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Intracellular Trafficking (intracellular + trafficking)
Selected AbstractsIntracellular trafficking and release of intact edible mushroom lectin from HT29 human colon cancer cellsFEBS JOURNAL, Issue 7 2000Lu-Gang Yu Our previous studies have shown that the Gal,1,3GalNAc,- (Thomsen,Friedenreich antigen)-binding lectin from the common edible mushroom Agaricus bisporus (ABL) reversibly inhibits cell proliferation, and this effect is a consequence of inhibition of nuclear localization sequence-dependent nuclear protein import after ABL internalization [Yu, L.G., Fernig, D.G., White, M.R.H., Spiller, D.G., Appleton, P., Evans, R.C., Grierson, I., Smith, J.A., Davies, H., Gerasimenko, O.V., Petersen, O.H., Milton, J.D. & Rhodes, J.M. (1999) J. Biol. Chem.274, 4890,4899]. Here, we have investigated further the intracellular trafficking and fate of ABL after internalization in HT29 human colon cancer cells. Internalization of 125I-ABL occurred within 30 min of the lectin being bound to the cell surface. Subcellular fractionation after pulse labelling of the cells with 125I-ABL for 2 h at 4 °C followed by culture of the cells at 37 °C demonstrated a steady increase in radioactivity in a crude nuclear extract. The radioactivity in this extract reached a maximum after 10 h and declined after 20 h. Release of ABL from the cell, after pulse labelling, was assessed using both fluorescein isothiocyanate-labelled ABL and 125I-ABL and was slow, with a t1/2 of 48 h. Most of the 125I-ABL both inside cells and in the medium remained intact, as determined by trichloroacetic acid precipitation and SDS/PAGE, and after 48 h only 22 ± 2% of ABL in the medium and 14 ± 2% inside the cells was degraded. This study suggests that the reversibility of the antiproliferative effect of ABL is associated with its release from cells after internalization. The internalization and subsequent slow release, with little degradation of ABL, reflects the tendency of lectins to resist biodegradation and implies that other endogenous or exogenous lectins may be processed in this way by intestinal epithelial cells. [source] Intracellular trafficking in neurones and glia of fibroblast growth factor-2, fibroblast growth factor receptor 1 and heparan sulphate proteoglycans in the injured adult rat cerebral cortexJOURNAL OF NEUROCHEMISTRY, Issue 4 2006W. E. Leadbeater Abstract The potent gliogenic and neurotrophic fibroblast growth factor (FGF)-2 signals through a receptor complex comprising high-affinity FGF receptor (FGFR)1 with heparan sulphate proteoglycans (HSPGs) as co-receptors. We examined the intracellular dynamics of FGF-2, FGFR1 and the HSPGs syndecan-2 and -3, glypican-1 and -2, and perlecan in neurones and glia in and around adult rat cerebral wounds. In the intact cerebral cortex, FGF-2 and FGFR1 mRNA and protein were constitutively expressed in astrocytes and neurones respectively. FGF-2 protein was localized exclusively to astrocyte nuclei. After injury, expression of FGF-2 mRNA was up-regulated only in astrocytes, whereas FGFR1 mRNA expression was increased in both glia and neurones, a disparity indicating that FGF-2 may act as a paracrine and autocrine factor for neurones and glia respectively. FGF-2 protein localized to both cytoplasm and nuclei of injury-responsive neurones and glia. There was weak or no staining of HSPGs in the normal cerebral neuropil and glia nuclei, with a few immunopositive neurones. Specific HSPGs responded to injury by differentially co-localizing with trafficked intracellular FGF-2 and FGFR1. The spatiotemporal dynamics of FGF-2,FGFR1,HSPG complex formation implies a role for individual HSPGs in regulating FGF-2 storage, nuclear trafficking and cell-specific injury responses in CNS wounds. [source] Intracellular trafficking of the Coxiella burnetii lipopolysaccharideCLINICAL MICROBIOLOGY AND INFECTION, Issue 2009L. Pretat No abstract is available for this article. [source] An adipocentric view of signaling and intracellular traffickingDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2002Silvia Mora Abstract Adipocytes have traditionally been considered to be the primary site for whole body energy storage mainly in the form of triglycerides and fatty acids. This occurs through the ability of insulin to markedly stimulate both glucose uptake and lipogenesis. Conventional wisdom held that defects in fuel partitioning into adipocytes either because of increased adipose tissue mass and/or increased lipolysis and circulating free fatty acids resulted in dyslipidemia, obesity, insulin resistance and perhaps diabetes. However, it has become increasingly apparent that loss of adipose tissue (lipodystrophies) in both animal models and humans also leads to metabolic disorders that result in severe states of insulin resistance and potential diabetes. These apparently opposite functions can be resolved by the establishment of adipocytes not only as a fuel storage depot but also as a critical endocrine organ that secretes a variety of signaling molecules into the circulation. Although the molecular function of these adipocyte-derived signals are poorly understood, they play a central role in the maintenance of energy homeostasis by regulating insulin secretion, insulin action, glucose and lipid metabolism, energy balance, host defense and reproduction. The diversity of these secretory factors include enzymes (lipoprotein lipase (LPL) and adipsin), growth factors [vascular endothelial growth factor (VEGF)], cytokines (tumor necrosis factor-,, interleukin 6) and several other hormones involved in fatty acid and glucose metabolism (leptin, Acrp30, resistin and acylation stimulation protein). Despite the large number of molecules secreted by adipocytes, our understanding of the pathways and mechanisms controlling intracellular trafficking and exocytosis in adipocytes is poorly understood. In this article, we will review the current knowledge of the trafficking and secretion processes that take place in adipocytes, focusing our attention on two of the best characterized adipokine molecules (leptin and adiponectin) and on one of the most intensively studied regulated membrane proteins, the GLUT4 glucose transporter. Copyright © 2002 John Wiley & Sons, Ltd. [source] Mechanisms and consequences of bladder cell invasion by uropathogenic Escherichia coliEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2008B. K. Dhakal ABSTRACT Strains of uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections worldwide. Multiple studies over the past decade have called into question the dogmatic view that UPEC strains act as strictly extracellular pathogens. Rather, bacterial expression of filamentous adhesive organelles known as type 1 pili and Afa/Dr fibrils enable UPEC to invade host epithelial cells within the urinary tract. Entry into bladder epithelial cells provides UPEC with a protected niche where the bacteria can persist quiescently for long periods, unperturbed by host defences and protected from many antibiotic treatments. Alternately, internalized UPEC can rapidly multiply, forming large intracellular inclusions that can contain several thousand bacteria. Initial work aimed at defining the host and bacterial factors that modulate the entry, intracellular trafficking, and eventual resurgence of UPEC suggests a high degree of host-pathogen crosstalk. Targeted disruption of these processes may provide a novel means to prevent and treat recurrent, relapsing and chronic infections within the urinary tract. [source] Mechanisms of substrate transport-induced clustering of a glial glutamate transporter GLT-1 in astroglial,neuronal culturesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2008Takayuki Nakagawa Abstract Glutamate uptake by the Na+ -dependent glutamate transporter GLT-1, which is predominantly expressed in astrocytes, is crucial for regulating glutamate concentration at the synaptic cleft and achieving proper excitatory neurotransmission. A body of evidence suggests that GLT-1 constitutively traffics between the plasma membrane and endosomes via an endocytosis/recycling pathway, and forms a cluster. Here, we report substrate transport via GLT-1-induced formation of GLT-1 cluster accompanied by intracellular trafficking in rat astroglial,neuronal cultures. We constructed a recombinant adenovirus expressing enhanced green fluorescence protein (EGFP)-tagged GLT-1. Adenoviral infection resulted in the expression of functional GLT-1,EGFP preferentially in astrocytes, partly as clusters. Treatment with glutamate, but not N -methyl-D-aspartate, dramatically increased the number of GLT-1 clusters within 1 h. The estimated EC50 value of glutamate was 240 ,m. In addition, glutamate decreased the cell surface expression and increased the intracellular expression of GLT-1. The GLT-1 clusters were found in early and recycling endosomes and partly in lysosomes, and were inhibited by blockade of endocytotic pathways. Ionotropic and metabotropic glutamate receptor antagonists had no effect on glutamate-induced GLT-1 clustering. The non-transportable glutamate uptake inhibitors (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate and dihydrokainate, as well as Na+ -free conditions, prevented the glutamate-induced GLT-1 clustering, whereas the competitive substrates, aspartate and L- trans -pyrrolidine-2,4-dicarboxylate, induced GLT-1 clustering. Furthermore, the Na+/K+ -ATPase inhibitor, ouabain, and the Na+ ionophores, gramicidin and monensin, produced GLT-1 clustering. Modulators of intracellular Ca2+signaling or membrane depolarization had no effect on GLT-1 clustering. Taken together, these results suggest that Na+ influx associated with GLT-1 substrate transport triggers the formation of GLT-1 clusters accompanied by intracellular trafficking via endocytotic pathways in astrocytes. [source] Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide libraryFEBS JOURNAL, Issue 21 2007Jeannine Mohrlüder 4-Aminobutyrate type A (GABAA) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABAA receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca2+ -dependent chaperone and a Ca2+ -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant Kd = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein. [source] Intracellular trafficking and release of intact edible mushroom lectin from HT29 human colon cancer cellsFEBS JOURNAL, Issue 7 2000Lu-Gang Yu Our previous studies have shown that the Gal,1,3GalNAc,- (Thomsen,Friedenreich antigen)-binding lectin from the common edible mushroom Agaricus bisporus (ABL) reversibly inhibits cell proliferation, and this effect is a consequence of inhibition of nuclear localization sequence-dependent nuclear protein import after ABL internalization [Yu, L.G., Fernig, D.G., White, M.R.H., Spiller, D.G., Appleton, P., Evans, R.C., Grierson, I., Smith, J.A., Davies, H., Gerasimenko, O.V., Petersen, O.H., Milton, J.D. & Rhodes, J.M. (1999) J. Biol. Chem.274, 4890,4899]. Here, we have investigated further the intracellular trafficking and fate of ABL after internalization in HT29 human colon cancer cells. Internalization of 125I-ABL occurred within 30 min of the lectin being bound to the cell surface. Subcellular fractionation after pulse labelling of the cells with 125I-ABL for 2 h at 4 °C followed by culture of the cells at 37 °C demonstrated a steady increase in radioactivity in a crude nuclear extract. The radioactivity in this extract reached a maximum after 10 h and declined after 20 h. Release of ABL from the cell, after pulse labelling, was assessed using both fluorescein isothiocyanate-labelled ABL and 125I-ABL and was slow, with a t1/2 of 48 h. Most of the 125I-ABL both inside cells and in the medium remained intact, as determined by trichloroacetic acid precipitation and SDS/PAGE, and after 48 h only 22 ± 2% of ABL in the medium and 14 ± 2% inside the cells was degraded. This study suggests that the reversibility of the antiproliferative effect of ABL is associated with its release from cells after internalization. The internalization and subsequent slow release, with little degradation of ABL, reflects the tendency of lectins to resist biodegradation and implies that other endogenous or exogenous lectins may be processed in this way by intestinal epithelial cells. [source] Molecular analyses and identification of promising candidate genes for loci on mouse chromosome 1 affecting alcohol physical dependence and associated withdrawalGENES, BRAIN AND BEHAVIOR, Issue 5 2008D. L. Denmark We recently mapped quantitative trait loci (QTLs) with large effects on predisposition to physical dependence and associated withdrawal severity following chronic and acute alcohol exposure (Alcdp1/Alcw1) to a 1.1-Mb interval of mouse chromosome 1 syntenic with human chromosome 1q23.2-23.3. Here, we provide a detailed analysis of the genes within this interval and show that it contains 40 coding genes, 17 of which show validated genotype-dependent transcript expression and/or non-synonymous coding sequence variation that may underlie the influence of Alcdp1/Alcw1 on ethanol dependence and associated withdrawal. These high priority candidates are involved in diverse cellular functions including intracellular trafficking, oxidative homeostasis, mitochondrial respiration, and extracellular matrix dynamics, and indicate both established and novel aspects of the neurobiological response to ethanol. This work represents a substantial advancement toward identification of the gene(s) that underlies the phenotypic effects of Alcdp1/Alcw1. Additionally, a multitude of QTLs for a variety of complex traits, including diverse behavioral responses to ethanol, have been mapped in the vicinity of Alcdp1/Alcw1, and as many as four QTLs on human chromosome 1q have been implicated in human mapping studies for alcoholism and associated endophenotypes. Thus, our results will be primary to further efforts to identify genes involved in a wide variety of behavioral responses to alcohol and may directly facilitate progress in human alcoholism genetics. [source] The neuronal ceroid lipofuscinosis protein CLN5: new insights into cellular maturation, transport, and consequences of mutations,HUMAN MUTATION, Issue 3 2010Mia-Lisa Schmiedt Abstract Neuronal ceroid lipofuscinoses (NCLs) represent a group of children's inherited neurodegenerative disorders caused by mutations in at least eight different genes. Mutations in the CLN5 gene result in the Finnish variant late infantile NCL characterized by gradual loss of vision, epileptic seizures, and mental deterioration. The CLN5 gene encodes a lysosomal glycoprotein of unidentified function. In this study, we have used both transient and stable expression systems for the characterization of CLN5, focusing on the localization, processing, and intracellular trafficking. We show that CLN5 is proteolytically cleaved, and that the mature polypeptide is transported to the lysosomes. Our data provide the first evidence that soluble CLN5 protein can also undergo mannose-6-phosphate receptor-independent trafficking to the lysosomes. We studied the localization and maturation of the CLN5 carrying the previously uncharacterized vLINCL disease causing mutations in HeLa cells. All analyzed disease mutations disturb the lysosomal trafficking of the mutated CLN5 proteins. The level of lysosomal targeting does not correlate, however, to disease onset, indicating that CLN5 may also function outside lysosomes. This study furthers our understanding of the basic properties of the CLN5 protein, necessary for the characterization of the consequences of disease mutations and for the planning of future therapies for vLINCL. Hum Mutat 31:356,365, 2010. © 2010 Wiley-Liss, Inc. [source] Interaction of 14-3-3 with Bid during seizure-induced neuronal deathJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Sachiko Shinoda Abstract Seizure-induced neuronal death may involve coordinated intracellular trafficking and protein,protein interactions of members of the Bcl-2 family. The 14-3-3 proteins are known to sequester certain pro-apoptotic members of this family. BH3-interacting domain death agonist (Bid) may contribute to seizure-induced neuronal death, although regulation by 14-3-3 has not been reported. In this study we examined whether 14-3-3 proteins interact with Bid during seizure-induced neuronal death. Brief seizures were evoked in rats by intraamygdala microinjection of kainic acid to elicit unilateral hippocampal CA3 neuronal death. Coimmunoprecipitation analysis demonstrated that although Bcl-2-associated death promoter (Bad) constitutively bound 14-3-3, there was no interaction between Bid and 14-3-3 in control brain. Seizures triggered Bid cleavage and a commensurate increase in binding of Bid to 14-3-3 within injured hippocampus. Casein kinases I and II, which can inactivate Bid by phosphoserine/threonine modification, did not coimmunoprecipitate with Bid. The largely uninjured contralateral hippocampus did not exhibit Bid cleavage or binding of 14-3-3 to Bid. In vitro experiments confirmed that 14-3-3, is capable of binding truncated Bid, likely in the absence of phosphoserine/threonine modification. These data suggest 14-3-3 proteins may target active as well as inactive conformations of pro-apoptotic Bcl-2 death agonists, highlighting novel targets for intervention in seizure-induced neuronal death. [source] Regulation of intracellular HAP1 traffickingJOURNAL OF NEUROSCIENCE RESEARCH, Issue 14 2007Juan Rong Abstract A number of proteins are known to interact with huntingtin (htt), the Huntington's disease (HD) protein. Understanding the function of these htt-associated proteins could help elucidate the pathogenesis of HD and the role that htt plays in the disease process. The present review focuses on one such protein, huntingtin-associated protein 1 (HAP1), which has proved to be involved in intracellular trafficking. Unlike huntingtin, which is expressed ubiquitously throughout the brain and body, HAP1 is enriched in neurons, suggesting that its dysfunction could contribute to the selective neuropathology in HD. HAP1 binds microtubule-dependent transporters that engage anterograde or retrograde transport and also associates with a variety of organelles and membrane receptors. This raises the interesting issue of how the HAP1 trafficking function is regulated. We discuss recent evidence for the involvement of HAP1 in intracellular trafficking as well as potential mechanisms that may regulate its trafficking. © 2007 Wiley-Liss, Inc. [source] Presenilin 1 is involved in the maturation of ,-site amyloid precursor protein-cleaving enzyme 1 (BACE1)JOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2007Akira Kuzuya Abstract One of the pathologic hallmarks of Alzheimer's disease is the excessive deposition of ,-amyloid peptides (A,) in senile plaques. A, is generated when ,-amyloid precursor protein (APP) is cleaved sequentially by ,-secretase, identified as ,-site APP-cleaving enzyme 1 (BACE1), and ,-secretase, a putative enzymatic complex containing presenilin 1 (PS1). However, functional interaction between PS1 and BACE1 has never been known. In addition to this classical role in the generation of A, peptides, it has also been proposed that PS1 affects the intracellular trafficking and maturation of selected membrane proteins. We show that the levels of exogenous and endogenous mature BACE1 expressed in presenilin-deficient mouse embryonic fibroblasts (PS,/,MEFs) were reduced significantly compared to those in wild-type MEFs. Moreover, the levels of mature BACE1 were increased in human neuroblastoma cell line, SH-SY5Y, stably expressing wild-type PS1, compared to native cells. Conversely, the maturation of BACE1 was compromised under the stable expression of dominant,negative mutant PS1 overexpression. Immunoprecipitation assay showed that PS1 preferably interacts with proBACE1 rather than mature BACE1, indicating that PS1 can be directly involved in the maturation process of BACE1. Further, endogenous PS1 was immunoprecipitated with endogenous BACE1 in SH-SY5Y cells and mouse brain tissue. We conclude that PS1 is directly involved in the maturation of BACE1, thus possibly functioning as a regulator of both ,- and ,-secretase in A, generation. © 2006 Wiley-Liss, Inc. [source] Cell adhesion molecules for targeted drug deliveryJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2006Alison L. Dunehoo Abstract Rapid advancement of the understanding of the structure and function of cell adhesion molecules (i.e., integrins, cadherins) has impacted the design and development of drugs (i.e., peptide, proteins) with the potential to treat cancer and heart and autoimmune diseases. For example, RGD peptides/peptidomimetics have been marketed as anti-thrombic agents and are being investigated for inhibiting tumor angiogenesis. Other cell adhesion peptides derived from ICAM-1 and LFA-1 sequences were found to block T-cell adhesion to vascular endothelial cells and epithelial cells; these peptides are being investigated for treating autoimmune diseases. Recent findings suggest that cell adhesion receptors such as integrins can internalize their peptide ligands into the intracellular space. Thus, many cell adhesion peptides (i.e., RGD peptide) were used to target drugs, particles, and diagnostic agents to a specific cell that has increased expression of cell adhesion receptors. This review is focused on the utilization of cell adhesion peptides and receptors in specific targeted drug delivery, diagnostics, and tissue engineering. In the future, more information on the mechanism of internalization and intracellular trafficking of cell adhesion molecules will be exploited for delivering drug molecules to a specific type of cell or for diagnosis of cancer and heart and autoimmune diseases. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95: 1856,1872, 2006 [source] Intracellular uptake and trafficking of Pluronic micelles in drug-sensitive and MDR cells: Effect on the intracellular drug localizationJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2002Natalya Rapoport Abstract The intracellular uptake and localization of a fluorescently labeled Pluronic P-105 in HL-60 leukemia cells and in A2780 drug-sensitive and A2780/ADR MDR ovarian carcinoma cells were characterized by flow cytometry and fluorescence microscopy. Pluronic P-105 molecules were labeled with a pH-sensitive fluorescent label, 5-(and 6-)carboxy-2,7,-dichlorofluorescein. The fluorescence intensity of labeled Pluronic was about twofold higher at pH 7.4 than at pH 5.5. At Pluronic concentrations exceeding the critical micelle concentration (cmc), flow cytometry histograms manifested bimodal distribution of cell fluorescence for all types of cells. Cell population characterized by higher fluorescence intensity presumably resulted from Pluronic transfer from the acidic environment of cytoplasmic vesicles (endosomes or lysosomes) into the neutral environment of the cytoplasm and cell nuclei, which suggested the permeabilization of the membranes of acidic vesicle by Pluronic molecules. For the MDR cells, the bimodal distribution of cell fluorescence was already observed at very low Pluronic concentrations in the incubation medium (i.e., below the cmc). The data suggest that the membranes of acidic vesicles of MDR cells are more susceptible to the action of polymeric surfactants than those of drug-sensitive cells. Permeabilization of acidic vesicles had a dramatic effect on the intracellular trafficking of drugs: when delivered in PBS, the anthracyclin drug ruboxyl (Rb) sequestered in cytoplasmic vesicles and was excluded from cell nuclei; however, when delivered in Pluronic micelles, drug accumulated in cell nuclei. Drug uptake from/with Pluronic micelles was substantially enhanced by ultrasound. These findings suggest that the nuclear accumulation of drugs internalized via fluid-phase endocytosis can be enhanced by the application of Pluronic micelles and can be further augmented by ultrasonic irradiation. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:157,170, 2002 [source] Alterations of chaperone protein expression in presenilin mutant neurons in response to glutamate excitotoxicityPATHOLOGY INTERNATIONAL, Issue 9 2002Izumi Maezawa Mutations in the presenilin-1 (PS1) gene underlie the most common form of familial dementia of the Alzheimer type (DAT). We demonstrated previously that the expression of PS1 with a M146V mutation in transgenic mice potentiates glutamate toxicity to neurons, due to an altered calcium homeostasis. Here, using a subtractive cDNA library approach, we report the identification of several genes, the altered expression of which may be associated with this unique PS1 -related vulnerability to glutamate. The identified genes, including chaperonin subunit 2 and nucleophosmin 1/B23, are involved in the intracellular trafficking of proteins and ions. Northern blot analysis revealed that the effect of glutamate on calcium-binding proteins was augmented in neurons from PS1 mutation mice, compared with neurons from mice lacking other genes relevant to the pathogenesis of DAT (FE65 and APOE) or neurons from control wild-type mice. Interestingly, mRNA for two chaperone proteins were expressed at lower levels specifically in neurons from PS1 mutant mice. These findings suggest that PS1 mutations may, in part, contribute to the development of DAT via altered expression of chaperone proteins. [source] Fluorescent protein fusions to a human immunodeficiency virus monoclonal antibody reveal its intracellular transport through the plant endomembrane systemPLANT BIOTECHNOLOGY JOURNAL, Issue 7 2008Sarah L. Irons Summary In order to further understand the production and intracellular trafficking of pharmaceutical proteins in plants, the light and heavy chains (LC and HC) of the human immunodeficiency virus neutralizing monoclonal antibody 2G12 were fused to fluorescent proteins [Venus and monomeric red fluorescent protein (mRFP)] to enable the visualization of their passage through the plant cell. Co-expression of LC and HC with various markers of the endomembrane system demonstrated that LC fusions were found in mobile punctate structures, which are likely to be pre-vacuolar compartments (PVCs) as a proportion of the LC fusions were found to be located in the vacuole. In addition, apoplast labelling was also observed with a 2G12LC-RFP fusion. The HC fusion expressed alone was found only in the endoplasmic reticulum (ER). When the LC and HC fusions were expressed together, they were found to co-locate to larger punctate structures, which were morphologically distinct from any observed on expression of LC or HC alone. These structures appeared to be in close association with the ER and their labelling partially overlapped with PVC marker fluorescence, but no increase in apoplast labelling was observed. Co-immunoprecipitation data demonstrated that the presence of the fluorescent proteins did not affect the assembly of the antibody, and also showed the association of BiP with the antibody chains. The antigen-binding activity of the Venus-fused 2G12 antibody was confirmed by enzyme-linked immunosorbent assay. [source] Lactosylated polyethylenimine for gene transfer into airway epithelial cells: role of the sugar moiety in cell delivery and intracellular trafficking of the complexesTHE JOURNAL OF GENE MEDICINE, Issue 3 2004Stéphanie Grosse Abstract Background As we have previously shown that lactosylated polyethylenimine (PEI) is the most efficient glycosylated PEI for gene transfer into human airway epithelial cells in primary culture, we have studied here the role of the lactose residue in the enhancement of gene transfer efficiency observed with lactosylated PEI as compared with unsubstituted PEI in immortalized (,CFTE29o- cells) and primary human airway epithelial cells. Methods and results After three transfections of 1 h performed daily, 60% of ,CFTE29o- cells were transfected with lactosylated PEI, whereas 25% of cells were transfected with unsubstituted PEI (p < 0.05). Cell viability was 1.8-fold greater with lactosylated PEI as compared with unsubstituted PEI (p < 0.05). As assessed by flow cytometry, the cellular uptake of lactosylated complexes was greater than that of complexes made with unsubstituted PEI (p < 0.05) and involved mostly a receptor-mediated endocytosis. The study of the intracellular trafficking in airway epithelial cells of complexes showed an endosomal and lysosomal accumulation of lactosylated complexes. In the presence of a proton pump inhibitor, the level of lactosylated and unsubstituted PEI-mediated gene expression was reduced more than 20-fold, whereas the cell viability increased to almost 100%. For both complexes, a nuclear localization was observed for less than 5% of intracellular complexes. Conclusions Our results show that the greater gene transfer efficiency observed for lactosylated complexes may be attributed to a higher amount of lactosylated complexes incorporated by airway epithelial cells and a lower cytotoxicity that might be related to reduced endosomolytic properties. However, the lactose residues substituting the PEI did not promote the entry of the plasmid into the nucleus. Copyright © 2004 John Wiley & Sons, Ltd. [source] Fusion of PDGFRB to two distinct loci at 3p21 and a third at 12q13 in imatinib-responsive myeloproliferative neoplasmsBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2010Claire Hidalgo-Curtis Summary We identified four patients who presented with BCR-ABL1 negative myeloproliferative neoplasms and cytogenetically visible abnormalities of chromosome band 5q31-35. Fluorescence in situ hybridization indicated that the platelet-derived growth factor receptor , gene (PDGFRB) was disrupted in all four cases and 5, rapid amplification of cDNA ends identified in-frame mRNA fusions between PDGFRB and WDR48 (3p21), GOLGA4 (3p21) and BIN2 (12q13). Strikingly, all three genes encode proteins involving intracellular trafficking. Imatinib, a known inhibitor of PDGFR,, selectively blocked the growth of t(3;5) myeloid colonies and produced clinically significant responses in all patients. We conclude that PDGFRB fuses to diverse partner genes in atypical myeloproliferative neoplasms (MPNs). Although very rare, identification of these fusions is critical for proper management of affected individuals. [source] Activation of the transient receptor potential vanilloid-1 (TRPV1) channel opens the gate for pain reliefBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2008G Jancsó Pharmacological modulation of the transient receptor potential vanilloid-1 (TRPV1) receptor function offers a promising means of producing pain relief at the level of the primary sensory neuron. In this issue of the BJP, the pharmacological approaches and the available experimental data that focus on the TRPV1 receptor to achieve therapeutically useful alleviation of pain and inflammation are reviewed. The potentials to inactivate TRPV1 receptor function by site- and modality-specific TRPV1 antagonists, uncompetitive TRPV1 blockers and drugs interfering with TRPV1 sensitization, are evaluated. A crucial issue of producing pain relief at the level of the nocisensor remains whether it can be achieved solely through inactivation of the TRPV1 receptor or TRPV1 agonist-induced defunctionalization of the whole primary afferent neuron is required. The accumulated evidence indicates that both pharmacological modulation of the intracellular trafficking of the TRPV1 receptor and defunctionalization of the nocisensors by TRPV1 agonists are promising novel approaches to tame the TRPV1 receptor. British Journal of Pharmacology (2008) 155, 1139,1141; doi:fn1; published online 10 November 2008 [source] Sorting nexin 3 (SNX3) is a component of a tubular endosomal network induced by Salmonella and involved in maturation of the Salmonella -containing vacuoleCELLULAR MICROBIOLOGY, Issue 9 2010Virginie Braun Summary Salmonella enterica serovar Typhimurium is an intracellular pathogen that grows within a modified endomembrane compartment, the Salmonella -containing vacuole (SCV). Maturation of nascent SCVs involves the recruitment of early endosome markers and the remodelling of phosphoinositides at the membrane of the vacuole, in particular the production of phosphatidylinositol 3-phosphate [PI(3)P]. Sorting nexins (SNXs) are a family of proteins characterized by the presence of a phox homology (PX) domain that binds to phosphoinositides and are involved in intracellular trafficking in eukaryotic cells. We therefore studied whether sorting nexins, particularly sorting nexin 3 (SNX3), play a role in Salmonella infection. We found that SNX3 transiently localized to SCVs at early times post invasion (10 min) and presented a striking tubulation phenotype in the vicinity of SCVs at later times (30,60 min). The bacterial effector SopB, which is known to promote PI(3)P production on SCVs, was required for the formation of SNX3 tubules. In addition, RAB5 was also required for the formation of SNX3 tubules. Depletion of SNX3 by siRNA impaired RAB7 and LAMP1 recruitment to the SCV. Moreover, the formation of Salmonella -induced filaments (Sifs) was altered by SNX3 knock-down. Therefore, SNX3 plays a significant role in regulating the maturation of SCVs. [source] Verotoxin 1 binding to intestinal crypt epithelial cells results in localization to lysosomes and abrogation of toxicityCELLULAR MICROBIOLOGY, Issue 2 2003D. E. Elaine Hoey Summary Verotoxins (VTs) are important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), a group of bacteria associated with severe disease sequelae in humans. The potent cytotoxic activity of VTs is important in pathogenicity, resulting in the death of cells expressing receptor Gb3 (globotriaosylceramide). EHEC, particularly serotype O157:H7, frequently colonize reservoir hosts (such as cattle) in the absence of disease, however, the basis to avirulence in this host has been unclear. The objective of this study was assessment of interaction between VT and intestinal epithelium, which represents the major interface between the host and enteric organisms. Bovine intestinal epithelial cells expressed Gb3 in vitro in primary cell cultures, localizing specifically to proliferating crypt cells in corroboration with in situ immunohistological observations on intestinal mucosa. Expression of receptor by these cells contrasts with the absence of Gb3 on human intestinal epithelium in vivo. Despite receptor expression, VT exhibited no cytotoxic activity against bovine epithelial cells. Sub-cellular localization of VT indicated that this toxin was excluded from endoplasmic reticulum but localized to lysosomes, corresponding with abrogation of cytotoxicity. VT intracellular trafficking was unaffected by treatment of primary cell cultures with methyl-,-cyclodextrin, indicating that Gb3 in these cells is not associated with lipid rafts but is randomly distributed in the membrane. The combination of Gb3 isoform, membrane distribution and VT trafficking correlate with observations of other receptor-positive cells that resist verocytotoxicity. These studies demonstrate that intestinal epithelium is an important determinant in VT interaction with major implications for the differential consequences of EHEC infection in reservoir hosts and humans. [source] | ||||||||||||||||