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Extracellular Medium (extracellular + medium)
Selected AbstractsTwo distinct P2Y receptors are involved in purine- and pyrimidine-evoked Ca2+ elevation in mammalian brain astrocytic culturesDRUG DEVELOPMENT RESEARCH, Issue 1-2 2001Chiara Bolego Abstract ATP and 2-methyl-thio-ATP (2-Me-SATP) increase cytosolic calcium concentrations ([Ca2+]i) in rat striatal astrocytes (Centemeri et al. [1997] Br J Pharmacol 121:1700,1706). The aim of the present study was to: (1) characterize pyrimidine-induced [Ca2+]i increases in the same experimental system, and (2) try to identify the multiple P2Y receptor subtypes mediating Ca2+ mobilization. UDP and UTP triggered a concentration-dependent [Ca2+]i elevation (EC50s = 0.58 ,M ± 0.4 and 31 ,M ± 6, respectively). Pyrimidine-evoked [Ca2+]i elevation was solely due to mobilization from intracellular stores, because: (1) removing calcium from extracellular medium or (2) blocking its influx with Ni2+ did not modify UTP responses; (3) the store-depleting agent thapsigargin completely abolished UTP-evoked [Ca2+]i increments. Guanosine-5,-O-(2-thiodiphosphate) partially inhibited the UTP response, whereas pertussis toxin (PTx) had no effect. The phospholipase C inhibitor U-73122 significantly reduced the UTP-evoked [Ca2+]i rise. Computer-assisted analysis indicated that the UTP and UDP responses are mediated by a single receptor, while ATP and 2-Me-SATP interact with two distinct receptors. The selective P2Y1 receptor antagonist MRS2179 abolished the ATP higher potency component. Sequential challenges with the same nucleotides resulted in almost complete homologous desensitization. Pre-exposure to UTP lowered the subsequent responses to either ATP or 2-Me-SATP. Maximally active concentrations of UTP and ATP were not additive. In conclusion, [Ca2+]i elevation in astrocytes by purines and pyrimidines is mediated by two distinct P2Y receptors, likely the P2Y1 and P2Y6 subtypes. Drug Dev. Res. 52:122,132, 2001. © 2001 Wiley-Liss, Inc. [source] On-line sample preconcentration with chemical derivatization of bacterial biomarkers by capillary electrophoresis: A dual strategy for integrating sample pretreatment with chemical analysisELECTROPHORESIS, Issue 21 2005Adam S. Ptolemy Abstract Simple, selective yet sensitive methods to quantify low-abundance bacterial biomarkers derived from complex samples are required in clinical, biological, and environmental applications. In this report, a new strategy to integrate sample pretreatment with chemical analysis is investigated using on-line preconcentration with chemical derivatization by CE and UV detection. Single-step enantioselective analysis of muramic acid (MA) and diaminopimelic acid (DAP) was achieved by CE via sample enrichment by dynamic pH junction with ortho -phthalaldehyde/N -acetyl- L -cysteine labeling directly in-capillary. The optimized method resulted in up to a 100-fold enhancement in concentration sensitivity compared to conventional off-line derivatization procedures. The method was also applied toward the detection of micromolar levels of MA and DAP excreted in the extracellular medium of Escherichia coli bacterial cell cultures. On-line preconcentration with chemical derivatization by CE represents a unique approach for conducting rapid, sensitive, and high-throughput analyses of other classes of amino acid and amino sugar metabolites with reduced sample handling, where the capillary functions simultaneously as a concentrator, microreactor, and chiral selector. [source] Impact of basic FGF expression in astrocytes on dopamine neuron synaptic function and developmentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2006Caroline Forget Abstract Behavioural sensitization to amphetamine (AMPH) requires action of the drug in the ventral midbrain where dopamine (DA) neurons are located. In vivo studies suggest that AMPH sensitization requires enhanced expression of basic fibroblast growth factor (bFGF) in the nucleus of midbrain astrocytes. One idea is that the AMPH-induced increase in bFGF expression in astrocytes leads to enhanced secretion of this peptide and to long-term plasticity in DA neurons. To study directly the effects of astrocytic expression of bFGF on DA neurons, we established a cell-culture model of mesencephalic astrocytes and DA neurons. Immunolabelling showed that even in the absence of a pharmacological stimulus, the majority of mesencephalic astrocytes in culture express bFGF at a nuclear level. Arguing against the idea that bFGF was secreted, bFGF was undetectable in the extracellular medium (below 10 pg/mL). However, supplementing culture medium with exogenous bFGF at standard concentrations (20 ng/mL) led to a dramatic change in the morphology of astrocytes, increased spontaneous DA release, and inhibited synapse formation by individual DA neurons. RNA interference (siRNA) against bFGF mRNA, caused a reduction in DA release but produced no change in synaptic development. Together these data demonstrate that under basal conditions (in the absence of a pharmacological stimulus such as amphetamine) bFGF is not secreted even though there is abundant nuclear expression in astrocytes. The effects of bFGF seen here on DA neurons are thus likely to be mediated through more indirect glial,neuronal interactions, leading to enhanced DA release without a necessary change in synapse number. [source] Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stressFEBS JOURNAL, Issue 3 2000Barbara Tavazzi To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5,10 mm) of hydrogen peroxide for 1 h at 37 °C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mm H2O2), oxidative stress produced, starting from 1 mm H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mm). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mm H2O2 -treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mm H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress. [source] A xylose-inducible expression system for Lactococcus lactisFEMS MICROBIOLOGY LETTERS, Issue 2 2004Anderson Miyoshi Abstract A new controlled production system to target heterologous proteins to cytoplasm or extracellular medium is described for Lactococcus lactis NCDO2118. It is based on the use of a xylose-inducible lactococcal promoter, PxylT. The capacities of this system to produce cytoplasmic and secreted proteins were tested using the Staphylococcus aureus nuclease gene (nuc) fused or not to the lactococcal Usp45 signal peptide. Xylose-inducible nuc expression is tightly controlled and resulted in high-level and long-term protein production, and correct targeting either to the cytoplasm or to the extracellular medium. Furthermore, this expression system is versatile and can be switched on or off easily by adding either xylose or glucose, respectively. These results confirm the potential of this expression system as an alternative and useful tool for the production of proteins of interest in L. lactis. [source] Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activityFEMS MICROBIOLOGY LETTERS, Issue 2 2001Francisco J del Castillo Abstract The Escherichia coli K-12 sheA gene encodes a pore-forming hemolysin that is secreted to the medium by a hitherto unidentified mechanism. To study SheA secretion, we constructed fusions between SheA and the mature form of the periplasmic enzyme ,-lactamase, and performed site-directed mutagenesis on these constructs. The SheA-Bla and Bla-SheA hybrid proteins displayed hemolytic activity and were efficiently exported to the extracellular medium. Our results with mutant hybrid proteins show that secretion of SheA is independent of its cytolytic activity, that secretion is paralleled by a transient leakage of periplasmic contents to the extracellular medium, and that deletion of the 11 C-terminal residues of SheA has no effect on its secretion and cytolytic activity. [source] Listeriolysin O: a key protein of Listeria monocytogenes with multiple functionsFEMS MICROBIOLOGY REVIEWS, Issue 4 2006Samer Kayal Abstract Cholesterol-dependent cytolysins (CDCs) are produced by a large number of pathogenic Gram-positive bacteria. Most of these single-chain proteins are secreted in the extracellular medium. Among the species producing CDCs, only two species belonging to the genus Listeria (Listeria monocytogenes and Listeria ivanovii) are able to multiply intracellularly and release their toxins in the phagosomal compartment of the infected host cell. This review provides an updated overview on the importance of listeriolysin O (LLO) in the pathogenicity of L. monocytogenes, focusing mainly on two aspects: (1) the structure,function relationship of LLO and (2) its role in intra- and extracellular signalling. We first examine the specific sequence determinants, or protein domains, that make this cytolysin so well adapted to the intracellular lifestyle of L. monocytogenes. The roles that LLO has in cellular signalling events in the context of relations to pathogenesis are also discussed. [source] Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptorJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Sannah Zoffmann Abstract Neurokinin A stimulates physiological responses in the peripheral and central nervous systems upon interacting primarily with the tachykinin NK2 receptor (NK2R). In this study, the structure of NKA bound to the NK2R is characterised by use of fluorescence resonance energy transfer. Four fluorescent NKA analogues with Texas red introduced at amino acid positions 1, 4, 7 and 10 were prepared. When bound to a NK2R carrying enhanced green fluorescent protein at the N-terminus, all peptides reduce green fluorescent protein fluorescence from 10% to 50% due to energy transfer. The derived donor-acceptor distances are 46, 55, 59 and 69 Å for the fluorophore linked to positions 1,10, respectively. The monotonic increase in distance clearly indicates that the peptide adopts an extended structure when bound to its receptor. The present data are used, in combination with rhodopsin structure, fluorescence studies, photoaffinity labelling and site-directed mutagenesis data to design a computer model of the NKA-NK2R complex. We propose that the N-terminus of NKA is exposed and accessible to the extracellular medium. Subsequent amino acids of the NKA peptide become progressively more buried residues up to approximately one-third of the transmembrane-spanning domain. [source] Upregulation of Serotonin Transporter by Alcohol in Human Dendritic Cells: Possible Implication in Neuroimmune DeregulationALCOHOLISM, Issue 10 2009Dakshayani Kadiyala Babu Background:, Alcohol is the most widely abused substance and its chronic consumption causes neurobehavioral disorders. It has been shown that alcohol affects the function of immune cells. Dendritic cells (DC) serve as the first line of defense against infections and are known to accumulate neurotransmitters such as 5-hydroxytryptamine (5-HT). The enzyme monoamine oxidase-A (MAO-A) degrades 5-HT that is associated with clinical depression and other neurological disorders. 5-HT is selectively transported into neurons through the serotonin transporter (SERT), which is a member of the sodium- and chloride-dependent neurotransmitter transporter (SLC6) family. SERT also serves as a receptor for psychostimulant recreational drugs. It has been demonstrated that several drugs of abuse such as amphetamine and cocaine inhibit the SERT expression; however, the role of alcohol is yet to be elucidated. We hypothesize that alcohol can modulate SERT and MAO-A expression in DC, leading to reciprocal downregulation of 5-HT in extracellular medium. Methods:, Dendritic cells were treated with different concentrations (0.05% to 0.2%v/v) of alcohol for 24,72 hours and processed for SERT and MAO-A expression using Q-PCR and Western blots analysis. In addition, SERT function in DC treated with alcohol both in the presence and absence of imipramine, a SERT inhibitor was measured using 4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide uptake assay. 5-HT levels in culture supernatant and intracellular 5-hydroxy indole acetic acid (5-HIAA) and cyclic AMP were also quantitated using ELISA. Results:, Dendritic cells treated with 0.1% alcohol for 24 hours showed significant upregulation of SERT and MAO-A expression compared with untreated DC. We also observed that 0.1% alcohol enhanced the function of SERT and decreased extracellular 5-HT levels compared with untreated DC cultures, and this was associated with the elevation of intracellular 5-HIAA and cyclic AMP levels. Conclusions:, Our study suggests that alcohol upregulates SERT and MAO-A by elevating cyclic AMP, which may lead to decreased concentration of 5-HT in the extracellular medium. As 5-HT is a major neurotransmitter and an inflammatory mediator, its alcohol-mediated depletion may cause both neurological and immunological deregulation. [source] GABAmimetic intravenous anaesthetics inhibit spontaneous Ca2+ -oscillations in cultured hippocampal neuronsACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 6 2006B. Sinner Background:, Spontaneous Ca2+ -oscillations are a possible mechanism of Ca2+ -mediated signal transduction in neurons. They develop by a periodical interplay of Ca2+, which enters the neuron from the extracellular medium and triggers Ca2+ release from the endoplasmic reticulum (ER). Ca2+ -oscillations are terminated by reuptake into the ER or plasmalemmal extrusion. Spontaneous Ca2+ -oscillations are glutamate dependent and appear to be responsible for neuronal plasticity and integration of information. Here, we examined the role of the gamma-aminobutyric acid (GABAA) receptor on spontaneous Ca2+ -oscillations and studied the effects of the anaesthetics midazolam, thiopental and the non-anesthetic barbituric acid on spontaneous Ca2+ -oscillations. Methods:, Hippocampal neuronal cell cultures of 19-day-old embryonic Wistar rats 17,18 days in culture were loaded with the Ca2+ -sensitive dye Fura-2AM. Experiments were performed using dual wave-length excitation fluorescence microscopy and calibration constants were obtained from in situ calibration. Results:, Spontaneous Ca2+ -oscillations are influenced by the GABAA receptor. The intravenous anaesthetics midazolam and thiopental suppressed the amplitude and frequency reversibly in a dose-dependent manner with EC50 in clinically relevant concentrations. This effect was mediated via the GABAA receptor as it could be reversed by the GABAA receptor antagonist bicuculline. In contrast, the application of barbituric acid had no effects on the spontaneous Ca2+ -oscillations. Conclusion:, Spontaneous Ca2+ -oscillations are influenced by the GABAA receptor. Spontaneous Ca2+ -oscillations might represent an interesting model system to study anaesthetic mechanisms on neuronal information processing. [source] Preservation of mouse liver tissue during cold storage in experimental solutions assessed by x-ray microanalysisLIVER TRANSPLANTATION, Issue 3 2003Inna Kozlova The increasing use of organs for transplantation necessitates the development of optimal preservation techniques. The goal of this study was to investigate changes in elemental content in mouse liver cells during cold storage by x-ray microanalysis in parallel with morphologic studies. Tissue was stored at 4°C for 4 to 12 hours in normal Krebs-Ringer solution (high sodium/potassium ratio), modified Krebs-Ringer solution (low Na+/K+ ratio), Euro-Collins solution, University of Wisconsin (UW) solution, or seven modified versions of the UW solution. Incubation of liver in normal Krebs-Ringer solution caused a significant increase in sodium and decrease in potassium concentrations in contrast to incubation in other solutions. The concentration of sodium, potassium, and chlorine in the cells closely followed the concentration in the storage solution, indicating that the intracellular concentration of these ions during storage is entirely dependent on diffusion processes. The calcium concentration was independent of the storage solution used. Studies by light and transmission electron microscopy showed good preservation of hepatocytes after storage for 8 and 12 hours in UW solution and its variants, modified Krebs-Ringer solution and Euro-Collins solution, but showed moderate damage to mitochondria and swelling of the endoplasmic reticulum in normal Krebs-Ringer solution. In addition, damage to the sinusoidal endothelial cells was observed after 4 hours in normal Krebs-Ringer solution and after 8 to 12 hours in the other solutions. In conclusion, the only factor determining the intracellular concentration of diffusible ions after cold tissue storage is the ionic composition of the extracellular medium. X-ray microanalysis provides an objective method for assessing whether the intracellular ionic composition of tissue is maintained during storage. [source] Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coliMOLECULAR MICROBIOLOGY, Issue 4 2004Sofía Fraile Summary The tolerance of the haemolysin transport system (Hly) for exporting dimeric protein substrates to the supernatants of Escherichia coli cultures was examined. A strong dimerization domain (i.e. an amphipathic ,-helix capable of forming a leucine zipper in the yeast transcription factor GCN4) was inserted into an epitope-tagged version of the 23 kDa C-terminal secretion signal of haemolysin (EHlyA). The zipper-containing polypeptide (ZEHlyA) was effectively secreted by E. coli cells carrying the HlyBD transporter and accumulated in the culture media as a stable dimer as determined by gel filtration chromatography. In vivo protein cross-linking experiments and coexpression with a secretion-deficient derivative of ZEHlyA indicated that leucine zipper-dependent dimerization occurs following secretion. To test whether dimerization allows the correct folding of the secreted polypeptide, immunoglobulin VHH -domains obtained from camel antibodies were fused to EHlyA and ZEHlyA. Functional dimerization of the ZEHlyA hybrid was anticipated to increase the apparent binding affinity (i.e. avidity) of the VHH moiety, thus becoming an excellent reporter of correct protein folding and dimerization. Both VHH -EHlyA and VHH -ZEHlyA hybrids were quantitatively secreted and found in the extracellular medium as active monomers and dimers respectively. When compared with their monomeric counterparts, the dimeric VHH -ZEHlyA molecules showed superior binding properties to their cognate antigen, with a 10-fold increase in their avidity. These data reveal a non-anticipated permissiveness of the Hly type I transport machinery for the secretion of substrates with dimerization capacity. [source] IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formationMOLECULAR MICROBIOLOGY, Issue 1 2000Kirsten Niebuhr Invasion of epithelial cells by Shigella flexneri involves entry and intercellular dissemination. Entry of bacteria into non-phagocytic cells requires the IpaA,D proteins that are secreted by the Mxi,Spa type III secretion machinery. Type III secretion systems are found in several Gram-negative pathogens and serve to inject bacterial effector proteins directly into the cytoplasm of host cells. In this study, we have analysed the IpgD protein of S. flexneri, the gene of which is located on the virulence plasmid at the 5, end of the mxi,spa locus. We have shown that IpgD (i) is stored in the bacterial cytoplasm in association with a specific chaperone, IpgE; (ii) is secreted by the Mxi,Spa type III secretion system in amounts similar to those of the IpaA,D proteins; (iii) is associated with IpaA in the extracellular medium; and (iv) is involved in the modulation of the host cell response after contact of the bacterium with epithelial cells. This suggests that IpgD is an effector that might be injected into host cells to manipulate cellular processes during infection. [source] Utilization of citrate and lactate through a lactate dehydrogenase and ATP-regulated pathway in boar spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2006Antonio Medrano Abstract Incubation of boar spermatozoa in Krebs,Ringer,Henseleit medium with either 10 mM lactate or 10 mM citrate induced a fast and robust increase in the intracellular levels of ATP in both cases, which reached a peak after 30 sec of incubation. Utilization of both citrate and lactate resulted in the export of CO2 to the extracellular medium, indicating that both substrates were metabolized through the Krebs cycle. Incubation with citrate resulted in the generation of extracellular lactate, which was inhibited in the presence of phenylacetic acid. This indicates that lactate is produced through the pyruvate carboxylase step. In addition, there was also a significant increase in tyrosine phosphorylation induced by both citrate and lactate. Boar sperm has a sperm-specific isoform of lactate dehydrogenase (LDH), mainly located in the principal piece of the tail. Kinetic studies showed that boar sperm has at least two distinct LDH activities. The major activity (with an estimated Km of 0.51 mM) was located in the supernatants of sperm extracts. The minor LDH activity (with an estimated Km of 5.9 mM) was associated with the nonsoluble fraction of sperm extracts. Our results indicate that boar sperm efficiently metabolizes citrate and lactate through a metabolic pathway regulated by LDH. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometryPROTEIN SCIENCE, Issue 2 2000Randy M. Whittal Abstract Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, KD's are <100 nm. n-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both ph's. cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes. [source] Proteomic analysis of exoproteins expressed by enterotoxigenic Staphylococcus aureus strainsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2008Gabriella Pocsfalvi Dr. Abstract Pathogenic bacteria excrete a variety of virulence factors into extracellular medium and to the cell surface which have essential roles in the colonization and insurrection of the host cells, and thus reflect the degree of bacterial pathogenicity. For the exploration of virulence factors expressed in the secreted proteome fraction, different Staphylococcus aureus strains were analyzed using gel-based bottom-up proteomic approach. A total of 119 distinct proteins were identified for the enterotoxin gene cluster (egc) negative and seb gene positive S. aureus American Type Culture Collection (ATCC) 14458 strain by the use of one- and 2-DE based proteomics. Detailed analysis of enterotoxin region of the 2-D map confirmed, beside the highly expressed staphylococcal enterotoxin B (SEB), the presence of enterotoxin-like proteins SElK and SElQ previously predicted by genotyping (Sergeev et al.., J. Clin. Microbiol. 2004, 42, 2134,2143). Exoprotein patterns at the late-exponential (7,h) and stationary (24,h) phases of cellular growth show a high-level similarity in this region. Comparative analysis of enterotoxin region of five S. aureus strains including two clinical isolates (RIMD 31092 and A900322), a food derived strain (AB-8802) with highly prevalent egc positive operon and a nonenterotoxigenic reference strain (ROS) revealed the presence of different known enterotoxins and other virulence factors along with a number of core exoproteins. In addition, production of SElL (RIMD 31092) and SElP (A900322) was demonstrated for the first time at the protein level. Under the experimental conditions applied none of the enterotoxins encoded by the genes of egc operon was identified. [source] The nuclear autoantigen CENP-B displays cytokine-like activities toward vascular smooth muscle cellsARTHRITIS & RHEUMATISM, Issue 11 2007Geneviève Robitaille Objective A growing number of intracellular autoantigenic polypeptides have been found to play a second biologic role when they are present in the extracellular medium. We undertook this study to determine whether the CENP-B nuclear autoantigen could be added to this set of bifunctional molecules. Methods Purified CENP-B or CENP-B released from apoptotic cells was tested for surface binding to a number of human cell types by cell-based enzyme-linked immunosorbent assay, flow cytometry, and indirect immunofluorescence. The biologic effects of CENP-B on the migration, interleukin secretion, and signaling pathways of its specific target cells were evaluated. Results CENP-B was found to bind specifically to the surface of human pulmonary artery smooth muscle cells (SMCs) and not to fibroblasts or endothelial cells (ECs). Furthermore, CENP-B bound preferentially to SMCs of the contractile type rather than to SMCs of the synthetic type. Binding of CENP-B to SMCs stimulated their migration during in vitro wound healing assays, as well as their secretion of interleukins 6 and 8. The mechanism by which CENP-B mediated these effects involved the focal adhesion kinase, Src, ERK-1/2, and p38 MAPK pathways. Finally, CENP-B released from apoptotic ECs was found to bind to SMCs, thus indicating a plausible in vivo source of extracellular CENP-B. Conclusion These novel biologic roles of the nuclear autoantigen CENP-B open up a new perspective for studying the pathogenic role of anti,CENP-B autoantibodies. [source] Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum,BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Rutger D. Douma Abstract Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra- and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration-based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration-based washing. In the centrifugation-based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin-G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3,4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin-G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood. Biotechnol. Bioeng. 2010;107: 105,115. © 2010 Wiley Periodicals, Inc. [source] Extracellular matrix alters the relationship between tritiated thymidine incorporation and proliferation of MC3T3-E1 cells during osteogenesis in vitroCELL PROLIFERATION, Issue 1 2002W. J. Peterson Bone cells in vivo exist in direct contact with extracellular matrix, which regulates their basic biological processes including metabolism, development, growth and differentiation. Thus, the in vitro activity of cells cultured on tissue culture treated plastic could be different from the activity of cells cultured on their natural substrate. We selected MC3T3-E1 pre-osteoblastic cells to study the effect of extracellular matrix on cell proliferation because these cells undergo a progressive developmental sequence of proliferation and differentiation. MC3T3-E1 cells were cultured on plastic or plastic coated with ECM, fibronectin, collagen type I, BSA or poly l -lysine and their ability to proliferate was assessed by incorporation of [3H]dT or by enumeration of cells. Our results show that (1) ECM inhibits incorporation of [3H]dT by MC3T3-E1 cells; (2) collagen type I, but not BSA, poly l -lysine or fibronectin also inhibits incorporation of [3H]dT; (3) the level of ECM inhibition of [3H]dT incorporation is directly related to the number of cells cultured, but unrelated to the cell cycle distribution or endogenous thymidine content; (4) the kinetic profile of [3H]dT uptake suggest that ECM inhibits transport of [3H]dT from the extracellular medium, and (5) cell counts are similar in cultures whether cells are grown on plastic or ECM. These results suggest that decreased incorporation of [3H]dT by cells cultured on ECM is not reflective of bone cell proliferation. [source] Human cytomegalovirus final envelopment on membranes containing both trans -Golgi network and endosomal markersCELLULAR MICROBIOLOGY, Issue 3 2010Victoria Cepeda Summary The human cytomegalovirus (HCMV) has been shown to complete its final envelopment on cytoplasmic membranes prior to its secretion to the extracellular medium. However, the nature of these membranes has not been characterized. It is thought that HCMV acquires its final envelope from the trans -Golgi network (TGN), though we and others have previously reported a role for endocytic membranes. Here we studied the localization of cellular markers in HCMV-infected cells and in isolated viruses. Immunofluorescence staining indicated that HCMV induces the recruitment of TGN and endosomal markers to the virus factory. Immuno-gold labelling of isolated viral particles and electron microscopy demonstrated the incorporation of TGN46, endosomal markers early endosomal antigen 1, annexin I, transferrin receptor and CD63, and the cation-independent mannose 6-phosphate receptor, which traffics between the TGN and endosomes into the viral envelope. Virus immunoprecipitation assays demonstrated that virions containing TGN46 and CD63 were infectious. This study reconciles the apparent controversy regarding the nature of the HCMV assembly site and suggests that HCMV has the ability to generate a novel membrane compartment containing markers for both TGN and endosomes, or that the membranes that HCMV uses for its envelope may be vesicles in transit between the TGN and endosomes. [source] The ESCRT machinery is not required for human cytomegalovirus envelopmentCELLULAR MICROBIOLOGY, Issue 12 2007Alberto Fraile-Ramos Summary The human cytomegalovirus (HCMV) has been proposed to complete its final envelopment on cytoplasmic membranes prior to its release to the extracellular medium. The nature of these membranes and the mechanisms involved in virus envelopment and release are poorly understood. Here we show by immunogold-labelling and electron microscopy that CD63, a marker of multivesicular bodies (MVBs), is incorporated into the viral envelope, supporting the notion that HCMV uses endocytic membranes for its envelopment. We therefore investigated a possible role for the cellular endosomal sorting complex required for transport (ESCRT) machinery in HCMV envelopment. Depletion of tumour suppressor gene 101 and ALIX/AIP1 with small interfering RNAs (siRNAs) in HCMV-infected cells did not affect virus production. In contrast, siRNAs against the vacuolar protein sorting 4 (VPS4) proteins silenced the expression of VPS4A and VPS4B, inhibited the sorting of epidermal growth factor to lysosomes, the formation of HIV Gag-derived virus-like particles and vesicular stomatitis virus infection, but enhanced the number of HCMV viral particles produced. Treatment of infected cells with protease inhibitors also increased viral production. These studies indicate that, in contrast to some enveloped RNA viruses, HCMV does not require the cellular ESCRT machinery to complete its envelopment. [source] Substrate recognition of type III secretion machines ,testing the RNA signal hypothesisCELLULAR MICROBIOLOGY, Issue 9 2005Joseph A. Sorg Summary Secretion by the type III pathway of Gram-negative microbes transports polypeptides into the extracellular medium or into the cytoplasm of host cells during infection. In pathogenic Yersinia spp., type III machines recognize 14 different Yop protein substrates via discrete signals genetically encoded in 7,15 codons at the 5, portion of yop genes. Although the signals necessary and sufficient for substrate recognition of Yop proteins have been mapped, a clear mechanism on how proteins are recognized by the machinery and then initiated into the transport pathway has not yet emerged. As synonymous substitutions, mutations that alter mRNA sequence but not codon specificity, affect the function of some secretion signals, recent work with several different microbes tested the hypothesis of an RNA-encoded secretion signal for polypeptides that travel the type III pathway. This review summarizes experimental observations and mechanistic models for substrate recognition in this field. [source] HeLa Cell Entry by Guanidinium-Rich ,-Peptides: Importance of Specific Cation,Cell Surface InteractionsCHEMBIOCHEM, Issue 8 2007Terra B. Potocky Abstract Short cationic oligomers, including arginine-rich peptides and analogous ,-amino acid oligomers (", -peptides"), can enter the cytoplasm and nucleus of a living cell from the extracellular medium. It seems increasingly clear that multiple entry pathways are possible, depending upon the structure of the guanidinium-rich molecule, the type of cell, and other factors. We have previously shown that conformational stability and spatial clustering of guanidinium groups increase the HeLa cell entry efficiency of short helical , -peptides bearing six guanidinium groups, results that suggest that these , -peptides could be useful tools for studying the entry process. Here we describe studies intended to identify the point in the entry process at which helix stability and spatial arrangement of guanidinium groups exert their effect. Our results suggest that key distinctions involve the mode of interaction between different guanidinium-rich ,-peptides and the HeLa cell surface. A specific guanidinium display appears to be required for proper engagement of cell-surface heparan sulfate proteoglycans and concomitant induction of endocytic uptake. [source] | ||||||||||||||||||||||||||||