqereliana mitochondria decrease superoxide anion production. We elucidated whether the two energy-dissipating systems work together on a modulation of superoxide level in cockroach mitochondria. Our data show that the simultaneous activation of UCP4 by palmitic acid and mKATP channel by pinacidil revealed a cumulative effect on weakening mitochondrial superoxide formation. The inhibition of UCP4 by GTP (and/or ATP) and mKATP channel by ATP elevated superoxide production. These results suggest a functional cooperation of both energy-dissipating systems in protection against oxidative stress in insects.

  17. Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    L. Avilán

    1997-12-01

    Full Text Available We cloned the streptokinase (STK gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

  18. Imaging bacterial protein expression using genetically encoded sensors composed of RNA

    OpenAIRE

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

    2013-01-01

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

  19. A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D.; Jin, Jian

    2009-10-02

    To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.

  20. Down Regulated Expression of the β1 Subunit of the Big-conductance Ca2+ Sensitive K+ Channel in Sphincter of Oddi Cells from Rabbits Fed with a High Cholesterol Diet

    Institute of Scientific and Technical Information of China (English)

    Pang DU; Guang-Bin CUI; Ya-Rong WANG; Xiao-Yong ZHANG; Ke-Jun MA; Jing-Guo WEI

    2006-01-01

    Hypercholesterolemia, which is closely related to gallbladder bile stasis, can cause sphincter of Oddi dysfunction (SOD) by increasing the tension of sphincter of Oddi (SO). Intracellular calcium ion concentration ([Ca2+]i) could influence the tension of SO. The β1 subunit of the big-conductance Ca2+sensitive K+ channel (BKCa) can enhance the sensitivity of the BKCa channel to [Ca2+]i. Absence and decline of the BKCa channel subunit β1 could lead to many diseases. However, the relationship between hypercholesterolemia and the expression of β1 subunit is not well understood. In this study, we successfully expressed and purified the rabbit BKCa β1 subunit protein and prepared its polyclonal antibody. The specificity of the prepared antibody was determined by western blotting. A SOD rabbit model induced by a high cholesterol diet was established and the expression of the β1 subunit of SO was determined by immunohistochemical staining and western blotting. Compared with the controls, our results demonstrated that hypercholesterolemia could decrease the expression of the β1 subunit in the SO cells from rabbits. This indicates that lower expression of BKCa channel β1 subunit might induce SOD.

  1. Differentially expressed protein markers in human submandibular and sublingual secretions.

    Science.gov (United States)

    Hu, Shen; Denny, Patricia; Denny, Paul; Xie, Yongming; Loo, Joseph A; Wolinsky, Lawrence E; Li, Yang; McBride, Jim; Ogorzalek Loo, Rachel R; Navazesh, Mavash; Wong, David T

    2004-11-01

    Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

  2. Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

    DEFF Research Database (Denmark)

    Rougier, Jean-Sébastien; van Bemmelen, Miguel X; Bruce, M Christine;

    2004-01-01

    The voltage-gated Na(+) channels (Na(v)) form a family composed of 10 genes. The COOH termini of Na(v) contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein...

  3. Replacement of fish meal in juvenile channel catfish, Ictalurus punctatus, diets using a yeast-derived protein source

    Science.gov (United States)

    We examined the effects of a yeast-derived protein source (NuPro) as a replacement for menhaden fish meal on weight gain, specific growth rate (SGR), food conversion ratio (FCR), whole-body composition, and disease resistance in juvenile channel catfish. NuPro replaced 0, 20, 40, 60, 80, and 100% o...

  4. Students' Understanding of External Representations of the Potassium Ion Channel Protein Part II: Structure-Function Relationships and Fragmented Knowledge

    Science.gov (United States)

    Harle, Marissa; Towns, Marcy H.

    2012-01-01

    Research that has focused on external representations in biochemistry has uncovered student difficulties in comprehending and interpreting external representations. This study focuses on students' understanding of three external representations (ribbon diagram, wireframe, and hydrophobic/hydrophilic) of the potassium ion channel protein. Analysis…

  5. Biomimetic membranes with aqueous nano channels but without proteins: impedance of impregnated cellulose ester filters.

    Science.gov (United States)

    Kocherginsky, Nikolai M; Lvovich, Vadim F

    2010-12-01

    Earlier we have shown that many important properties of ionic aqueous channels in biological membranes can be imitated using simple biomimetic membranes. These membranes are composed of mixed cellulose ester-based filters, impregnated with isopropyl myristate or other esters of fatty acids, and can be used for high-throughput drug screening. If the membrane separates two aqueous solutions, combination of relatively hydrophilic polymer support with immobilized carboxylic groups results in the formation of thin aqueous layers covering inner surface of the pores, while the pore volume is filled by lipid-like substances. Because of these aqueous layers biomimetic membranes even without proteins have a cation/anion ion selectivity and specific (per unit of thickness) electrical properties, which are similar to typical properties of biological membranes. Here we describe frequency-dependent impedance of the isopropyl myristate-impregnated biomimetic membranes in the 4-electrode arrangement and present the results as Bode and Nyquist diagrams. When the membranes are placed in deionized water, it is possible to observe three different dispersion processes in the frequency range 0.1 Hz to 30 kHz. Only one dispersion is observed in 5 mM KH(2)PO(4) solution. It is suggested that these three dispersion features are determined by (a) conductivity in aqueous structures/channels, formed near the internal walls of the filter pores at high frequencies, (b) dielectric properties of the whole membrane at medium frequencies, determined by polymer support, aqueous layers and impregnating oil, and, finally, (c) by the processes in hydrated liquid crystal structures formed in pores by impregnating oil in contact with water at low frequencies.

  6. Cellular prion protein expression is not regulated by the Alzheimer's amyloid precursor protein intracellular domain.

    Directory of Open Access Journals (Sweden)

    Victoria Lewis

    Full Text Available There is increasing evidence of molecular and cellular links between Alzheimer's disease (AD and prion diseases. The cellular prion protein, PrP(C, modulates the post-translational processing of the AD amyloid precursor protein (APP, through its inhibition of the β-secretase BACE1, and oligomers of amyloid-β bind to PrP(C which may mediate amyloid-β neurotoxicity. In addition, the APP intracellular domain (AICD, which acts as a transcriptional regulator, has been reported to control the expression of PrP(C. Through the use of transgenic mice, cell culture models and manipulation of APP expression and processing, this study aimed to clarify the role of AICD in regulating PrP(C. Over-expression of the three major isoforms of human APP (APP(695, APP(751 and APP(770 in cultured neuronal and non-neuronal cells had no effect on the level of endogenous PrP(C. Furthermore, analysis of brain tissue from transgenic mice over-expressing either wild type or familial AD associated mutant human APP revealed unaltered PrP(C levels. Knockdown of endogenous APP expression in cells by siRNA or inhibition of γ-secretase activity also had no effect on PrP(C levels. Overall, we did not detect any significant difference in the expression of PrP(C in any of the cell or animal-based paradigms considered, indicating that the control of cellular PrP(C levels by AICD is not as straightforward as previously suggested.

  7. Different protein expression of myocardium from Chinese mini-swine model of myocardial infarct

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yanfeng; YE Nengsheng; ZHANG Rongli; FENG Xue; LUO Guoan; WANG Yiming

    2007-01-01

    High-resolution two-dimensional gel electropho-resis (2-DE), followed by computer-assisted image analysis was used to screen protein patterns of normal and infarcted myocardial tissues for quantitative and qualitative differencesin protein expression. In the gels of pH 5-8 immobilizedpH gradient (IPG) strips, 851 protein spots were detected in normal myocardial tissue and 1 032 protein spots were resolved in infarcted myocardial tissue. Thirteen protein spots only expressed in normal myocardial tissue, and 14 protein spots only expressed in infarcted myocardial tissue. Results also showed that 49 protein spots displayed quantitative changes in expression between normal and infarcted myocar-dial tissue. Eleven protein spots were subjected to mass spectrometry (MS) analysis and seven proteins were identi-fied by peptide mass fingerprinting (PMF). These proteins may be involved in cardiovascular injury, and could play an important role in the treatment of coronary heart disease.

  8. Regulation of voltage-gated sodium channel expression in cancer: hormones, growth factors and auto-regulation.

    Science.gov (United States)

    Fraser, Scott P; Ozerlat-Gunduz, Iley; Brackenbury, William J; Fitzgerald, Elizabeth M; Campbell, Thomas M; Coombes, R Charles; Djamgoz, Mustafa B A

    2014-03-19

    Although ion channels are increasingly being discovered in cancer cells in vitro and in vivo, and shown to contribute to different aspects and stages of the cancer process, much less is known about the mechanisms controlling their expression. Here, we focus on voltage-gated Na(+) channels (VGSCs) which are upregulated in many types of carcinomas where their activity potentiates cell behaviours integral to the metastatic cascade. Regulation of VGSCs occurs at a hierarchy of levels from transcription to post-translation. Importantly, mainstream cancer mechanisms, especially hormones and growth factors, play a significant role in the regulation. On the whole, in major hormone-sensitive cancers, such as breast and prostate cancer, there is a negative association between genomic steroid hormone sensitivity and functional VGSC expression. Activity-dependent regulation by positive feedback has been demonstrated in strongly metastatic cells whereby the VGSC is self-sustaining, with its activity promoting further functional channel expression. Such auto-regulation is unlike normal cells in which activity-dependent regulation occurs mostly via negative feedback. Throughout, we highlight the possible clinical implications of functional VGSC expression and regulation in cancer. PMID:24493753

  9. Endogenous and exogenous hydrogen sulfide facilitates T-type calcium channel currents in Cav3.2-expressing HEK293 cells.

    Science.gov (United States)

    Sekiguchi, Fumiko; Miyamoto, Yosuke; Kanaoka, Daiki; Ide, Hiroki; Yoshida, Shigeru; Ohkubo, Tsuyako; Kawabata, Atsufumi

    2014-02-28

    Hydrogen sulfide (H2S), a gasotransmitter, is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE). We have shown that an H2S donor, NaHS, causes hyperalgesia in rodents, an effect inhibited by knockdown of Cav3.2 T-type Ca(2+) channels (T-channels), and that NaHS facilitates T-channel-dependent currents (T-currents) in NG108-15 cells that naturally express Cav3.2. In the present study, we asked if endogenous and exogenous H2S participates in regulation of the channel functions in Cav3.2-transfected HEK293 (Cav3.2-HEK293) cells. dl-Propargylglycine (PPG), a CSE inhibitor, significantly decreased T-currents in Cav3.2-HEK293 cells, but not in NG108-15 cells. NaHS at 1.5mM did not affect T-currents in Cav3.2-HEK293 cells, but enhanced T-currents in NG108-15 cells. In the presence of PPG, NaHS at 1.5mM, but not 0.1-0.3mM, increased T-currents in Cav3.2-HEK293 cells. Similarly, Na2S, another H2S donor, at 0.1-0.3mM significantly increased T-currents in the presence, but not absence, of PPG in Cav3.2-HEK293 cells. Expression of CSE was detected at protein and mRNA levels in HEK293 cells. Intraplantar administration of Na2S, like NaHS, caused mechanical hyperalgesia, an effect blocked by NNC 55-0396, a T-channel inhibitor. The in vivo potency of Na2S was higher than NaHS. These results suggest that the function of Cav3.2 T-channels is tonically enhanced by endogenous H2S synthesized by CSE in Cav3.2-HEK293 cells, and that exogenous H2S is capable of enhancing Cav3.2 function when endogenous H2S production by CSE is inhibited. In addition, Na2S is considered a more potent H2S donor than NaHS in vitro as well as in vivo.

  10. Stable Surface Expression of a Gene for Helicobacter pylori Toxic Porin Protein with pBAD Expression System

    Institute of Scientific and Technical Information of China (English)

    Zhixiang PENG; Xi WEI; Zhengmei LIN

    2009-01-01

    successive passages could express Hope protein, while only 1 from 5 E. coli colonies that contained lac operon-regulated plasmid encoding hopE gene could express HopE. Indi-rect immunofluorescence confirmed the expression of HopE on E. coli cell surface.

  11. [Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

    Science.gov (United States)

    Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

    2016-01-01

    We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

  12. Rational Design of Analyte Channels of the Green Fluorescent Protein for Biosensor Applications

    Directory of Open Access Journals (Sweden)

    Natta Tansila, Tanawut Tantimongcolwat, Chartchalerm Isarankura-Na-Ayudhya, Chanin Nantasenamat, Virapong Prachayasittikul

    2007-01-01

    Full Text Available A novel solvent-exposed analyte channel, generated by F165G substitution, on the surface of green fluorescent protein (designated His6GFPuv/F165G was successfully discovered by the aid of molecular modeling software (PyMOL in conjunction with site-directed mutagenesis. Regarding the high predictive performance of PyMOL, two pore-containing mutants namely His6GFPuv/H148G and His6GFPuv/H148G/F165G were also revealed. The pore sizes of F165G, H148G, and the double mutant H148G/F165G were in the order of 4, 4.5 and 5.5 Å, respectively. These mutants were subjected to further investigation on the effect of small analytes (e.g. metal ions and hydrogen peroxide as elucidated by fluorescence quenching experiments. Results revealed that the F165G mutant exhibited the highest metal sensitivity at physiological pH. Meanwhile, the other 2 mutants lacking histidine at position 148 had lower sensitivity against Zn2+ and Cu2+ than those of the template protein (His6GFPuv. Hence, a significant role of this histidine residue in mediating metal transfer toward the GFP chromophore was proposed and evidently demonstrated by testing in acidic condition. Results revealed that at pH 6.5 the order of metal sensitivity was found to be inverted whereby the H148G/F165G became the most sensitive mutant. The dissociation constants (Kd to metal ions were in the order of 4.88×10-6 M, 16.67×10-6 M, 25×10-6 M, and 33.33×10-6 M for His6GFPuv/F165G, His6GFPuv, His6GFPuv/H148G/F165G and His6GFPuv/H148G, respectively. Sensitivity against hydrogen peroxide was in the order of H148G/F165G > H148G > F165G indicating the crucial role of pore diameters. However, it should be mentioned that H148G substitution caused a markedly decrease in pH- and thermo-stability. Taken together, our findings rendered the novel pore of GFP as formed by F165G substitution to be a high impact channel without adversely affecting the intrinsic fluorescent properties. This opens up a great potential of

  13. Functional coupling between heterologously expressed dopamine D(2) receptors and KCNQ channels

    DEFF Research Database (Denmark)

    Ljungstrom, Trine; Grunnet, Morten; Jensen, Bo Skaaning;

    2003-01-01

    -channel interaction. The KCNQ4 current was investigated in further detail and was increased by 19.9+/-1.6% ( n=20) by D(2L) receptor stimulation. The effect could be mimicked by injection of GTPgammaS and prevented by injection of Bordetella pertussis toxin, indicating that channel stimulation was mediated via a G...

  14. Trafficking and surface expression of hyperpolarization-activated cyclic nucleotide-gated channels in hippocampal neurons

    NARCIS (Netherlands)

    Y. Noam; Q. Zha; L. Phan; R.L. Wu; D.M. Chetkovich; W.J. Wadman; T.Z. Baram

    2010-01-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current I(h) and thus play important roles in the regulation of brain excitability. The subcellular distribution pattern of the HCN channels influences the effects that they exert on the proper

  15. Finding differentially expressed genes in two-channel DNA microarray datasets: how to increase reliability of data preprocessing.

    Science.gov (United States)

    Rotter, Ana; Hren, Matjaz; Baebler, Spela; Blejec, Andrej; Gruden, Kristina

    2008-09-01

    Due to the great variety of preprocessing tools in two-channel expression microarray data analysis it is difficult to choose the most appropriate one for a given experimental setup. In our study, two independent two-channel inhouse microarray experiments as well as a publicly available dataset were used to investigate the influence of the selection of preprocessing methods (background correction, normalization, and duplicate spots correlation calculation) on the discovery of differentially expressed genes. Here we are showing that both the list of differentially expressed genes and the expression values of selected genes depend significantly on the preprocessing approach applied. The choice of normalization method to be used had the highest impact on the results. We propose a simple but efficient approach to increase the reliability of obtained results, where two normalization methods which are theoretically distinct from one another are used on the same dataset. Then the intersection of results, that is, the lists of differentially expressed genes, is used in order to get a more accurate estimation of the genes that were de facto differentially expressed.

  16. Patagonfibrase modifies protein expression of tissue factor and protein disulfide isomerase in rat skin.

    Science.gov (United States)

    Peichoto, María Elisa; Santoro, Marcelo Larami

    2016-09-01

    Patagonfibrase is a hemorrhagic metalloproteinase isolated from the venom of the South American rear-fanged snake Philodryas patagoniensis, and is an important contributor to local lesions inflicted by this species. The tissue factor (TF)-factor VIIa complex, besides triggering the coagulation cascade, has been demonstrated to be involved in inflammatory events. Our aim was to determine whether patagonfibrase affects the expression of TF and protein disulfide isomerase (PDI), an enzyme that controls TF biological activity, at the site of patagonfibrase injection, and thus if they may play a role in hemostatic and inflammatory events induced by snake venoms. Patagonfibrase (60 μg/kg) was administered s.c. to rats, and after 3 h blood was collected to evaluate hemostasis parameters, and skin fragments close to the site of injection were taken to assess TF and PDI expression. Patagonfibrase did not alter blood cell counts, plasma fibrinogen levels, or levels of TF activity in plasma. However, by semiquantitative Western blotting, patagonfibrase increased TF expression by 2-fold, and decreased PDI expression by 3-fold in skin samples. In agreement, by immunohistochemical analyses, prominent TF expression was observed in the subcutaneous tissue. Thus, patagonfibrase affects the local expression of TF and PDI without inducing any systemic hemostatic disturbance, although that they may be involved in the local inflammatory events induced by hemorrhagic metalloproteinases. Once antivenom therapy is not totally effective to treat the local injury induced by snake venoms, modulation of the activity and expression of TF and/or PDI might become a strategy for treating snake envenomation. PMID:27390042

  17. Expression of interferon inducible protein-10 in pancreas of mice

    Institute of Scientific and Technical Information of China (English)

    Dong Li; Su-Wen Zhu; Dong-Juan Liu; Guo-Liang Liu

    2005-01-01

    AIM: To investigate the expression of interferon inducible protein-10 (IP-10) in pancreas of mice and to discuss its possible role in the pathogenesis of type 1 diabetes.METHODS: Non-obese diabetic (NOD) mice were used as experiment group and BALB/c mice as non-diabetic prone model. Immunohistochemistry method was used to evaluate the expression of IP-10 in the pancreas of NOD mice and BALB/c mice. Immunoelectron microscope was used to show the location of IP-10 in pancreatic islet β cells.RESULTS: Pancreatic islets were positively stained in all the NOD mice. Insulitis could be found in mice at the age of 4 wk. The weakly positive results were found in control group with no insulitis. Immunoelectron microscopy further demonstrated that IP-10 was produced by pancreatic β cells and stored in cytoplasm of the cells.CONCLUSION: IP-10 can be largely produced in pancreatic islets of NOD mice at the age of 2 wk when there is no significant insulitis, and may play an important part in the pathogenesis of type 1 diabetes by attracting immune cells to infiltrate the pancreatic islets.

  18. Plasma-assisted quadruple-channel optosensing of proteins and cells with Mn-doped ZnS quantum dots

    Science.gov (United States)

    Li, Chenghui; Wu, Peng; Hou, Xiandeng

    2016-02-01

    Information extraction from nano-bio-systems is crucial for understanding their inner molecular level interactions and can help in the development of multidimensional/multimodal sensing devices to realize novel or expanded functionalities. The intrinsic fluorescence (IF) of proteins has long been considered as an effective tool for studying protein structures and dynamics, but not for protein recognition analysis partially because it generally contributes to the fluorescence background in bioanalysis. Here we explored the use of IF as the fourth channel optical input for a multidimensional optosensing device, together with the triple-channel optical output of Mn-doped ZnS QDs (fluorescence from ZnS host, phosphorescence from Mn2+ dopant, and Rayleigh light scattering from the QDs), to dramatically improve the protein recognition and discrimination resolution. To further increase the cross-reactivity of the multidimensional optosensing device, plasma modification of proteins was explored to enhance the IF difference as well as their interactions with Mn-doped ZnS QDs. Such a sensor device was demonstrated for highly discriminative and precise identification of proteins in human serum and urine samples, and for cancer and normal cells as well.Information extraction from nano-bio-systems is crucial for understanding their inner molecular level interactions and can help in the development of multidimensional/multimodal sensing devices to realize novel or expanded functionalities. The intrinsic fluorescence (IF) of proteins has long been considered as an effective tool for studying protein structures and dynamics, but not for protein recognition analysis partially because it generally contributes to the fluorescence background in bioanalysis. Here we explored the use of IF as the fourth channel optical input for a multidimensional optosensing device, together with the triple-channel optical output of Mn-doped ZnS QDs (fluorescence from ZnS host, phosphorescence from Mn2

  19. An increased expression of Ca(2+) channel alpha(1A) subunit immunoreactivity in deep cerebellar neurons of rolling mouse Nagoya.

    Science.gov (United States)

    Sawada, K; Sakata-Haga, H; Ando, M; Takeda, N; Fukui, Y

    2001-12-01

    Rolling mouse Nagoya (RMN) is an ataxic mutant and carries a mutation in the gene coding for the alpha(1A) subunit of the P/Q-type Ca(2+) channel. We examined the immunohistochemical expression of the alpha(1A) subunit in deep cerebellar nuclei of RMN. The antibody used recognized residues 865-883 of the mouse alpha(1A) subunit not overlapping the altered sequences in RMN. In RMN, many neurons exhibited definite alpha(1A) subunit-staining in the medial nucleus, interposed nucleus, and lateral nucleus of deep cerebellar nuclei. The number of positive neurons in these nuclei was significantly higher in RMN than in controls. Increased expression of the alpha(1A) subunit in deep cerebellar neurons might compensate for the altered function of the P/Q-type Ca(2+) channel of RMN.

  20. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    Science.gov (United States)

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.

  1. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    Science.gov (United States)

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. PMID:26805756

  2. Expression of Yes-associated protein 1 gene and protein in oral squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    LI Song-ying; HU Ji-an; WANG Hui-ming

    2013-01-01

    Background Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the oral and maxillofaoial region.Yes-associated protein 1 (YAP1) has been implicated as a bona fide oncogene in solid tumors.We seek to elucidate the role of YAP1 in OSCC tissue.Methods We identified YAP1 gene and protein overexpression in 30 OSCC patients and 10 normal oral mucosa tissues by immunohistochemistry,Western blotting and reverse transcription polymerase chain reaction (RT-PCR).Results In the normal oral mucosa by immunohistochemical staining,YAP1 mainly located in both the cytoplasm and nucleus mainly the nuclei of the basal cells.In OSCC,the expression of YAP1 translocated from the nucleus to cytoplasm;YAP1 being mainly located in both the cytoplasm and nucleus of the adjacent mucosa.The expression of YAP1 gradual increased in normal oral mucosa,tumor adjacent mucosa and low grade,middle grade,high grade OSCC tissue by Western blotting.Significant difference was found between the expressions of the normal oral mucosa and OSCC tissue (P <0.05).The coincidence was detected between the normal oral mucosa and OSCC tissue by RT-PCR (P <0.05).Conclusions YAP1 is involved in the carcinogenesis and development of OSCC.There is a transformation between nucleus and cytoplasm.

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

    Science.gov (United States)

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

    2013-03-01

    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. PMID:23376073

  4. C-reactive protein inhibits survivin expression via Akt/mTOR pathway downregulation by PTEN expression in cardiac myocytes.

    Directory of Open Access Journals (Sweden)

    Beom Seob Lee

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

  5. Influence of dexamethasone on the expression and distribution of transient receptor potential cation channel 6 in glomerular podocytes

    Institute of Scientific and Technical Information of China (English)

    王辉阳

    2014-01-01

    Objective To observe the changes of foot processes,expression and distribution of transient receptor potential cation channel 6(TRPC6)in podocytes by puromycin aminonucleoside(PAN)and dexamethasone(DEX)intervention,then to investigate the function of TRPC6 in podocytes and its relation to proteinuria in kidney diseases.Methods Podocytes cultured in vitro were divided into three group:control group,PAN stimulation group and DEX intervention group.Mouse podocyte cell line

  6. Molecular cloning, expression and the adjuvant effects of interleukin-8 of channel catfish (Ictalurus Punctatus) against Streptococcus iniae

    OpenAIRE

    Erlong Wang; Jun Wang; Bo Long; Kaiyu Wang; Yang He; Qian Yang; Defang Chen; Yi Geng; Xiaoli Huang; Ping Ouyang; Weimin Lai

    2016-01-01

    Interleukin-8 (IL-8) as an important cytokine involving in inflammatory and immune response, has been studied as effective adjuvants for vaccines in mammals. However, there are fewer reports about the characterization and adjuvant effects of IL-8 in fish. In this study, cloning and sequence analysis of IL-8 coding region of channel catfish (Ictalurus punctatus) were conducted, mature IL-8(rtIL-8) was expressed and evaluated for its adjuvant effects on the immunoprotection of subunit vaccine e...

  7. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

    Science.gov (United States)

    Veit, Guido; Oliver, Kathryn; Apaja, Pirjo M; Perdomo, Doranda; Bidaud-Meynard, Aurélien; Lin, Sheng-Ting; Guo, Jingyu; Icyuz, Mert; Sorscher, Eric J; Hartman Iv, John L; Lukacs, Gergely L

    2016-05-01

    The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

  8. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

    Directory of Open Access Journals (Sweden)

    Guido Veit

    2016-05-01

    Full Text Available The most common cystic fibrosis (CF causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del, results in functional expression defect of the CF transmembrane conductance regulator (CFTR at the apical plasma membrane (PM of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER. Deletion of phenylalanine 670 (ΔF670 in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

  9. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

    Science.gov (United States)

    Veit, Guido; Oliver, Kathryn; Apaja, Pirjo M; Perdomo, Doranda; Bidaud-Meynard, Aurélien; Lin, Sheng-Ting; Guo, Jingyu; Icyuz, Mert; Sorscher, Eric J; Hartman Iv, John L; Lukacs, Gergely L

    2016-05-01

    The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect. PMID:27168400

  10. Upregulated TRPC3 and downregulated TRPC1 channel expression during hypertension is associated with increased vascular contractility in rat

    Directory of Open Access Journals (Sweden)

    Muzamil M Noorani

    2011-07-01

    Full Text Available Transient receptor potential C1 and C3 (TRPC1 and TRPC3 are expressed in vascular smooth muscle cells and are thought to be involved in vascular contractility. In the present study, we determined the effect of systemic hypertension on TRPC1/TRPC3 channel expression and vascular contractility in rat carotid artery (CA. CA were studied from male spontaneously hypertensive (SHR, Wistar Kyoto (WKY and Long-Evans (LE rats. TRPC1/3 expression was determined by RT-PCR and Western blot. TRP channel function was evaluated by whole cell patch clamp, using UTP (60 µM to stimulate TRPC1/3 channels. Contractions of endothelium-denuded CA segments to UTP (1 – 300 µM and phenylephrine (Phe; 0.1 nM-10 µM were measured in an isometric tension bath. TRPC1 and TRPC3 mRNA was present in CA of both WKY and SHR. Western blot demonstrated 3.1 ± 1.2 times greater TRPC3 expression and 0.5 ± 0.2 times TRPC1 in SHR versus WKY CA. Isolated CA showed potentiated contraction to UTP in the SHR versus WKY. Activation of voltage-dependent Ca2+ channels (VDCC in UTP-mediated constriction only occurred in SHR CA. Contraction to Phe was unaltered between WKY and SHR CA and involved equal significant VDCC activation in both groups. Patch clamp demonstrated that the UTP-stimulated current (Iutp was greater in SHR compared to the normotensive WKY and LE rats with peak Iutp (at -110 mV of -63 ± 24 pA compared to -25 ± 4 pA, respectively. We demonstrate that UTP-mediated but not Phe-mediated constrictions are potentiated in the CA during hypertension. Expression of TRPC1 is decreased whereas TRPC3 is increased in SHR CA. Interestingly, VDCC activation only contributes to UTP-mediated contraction of SHR CAs whereas it contributes substantially and equally in Phe-mediated contraction. We speculate that the alteration of TRPC channel expression in hypertension leads to greater smooth muscle depolarization, VDCC activation, and vascular contractility in the UTP (but not Phe

  11. Comment on: Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in the gastrointestinal system

    Institute of Scientific and Technical Information of China (English)

    Ali Mobasheri

    2006-01-01

    @@ TO THE EDITOR Sir, I read with great interest the recently published article in the World Journal of Gastroenterology by Jin and co-workers[1] on the cloning and characterization of porcine aquaporin 1 water channel from the pig liver and studies on its expression in the porcine gastrointestinal system. The authors should be congratulated for making this important and valuable contribution to the field of aquaporin biology and porcine gastrointestinal physiology.However, there are a number of unresolved issues and controversies concerning the expression of aquaporins (especially aquaporin 1) in the gastrointestinal system that are worthy of additional comment and discussion by Jin and co-workers.

  12. Heterogeneous Expression of T-type Ca2+ Channels Defines Different Neuronal Populations in the Inferior Olive of the Mouse

    Science.gov (United States)

    Bazzigaluppi, Paolo; de Jeu, Marcel T. G.

    2016-01-01

    The neurons in the inferior olive express subthreshold oscillations in their membrane potential. This oscillatory activity is known to drive synchronous activity in the cerebellar cortex and plays a role in motor learning and motor timing. In the past years, it was commonly thought that olivary neurons belonged to a unique population of oscillating units and that oscillation properties were exclusively dependent on network settings and/or synaptic inputs. The origin of olivary oscillations is now known to be a local phenomenon and is generated by a combination of conductances. In the present work, we show the existence of at least two neuronal populations that can be distinguished on the basis of the presence or absence of low-voltage activated Ca2+ channels. The expression of this channel determines the oscillatory behavior of olivary neurons. Furthermore, the number of cells that express this channel is different between sub nuclei of the inferior olive. These findings clearly indicate the functional variability within and between olivary sub nuclei.

  13. Heterogeneous Expression of T-type Ca2+ Channels Defines Different Neuronal Populations in the Inferior Olive of the Mouse

    Science.gov (United States)

    Bazzigaluppi, Paolo; de Jeu, Marcel T. G.

    2016-01-01

    The neurons in the inferior olive express subthreshold oscillations in their membrane potential. This oscillatory activity is known to drive synchronous activity in the cerebellar cortex and plays a role in motor learning and motor timing. In the past years, it was commonly thought that olivary neurons belonged to a unique population of oscillating units and that oscillation properties were exclusively dependent on network settings and/or synaptic inputs. The origin of olivary oscillations is now known to be a local phenomenon and is generated by a combination of conductances. In the present work, we show the existence of at least two neuronal populations that can be distinguished on the basis of the presence or absence of low-voltage activated Ca2+ channels. The expression of this channel determines the oscillatory behavior of olivary neurons. Furthermore, the number of cells that express this channel is different between sub nuclei of the inferior olive. These findings clearly indicate the functional variability within and between olivary sub nuclei. PMID:27540355

  14. Adenosine regulates a chloride channel via protein kinase C and a G protein in a rabbit cortical collecting duct cell line.

    OpenAIRE

    Schwiebert, E. M.; Karlson, K H; Friedman, P A; Dietl, P.; Spielman, W S; Stanton, B.A.

    1992-01-01

    We examined the regulation by adenosine of a 305-pS chloride (Cl-) channel in the apical membrane of a continuous cell line derived from rabbit cortical collecting duct (RCCT-28A) using the patch clamp technique. Stimulation of A1 adenosine receptors by N6-cyclohexyladenosine (CHA) activated the channel in cell-attached patches. Phorbol 12,13-didecanoate and 1-oleoyl 2-acetylglycerol, activators of protein kinase C (PKC), mimicked the effect of CHA, whereas the PKC inhibitor H7 blocked the ac...

  15. Protein-directed synthesis of Mn-doped ZnS quantum dots: a dual-channel biosensor for two proteins.

    Science.gov (United States)

    Wu, Peng; Zhao, Ting; Tian, Yunfei; Wu, Lan; Hou, Xiandeng

    2013-06-01

    Proteins typically have nanoscale dimensions and multiple binding sites with inorganic ions, which facilitates the templated synthesis of nanoparticles to yield nanoparticle-protein hybrids with tailored functionality, water solubility, and tunable frameworks with well-defined structure. In this work, we report a protein-templated synthesis of Mn-doped ZnS quantum dots (QDs) by exploring bovine serum albumin (BSA) as the template. The obtained Mn-doped ZnS QDs give phosphorescence emission centered at 590 nm, with a decay time of about 1.9 ms. A dual-channel sensing system for two different proteins was developed through integration of the optical responses (phosphorescence emission and resonant light scattering (RLS)) of Mn-doped ZnS QDs and recognition of them by surface BSA phosphorescent sensing of trypsin and RLS sensing of lysozyme. Trypsin can digest BSA and remove BSA from the surface of Mn-doped ZnS QDs, thus quenching the phosphorescence of QDs, whereas lysozyme can assemble with BSA to lead to aggregation of QDs and enhanced RLS intensity. The detection limits for trypsin and lysozyme were 40 and 3 nM, respectively. The selectivity of the respective channel for trypsin and lysozyme was evaluated with a series of other proteins. Unlike other protein sensors based on nanobioconjugates, the proposed dual-channel sensor employs only one type of QDs but can detect two different proteins. Further, we found the RLS of QDs can also be useful for studying the BSA-lysozyme binding stoichiometry, which has not been reported in the literature. These successful biosensor applications clearly demonstrate that BSA not only serves as a template for growth of Mn-doped ZnS QDs, but also impacts the QDs for selective recognition of analyte proteins. PMID:23576296

  16. CLONING SEGMENT SPIKE PROTEIN GENE OF SARS-COV AND ITS EXPRESSION IN ESCHERICHIA COLI

    Institute of Scientific and Technical Information of China (English)

    刘中华; 许文波; 毛乃颖; 张燕; 朱贞; 崔爱利; 杨建国; 胡海涛

    2004-01-01

    Objective Expressing and purifying the segment of SARS-CoV spike protein in E.Coli. Methods The target gene was obtained by RT-PCR. The PCR product was cloned into pEGM- T Easy Vector, sequencing and double restriction digestion ( BamHⅠ,PstⅠ) were performed. The target gene was subcloned into PQE30 expression vector. The gene was expressed in the E.coli strain M15 cells induced by IPTG. The protein was purified with a nickel HiTrap chelating metal affinity column. Results The recombinant expression plasmid was successfully constructed and the protein was well expressed in E. coli strain M15 cells. The ideal pure protein was obtained by purification. Western blotting analysis suggested the protein could act with the convalescent sera of lab confirmed SARS patients. Conclusion The segment of SARS-CoV spike protein was well expressed and purified, and can be applied in diagnosis and immunological research of SARS.

  17. High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Fei Zhong; Zhen-Yu Zhong; Shuang Liang; Xiu-Jin Li

    2006-01-01

    AIM: To develop a highly efficacious method for preparation of soluble SARS S-protein using adenovirus vector to meet the requirement for S-protein investigation.METHODS: The human adenovirus vector was used to express the soluble S-protein (corresponding to 1~1190 amino acids) fused with Myc/His tag using codon-optimized gene construct in HEK239 cells. The recombinant adenovirus bearing S-protein gene was generated by ligation method. The expressed S-protein with Myc/His tag was purified from culture medium with Ni-NTA agarose beads followed by dialysis. The S-protein was detected by Western blot and its biologic activity was analyzed by binding to Vero cells.RESULTS: Under the conditions of infection dose (MOI of 50) and expression time (48 h), the high-level expression of S-protein was obtained. The expression level was determined to be approximately 75 μg/106cells after purification. Purified soluble S-protein was readily detected by Western blot with anti-Myc antibody and showed the ability to bind to surface of Vero cells,demonstrating that the soluble S-protein could remain the biologic activity in the native molecule.CONCLUSION: The high-level expression of S-protein in HEK293 cells mediated by adenovirus can be achieved under the optimized expression conditions. The proteins possess the biologic activity, which lays a foundation for further investigation of S-protein biological function.

  18. When children express their preferences regarding sales channels: Online or offline or online and offline?

    OpenAIRE

    Boulay, Jacques; Faultrier, Brigitte de; Feenstra, Florence; Muzellec, Laurent

    2014-01-01

    Purpose: The purpose of this paper is to investigate the preferences of children under the age of 12 regarding sales channels: how young consumers perceive online vs offline shopping in terms of advantages and disadvantages. Within a cross channel perspective, it also analyses the connections they make between brick-and-mortar and online stores. Design/methodology/approach: Results are drawn from an exploratory and qualitative study based on a multi-category approach. In all, 62 children (34 ...

  19. Promoters of Cancer Genes for Recombinant Protein Expression in Human Cancer Cell Lines

    OpenAIRE

    Behrouz Farhadi; Tamouchin Moharrami; Mohammad Pourhassan-Moghaddam; Kazem Nejati-Koshki

    2012-01-01

    Introduction: Production of complex human recombinant proteins is an important issue in medical biotechnology. These proteins are mostly expressed in non-human mammalian host cells. This has some problems including non-human post-translational modifications, application of high-cost agents for inducing protein expression and low yields. Therefore, it is necessary to use new expression systems to overcome the indicated challenges. Methods: In this paper, we hypothesize the application of promo...

  20. You're one in a googol: optimizing genes for protein expression

    OpenAIRE

    Welch, Mark; Villalobos, Alan; Gustafsson, Claes; Minshull, Jeremy

    2009-01-01

    A vast number of different nucleic acid sequences can all be translated by the genetic code into the same amino acid sequence. These sequences are not all equally useful however; the exact sequence chosen can have profound effects on the expression of the encoded protein. Despite the importance of protein-coding sequences, there has been little systematic study to identify parameters that affect expression. This is probably because protein expression has largely been tackled on an ad hoc basi...

  1. COOH-terminal association of human smooth muscle calcium channel Ca(v)1.2b with Src kinase protein binding domains: effect of nitrotyrosylation.

    Science.gov (United States)

    Kang, Minho; Ross, Gracious R; Akbarali, Hamid I

    2007-12-01

    The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Ca(v)) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa(v)1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCa(v)1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y(2134) prevented SH2 binding and resulted in reduced phosphorylation of hCa(v)1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y(1837), Y(1861), and Y(2134) were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y(1837-2134F). These data demonstrate that the COOH terminus of hCa(v)1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.

  2. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

    Science.gov (United States)

    Chen, Ailu; Wang, Ruijia; Liu, Shikai; Peatman, Eric; Sun, Luyang; Bao, Lisui; Jiang, Chen; Li, Chao; Li, Yun; Zeng, Qifan; Liu, Zhanjiang

    2016-06-01

    Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish. PMID:26747053

  3. A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.

    Science.gov (United States)

    Chanda, Pranab K; Edris, Wade A; Kennedy, Jeffrey D

    2006-05-01

    A set of ligation-independent expression vectors system has been developed for co-expression of proteins in Escherichia coli. These vectors contain a strong T7 promoter, different drug resistant genes, and an origin of DNA replication from a different incompatibility group, allowing combinations of these plasmids to be stably maintained together. In addition, these plasmids also contain the lacI gene, a transcriptional terminator, and a 3' polyhistidine (6x His) affinity tag (H6) for easy purification of target proteins. All of these vectors contain an identical transportable cassette flanked by suitable restriction enzyme cleavage sites for easy cloning and shuttling among different vectors. This cassette incorporates a ligation-independent cloning (LIC) site for LIC manipulations, an optimal ribosome binding site for efficient protein translation, and a 6x His affinity tag for protein purification Therefore, any E. coli expression vector of choice can be easily converted to LIC type expression vectors by shuttling the cassette using the restriction enzyme cleavage sites at the ends. We have demonstrated the expression capabilities of these vectors by co-expressing three bacterial (dsbA, dsbG, and Trx) and also two other mammalian proteins (KChIP1 and Kv4.3). We further show that co-expressed KChIP1/Kv4.3 forms soluble protein complexes that can be purified for further studies. PMID:16325426

  4. How is mRNA expression predictive for protein expression? A correlation study on human circulating monocytes

    Institute of Scientific and Technical Information of China (English)

    Yanfang Guo; Yuan Chen; Hui Jiang; Lijun Tan; Jingyun Xie; Xuezhen Zhu; Songping Liang; Hongwen Deng; Peng Xiao; Shufeng Lei; Feiyan Deng; Gary Guishan Xiao; Yaozhong Liu; Xiangding Chen; Liming Li; Shan Wu

    2008-01-01

    A key assumption in studying mRNA expression is that it is informative in the prediction of protein expression. However,only limited studies have explored the mRNA-protein expression correlation in yeast or human tissues and the results have been relatively inconsistent. We carried out correlation analyses on mRNA-protein expressions in freshly isolated human circulating monocytes from 30 unrelated women. The expressed proteins for 71 genes were quantified and identified by 2-D electrophoresis coupled with mass spectrometry. The corresponding mRNA expressions were quantified by Affymetrix gene chips. Significant correlation (r=0.235, P<0.0001) was observed for the whole dataset including all studied genes and all samples. The correlations varied in different biological categories of gene ontology. For example, the highest correlation was achieved for genes of the extracellular region in terms of cellular component (r=0.643, P<0.0001) and the lowest correlation was obtained for genes of regulation (r=0.099, P=0.213) in terms of biological process. In the genome, half of the samples showed significant positive correlation for the 71 genes and significant correlation was found between the average mRNA and the average protein expression levels in all samples (r=0.296, P<0.01). However, at the study group level, only five studied genes had significant positive correlation across all the samples. Our results showed an overall positive correlation between mRNA and protein expression levels.However, the moderate and varied correlations suggest that mRNA expression might be sometimes useful, but certainly far from perfect, in predicting protein expression levels.

  5. Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimer's disease

    NARCIS (Netherlands)

    Kamphuis, W.; Middeldorp, Jinte; Kooijman, Lieneke; Sluijs, Jacqueline A; Kooi, Evert-Jan; Moeton, Martina; Freriks, Michel; Mizee, Mark R; Hol, Elly M

    2014-01-01

    In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of ast

  6. Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model

    Directory of Open Access Journals (Sweden)

    Wall Susan M

    2004-08-01

    Full Text Available Abstract Background Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+ or lack a complete Slc26a4 gene (Slc26a4-/-. Methods Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry. Results Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+