Synthesis, characterisation, and biological studies of CdTe quantum dot-naproxen conjugates.
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Animals↗
Biomedical subjects
Yuri Volkov
Publications and source records attributed to Yuri Volkov.
Fluorescent immunoprecipitation analysis of cell surface proteins: a methodology compatible with mass-spectrometry.
Radiolabelling and biotinylation of cell proteins followed by immunoprecipitation is a common procedure for biochemical characterization of cell-surface antigens recognized by monoclonal antibodies. Here we present a new method of cell labelling with fluorescent dyes followed by immunoprecipitation and SDS-PAGE with subsequent detection of specific bands by fluorescence imaging devices. Fluorescent immunoprecipitation analysis (FIPA) of cell surface proteins is a fast and sensitive alternative to conventional immunoprecipitation methods, eliminating the need to employ radioactive or biotin labels. The proposed method is compatible with mass spectrometry analysis and permits the identification of immunoprecipitated proteins.
Amino Acid Sequence↗
Ursodeoxycholic acid inhibits interleukin 1 beta [corrected] and deoxycholic acid-induced activation of NF-kappaB and AP-1 in human colon cancer cells.
Deoxycholic acid (DCA) has been implicated in colorectal carcinogenesis in humans with effects on proliferation and apoptosis, mediated at least in part by activation of transcription factors nuclear factor kappa B (NF-kappaB), activator protein 1 (AP-1) and protein kinase C (PKC) enzymes. Ursodeoxycholic acid (UDCA) is reported to reduce the frequency of colonic carcinogenesis in ulcerative colitis patients. Hence, we postulated that it might differ from DCA in its regulation of these transcription factors. The aim of the study was to determine effects of DCA and UDCA on NF-kappaB and AP-1 activation and explore its relationship to PKC. Human colonic tumour cell lines HCT116 were treated with DCA, UDCA, alone or pretreated with UDCA followed by DCA or IL-1beta. In other experiments, cells were pretreated with PKC inhibitors and then stimulated with DCA and IL-1beta or PMA. Gel shift assays were performed on nuclear extracts of the cells for NF-kappaB and AP-1 analysis. Western blot analyses and immunofluorescence were performed for Rel A (p65) and IkappaB-alpha levels on the treated cells. DCA increased NF-kappaB and AP-1 DNA binding. UDCA did not increase DNA binding of NF-kappaB and AP-1 and UDCA pretreatment inhibited DCA-induced NF-kappaB and AP-1 DNA binding. PKC inhibitors blocked DCA-induced NF-kappaB and AP-1 activation. These results were validated by Western blot analysis for RelA and IkappaB-alpha. In conclusion, UDCA did not induce NF-kappaB and AP-1 DNA binding but also blocked DCA-induced NF-kappaB and AP-1 activation. These findings suggest a possible mechanistic role for UDCA in blocking pathways thought to be involved in colon carcinogenesis.
Binding Sites↗
The hepatitis C envelope 2 protein inhibits LFA-1-transduced protein kinase C signaling for T-lymphocyte migration.
BACKGROUND & AIMS: The ability of viruses to escape the host immune response represents a globally important problem related to a wide variety of pathogens. Hepatitis C is one of the major causes of liver disease worldwide. Clearance rates of this virus are low, and this condition normally involves a chronic inflammatory process. This raises a possibility that the virus may have developed mechanisms enabling it to evade T-cell-mediated immune surveillance. The aim of this study was to investigate the effect of the hepatitis C envelope protein E2 on LFA-1-stimulated T-cell migration and macrophage inflammatory protein (MIP-1alpha, MIP-1beta) secretion. METHODS: T cells were stimulated through the leukocyte function-associated molecule-1 (LFA-1) receptor by incubating with either intracellular adhesion molecule 1 (ICAM-1)-Fc fusion protein or anti-LFA-1 immobilized on 8-well chamber slides. Subcellular localization of protein kinase C (PKC)-beta, CD81, and LFA-1 was determined by immunofluorescence analysis. Lipid raft formation was assessed using the Cellomics Kineticscan reader. MIP-1alpha and MIP-1beta levels were detected by enzyme-linked immunosorbent assay. RESULTS: We report that the hepatitis C envelope protein E2 can dramatically inhibit T-lymphocyte motility and chemokine release induced via LFA-1 integrin ligation. We have demonstrated a novel T-lymphocyte-directed viral inhibitory mechanism involving the PKC-beta enzyme as a definitive intracellular target. E2-CD81 interaction stimulates translocation of PKC-beta to lipid rafts, thereby preventing its association with the centrosome and microtubule cytoskeleton, which is crucial to the process of T-cell migration. CONCLUSIONS: These studies identify a mechanism whereby the hepatitis C virus can evade the host immune response by inhibition of T-cell migration.
Cell Movement↗
The scaffolding protein CG-NAP/AKAP450 is a critical integrating component of the LFA-1-induced signaling complex in migratory T cells.
T cell migration represents a complex highly coordinated process involving participation of surface receptor/ligand interactions, cytoskeletal rearrangements, and phosphorylation-dependent signaling cascades. Members of the A-kinase anchoring protein (AKAP) family of giant scaffolding proteins can assemble and compartmentalize multiple signaling and structural molecules thereby providing a platform for their targeted positioning and efficient interactions. We characterize here the expression, intracellular distribution, and functional role of the scaffolding protein CG-NAP (centrosome and Golgi localized protein kinase N-associated protein)/AKAP450 in the process of active T cell motility induced via LFA-1 integrins. This protein is predominantly localized at the centrosome and Golgi complex. T cell locomotion triggered by LFA-1 ligation induces redistribution of CG-NAP/AKAP450 along microtubules in trailing cell extensions. Using an original in situ immunoprecipitation approach, we show that CG-NAP/AKAP450 is physically associated with LFA-1 in the multimolecular signaling complex also including tubulin and the protein kinase C beta and delta isoenzymes. CG-NAP/AKAP450 recruitment to this complex was specific for the T cells migrating on LFA-1 ligands, but not on the beta(1) integrin ligand fibronectin. Using the GFP-tagged C-terminal CG-NAP/AKAP450 construct, we demonstrate that expression of the intact CG-NAP/AKAP450 and its recruitment to the LFA-1-associated multimolecular complex is critically important for polarization and migration of T cells induced by this integrin.
A Kinase Anchor Proteins↗
Stimulus-induced phosphorylation of PKC theta at the C-terminal hydrophobic-motif in human T lymphocytes.
Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC beta(I) and delta are constitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC theta is constitutively phosphorylated at the activation-loop and turn-motif sites in T cells, PMA or anti-CD3/CD28 stimulation results in an increase in phosphorylation at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the membrane. Studies on the stimulus-induced phosphorylation of PKC theta demonstrate that an upstream kinase activity involving a conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mTOR pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane translocation, suggesting that this site may control other aspects of PKC theta signalling.
Amino Acid Motifs↗
Bile acid deoxycholate induces differential subcellular localisation of the PKC isoenzymes beta 1, epsilon and delta in colonic epithelial cells in a sodium butyrate insensitive manner.
Elevated levels of bile acids have been implicated in the abnormal morphogenesis of the colonic epithelium thus contributing to colorectal cancer (CRC). Alternatively sodium butyrate (NaB) produced by anaerobic fermentation of dietary fibre is regarded as being protective against colon cancer. Bile acids such as deoxycholic acid (DCA) are thought to mediate some of their actions by differentially activating protein kinase C (PKC). We examined the effects of DCA on the subcellular localisation of PKC-beta(1), -epsilon and -delta and whether these responses could be modulated by NaB. HCT116 cells endogenously express PKC-epsilon and -delta but not PKC-beta. DCA treatment results in endogenous PKC-epsilon translocation but not PKC-delta after 1 hr. To study the subcellular localisation of PKC isoforms in response to DCA in real time, PKC-beta(1), PKC-epsilon and PKC-delta functionally intact green fluorescent protein (GFP) fusion constructs were used. Stimulation with 300 microM DCA induces rapid translocation of PKC-beta(1)-GFP and PKC-epsilon-GFP but not PKC-delta-GFP from the cytosol to the plasma membrane in 15 min. Interestingly, pretreatment with 4mM NaB does not modify the response of the PKC isoenzymes to DCA as PKC-beta(1)-GFP and PKC-epsilon-GFP translocates to the plasma membrane in 15 min whereas PKC-delta-GFP localisation remains unaltered. Immunofluorescence shows that PKC-beta(1)-GFP and PKC-epsilon-GFP cells treated with DCA colocalise with the cytoskeletal elements actin and tubulin adjacent to the plasma membrane. Our findings demonstrate that the differential activation of the PKC isoenzymes by DCA may be of critical importance for the functional responses of colonic epithelial cells. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.
Butyrates↗
CD44 cross-linking induces protein kinase C-regulated migration of human T lymphocytes.
The cell surface receptor CD44 is widely implicated in leukocyte migration to inflammatory sites. In this study, the responses of human T cells following cross-linking of CD44 were examined. We demonstrate that engagement of CD44 using immobilized mAbs or hyaluronan-enriched extracellular matrix lattices induces active migration in T lymphocytes accompanied by cycles of cytoskeletal rearrangement and cell polarization. We have investigated the functional impact and subcellular localization of protein kinase C (PKC) isoenzymes, beta and delta, previously shown by our group to be involved in active T cell locomotion induced by leukocyte function-associated antigen-1 (LFA-1) integrin receptors. PKCbeta was associated with the centrosome and the microtubule-rich tail of the polarized cell and PKCdelta was predominantly located about the region of the microtubule organizing center. A selective pharmacological inhibitor of classical PKC isoforms, Go6976, suppressed lymphocyte polarization and migration following CD44 ligation. Selective targeting of PKCdelta using the pharmacological inhibitor rottlerin or a pseudosubstrate-blocking peptide reduced CD44-activated cell migration but did not completely ablate it. Our data demonstrate that ligation of CD44 induces phenotypic changes, cytoskeletal rearrangements and redistribution of PKC isoforms beta and delta, resulting in cell migration, as previously described for the cell surface receptor, LFA-1. This suggests potential convergence of intracellular signaling pathways induced via CD44 and LFA-1 integrin.
Cell Movement↗
Ursodeoxycholic acid inhibits translocation of protein kinase C in human colonic cancer cell lines.
Deoxycholic acid (DCA) has been implicated in colonic carcinogenesis through effects mediated by protein kinase C (PKC) activation. By contrast, ursodeoxycholic acid (UDCA) is reported to reduce colon cancer incidence in ulcerative colitis. The aim of this study was to investigate whether UDCA modulated DCA-induced PKC isoenzyme translocation to its site of activity. HCT116 cells were treated with DCA, UDCA alone or pre-treated with UDCA followed by DCA. Analysis of translocation of endogenous and enhanced green fluorescent protein (EGFP) constructs of PKC isoenzymes was performed. Both DCA and phorbol myristate acetate (PMA) but not UDCA caused translocation of endogenous PKC alpha, epsilon and delta and transfected PKC beta1-, epsilon- and delta-EGFP from cytosol to plasma membrane, reflecting isoenzyme activation. Furthermore, UDCA inhibited DCA-induced translocation of PKC isoenzymes. Inhibition of DCA-induced PKC translocation may be a mechanism for UDCA-mediated chemoprevention of colon carcinogenesis.
Anticarcinogenic Agents↗
In-situ observation of nanowire growth from luminescent CdTe nanocrystals in a phosphate buffer solution.
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Journal Article↗
A multidisciplinary approach to the study of T cell migration.
Active T cell locomotion depends on efficient repeated cycles of integrin receptor/ligand interactions mediating cell adhesion and detachment, intracellular signaling cascades orchestrating posttranslation modifications of interacting proteins, dynamic reassembly of participating cytoskeletal elements, and structural support of associated scaffolding molecules. Using an integrated approach based on novel cutting edge technologies of live cell imaging, cell transfection, proteomics, and nanotechnology, we provide here a detailed characterisation of crucial mechanisms involved in LFA-1 integrin-mediated T cell migration. Polarization and phenotypic changes associated with LFA-1-triggered T cell locomotion is largely dependent on the intact functioning of the microtubule cytoskeleton. Experiments utilizing 4-D (3-D over time) confocal live imaging of T cells, microinjected with fully functional constructs encoding protein kinase C beta (PKC-beta) isoenzyme tagged with enhanced green fluorescent protein (GFP), elucidate that LFA-1-induced activation is associated with translocation of PKC-beta to sites associated with centrosomes and tubulin cytoskeleton in locomotory T lymphocytes. We also provide here a characterization of a novel microfluidics-based multichannel platform enabling detailed analysis of leukocyte adhesion and migration under regulated shear stress conditions. Using precision machined surfaces, we demonstrate that the substrate topography can influence the motile response of the two different T cell types in different ways, and this can be quantified in terms of specified motility parameters. Finally, using an original in situ immunoprecipitation method, in which LFA-1 antibodies are utilized to induce intracellular association of proteins in the cytoskeletal/signaling complex, we demonstrate that this complex includes a number of structural and signaling proteins, which have been identified by 2-D electrophoresis and MALDI-TOF protein sequencing.
Cell Adhesion↗
Direct and indirect antibody-induced TX-100 resistance of cell surface antigens.
Translocation into detergent-insoluble microdomains (rafts) represents one of the earliest events in the process of cell activation, which follows the binding of surface receptor with natural ligand or mimicking antibody. In this study, the antibody-induced TX-100 resistance of surface antigens has been studied utilizing flow cytometry on TX-100 extracted cells. TX-100 resistance was evaluated by the ratio of antigen retained on the cells after detergent extraction compared with mAb-pretreated and untreated cells. All the antigens under study except CD98 demonstrated antibody-induced TX-100 resistance if the cells were treated with monoclonal antibodies and further cross-linked with secondary antibodies prior to lysis. CD20, CD5, and sIgM molecules were capable of transferring into TX-100-insoluble state in the absence of additional cross-linking. The experiments on modification of raft and cytoskeletal components of the cell, as well as the data on co-localization of TX-100-resistant antigens with raft and cytoskeletal markers strongly indicate that antibody-induced TX-100 resistance of antigens is mainly related to the translocation of antigens into lipid microdomains.
Animals↗
Decrease in hepatic CD56(+) T cells and V alpha 24(+) natural killer T cells in chronic hepatitis C viral infection.
BACKGROUND/AIMS: The intrahepatic immune system is likely to play a key role in determining the outcome of hepatitis C virus (HCV) infection. The hepatic lymphocyte repertoire is characterised by high CD8/CD4 T cell ratios and large numbers of gamma delta T cells, natural killer (NK) cells, NK T cells and NK receptor-positive T cells. It is not known which of these populations contribute to immunity against HCV or immune pathology. METHODS: To explore the relative contributions of lymphocyte subpopulations, we have compared the intrahepatic lymphocyte repertoires and cytokine expression in 13 patients with mild chronic hepatitis C infection, 14 with end-stage hepatitis C cirrhosis and five histologically normal livers by flow cytometry and immunohistochemistry. RESULTS: CD4(+) T cells bearing alpha beta T cell receptors (TCR) were significantly expanded in livers with chronic HCV infection while CD56(+) alpha beta T cells and V alpha 24 TCR-positive T cells were significantly depleted. Expanded CD4(+)T cells were predominantly Th1 cells, producing interferon-gamma but not interleukin-4. CONCLUSIONS: Failure to resolve HCV infection may be due to deficient innate and/or memory immune responses, while Th1 cells may mediate immune pathology.
Adult↗