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By admin, on May 5th, 2012
Oncol Rep. 2012 Apr 30;
Solár P, Hrčková G, Varinská L, Solárová Z, Kriška J, Uhrínová I, Kello M, Mojžiš J, Fedoročko P, Sytkowski AJ
Erythropoietin (Epo) is a critical regulator of erythroid cell proliferation, differentiation and apoptosis. In the form of a recombinant protein, it is widely used to treat various forms of anemia, including that associated with cancer and with the myelosuppressive effects of chemotherapy. Studies of ovarian cancer cell lines have demonstrated the presence of the Epo receptor (EpoR), but there are disagreements regarding its localization and functionality in these cells. Using fluorescence microscopy, we were not able to identify the EpoR on the surface of A2780 cells, in contrast to the positive control K562 cells. Flow cytometry did reveal a weak surface EpoR signal in A2780 cells. Interestingly, most of the EpoR in A2780 cells was found in the cytoplasm, more abundantly as an intracellular membrane-associated protein than a soluble one. Silencing EpoR expression by lentiviral-mediated shRNA resulted in reduced A2780 prolife-ration as well as reduction in Epo-induced phosphorylation of Erk1/2. Our findings provide important insights into the biology of the EpoR in ovarian cancer cells.
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Location and the functionality of erythropoietin receptor(s) in A2780 cells.
By admin, on May 5th, 2012
Oncol Rep. 2012 Apr 30;
Solár P, Hrčková G, Varinská L, Solárová Z, Kriška J, Uhrínová I, Kello M, Mojžiš J, Fedoročko P, Sytkowski AJ
Erythropoietin (Epo) is a critical regulator of erythroid cell proliferation, differentiation and apoptosis. In the form of a recombinant protein, it is widely used to treat various forms of anemia, including that associated with cancer and with the myelosuppressive effects of chemotherapy. Studies of ovarian cancer cell lines have demonstrated the presence of the Epo receptor (EpoR), but there are disagreements regarding its localization and functionality in these cells. Using fluorescence microscopy, we were not able to identify the EpoR on the surface of A2780 cells, in contrast to the positive control K562 cells. Flow cytometry did reveal a weak surface EpoR signal in A2780 cells. Interestingly, most of the EpoR in A2780 cells was found in the cytoplasm, more abundantly as an intracellular membrane-associated protein than a soluble one. Silencing EpoR expression by lentiviral-mediated shRNA resulted in reduced A2780 prolife-ration as well as reduction in Epo-induced phosphorylation of Erk1/2. Our findings provide important insights into the biology of the EpoR in ovarian cancer cells.
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Location and the functionality of erythropoietin receptor(s) in A2780 cells.
By admin, on April 21st, 2012
Nat Protoc. 2012 May; 7(5): 903-20
Karatekin E, Rothman JE
Many biological processes rely on membrane fusion, and therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, and even to single-molecule events using fluorescently labeled vesicle-associated v-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) liposomes and target-membrane-associated t-SNARE-reconstituted planar, supported bilayers (t-SBLs). Docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorescence microscopy. In this assay, fusion is dependent on SNAP-25, one of the t-SNARE subunits that is required for fusion in vivo. The success of the assay is due to the use of: (i) bilayers covered with a thin layer of poly(ethylene glycol) (PEG) to control bilayer-bilayer and bilayer-substrate interactions, and (ii) microfluidic flow channels that present many advantages, such as the removal of nonspecifically bound liposomes by flow. The protocol takes 6-8 d to complete. Analysis can take up to 2 weeks.
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Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells.
By admin, on April 21st, 2012
Nat Protoc. 2012 May; 7(5): 903-20
Karatekin E, Rothman JE
Many biological processes rely on membrane fusion, and therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, and even to single-molecule events using fluorescently labeled vesicle-associated v-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) liposomes and target-membrane-associated t-SNARE-reconstituted planar, supported bilayers (t-SBLs). Docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorescence microscopy. In this assay, fusion is dependent on SNAP-25, one of the t-SNARE subunits that is required for fusion in vivo. The success of the assay is due to the use of: (i) bilayers covered with a thin layer of poly(ethylene glycol) (PEG) to control bilayer-bilayer and bilayer-substrate interactions, and (ii) microfluidic flow channels that present many advantages, such as the removal of nonspecifically bound liposomes by flow. The protocol takes 6-8 d to complete. Analysis can take up to 2 weeks.
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Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells.
By admin, on April 21st, 2012
Anim Reprod Sci. 2012 Apr 4;
Rodriguez PC, Satorre MM, Beconi MT
Spermatozoa require a preparatory process called capacitation to fertilize mature oocytes. Two events related to capacitation of mammalian spermatozoa are an increase in intracellular Ca(2+) and protein tyrosine phosphorylation. The sites that regulate intracellular Ca(2+) concentration are plasma membrane and mitochondria. There are different systems for mitochondrial Ca(2+) influx and efflux. Our aim was to study the involvement of mitochondrial Ca(2+) cycle during heparin-induced capacitation in cryopreserved bovine spermatozoa. Samples were incubated at 38°C for 45min, in TALP medium, in the presence of: (a) heparin (H), a well known capacitation inducer; (b) H+CGP 37157, a specific inhibitor of mitochondrial Ca(2+) efflux; (c) H+RU 360, a specific inhibitor of Ca(2+) influx to the mitochondria and (d) H+CGP 37157+RU 360. In every treatment, capacitation (by CTC), progressive motility (by optical microscopy), viability (by the eosin/nigrosin technique) and protein tyrosine phosphorylation (by Western Immuno-blotting), were evaluated. The addition of CGP 37157 (20μM) decreased progressive motility (p<0.05), without affecting capacitation or protein tyrosine phosphorylation, indicating the importance of calcium efflux for maintaining progressive motility. RU 360 (5μM) significantly reduced capacitation without affecting progressive motility, sperm viability or protein tyrosine phosphorylation, showing that inhibition of the mitochondrial calcium uptake, negatively affect the capacitation process. The addition of both inhibitors showed the effect of RU 360. According with these results, there would exist a differential participation of the income and outcome mitochondrial calcium carriers, in the capacitation process. In conclusion, this research demonstrates the importance of normal mitochondrial calcium cycle in the achievement of sperm capacitation and the maintenance of progressive motility in cryopreserved bovine spermatozoa.
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Effect of two intracellular calcium modulators on sperm motility and heparin-induced capacitation in cryopreserved bovine spermatozoa.
By admin, on April 21st, 2012
Anim Reprod Sci. 2012 Apr 4;
Rodriguez PC, Satorre MM, Beconi MT
Spermatozoa require a preparatory process called capacitation to fertilize mature oocytes. Two events related to capacitation of mammalian spermatozoa are an increase in intracellular Ca(2+) and protein tyrosine phosphorylation. The sites that regulate intracellular Ca(2+) concentration are plasma membrane and mitochondria. There are different systems for mitochondrial Ca(2+) influx and efflux. Our aim was to study the involvement of mitochondrial Ca(2+) cycle during heparin-induced capacitation in cryopreserved bovine spermatozoa. Samples were incubated at 38°C for 45min, in TALP medium, in the presence of: (a) heparin (H), a well known capacitation inducer; (b) H+CGP 37157, a specific inhibitor of mitochondrial Ca(2+) efflux; (c) H+RU 360, a specific inhibitor of Ca(2+) influx to the mitochondria and (d) H+CGP 37157+RU 360. In every treatment, capacitation (by CTC), progressive motility (by optical microscopy), viability (by the eosin/nigrosin technique) and protein tyrosine phosphorylation (by Western Immuno-blotting), were evaluated. The addition of CGP 37157 (20μM) decreased progressive motility (p<0.05), without affecting capacitation or protein tyrosine phosphorylation, indicating the importance of calcium efflux for maintaining progressive motility. RU 360 (5μM) significantly reduced capacitation without affecting progressive motility, sperm viability or protein tyrosine phosphorylation, showing that inhibition of the mitochondrial calcium uptake, negatively affect the capacitation process. The addition of both inhibitors showed the effect of RU 360. According with these results, there would exist a differential participation of the income and outcome mitochondrial calcium carriers, in the capacitation process. In conclusion, this research demonstrates the importance of normal mitochondrial calcium cycle in the achievement of sperm capacitation and the maintenance of progressive motility in cryopreserved bovine spermatozoa.
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Effect of two intracellular calcium modulators on sperm motility and heparin-induced capacitation in cryopreserved bovine spermatozoa.
By admin, on April 19th, 2012
Cancer Res . 2012 Apr 16; Earley S, Vinegoni C, Dunham J, Gorbatov R, Fumene Feruglio P, Weissleder R Observing drug responses in the tumor microenvironment in vivo can be technically challenging.
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In vivo imaging of drug-induced mitochondrial outer membrane permeabilization at single cell resolution.
By admin, on April 19th, 2012
Cancer Res . 2012 Apr 16; Earley S, Vinegoni C, Dunham J, Gorbatov R, Fumene Feruglio P, Weissleder R Observing drug responses in the tumor microenvironment in vivo can be technically challenging. As a result, cellular responses to molecularly targeted cancer drugs are often studied in cell culture, which does not accurately represent the behavior of cancer cells growing in vivo.
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In vivo imaging of drug-induced mitochondrial outer membrane permeabilization at single cell resolution.
By admin, on April 8th, 2012
Fish Shellfish Immunol . 2012 Mar 28; Sun Y, Zhang M, Liu CS, Qiu R, Sun L Streptococcosis and vibriosis caused by Streptococcus iniae and Vibrio anguillarum respectively have affected fish culture industries around the world.
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A divalent DNA vaccine based on Sia10 and OmpU induces cross protection against Streptococcus iniae and Vibrio anguillarum in Japanese flounder.
By admin, on April 7th, 2012
J Vis Exp. 2012;
Crites TJ, Chen L, Varma R
Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCRαβ heterodimer that associates non-covalently with CD3 dimers (the εδ and εγ heterodimers and the ζζ homodimer)(1). Upon engagement of the receptor, the CD3 ζ chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules(2). The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation(2). TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors (3-4). Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways. One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously(5-6). They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface (7-8). This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCRβ, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands (9-11) as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ(12-16).
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A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins.
By admin, on April 7th, 2012
J Vis Exp. 2012;
Crites TJ, Chen L, Varma R
Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCRαβ heterodimer that associates non-covalently with CD3 dimers (the εδ and εγ heterodimers and the ζζ homodimer)(1). Upon engagement of the receptor, the CD3 ζ chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules(2). The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation(2). TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors (3-4). Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways. One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously(5-6). They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface (7-8). This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCRβ, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands (9-11) as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ(12-16).
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A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins.
By admin, on April 6th, 2012
Background: Pseudomonas aeruginosa mainly achieves multidrug resistance by use of the MexAB-OprM pump. Results: We determined the crystal structure of MexA. Electron microscopy work using MexA and OprM reveals …
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Assembly and Channel Opening of Outer Membrane Protein in …
By admin, on April 5th, 2012
Infect Immun . 2012 Apr 2; Forbes SJ, Martinelli D, Hsieh C, Ault JG, Marko M, Mannella CA, Mantis NJ Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium is an energetically demanding process, involving the transfer of effector proteins from invading bacteria into host cells via a specialized organelle known as the SPI-1 type 3-secretion system (T3SS). By a mechanism that remains poorly understood, entry of S
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Association of a Protective Monoclonal IgA with the O-Antigen of Salmonella enterica serovar Typhimurium Impacts Type 3 Secretion and Outer Membrane Integrity.
By admin, on April 5th, 2012
Infect Immun . 2012 Apr 2; Forbes SJ, Martinelli D, Hsieh C, Ault JG, Marko M, Mannella CA, Mantis NJ Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium is an energetically demanding process, involving the transfer of effector proteins from invading bacteria into host cells via a specialized organelle known as the SPI-1 type 3-secretion system (T3SS). By a mechanism that remains poorly understood, entry of S
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Association of a Protective Monoclonal IgA with the O-Antigen of Salmonella enterica serovar Typhimurium Impacts Type 3 Secretion and Outer Membrane Integrity.
By admin, on April 5th, 2012
Mol Microbiol . 2012 Apr 2; Lamsa A, Liu WT, Dorrestein PC, Pogliano K Bacillus subtilis SDP is a peptide toxin that kills cells outside the biofilm to support continued growth. We show that purified SDP acts like endogenously produced SDP; it delays sporulation, and the SdpI immunity protein confers SDP resistance
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The Bacillus subtilis cannibalism toxin SDP collapses the proton motive force and induces autolysis.
By admin, on March 31st, 2012
Biol Cell. 2012 Mar 22;
Thayanidhi N, Liang Y, Hasegawa H, Nycz DC, Oorschot V, Klumperman J, Hay JC
BACKGROUND INFORMATION: The arginine-type soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) ykt6 possesses several atypical properties including selective high expression in neurons and neuroendocrine cells, a lipidated C-terminus rather than a transmembrane anchor, localization to punctae that do not correspond with known endomembrane markers, a potent ability to protect the secretory pathway from insults such as alpha-synuclein over-expression, and a propensity for specific upregulation in tumors. We have followed up on several of these features that together suggest nontraditional SNARE structures and functions. RESULTS: A significant portion of ykt6 in PC12 cells was found in a protease-resistant state suggestive of a large complex or aggregate. Other ER/Golgi SNAREs were not protease resistant, demonstrating that SNARE complexes per se did not cause protease resistance. Mutagenesis indicated that lipidation of the ykt6 C-terminus was also not involved, implicating its longin domain in particle formation. Gel filtration demonstrated that a smaller fraction of particulate ykt6 participated in SNARE complexes than other ER/Golgi SNAREs, suggesting that protease resistant ykt6 may not be available for SNARE interactions. Previously observed ykt6 fluorescent punctae were validated as bona fide particulate structures. Immunogold electron microscopy revealed ykt6 labeling of ~100 nm electron densities associated with diverse membranes. Density gradient analysis of the protease resistant structures confirmed their tight association with membranes. Since excess ykt6 has been correlated with tumorigenesis, we tested whether ykt6 over-expression in NRK cells that normally express little ykt6 affected the cell cycle. Ykt6 over-expression was found to result in altered cell division cycles as evidenced by significantly smaller cells, a higher mitotic index, and increased DNA synthesis. Mutagenesis studies dis-correlated SNARE function with the cell cycle effects; instead, the cell cycle effects correlated better with ykt6 properties related to the longin domain or particle formation. CONCLUSIONS: The ykt6 particles/aggregates may represent ykt6 engaged in a non-SNARE function(s) or else nonfunctional, stored, and/or excess ykt6. Whether the particulate ykt6 structures represents a means of buffering the apparent proliferative activity or are in fact mechanistically related to this activity will be of future interest in neuroscience and cancer biology.
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R-SNARE YKT6 Resides in Membrane-Associated Protease-Resistant Protein Particles and Modulates Cell Cycle Progression When Over-Expressed.
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