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Intact Cells (intact + cell)
Selected AbstractsSperm morphology in the black coral Cirrhipathes sp. (Anthozoa, Antipatharia)INVERTEBRATE BIOLOGY, Issue 3 2008Elda Gaino Abstract. Male polyps of the antipatharian Cirrhipathes sp., collected along the coral reef of Siladen Island (Sulawesi, Indonesia), were studied in order to gain an insight into the reproductive biology. Spermatocysts (maximum size 120 ,m) are located within the primary gametogenic mesenteries and are separated by mesenteric cell cytoplasmic extensions. Sperm, maturing along radial rows, have a fairly round shape and contain a series of electron-dense vesicles in the apical nuclear region. A single mitochondrion flanks the nucleus. A peculiar cup-like electron-dense body, edged with regularly spaced electron-dense granules, is interposed between the nucleus and the tail, and delimits a central region that includes two centrioles. Cross-sections of the cup-like body reveal that the distal centriole has a pericentriolar system, consisting of nine arms arranged in a radial pattern. Each arm branches into three processes that are connected to the electron-dense granules. Indirect evidence of spawning is derived from the accumulation of sperm in the gastric cavity. This process takes place through the lysis of the cells bordering the mesenteries. Intact cells of this bordering layer appear to be involved in the phagocytosis of non-expelled gametes. [source] Plasma membrane permeabilization by 60- and 600-ns electric pulses is determined by the absorbed doseBIOELECTROMAGNETICS, Issue 2 2009Bennett L. Ibey Abstract We explored how the effect of plasma membrane permeabilization by nanosecond-duration electric pulses (nsEP) depends on the physical characteristics of exposure. The resting membrane resistance (Rm) and membrane potential (MP) were measured in cultured GH3 and CHO cells by conventional whole-cell patch-clamp technique. Intact cells were exposed to a single nsEP (60 or 600 ns duration, 0,22 kV/cm), followed by patch-clamp measurements after a 2,3 min delay. Consistent with earlier findings, nsEP caused long-lasting Rm decrease, accompanied by the loss of MP. The threshold for these effects was about 6 kV/cm for 60 ns pulses, and about 1 kV/cm for 600 ns pulses. Further analysis established that it was neither pulse duration nor the E-field amplitude per se, but the absorbed dose that determined the magnitude of the biological effect. In other words, exposure to nsEP at either pulse duration caused equal effects if the absorbed doses were equal. The threshold absorbed dose to produce plasma membrane effects in either GH3 or CHO cells at either pulse duration was found to be at or below 10 mJ/g. Despite being determined by the dose, the nsEP effect clearly is not thermal, as the maximum heating at the threshold dose is less than 0.01 °C. The use of the absorbed dose as a universal exposure metric may help to compare and quantify nsEP sensitivity of different cell types and of cells in different physiological conditions. The absorbed dose may also prove to be a more useful metric than the incident E-field in determining safety limits for high peak, low average power EMF emissions. Bioelectromagnetics 30:92,99, 2009. © 2008 Wiley-Liss, Inc. [source] Progress toward a biomimetic leaf: 4,000 h of hydrogen production by coating-stabilized nongrowing photosynthetic Rhodopseudomonas palustrisBIOTECHNOLOGY PROGRESS, Issue 4 2010Jimmy L. Gosse Abstract Intact cells are the most stable form of nature's photosynthetic machinery. Coating-immobilized microbes have the potential to revolutionize the design of photoabsorbers for conversion of sunlight into fuels. Multi-layer adhesive polymer coatings could spatially combine photoreactive bacteria and algae (complementary biological irradiance spectra) creating high surface area, thin, flexible structures optimized for light trapping, and production of hydrogen (H2) from water, lignin, pollutants, or waste organics. We report a model coating system which produced 2.08 ± 0.01 mmol H2 m,2 h,1 for 4,000 h with nongrowing Rhodopseudomonas palustris, a purple nonsulfur photosynthetic bacterium. This adhesive, flexible, nanoporous Rps. palustris latex coating produced 8.24 ± 0.03 mol H2 m,2 in an argon atmosphere when supplied with acetate and light. A simple low-pressure hydrogen production and trapping system was tested using a 100 cm2 coating. Rps. palustris CGA009 was combined in a bilayer coating with a carotenoid-less mutant of Rps. palustris (CrtI,) deficient in peripheral light harvesting (LH2) function. Cryogenic field emission gun scanning electron microscopy (cryo-FEG-SEM) and high-pressure freezing were used to visualize the microstructure of hydrated coatings. A light interaction and reactivity model was evaluated to predict optimal coating thickness for light absorption using the Kubelka-Munk theory (KMT) of reflectance and absorptance. A two-flux model predicted light saturation thickness with good agreement to observed H2 evolution rate. A combined materials and modeling approach could be used for guiding cellular engineering of light trapping and reactivity to enhance overall photosynthetic efficiency per meter square of sunlight incident on photocatalysts. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] A CALCIUM-DEPENDENT PROTEIN KINASE FUNCTIONS IN WOUND HEALING IN VENTRICARIA VENTRICOSA (CHLOROPHYTA)JOURNAL OF PHYCOLOGY, Issue 6 2000Koh-ichi Sugiyama The cytoplasm around a wound made in the multinucleate unicellular green alga Ventricaria ventricosa ( J. Agardh) Olsen et West formed an aggregation-ring surrounding the wound immediately after injury. A contraction of the ring then brought about wound healing in culture medium containing Ca2+. Involvement of a calcium-dependent protein kinase (CDPK) as a regulator of wound healing was examined using an anti- Dunaliella tertiolecta CDPK antibody. A 52-kDa protein cross-reacting with the antibody was detected by Western blotting. Protein kinases of 60 kDa and 52 kDa, which were markedly activated by Ca2+, and a 40-kDa Ca2+ -independent protein kinase were detected by an in-gel protein kinase assay using myelin basic protein as the substrate. A 52-kDa band with Ca2+ -dependent protein kinase activity was immunoprecipitated from the cytoplasmic extract, indicating that these 52-kDa proteins are identical and possess CDPK activity. Microscopic observation showed that the contraction of the aggregation ring was suppressed by application of the anti-CDPK to the culture medium. A protein kinase inhibitor, K-252a, and the calmodulin inhibitors, calmidazolium and compound 48 / 80, which inhibit CDPK activity, also suppressed the contraction of the aggregation-ring. Immunofluorescence microscopy showed a similar distribution of 52-kDa CDPK to the distribution of f-actin, which was randomly distributed in an intact cell and formed a bundle during wound healing. Further, f-actin was not recruited after injury in the presence of the antibody to CDPK. These results suggest that the 52-kDa CDPK functions as a Ca2+ receptor in wound healing and simultaneously participates in the organization and contraction of f-actin to heal the wound. [source] Unsupervised immunophenotypic profiling of chronic lymphocytic leukemiaCYTOMETRY, Issue 3 2006Luzette K. Habib Abstract Background Proteomics and functional genomics have revolutionized approaches to disease classification. Like proteomics, flow cytometry (FCM) assesses concurrent expression of many proteins, with the advantage of using intact cells that may be differentially selected during analysis. However, FCM has generally been used for incremental marker validation or construction of predictive models based on known patterns, rather than as a tool for unsupervised class discovery. We undertook a retrospective analysis of clinical FCM data to assess the feasibility of a cell-based proteomic approach to FCM by unsupervised cluster analysis. Methods Multicolor FCM data on peripheral blood (PB) and bone marrow (BM) lymphocytes from 140 consecutive patients with B-cell chronic lymphoproliferative disorders (LPDs), including 81 chronic lymphocytic leukemia (CLLs), were studied. Expression was normalized for CD19 totals, and recorded for 10 additional B-cell markers. Data were subjected to hierarchical cluster analysis using complete linkage by Pearson's correlation. Analysis of CLL in PB samples (n = 63) discovered three major clusters. One cluster (14 patients) was skewed toward "atypical" CLL and was characterized by high CD20, CD22, FMC7, and light chain, and low CD23. The remaining two clusters consisted almost entirely (48/49) of cases recorded as typical BCLL. The smaller "typical" BCLL cluster differed from the larger cluster by high CD38 (P = 0.001), low CD20 (P = 0.001), and low CD23 (P = 0.016). These two typical BCLL clusters showed a trend toward a difference in survival (P = 0.1090). Statistically significant cluster stability was demonstrated by expanding the dataset to include BM samples, and by using a method of random sampling with replacement. Conclusions This study supports the concept that unsupervised immunophenotypic profiling of FCM data can yield reproducible subtypes of lymphoma/chronic leukemia. Expanded studies are warranted in the use of FCM as an unsupervised class discovery tool, akin to other proteomic methods, rather than as a validation tool. © 2006 International Society for Analytical Cytology [source] C-peptide makes a comebackDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2003John Wahren Proinsulin C-peptide was for long considered to be without biological activity of its own. New findings demonstrate, however, that it is capable of eliciting both molecular and physiological effects, suggesting that C-peptide is in fact a bioactive peptide. When administered in replacement doses to animal models or to patients with type 1 diabetes, C-peptide ameliorates diabetes-induced functional and structural changes in both the kidneys and the peripheral nerves. It augments blood flow in a number of tissues, notably skeletal muscle, myocardium, skin and nerve. These effects are thought to be mediated via a stimulatory influence on Na+,K+ -ATPase and on endothelial nitric oxide synthase. Specific binding of C-peptide to cell membranes of intact cells and to detergent-solubilized cellular components has been demonstrated, indicating the existence of cell-surface binding sites for C-peptide. A number of intracellular responses are elicited by C-peptide, including a rise in Ca2+ concentration and activation of MAP-kinase signaling pathways. Many but not all of C-peptide's intracellular effects can be inhibited by pertussis toxin, supporting the notion that C-peptide may interact via a G-protein-coupled receptor. Additional data suggest that C-peptide may interact synergistically also in the insulin signaling pathway. Combined, the available observations show conclusively that C-peptide is biologically active, even though its molecular mechanism of action is not as yet fully understood. The possibility that replacement of C-peptide in patients with type 1 diabetes may serve to retard or prevent the development of long-term complications should be evaluated. Copyright © 2003 John Wiley & Sons, Ltd. [source] Determination of the bacterial pathogen Edwardsiella tarda in fish species by capillary electrophoresis with blue light-emitting diode-induced fluorescenceELECTROPHORESIS, Issue 18-19 2004Lijun Yu Abstract High-performance capillary electrophoresis (HPCE) has been applied to the identification, separation, and quantitation of intact bacteria. We demonstrate that a pathogen (Edwardsiella tarda) which causes systemic infection in commercially important fish species can be rapidly identified and determined (<10 min) after direct injection into fish fluid by CE blue light-emitting diode (LED)-induced fluorescence. SYTO 13 (488 nm/509 nm), a cell-permeable green nucleic acid stain, was used to stain the cells. Remarkably high efficiency (>1 200,000 theoretical plates/m) was achieved with this rapid and efficient CE method. It was found that proper sample vortexing (90 s) would be beneficial to disperse aggregated cells and facilitate the focusing of intact cells during electrophoresis. Ionization of the surface constituents of Edwardsiella tarda cells provided efficient surface charges for the intact cells to be separated from the EOF and damaged or lysed cells when the separation was performed in running buffer (3.94 mM Tris, 0.56 mM borate, 0.013 mM EDTA) at pH 10.5. The limit of detection (LOD) and recovery were found to be 4.2×104 cells/mL and 70.0%, respectively. This proposed CE method could become an effective tool for diagnosis and tracking of certain diseases caused by bacteria in fish species as well as in human beings. [source] Toward a greater appreciation of noncovalent chemical/DNA interactions: Application of biological and computational approaches,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2-3 2005Ronald D. Snyder Abstract Noncovalent DNA interactions, e.g., DNA intercalation and DNA groove-binding, have not been well studied relative to covalent interactions largely due to the inability of predicting and detecting such events in intact cells. We have adapted an in vitro bleomycin amplification method for DNA intercalation for use in cultured V79 Chinese hamster cells and have validated this approach through the use of a three-dimensional DNA computational docking model that quantifies potential strength of DNA intercalative binding based on electrostatics and hydrogen bonding. For many structural classes of molecules, DNA intercalation is necessary but not sufficient for genotoxicity. The present article reviews our progress to date in predicting and confirming noncovalent binding of drugs and other chemicals and in understanding the mechanistic relationship between intercalation and genotoxicity. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc. [source] Reduction of fumarate, mesaconate and crotonate by Mfr, a novel oxygen-regulated periplasmic reductase in Campylobacter jejuniENVIRONMENTAL MICROBIOLOGY, Issue 3 2010Edward Guccione Summary Methylmenaquinol : fumarate reductase (Mfr) is a newly recognized type of fumarate reductase present in some ,-proteobacteria, where the active site subunit (MfrA) is localized in the periplasm, but for which a physiological role has not been identified. We show that the Campylobacter jejuni mfrABE operon is transcribed from a single promoter, with the mfrA gene preceded by a small open reading-frame (mfrX) encoding a C. jejuni -specific polypeptide of unknown function. The growth characteristics and enzyme activities of mutants in the mfrA and menaquinol : fumarate reductase A (frdA) genes show that the cytoplasmic facing Frd enzyme is the major fumarate reductase under oxygen limitation. The Mfr enzyme is shown to be necessary for maximal rates of growth by fumarate respiration and rates of fumarate reduction in intact cells measured by both viologen assays and 1H-NMR were slower in an mfrA mutant. As periplasmic fumarate reduction does not require fumarate/succinate antiport, Mfr may allow more efficient adaptation to fumarate-dependent growth. However, a further rationale for the periplasmic location of Mfr is suggested by the observation that the enzyme also reduces the fumarate analogues mesaconate and crotonate; fermentation products of anaerobes with which C. jejuni shares its gut environment, that are unable to be transported into the cell. Both MfrA and MfrB subunits were localized in the periplasm by immunoblotting and 2D-gel electrophoresis, but an mfrE mutant accumulated unprocessed MfrA in the cytoplasm, suggesting a preassembled MfrABE holoenzyme has to be recognized by the TAT system for translocation to occur. Gene expression studies in chemostat cultures following an aerobic-anaerobic shift showed that mfrA is highly upregulated by oxygen limitation, as would be experienced in vivo. Our results indicate that in addition to a role in fumarate respiration, Mfr allows C. jejuni to reduce analogous substrates specifically present in the host gut environment. [source] Biological significance of metals partitioned to subcellular fractions within earthworms (Aporrectodea caliginosa),ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2006Martina G. Vijver Abstract Metal ions in excess of metabolic requirements are potentially toxic and must be removed from the vicinity of important biological molecules to protect organisms from adverse effects. Correspondingly, metals are sequestrated in various forms, defining the accumulation pattern and the magnitude of steady-state levels reached. To investigate the subcellular fractions over which Ca, Mg, Fe, Cu, Zn, Cd, Pb, Ni, and As are distributed, earthworms (Aporrectodea caliginosa) collected from the field were analyzed by isolating metal-rich granules and tissue fragments from intracellular microsomal and cytosolic fractions (i.e., heat-stable proteins and heat-denatured proteins). The fractions showed metal-specific binding capacity. Cadmium was mainly retrieved from the protein fractions. Copper was equally distributed over the protein fraction and the fraction comprising tissue fragments, cell membranes, and intact cells. Zinc, Ca, Mg, and As were mainly found in this fraction as well. Lead, Fe, and Ni were mainly isolated from the granular fraction. To study accumulation kinetics in the different fractions, three experiments were conducted in which earthworms were exposed to metal-spiked soil and a soil contaminated by anthropogenic inputs and, indigenous earthworms were exposed to field soils. Although kinetics showed variation, linear uptake and steady-state types of accumulation patterns could be understood according to subcellular compartmentalization. For risk assessment purposes, subcellular distribution of metals might allow for a more precise estimate of effects than total body burden. Identification of subcellular partitioning appears useful in determining the biological significance of steady-state levels reached in animals. [source] Functional Characterisation of the Volume-Sensitive Anion Channel in Rat Pancreatic ,-CellsEXPERIMENTAL PHYSIOLOGY, Issue 2 2001L. Best The whole-cell and perforated patch configurations of the patch-clamp technique were used to characterise the volume-sensitive anion channel in rat pancreatic ,-cells. The channel showed high permeability (P) relative to Cl, to extracellular monovalent organic anions (PSCN/PCll= 1.73, Pacetate/PCll= 0.39, Plactate/PCll= 0.38, Pacetoacetate/PCll= 0.32, Pglutamate/PCll= 0.28) but was less permeable to the divalent anion malate (Pmalate/PCll= 0.14). Channel activity was inhibited by a number of putative anion channel inhibitors, including extracellular ATP (10 mM), 1,9-dideoxyforskolin (100 ,M) and 4-OH tamoxifen (10 ,M). Inclusion of the catalytic subunit of protein kinase A in the pipette solution did not activate the volume-sensitive anion channel in non-swollen cells. Furthermore, addition of 8-bromoadenosine 3,,5,-cyclic monophosphate (8-BrcAMP) or forskolin failed to activate the channel in intact cells under perforated patch conditions. Addition of phorbol 12,13-dibutyrate (200 nM), either before or after cell swelling, also failed to affect channel activation. Our findings do not support the suggestion that the volume-sensitive anion channel in pancreatic ,-cells can be activated by protein kinase A. Furthermore, the ,-cell channel does not appear to be subject to regulation via protein kinase C. [source] Interaction of the alpha-helical H6 peptide from the pro-apoptotic protein tBid with cardiolipinFEBS JOURNAL, Issue 21 2009Patrice X. Petit BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices ,H6 and ,H7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c -releasing activity. In the present study, we focused on the interaction between the ,H6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the ,H6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of ,H6 into the phospholipid monolayer. The modification of two charged amino acid residues in ,H6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and ,H6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that ,H6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via ,H6. [source] Characterization of 1H NMR detectable mobile lipids in cells from human adenocarcinomasFEBS JOURNAL, Issue 5 2009Anna Maria Luciani Magnetic resonance spectroscopy studies are often carried out to provide metabolic information on tumour cell metabolism, aiming for increased knowledge for use in anti-cancer treatments. Accordingly, the presence of intense lipid signals in tumour cells has been the subject of many studies aiming to obtain further insight on the reaction of cancer cells to external agents that eventually cause cell death. The present study explored the relationship between changes in neutral lipid signals during cell growth and after irradiation with gamma rays to provide arrest in cell cycle and cell death. Two cell lines from human tumours were used that were differently prone to apoptosis following irradiation. A sub-G1 peak was present only in the radiosensitive HeLa cells. Different patterns of neutral lipids changes were observed in spectra from intact cells, either during unperturbed cell growth in culture or after radiation-induced growth arrest. The intensities of triglyceride signals in the spectra from extracted total lipids changed concurrently. The increase in lipid peak intensities did not correlate with the apoptotic fate. Modelling to fit the experimental data revealed a dynamic equilibrium between the production and depletion of neutral lipids. This is observed for the first time in cells that are different from adipocytes. [source] Interaction between catalytically inactive calpain and calpastatinFEBS JOURNAL, Issue 8 2006Evidence for its occurrence in stimulated cells Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca2+, substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca2+] or extensive diffusion of calpastatin into the cytosol without affecting Ca2+ homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain,calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action. [source] Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cellsFEBS JOURNAL, Issue 4 2006Jorge Alegre-Cebollada Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS-7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose-dependent and time-dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent-resistant cellular membranes showed that sticholysin II colocalizes with caveolin-1 in fractions corresponding to raft-like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane-associated protein. Toxin binding to raft-like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS-7 cells through interaction with membrane domains which behave like cellular rafts. [source] The function of D1-H332 in Photosystem II electron transport studied by thermoluminescence and chlorophyll fluorescence in site-directed mutants of Synechocystis 6803FEBS JOURNAL, Issue 17 2004Yagut Allahverdiyeva The His332 residue of the D1 protein has been identified as the likely ligand of the catalytic Mn ions in the water oxidizing complex (Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J. & Iwata, S. (2004) Science 303, 1831,1838). However, its function has not been fully clarified. Here we used thermoluminescence and flash-induced chlorophyll fluorescence measurements to characterize the effect of the D1-H333E, D1-H332D and D1-H332S mutations on the electron transport of Photosystem II in intact cells of the cyanobacterium Synechocystis 6803. Although the mutants are not photoautotrophic they all show flash-induced thermoluminescence and chlorophyll fluorescence, which originate from the S2QA, and S2QB, recombinations demonstrating that charge stabilization takes place in the water oxidizing complex. However, the conversion of S2 to higher S states is inhibited and the energetic stability of the S2QA, charge pair is increased by 75, 50 and 7 mV in the D1-H332D, D1-H332E and D1-H332S mutants, respectively. This is most probably caused by a decrease of Em(S2/S1). Concomitantly, the rate of electron donation from Mn to Tyr-Z, during the S1 to S2 transition is slowed down, relative to the wild type, 350- and 60-fold in the D1-H332E and D1-H332D mutants, respectively, but remains essentially unaffected in D1-H332S. A further effect of the D1-H332E and D1-H332D mutations is the retardation of the QA to QB electron transfer step as an indirect consequence of the donor side modification. Our data show that although the His residue in the D1-332 position can be substituted by other metal binding residues for binding photo-oxidisable Mn it is required for controlling the functional redox energetics of the Mn cluster. [source] Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cellsFEBS JOURNAL, Issue 2 2004Dominique Legrand Lactoferrin (Lf), a multifunctional molecule present in mammalian secretions and blood, plays important roles in host defense and cancer. Indeed, Lf has been reported to inhibit the proliferation of cancerous mammary gland epithelial cells and manifest a potent antiviral activity against human immunodeficiency virus and human cytomegalovirus. The Lf-binding sites on the cell surface appear to be proteoglycans and other as yet undefined protein(s). Here, we isolated a Lf-binding 105 kDa molecular mass protein from cell extracts and identified it as human nucleolin. Medium,affinity interactions (, 240 nm) between Lf and purified nucleolin were further illustrated by surface plasmon resonance assays. The interaction of Lf with the cell surface-expressed nucleolin was then demonstrated through competitive binding studies between Lf and the anti-human immunodeficiency virus pseudopeptide, HB-19, which binds specifically surface-expressed nucleolin independently of proteoglycans. Interestingly, binding competition studies between HB-19 and various Lf derivatives in proteoglycan-deficient hamster cells suggested that the nucleolin-binding site is located in both the N- and C-terminal lobes of Lf, whereas the basic N-terminal region is dispensable. On intact cells, Lf co-localizes with surface nucleolin and together they become internalized through vesicles of the recycling/degradation pathway by an active process. Morever, a small proportion of Lf appears to translocate in the nucleus of cells. Finally, the observations that endocytosis of Lf is inhibited by the HB-19 pseudopeptide, and the lack of Lf endocytosis in proteoglycan-deficient cells despite Lf binding, point out that both nucleolin and proteoglycans are implicated in the mechanism of Lf endocytosis. [source] New insights into the P-glycoprotein-mediated effluxes of rhodaminesFEBS JOURNAL, Issue 3 2003Chatchanok Loetchutinat Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P-glycoprotein (P-gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, ka = VM/km, which is a measure of the efficiency of the P-gp-mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P-gp-mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P-gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P-gp-mediated efflux of rhodamine analogues in intact cells. [source] Effects of methylcyclodextrin on lysosomesFEBS JOURNAL, Issue 5 2001Michel Jadot The cholesterol complexing agent methyl-cyclodextrin (MCD) provides an efficient mean for the removal of cholesterol from biological membranes. In order to study the effects of this agent on the lysosomal membrane in situ, we treated HepG2 cells with MCD and studied the effects of this treatment on lysosomes in isolated fractions. We found that lysosomes prepared from treated cells are more sensitive to various membrane perturbing treatments such as: incubation of lysosomes in isotonic glucose, in hypotonic sucrose or in the presence of the lytic agent glycyl- l -phenylalanine 2-naphthylamide. The lysosomal membrane is also less resistant to increased hydrostatic pressure. Centrifugation methods were used to analyse the effect of MCD on lysosomes. Isopycnic centrifugation in sucrose density gradients demonstrates that the drug induces a reversible density increase of the lysosomes. Our study indicates that extracellularly added MCD can modify the properties of the lysosomal membrane in living cells. It suggests that MCD could be an effective tool to modulate the physical properties of lysosomes within intact cells and to monitor the cellular responses to such modifications. [source] Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformansFEMS YEAST RESEARCH, Issue 4 2007Richard A. Eigenheer Abstract Extracellular proteins of Cryptococcus neoformans are involved in the pathogenesis of cryptococcosis, and some are immunoreactive antigens that may potentially serve as candidates for vaccine development. To further study the extracellular proteome of the human fungal pathogen Cry. neoformans, we conducted a proteomic analysis of secreted and cell wall-bound proteins with an acapsular strain of Cry. neoformans. Proteins were identified from both intact cells and cell walls. In both cases, extracellular proteins were removed with trypsin or ,-glucanase, and then all proteins/peptides were purified by solid-phase extraction, spin dialysis, and HPLC, and identified by liquid chromatography,mass spectrometry. This study identified 29 extracellular proteins with a predicted N-terminal signal sequence and also a predicted glycosylphosphatidylinositol anchor motif in more than half. Among the novel proteins identified were five glycosylphosphatidylinositol-anchored proteins with extensive Ser/Thr-rich regions but no apparent functional domains, a glycosylphosphatidylinositol-anchored aspartic protease, and a metalloprotease with structural similarity to an elastinolytic metalloprotease of Aspergillus fumigatus. This study suggests that Cry. neoformans has the machinery required to target glycosylphosphatidylinositol-anchored proteins to the cell wall, and it confirms the extracellular proteolytic ability of Cry. neoformans. [source] Haemocytes of the hemlock woolly adelgid Adelges tsugae Annand (Hom., Adelgidae) and changes after exposure to low temperaturesJOURNAL OF APPLIED ENTOMOLOGY, Issue 3-4 2000V. Gouli The main cellular components of the haemolymph of Adelges tsugae Annand were identified from individuals collected in Massachusetts, USA in February and October 1997. The cell types present were formative cells, plasmatocytes, adipohaemocytes, vermiform cells, prohaemocytes and oenocytoids. Vermiform cells were the most abundant. Very few oenocytoids were found. They were significantly more cells of all types in the haemolymph of A. tsugae collected in October than in those collected in February. Adelges tsugae collected in February were exposed in the laboratory to -20, -25 and -30°C for 2, 4 and 8h. No intact cells were found in the haemolymph of A. tsugae exposed to -30°C. Haemocytes were damaged by exposure to -20 and -25°C. The effect of these temperatures on the different cell types is presented. [source] Synergistic effect of enterocin AS-48 in combination with outer membrane permeabilizing treatments against Escherichia coli O157:H7JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2005S. Ananou Abstract Aims:, To determine the effects of outer membrane (OM) permeabilizing agents on the antimicrobial activity of enterocin AS-48 against Escherichia coli O157:H7 CECT 4783 strain in buffer and apple juice. Methods and Results:, We determined the influence of pH, EDTA, sodium tripolyphosphate (STPP) and heat on E. coli O157:H7 CECT 4783 sensitivity to enterocin AS-48 in buffer and in apple juice. Enterocin AS-48 was not active against intact cells of E. coli O157:H7 CECT 4783 at neutral pH. However, cells sublethally injured by OM permeabilizing agents (EDTA, STPP, pH 5, pH 8·6 and heat) became sensitive to AS-48, decreasing the amount of bacteriocin required for inhibition of E. coli O157:H7 CECT 4783. Conclusions:, The results presented indicate that enterocin AS-48 could potentially be applied with a considerably wider range of protective agents, such as OM permeabilizing agents, with increased efficacy in inhibiting E. coli O157:H7. Significance and Impact of the Study:, Results from this study support the potential use of enterocin AS-48 to control E. coli O157:H7 in combination with other hurdles. [source] Dexamethasone inhibits lipopolysaccharide-induced hydrogen sulphide biosynthesis in intact cells and in an animal model of endotoxic shockJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Ling Li Abstract Dexamethasone (1 mg/kg, i.p.) administered either 1 hr before or 1 hr after E. coli lipopolysaccharide (LPS, 4 mg/kg, i.p.) in conscious rats inhibited the subsequent (4 hrs) rise in plasma cytokine (interleukin [IL]-1,, tumour necrosis factor [TNF]-,), nitrate/nitrite (NO×), soluble intercellular adhesion molecule-1 (sICAM-1) concentration and lung/liver myeloperoxidase activity indicative of an anti-inflammatory effect. Dexamethasone also reduced the LPS-evoked rise in plasma hydrogen sulphide (H2S) concentration, liver H2S synthesizing activity and expression of cystathionine , lyase (CSE) and inducible nitric oxide synthase (iNOS). Mifepristone (RU-486) inhibited these effects. Dexamethasone (1,10 ,M) reduced the LPS-evoked release of IL-1,, TNF-, and L-selectin, decreased expression of CSE and iNOS and diminished nuclear factor ,B (NF-,B)-DNA binding in isolated rat neutrophils. In contrast, NaHS (100 ,M) increased L-selectin release from rat neutrophils. Dexamethasone also reduced LPS-induced up-regulation of CSE in foetal liver cells. 6-amino-4-(4-phenoxyphenylethylamino) quinazoline (QNZ, 10 nM), a selective inhibitor of transcription via the NF-,B pathway, abolished LPS-induced up-regulation of CSE expression. We propose that inhibition of CSE expression and reduction in formation of the pro-inflammatory component of H2S activity contributes to the anti-inflammatory effect of dexamethasone in endotoxic shock. Whether H2S plays a part in the anti-inflammatory effect of this steroid in other forms of inflammation such as arthritis or asthma warrants further study. [source] Modulation of nucleocytoplasmic trafficking by retention in cytoplasm or nucleusJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Daniela M. Roth Abstract Nuclear protein transport processes have largely been studied using in vitro semi-intact cell systems where high concentrations of nuclear localizing substrates are used, and cytoplasmic components such as the microtubule (MT) network, are either absent or damaged. Here we use the fluorescence recovery after photobleaching (FRAP) technique to analyze the nucleocytoplasmic flux of distinct fluorescently tagged proteins over time in living cultured cells. FRAP was performed in different parts of the cell to analyze the kinetics of nucleocytoplasmic trafficking and intranuclear/cytoplasmic mobility of the tumor suppressor Rb protein and a SV40 large tumor antigen (T-ag) derivative containing the nuclear localization sequence (NLS), both fused to green fluorescent protein (GFP). The results indicate that proteins carrying the T-ag NLS are highly mobile in the nucleus and cytoplasm. Rb, in contrast, is largely immobile in both cellular compartments, with similar nuclear import and export kinetics. Rb nuclear export was CRM-1-mediated, with its reduced mobility in the cytoplasm in part due to association with MTs. Overall our results show that nuclear and cytoplasm retention modulates the rates of nuclear protein import and export in intact cells. J. Cell. Biochem. 107: 1160,1167, 2009. © 2009 Wiley-Liss, Inc. [source] COMPARATIVE STUDY OF SHELL SWAB AND SHELL CRUSH METHODS FOR THE RECOVERY OF SALMONELLA FROM SHELL EGGSJOURNAL OF FOOD SAFETY, Issue 4 2008T. KAWASAKI ABSTRACT Swabbing is the standard methodology for the recovery of resident microorganism from shell eggs in Japan. A comparative study of shell swab (SW) and shell crush (CR) techniques was performed to recover the laboratory-inoculated Salmonella from shell eggs. It was found that the recovery of Salmonella by CR methods was significantly higher (4.5,7.5 log cfu/egg) than that of SW methods (3.1,6.3 log cfu/egg). However, analyses with quantitative real-time polymerase chain reaction (invA as a target gene), fluorescent microscopic and quantitative analyses with a Live/Dead BacLight bacterial viability kit revealed that not all of the inoculated Salmonella spp. populations were recovered as intact cells by either method. The chemiluminescent bacterial viability assay showed that chemiluminescence intensity (CI) began to increase after 30 min in CR samples; on the other hand, SW samples did not show any increase in CI for 2 h. These results suggest that SW might cause more damage and lethality to cells than CR. In addition, to determine the most appropriate method for recovering resident aerobic bacteria, coliforms and Salmonella spp from shell eggs, 4,000 commercial eggs were collected and sampled by shell rinse (SR) and CR techniques using phosphate-buffered saline (PBS) warmed to different temperatures. PBS at 37C was found to be the best recovery solution and temperature, respectively, for recovering aerobic microorganisms from shell eggs by both methods and the CR methods recovered a higher population than did the SR methods (4.9 versus 5.8 log cfu/egg for SR and CR methods, respectively; n = 500 eggs/method). Therefore, the CR method along with recovery buffer (PBS) at 37C could be an effective technique for the recovery of microorganisms from post-processed shell eggs. PRACTICAL APPLICATIONS There is a need to develop a rapid and highly sensitive method for the recovery of microorganisms from shell eggs. Such recovery methods are also useful for evaluating the efficacy of newly developed shell egg disinfection techniques. Many methods involving rinsing, swabbing, and crushing of shell eggs have been reported; however, we performed a comparative study of the method used to recover the Salmonella from shell eggs. We found that the shell crush method (CR) was superior to the shell swab method (SW) for the recovery of Salmonella spp., and phosphate-buffered saline (PBS) at 37C was found to be the best recovery solution and temperature, respectively, for recovering microorganisms from shell eggs by both methods. Therefore, the CR method along with recovery buffer (PBS) at 37C could be an effective technique for the recovery of microorganisms from post-processed shells. Use of this method could be recommended for the microbial evaluation of post-processed shell eggs in industries. [source] Potential of ,flat' fibre evanescent wave spectroscopy to discriminate between normal and malignant cells in vitroJOURNAL OF MICROSCOPY, Issue 2 2007Z. HAMMODY Summary The present study focuses on evaluating the potential of flattened AgClBr fibre-optic evanescent wave spectroscopy (FTIR-FEWS) technique for detection and identification of cancer cells in vitro using cell culture as a model system. The FTIR-FEWS results are compared to those from FTIR-microspectroscopy (FTIR-MSP) method extensively used to identify spectral properties of intact cells. Ten different samples of control and malignant cells were measured in parallel by the above two methods. Our results show a significant similarity between the results obtained by the two methodologies. The absorbance level of Amide I/Amide II, phosphates and carbohydrates were significantly altered in malignant compared to the normal cells using both systems. Thus, common biomarkers such as Amide I/Amide II, phosphate and carbohydrate levels can be derived to discern between normal and cancer cells. However, marked differences are also noted between the two methodologies in the protein bands due to CH3 bending vibration (1480,1350 cm,1). The spectral differences may be attributed to the variation in the penetration depth of the two methodologies. The use of flattened fibre rather than the standard cylindrical fibre has several practical advantages and is considered as an important step towards in vivo measurements in real time, such as that of skin nevi and melanoma using special designs of fibre-optic,based sensors. [source] The catalytic domain of human neuropathy target esterase mediates an organophosphate-sensitive ionic conductance across liposome membranesJOURNAL OF NEUROCHEMISTRY, Issue 2 2001Philip J. Forshaw In humans and other vertebrates, reaction of organophosphates with a neuronal membrane protein, neuropathy target esterase (NTE), initiates events which culminate in axonal degeneration. The initiation process appears to involve modification of a property of the protein distinct from its esterase activity, subsequent to formation of a negatively charged adduct with the active site serine residue. Here, we show that membrane patches from liposomes containing NEST, a recombinant hydrophobic polypeptide comprising the esterase domain of human NTE, display a transmembrane ionic conductance with both stable and high-frequency flickering components. An asymmetric current,voltage relationship suggested that ion flow was favoured in one direction relative to the membrane and its associated NEST molecules. Flow of anions was slightly favoured compared with cations. The flickering current formed a much larger proportion of the overall conductance in patches containing wild-type NEST compared with the catalytically inactive S966A mutant form of the protein. The conductance across patches containing NEST, but not those with the S966A mutant, was significantly reduced after adding neuropathic organophosphates to the bathing medium. By contrast, non-neuropathic covalent inhibitors of the catalytic activity of NEST did not reduce NEST-mediated conductance. Future work may establish whether NTE itself mediates an organophosphate-sensitive ion flux across intracellular membranes within intact cells. [source] Intracellular glutathione mediates the denitrosylation of protein nitrosothiols in the rat spinal cordJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2009Jorge M. Romero Abstract Protein S-nitrosothiols (PrSNOs) have been implicated in the pathophysiology of neuroinflammatory and neurodegenerative disorders. Although the metabolically instability of PrSNOs is well known, there is little understanding of the factors involved in the cleavage of S-NO linkage in intact cells. To address this issue, we conducted chase experiments in spinal cord slices incubated with S-nitrosoglutathione (GSNO). The results show that removal of GSNO leads to a rapid disappearance of PrSNOs (t½ , 2 hr), which is greatly accelerated when glutathione (GSH) levels are raised with the permeable analogue GSH ethyl ester. Moreover, PrSNOs are stable in the presence of the GSH depletor diethyl maleate, indicating that GSH is critical for protein denitrosylation. Inhibition of GSH-dependent enzymes (glutathione S-transferase, glutathione peroxidase, and glutaredoxin) and enzymes that could mediate denitrosylation (alcohol dehydrogense-III, thioredoxin and protein disulfide isomerase) do not alter the rate of PrSNO decomposition. These findings and the lack of protein glutathionylation during the chase indicate that most proteins are denitrosylated via rapid transnitrosylation with GSH. The differences in the denitrosylation rate of individual proteins suggest the existence of additional structural factors in this process. This study is relevant to our recent discovery that PrSNOs accumulate in the central nervous system of patients with multiple sclerosis. © 2008 Wiley-Liss, Inc. [source] Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitationMICROSCOPY RESEARCH AND TECHNIQUE, Issue 4 2008Stanley Walter Botchway Abstract The real-time uptake of serotonin, a neurotransmitter, by rat leukemia mast cell line RBL-2H3 and 5-hydroxytryptophan by Chinese hamster V79 cells has been studied by fluorescence lifetime imaging microscopy (FLIM), monitoring ultraviolet (340 nm) fluorescence induced by two-photon subpicosecond 630 nm excitation. Comparison with two-photon excitation with 590 nm photons or by three-photon excitation at 740 nm shows that the use of 630 nm excitation provides optimal signal intensity and lowered background from auto-fluorescence of other cellular components. In intact cells, we observe using FLIM three distinct fluorescence lifetimes of serotonin and 5-hydroxytryptophan according to location. The normal fluorescence lifetimes of both serotonin (3.8 ns) and 5-hydroxytryptophan (3.5 ns) in solution are reduced to ,2.5 ns immediately on uptake into the cell cytosol. The lifetime of internalized serotonin in RBL-2H3 cells is further reduced to ,2.0 ns when stored within secretory vesicles. Microsc. Res. Tech., 2008. © 2007 Wiley-Liss, Inc. [source] Apple polyphenols diminish the phosphorylation of the epidermal growth factor receptor in HT29 colon carcinoma cellsMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 5 2007Diana Fridrich Abstract Previously, we showed that an apple juice extract (AE) potently inhibits the protein tyrosine kinase (PTK) activity of the epidermal growth factor receptor (EGFR). In the present study, an apple pomace extract (APE) was found to exceed the EGFR inhibitory properties of AE in a cell-free system. The impact of the extracts on the phosphorylation status of the EGFR in intact cells (HT29) was sensitive to catalase, added to suppress the accumulation of hydrogen peroxide. In the absence of catalase, the formation of hydrogen peroxide was observed, achieving 1.1 ± 0.1 ,M (AE) and 1.5 ± 0.1 ,M (APE) after 45 min of incubation. In the presence of catalase, suppressing the hydrogen peroxide level to the solvent control, APE effectively suppressed EGFR phosphorylation, even exceeding the effects of AE. Both extracts inhibited the growth of HT29 cells, albeit the enhanced EGFR inhibitory properties of APE compared to AE were not reflected by a higher growth inhibitory potential. The results clearly show that the effect of apple extracts on the EGFR and cell growth are not simply artefacts of hydrogen peroxide formation. However, the formation of hydrogen peroxide has to be considered to modulate and/or mask cellular responses to apple extracts. [source] | |||||||||||||||||||||||||||||||