Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of X-100

  • surfactant triton x-100
  • triton x-100

  • Terms modified by X-100

  • x-100 micelle

  • Selected Abstracts

    Deletion of mdmB impairs mitochondrial distribution and morphology in Aspergillus nidulans

    CYTOSKELETON, Issue 2 2003
    Katrin V. Koch
    Abstract Mitochondria form a dynamic network of interconnected tubes in the cells of Saccharomyces cerevisiae or filamentous fungi such as Aspergillus nidulans,Neurospora crassa, or Podospora anserina. The dynamics depends on the separation of mitochondrial fragments, their movement throughout the cell, and their subsequent fusion with the other parts of the organelle. Interestingly, the microtubule network is required for the distribution in N. crassa and S. pombe, while S. cerevisiae and A. nidulans appear to use the actin cytoskeleton. We studied a homologue of S. cerevisiae Mdm10 in A. nidulans, and named it MdmB. The open reading frame is disrupted by two introns, one of which is conserved in mdm10 of P. anserina. The MdmB protein consists of 428 amino acids with a predicted molecular mass of 46.5 kDa. MdmB shares 26% identical amino acids to Mdm10 from S. cerevisiae, 35% to N. crassa, and 32% to the P. anserina homologue. A MdmB-GFP fusion protein co-localized evenly distributed along mitochondria. Extraction of the protein was only possible after treatment with a non-ionic and an ionic detergent (1% Triton X-100; 0.5% SDS) suggesting that MdmB was tightly bound to the mitochondrial membrane fraction. Deletion of the gene in A. nidulans affected mitochondrial morphology and distribution at 20C but not at 37C. mdmB deletion cells contained two populations of mitochondria at lower temperature, the normal tubular network plus some giant, non-motile mitochondria. Cell Motil. Cytoskeleton 55:114,124, 2003. 2003 Wiley-Liss, Inc. [source]

    Measurement of the force and torque produced in the calcium response of reactivated rat sperm flagella

    CYTOSKELETON, Issue 1 2001
    Mark J. Moritz
    Abstract Rat sperm that are demembranated with Triton X-100 and reactivated with Mg-ATP show a strong mechanical response to the presence of free calcium ion. At pCa < 4, the midpiece region of the flagellum develops a strong and sustained curvature that gives the cell the overall appearance of a fishhook [Lindemann and Goltz, 1988: Cell Motil. Cytoskeleton 10:420,431]. In the present study, the force and torque that maintain the calcium-induced hook have been examined quantitatively. In addition, full-length and shortened flagella were manipulated to evaluate the plasticity of the hooks and determined the critical length necessary for maintaining the curvature. The hooks were found to be highly resilient, returning to their original configuration (>95%) after being straightened and released. The results from manipulating the shortened flagella suggest that the force holding the hook in the curved configuration is generated in the basal 60 ,m of the flagellum. The force required to straighten the calcium-induced hooks was measured with force-calibrated glass microprobes, and the bending torque was calculated from the measured force. The force and torque required to straighten the flagellum were found to be proportional to the change in curvature of the hooked region of the flagellum, suggesting an elastic-like behavior. The average torque to open the hooks to a straight position was 2.6 (1.4) 10 -7 dyne cm (2.6 10 -14 N m) and the apparent stiffness was 4.3 (1.3) 10 -10 dyne cm2 (4.3 10 -19 N m2). The stiffness of the hook was determined to be approximately one quarter the rigor stiffness of a rat sperm flagellum measured under comparable conditions. Cell Motil. Cytoskeleton 49:33,40, 2001. 2001 Wiley-Liss, Inc. [source]

    Comparative Studies of Tridentate Sulfur and Nitrogen-Containing Ligands as Ionophores for Construction of Cadmium Ion-Selective Membrane Sensors

    ELECTROANALYSIS, Issue 11 2007
    Ashok, Kumar Singh
    Abstract New polymeric membrane cadmium-ion selective sensors have been prepared by incorporating nitrogen and sulfur containing tridentate ligands as the ionophores into the plasticized PVC membranes. Poly(vinyl chloride) (PVC) based membranes of potassium hydrotris[N -(2,6-xylyl)thioimdazolyl) borate] (KTt2,6-xylyl) and potassium hydrotris(3-phenyl-5-methylpyrazolyl) borate (KTpPh,Me) with sodium tetraphenyl borate (NaTPB) as an anionic excluder and dibutylphthalate (DBP), tributylphthalate (TBP), dioctylsebacate (DOS), and o -nitrophenyloctyl ether (o -NPOE) as plasticizing solvent mediators were investigated in different compositions. KTt2,6-xylyl was found to be a selective and sensitive ion carrier for Cd(II) membrane sensor. A membrane composed of KTt2,6-xylyl:NaTPB:PVC:DBP with the % mole ratio 2.3,:,1.1,:,34.8,:,61.8 (w/w) works well over a very wide concentration range (7.810,8,1.010,2,M) with a Nernstian slope of 29.40.2,mV/decades of activity between pH values of 3.5 to 9.0 with a detection limit of 4.3710,8,M. The sensor displays very good discrimination toward Cd(II) ions with regard to most common cations. The proposed sensor shows a short response time for whole concentration range (ca. 8,s). The effects of the cationic (tetrabutylammonium chloride, TBC), anionic (sodium dodecyl sulfate, SDS) and nonionic (Triton X-100) surfactants were investigated on the potentiometric properties of proposed cadmium-selective sensor. The proposed sensor based on KTt2,6-xylyl ionophore has also been used for the direct determination of cadmium ions in different water samples and human urine samples. [source]

    Barrel Plating Rhodium Electrode: Application to Flow Injection Analysis of Hydrazine

    ELECTROANALYSIS, Issue 14 2005
    Jun-Wei Sue
    Abstract We introduce here the application of barrel plating technology for mass production of disposable-type electrodes. Easy for mass production, barrel plating rhodium electrode (Rh-BPE) is for the first time demonstrated for analytical application. Hydrazine was chosen as a model analyte to elucidate the electrocatalytic and analytical ability of the Rh-BPE system in pH,7 phosphate buffer solution. Flow injection analysis (FIA) of hydrazine showed a linear calibration range of 25,1000,ppb with a slope and a regression coefficient of 5,nA/ppb and 0.9946, respectively. Twenty-two replicate injections of 25,ppb hydrazine showed a relative standard deviation of 3.17% indicating a detection limit (S/N=3) of 2.5,ppb. The system can be continuously operated for 1 day without any alteration in the FIA signals and is tolerable to the interference of oxalic acid, gelatine, Triton X-100, and albumin for even up to 100 times excess in concentration with respect to 400,ppb hydrazine. Since the fabrication cost of the electrode is cheap, it is thus disposable in nature. Furthermore, barrel plating technique can be extendable to other transition metals for application in many fields of research interest. [source]

    Delivery of bioactive, gel-isolated proteins into live cells

    ELECTROPHORESIS, Issue 9 2003
    Jennifer E. Taylor
    Abstract The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein. [source]

    Adsorption of hydrophobic organic compounds onto a hydrophobic carbonaceous geosorbent in the presence of surfactants,

    Peng Wang
    Abstract The adsorption of hydrophobic organic compounds (HOCs; atrazine and diuron) onto lampblack was studied in the presence of nonionic, cationic, and anionic surfactants (Triton X-100), benzalkonium chloride [BC], and linear alkylbenzene sulfonate [LAS]) to determine the effect of the surfactant on HOC adsorption onto a hydrophobic carbonaceous geosorbent. Linear alkylbenzene sulfonate showed an adsorption capacity higher than that of BC but similar to that of Triton X-100, implying the charge property of a surfactant is not a useful indicator for predicting the surfactant's adsorption onto a hydrophobic medium. The results also indicated that the octanol-water partition coefficient (Kow) of a surfactant is not a good predictor of that surfactant's sorption onto a hydrophobic medium. Under subsaturation adsorption conditions (i.e., before sorption saturation is reached), surfactant adsorption reduced HOC adsorption to a significant extent, with the reduction in HOC adsorption increasing monotonically with the amount of surfactant adsorbed. Among the three surfactants, Triton X-100 was the most effective in reducing HOC adsorption, whereas BC and LAS showed similar effectiveness in this regard. Under the same amount of the surfactant sorbed, the reduction in atrazine adsorption was consistently greater than that for diuron because of atrazine's lower hydrophobicity. No significant difference was observed in the amount of the HOC adsorbed under different adsorption sequences. Our results showed that the presence of surfactant can significantly decrease HOC adsorption onto hydrophobic environmental media and, thus, is important in predicting HOC fate and transport in the environment. [source]

    Modulation of P-glycoprotein-mediated multidrug resistance by acceleration of passive drug permeation across the plasma membrane

    FEBS JOURNAL, Issue 23 2007
    Ronit Regev
    The drug concentration inside multidrug-resistant cells is the outcome of competition between the active export of drugs by drug efflux pumps, such as P-glycoprotein (Pgp), and the passive permeation of drugs across the plasma membrane. Thus, reversal of multidrug resistance (MDR) can occur either by inhibition of the efflux pumps or by acceleration of the drug permeation. Among the hundreds of established modulators of Pgp-mediated MDR, there are numerous surface-active agents potentially capable of accelerating drug transbilayer movement. The aim of the present study was to determine whether these agents modulate MDR by interfering with the active efflux of drugs or by allowing for accelerated passive permeation across the plasma membrane. Whereas Pluronic P85, Tween-20, Triton X-100 and Cremophor EL modulated MDR by inhibition of Pgp-mediated efflux, with no appreciable effect on transbilayer movement of drugs, the anesthetics chloroform, benzyl alcohol, diethyl ether and propofol modulated MDR by accelerating transbilayer movement of drugs, with no concomitant inhibition of Pgp-mediated efflux. At higher concentrations than those required for modulation, the anesthetics accelerated the passive permeation to such an extent that it was not possible to estimate Pgp activity. The capacity of the surface-active agents to accelerate passive drug transbilayer movement was not correlated with their fluidizing characteristics, measured as fluorescence anisotropy of 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene. This compound is located among the headgroups of the phospholipids and does not reflect the fluidity in the lipid core of the membranes where the limiting step of drug permeation, namely drug flip-flop, occurs. [source]

    Characterization of phycoviolobilin phycoerythrocyanin-,84-cystein-lyase-(isomerizing) from Mastigocladus laminosus

    FEBS JOURNAL, Issue 18 2002
    Kai-Hong Zhao
    Cofactor requirements and enzyme kinetics have been studied of the novel, dual-action enzyme, the isomerizing phycoviolobilin phycoerythrocyanin-,84-cystein-lyase(PVB-PEC-lyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo-,-subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg2+ or Mn2+, were required, and the reaction was aided by the detergent, Triton X-100. Phosphate buffer inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (Km = 12,16 m, depending on [PecA], kcat , 1.2 10,4s,1) and the apoprotein (Km = 2.4 m at 14 m PCB, kcat = 0.8 10,4s,1). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB-PEC-lyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore. [source]

    Identification of phospholipids as new components that assist in the in vitro trimerization of a bacterial pore protein

    FEBS JOURNAL, Issue 3 2001
    Hans De Cock
    The in vitro trimerization of folded monomers of the bacterial pore protein PhoE, into its native-like, heat- and SDS-stable form requires incubations with isolated cell envelopes and Triton X-100. The possibility that membranes could be isolated that are enriched in assembly factors required for assembly of the pore protein was now investigated. Fractionation of total cell envelopes of Escherichia coli via various techniques indeed revealed the existence of membrane fractions with different capacities to support assembly in vitro. Fractions containing mainly inner membrane vesicles supported the formation of trimers that were associated with these membrane vesicles. However, only a proportion of these trimers were heat- and SDS-stable and these were formed with slow kinetics. In contrast, fractions containing mainly outer membrane vesicles supported formation of high amounts of heat-stable trimers with fast kinetics. We identified phospholipids as active assembly components in these membranes that support trimerization of folded monomers in a process with similar characteristics as observed with inner membrane vesicles. Furthermore, phospholipids strongly stimulate the kinetics of trimerization and increase the final yield of heat-stable trimers in the context of outer membranes. We propose that lipopolysaccharides stabilize the assembly competent state of folded monomers as a lipochaperone. Phospholipids are involved in converting the folded monomer into new assembly competent intermediate with a short half-life that will form heat-stable trimers most efficiently in the context of outer membrane vesicles. These results provide biochemical evidence for the involvement of different lipidic components at distinct stages of the porin assembly process. [source]

    The assembly factor P17 from bacteriophage PRD1 interacts with positively charged lipid membranes

    FEBS JOURNAL, Issue 20 2000
    Juha M. Holopainen
    The interactions of the assembly factor P17 of bacteriophage PRD1 with liposomes were investigated by static light scattering, fluorescence spectroscopy, and differential scanning calorimetry. Our data show that P17 binds to positively charged large unilamellar vesicles composed of the zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine and sphingosine, whereas only a weak interaction is evident for 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles. P17 does not bind to negatively charged membranes composed of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and 1-palmitoyl-2-oleoyl-phosphatidylcholine. Our differential scanning calorimetry results reveal that P17 slightly perturbs the phase behaviour of neutral phosphatidylcholine and negatively charged multilamellar vesicles. In contrast, the phase transition temperature of positively charged dimyristoylphosphatidylcholine/sphingosine multilamellar vesicles (molar ratio 9 : 1, respectively) is increased by approximately 2.4 C and the half width of the enthalpy peak broadened from 1.9 to 5.6 C in the presence of P17 (protein : lipid molar ratio 1 : 47). Moreover, the enthalpy peak is asymmetrical, suggesting that lipid phase separation is induced by P17. Based on the far-UV CD spectra, the ,-helicity of P17 increases upon binding to positively charged micelles composed of Triton X-100 and sphingosine. We propose that P17 can interact with positively charged lipid membranes and that this binding induces a structural change on P17 to a more tightly packed and ordered structure. [source]

    Characterization of Xenopus egg membrane microdomains containing uroplakin Ib/III complex: roles of their molecular interactions for subcellular localization and signal transduction

    GENES TO CELLS, Issue 2 2007
    A.K.M. Mahbub Hasan
    A single-transmembrane protein uroplakin III (UPIII) and its tetraspanin binding-partner uroplakin Ib (UPIb) are members of the UP proteins that were originally identified in mammalian urothelium. In Xenopus laevis eggs, these proteins: xUPIII and xUPIb, are components of the cholesterol-enriched membrane microdomains or "rafts" and involved in the sperm,egg membrane interaction and subsequent egg activation signaling via Src tyrosine kinase at fertilization. Here, we investigate whether the xUPIII-xUPIb complex is in close proximity to CD9, a tetraspanin that has been implicated in the sperm,egg fusion in the mouse and GM1, a ganglioside typically enriched in egg rafts. Preparation of the egg membrane microdomains using different non-ionic detergents (Brij 98 and Triton X-100), chemical cross-linking, co-immunoprecipitation, in vitro kinase assay and in vitro fertilization experiments demonstrated that GM1, but not CD9, is in association with the xUPIII-xUPIb complex and contributes to the sperm-dependent egg activation. Transfection experiments using HEK293 cells demonstrated that xUPIII and xUPIb localized efficiently to the cholesterol-dependent membrane microdomains when they were co-expressed, whereas co-expression of xUPIII and CD9, instead of xUPIb, did not show this effect. Furthermore, xUPIII and xUPIb were shown to suppress kinase activity of the wild type, but not a constitutively active form of, Xenopus Src protein co-expressed in HEK293 cells. These results provide novel insight into the molecular architecture of the egg membrane microdomains containing xUPIII, xUPIb and Src, which may contribute to the understanding of sperm,egg interaction and signaling during Xenopus fertilization. [source]

    Characterization of human sperm N -acetylglucosaminidase

    S. L. Perez Martinez
    Summary N -acetylglucosaminidase (NAG) is particularly active in mammalian spermatozoa and appears to be involved in fertilization. Although it is assumed that this enzyme is acrosomal, previous results from our laboratory suggest the presence of NAG at the sperm plasma membrane level. The present study attempted to analyse the subcellular distribution of this enzyme in human spermatozoa. Sperm were incubated under different conditions and NAG activity measured in the soluble extracts and cell pellets using a specific fluorometric substrate. A significant proportion of NAG activity was released when sperm were incubated in culture medium, suggesting a weak association with the plasma membrane. This location was confirmed by western blot analysis of plasma membrane fractions and immunofluorescence on non-permeabilized sperm, which showed a positive signal mainly on the acrosomal domain. The distribution of NAG activity between plasma membrane and acrosome was analysed after cell disruption by freezing and thawing. Triton X-100 stimulated sperm and epididymal NAG activity but not the enzyme obtained from other sources. In addition, biotinylated human recombinant NAG was able to bind to human sperm. Finally, after sperm incubation under capacitating conditions, NAG total activity increased and the sperm enzyme lost its ability to be stimulated by Triton X-100. The possible connection of these results with sperm maturation, capacitation and NAG participation in primary binding to the zona pellucida, was discussed. [source]

    Microinjected neutrophils retain the ability to take up bacteria

    JOURNAL OF ANATOMY, Issue 5 2002
    M. M. Bird
    It is now possible to microinject protein to probe specific biochemical pathways and/or cell functions in small cells such as human neutrophils (Bird et al. J.Anat.198, 2001). We have shown that these cells retain their ability to modify their F-actin cytoskeleton following the microinjection procedure. The principal task of neutrophils is to hunt and kill bacteria by responding to chemotactic gradients which cause them to extend actin rich pseudopodia in the direction of the highest concentration of these molecules. On reaching their target the neutrophils make tight contact with the bacteria and phagocytosis ensues. Here we address the question of whether or not the microinjected cells are still able to maintain their normal phagocytic activities. Human neutrophils maintained in culture for 20 mins were confronted with Staphylococcus aureus (1 104 cells/mL) for 5 min and then injected with rat IgG as an exogenous protein that also serves as a marker for injected cells. After 30 min the cells were fixed for fluorescence or confocal microscopy in 3.7% formaldehyde and permeabilised for 5 min (0.2% Triton X-100 in PBS). They were then incubated for 45 min in 2.5 L FITC-anti rat IgG and 1 L TRITC-phalloidin (to show the F-actin cytoskeleton), in 996.5 L of PBS, washed 6 times in PBS and mounted on slides in 5 L Mowiol containing a grain of antiquench. For TEM cells were fixed in 1.5% glutaraldehyde in cacodylate buffer for 3 min at room temperature and then washed in 0.2 m cacodylate buffer 6 times before incubation with 1 mm NiCl2 and SIGMA fast DAB peroxidase tablets for 30 min. The cells were postfixed in a 2% solution of osmium tetroxide for 30 min, dehydrated through a series of graded ethanols, and embedded and sectioned for TEM. By TEM the injected neutrophils were observed to have taken up bacteria into vacuoles of varying size. At the earliest stages of this process, prior to and immediately following the initial release of granular contents and the initiation of mechanisms to rapidly destroy bacteria, the bacteria fitted more tightly in the vacuoles than at later stages. Injected neutrophils commonly contained several bacteria; more than one bacterium was frequently located within a single vacuole of substantial size. Confocal laser microscopic observations confirmed that cells containing ingested bacteria also contained IgG. Thus injected cells not only survive the microinjection procedure but also retain their ability to take up bacteria and initiate the digestive process. [source]

    Phytase production by Sporotrichum thermophile in a cost-effective cane molasses medium in submerged fermentation and its application in bread

    B. Singh
    Abstract Aims:, Phytase production by Sporotrichum thermophile in a cost-effective cane molasses medium in submerged fermentation and its application in bread. Methods and Results:, The production of phytase by a thermophilic mould S. thermophile was investigated using free and immobilized conidiospores in cane molasses medium in shake flasks, and stirred tank and air-lift fermenters. Among surfactants tested, Tweens (Tween-20, 40 and 80) and sodium oleate increased phytase accumulation, whereas SDS and Triton X-100 inhibited the enzyme production. The mould produced phytase optimally at aw 095, and it declined sharply below this aw value. The enzyme production was comparable in air-lift and stirred tank reactors with a marked reduction in fermentation time. Among the matrices tried, Ca-alginate was the best for conidiospore immobilization, and fungus secreted sustained levels of enzyme titres over five cycles. The phytic acid in the dough was efficiently hydrolysed by the enzyme accompanied by the liberation of soluble phosphate in the bread. Conclusions:, The phytase production by S. thermophile was enhanced in the presence of Tween-80 in cane molasses medium. A peak in enzyme production was attained in 48 h in the fermenter when compared with that of 96 h in shake flasks. Ca-alginate immobilized conidiospores germinated to produce fungal growth that secreted sustained levels of phytase over five cycles. The bread made with phytase contained reduced level of phytic acid and a high-soluble phosphate. Significance and Impact of the Study:, The phytase accumulation by S. thermophile was increased by the surfactants. The sustainability of enzyme production in stirred tank and air-lift fermenters suggested the possibility for scaling up of phytase. The bread made with phytase contained low level of antinutrient, i.e. phytic acid. [source]

    Novel method for clearing red blood cell debris from BacT/ALERT blood culture medium for improved microscopic and antimycobacterial drug susceptibility test results

    Krishnamoorthy Gopinath
    Abstract Even though automation in mycobacterial culture has immensely improved the detection of organisms, identification of species and antimycobacterial susceptibility testing from blood culture bottles remain cumbersome and error-prone due to the presence of intact red blood cells (RBCs). The removal or lysis of these RBCs and excessive protein from the blood components could theoretically help improve this process. The present study reports an effective method that uses ammonium chloride (NH4Cl) and Triton X-100 to lyse the RBCs in blood culture medium. The method was optimized by preparing various concentrations of NH4Cl and Triton X-100, and incubation conditions, leading to eight protocols. The lysis protocol with a concentration of 150,mM of NH4Cl, 0.5% Triton X-100, and 1% potassium bicarbonate, pH 7.0, and incubation at 37C for 15,min was found to be optimal. This method not only made the culture medium clear, the protein concentration decreased from 753.539.4 to 53.24.2,mg/mL in the M. tuberculosis -spiked culture medium and in the blood culture medium inoculated with the blood from tuberculosis patients. The method had no adverse effect on mycobacteria, and no depletion of M. tuberculosis colony-forming units was found. The lysate could be used for antimycobacterial susceptibility testing with no difficulty in setting the mycobacterial concentration of inoculum to 0.5 McFarland standards. Furthermore, this method had the added advantage in the microscopy and molecular methods for the speciation of Mycobacterium sp. J. Clin. Lab. Anal. 21:220,226, 2007. 2007 Wiley-Liss, Inc. [source]

    Novel rapid immunochromatographic test based on an enzyme immunoassay for detecting nucleocapsid antigen in SARS-associated coronavirus

    Hiroyuki Kogaki
    Abstract A novel severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been discovered. The detection of both antigens and antibodies in SARS-CoV from human specimens with suspected SARS plays an important role in preventing infection. We developed a novel rapid immunochromatographic test (RICT) based on the sandwich format enzyme immunoassay (EIA) with an all-in-one device for detecting the native nucleocapsid antigen (N-Ag) of SARS-CoV using monoclonal antibodies (MoAbs), which we produced by immunizing recombinant N-Ag to mice. RICT is a qualitative assay for respiratory aspirates and serum specimens. With this assay, a positive result can be judged subjectively by the appearance of a blue line on the device 15 min after the sample is applied. RICT with several pairs of MoAbs showed a high sensitivity for the detection of recombinant N-Ag as well as viral N-Ag of SARS-CoV. rSN122 and rSN21-2 were the best MoAbs for immobilized antibody and enzyme labeling, respectively. With regard to analytical sensitivity, RICT detected N-Ag at 31 pg/mL for recombinant N-Ag, and at 1.99102 TCID50/mL for SARS-CoV. The specificity of RICT was 100% when 150 human sera and 50 nasopharyngeal aspirates (NSPs) were used. RICT based on an EIA using the rSN122/rSN21-2 pair is a sensitive, specific, and reliable rapid assay for detecting N-Ag in SARS-CoV treated with either heat or Triton X-100. J. Clin. Lab. Anal. 19:150,159, 2005. 2005 Wiley-Liss, Inc. [source]

    Investigation of ion-pair precipitates of selected alkoxylates and complex salts of specific metal cations by liquid secondary ion mass spectrometry


    Abstract Liquid secondary ion (LSI) mass spectra of ion-pair precipitates obtained for Triton X-100 with strontium, lead, cadmium and mercury tetraphenylborates and for selected butoxylene,ethoxylene monoalkyl ethers with barium tetraiodobismuthate(III) are discussed. On the basis of LSI mass spectra, recorded in both positive and negative modes, the formulae of the ion-pair precipitates were determined. On the basis of B/E mass spectra, the fragmentation routes of [M , H + Ba]+ ions for butoxylene,ethoxylene monoalkyl ether complexes of barium and [M , H + Cd]+ ions for the Triton X-100 complex of cadmium are proposed. Copyright 2002 John Wiley & Sons, Ltd. [source]

    Optimized virus disruption improves detection of HIV-1 p24 in particles and uncovers a p24 reactivity in patients with undetectable HIV-1 RNA under long-term HAART

    Jrg Schpbach
    Abstract HIV-1 p24 antigen (p24) measurement by signal amplification-boosted ELISA of heat-denatured plasma is being evaluated as an alternative to HIV-1 RNA quantitation in resource-poor settings. Some observations suggested that virion-associated p24 is suboptimally detected using Triton X-100-based virus dissociation buffer (kit buffer). A new reagent (SNCR buffer) containing both denaturing and non-denaturing detergents was therefore developed and evaluated. The SNCR buffer increased the measured p24 concentration about 1.5- to 3-fold in HIV-negative plasma reconstituted with purified HIV-1 particles, while not increasing the background. Among 127 samples of HIV-1-positive patients with moderate to high concentrations of HIV-1 RNA the increase was about threefold across the entire concentration range (P,<,0.0001). Specificity before neutralization among prospectively tested clinical samples ruled HIV-negative was 828 of 845 (98.0%) for the SNCR buffer and 464 of 479 (96.9%) for kit buffer. Specificity after confirmatory neutralization of reactive samples or a follow-up test was 100% with either buffer. Surprisingly, the SNCR buffer revealed a p24 reactivity in 115 of 187 samples (61.5%) from adult patients exhibiting undetectable HIV-1 RNA below 5 copies/ml for a duration of 6,30 months under HAART (3.7% with kit buffer). The rate of p24 reactivity in these patients did not decrease with duration of HAART. In conclusion, the SNCR buffer improves the detection of particle-associated HIV-1 p24, thereby increasing the measured p24 concentration in samples with medium to high HIV-1 RNA. It also uncovers the presence of a p24 reactivity, whose identity remains to be determined, in a significant fraction of samples with undetectable HIV-1 RNA under long-term HAART. J. Med. Virol. 78:1003,1010, 2006. 2006 Wiley-Liss, Inc. [source]

    Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain

    Shintaro Besshoh
    Abstract Ischemia results in increased phosphorylation of NMDA receptors. To investigate the possible role of lipid rafts in this increase, lipid rafts and post-synaptic densities (PSDs) were isolated by the extraction of rat brain synaptosomes with Triton X-100 followed by sucrose density gradient centrifugation. Lipid rafts accounted for the majority of PSD-95, whereas SAP102 was predominantly located in PSDs. Between 50 and 60% of NMDA receptors were associated with lipid rafts. Greater than 85,90% of Src and Fyn were present in lipid rafts, whereas Pyk2 was mainly associated with PSDs. Lipid rafts and PSDs were isolated from animals subjected to 15 min of global ischemia followed by 6 h of recovery. Ischemia did not affect the yield, density, flotillin-1 or cholesterol content of lipid rafts. Following ischemia, the phosphorylation of NR1 by protein kinase C and tyrosine phosphorylation of NR2A and NR2B was increased in both lipid rafts and PSDs, with a greater increase in tyrosine phosphorylation occurring in the raft fraction. Following ischemia, NR1, NR2A and NR2B levels were elevated in PSDs and reduced in lipid rafts. The findings are consistent with a model involving close interaction between lipid rafts and PSDs and a role for lipid rafts in ischemia-induced signaling pathways. [source]

    Abundant Tissue Butyrylcholinesterase and Its Possible Function in the Acetylcholinesterase Knockout Mouse

    Bin Li
    Abstract: We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well. [source]

    Mutual effects of caveolin and nerve growth factor signaling in pig oligodendrocytes

    Matthias Schmitz
    Abstract Signaling of growth factors may depend on the recruitment of their receptors to specialized microdomains. Previous reports on PC12 cells indicated an interaction of raft-organized caveolin and TrkA signaling. Because porcine oligodendrocytes (OLs) respond to nerve growth factor (NGF), we were interested to know whether caveolin also plays a role in oligodendroglial NGF/TrkA signaling. OLs expressed caveolin at the plasma membrane but also intracellularly. This was partially organized in the classically ,-shaped invaginations, which may represent caveolae. We could show that caveolin and TrkA colocalize by using a discontinuous sucrose gradient (Song et al. [1996] J. Biol. Chem. 271:9690,9697), MACS technology, and immunoprecipitation. However, differential extraction of caveolin and TrkA with Triton X-100 at 4C indicated that caveolin and TrkA are probably not exclusively present in detergent-resistant, caveolin-containing rafts (CCRs). NGF treatment of OLs up-regulated the expression of caveolin-1 (cav-1) and stimulated tyrosine-14 phosphorylation of cav-1. Furthermore, OLs were transfected with cav-1-specific small interfering RNA (siRNA). A knockdown of cav-1 resulted in a reduced activation of downstream components of the NGF signaling cascade, such as p21Ras and mitogen-activated protein kinase (MAPK) after NGF exposure of OLs. Subsequently, increased oligodendroglial process formation via NGF was impaired. The present study indicates that CCRs/caveolin could play a modulating role during oligodendroglial differentiation and regeneration. 2009 Wiley-Liss, Inc. [source]

    Proteomic comparison of two fractions derived from the transsynaptic scaffold

    Greg R. Phillips
    Abstract A fraction derived from presynaptic specializations (presynaptic particle fraction; PPF) can be separated from postsynaptic densities (PSD) by adjusting the pH of Triton X-100 (TX-100) extraction of isolated transsynaptic scaffolds. Solubilization of the PPF corresponds to disruption of the presynaptic specialization. We show that the PPF is insoluble to repeated TX-100 extraction at pH 6.0 but becomes soluble in detergent at pH 8.0. By immunolocalization, we find that the major proteins of the PPF, clathrin and dynamin, are concentrated in the presynaptic compartment. By using multidimensional protein identification technology, we compared the protein compositions of the PPF and the PSD fraction. We identified a total of 341 proteins, 50 of which were uniquely found in the PPF, 231 in the PSD fraction, and 60 in both fractions. Comparison of the two fractions revealed a relatively low proportion of actin and associated proteins and a high proportion of vesicle or intracellular compartment proteins in the PPF. We conclude that the PPF consists of presynaptic proteins not connected to the actin-based synaptic framework; its insolubility in pH 6 and solubility in pH 8 buffered detergent suggests that clathrin might be an anchorage scaffold for many proteins in the PPF. 2005 Wiley-Liss, Inc. [source]

    Myelin proteolipid protein, basic protein, the small isoform of myelin-associated glycoprotein, and p42MAPK are associated in the Triton X-100 extract of central nervous system myelin

    Dina N. Arvanitis
    Abstract To further our understanding of the functions of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), and other myelin proteins, such as 2,3,-cyclic nucleotide 3,-phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG), bovine brain myelin was extracted with Triton X-100, and protein complexes in the detergent-soluble fraction were isolated by coimmunoprecipitation and sucrose density gradient sedimentation. MBP, PLP, and the small isoform of MAG (S-MAG) were coimmunoprecipitated from the detergent-soluble fraction by anti-PLP, anti-MBP or anti-MAG monoclonal antibodies. Additionally, a 30 kDa phosphoserine-containing protein and two phosphotyrosine-containing proteins (Mr 30 and 42 kDa) were found in the coimmunoprecipitates. The 42 kDa protein is probably p42MAPK, in that MAPK was shown also to be present in the immunoprecipitated complex. CNP, the small PLP isoform DM20, the large MAG isoform L-MAG, MOG, CD44, MEK, p44MAPK, and actin were not present in the immunoprecipitates, although they were present in the detergent-soluble fraction. Lipid analysis revealed that the PLP,MBP,S-MAG coimmunoprecipitated with some phospholipids and sulfatide but not cholesterol or galactosylceramide. However, the complex had a high density, indicating that the lipid/protein ratio is low, and it was retained on a Sepharose CL6B column, indicating that it is not a large membrane fragment. Given that MAG is localized mainly in the periaxonal region of myelin, where it interacts with axonal ligands, the PLP,MBP,S-MAG complex may come from these regions, where it could participate in dynamic functions in the myelin sheath and myelin,axonal interactions. 2002 Wiley-Liss, Inc. [source]

    Triton-X-100-modified polymer and microspheres for reversal of multidrug resistance

    Zhi Liu
    Triton X-100 is a non-ionic detergent capable of reversing multidrug resistance (MDR) due to its interaction with cell membranes. However, it interacts with cells in a non-specific way, causing cytotoxicity. This work aimed to develop polymeric chemosensitizers that possess the ability to reverse MDR and lower toxic side effects. When being delivered to tumours, the polymeric chemosensitizers may also have longer retention times in tumours than the free detergent. Triton-X-100-immobilized dextran microspheres (T-MS) and inulin (T-IN) were prepared and characterized. Their cytotoxicity against multidrug-resistant Chinese hamster ovary cells (CHRC5) was compared with that of free Triton X-100 solutions. The in-vitro effect of the products on 3H-vinblastine accumulation by CHRC5 cells was determined. Both T-MS and T-IN showed a marked decrease in the cytotoxicity, as compared with free Triton solutions at equivalent concentrations. Drug accumulation by CHRC5 cells was increased over two fold in the presence of T-MS or T-IN. These results suggest that polymeric drug carriers with MDR-reversing capability and lower cytotoxicity may be prepared by immobilization of chemosensitizers. [source]

    Kinetic study on the prooxidative effect of vitamin C on the autoxidation of glycerol trioleate in micelles

    Zai-Qun Liu
    Abstract Vitamin C (L -ascorbic acid) protects human health by scavenging toxic free radicals and other reactive oxygen species formed in cell metabolism. The surplus supplementation of vitamin C, however, may be harmful to health because the level of 8-oxoguanine and 8-oxoadenine in lymphocyte DNA varies remarkably. In the process of the kinetic investigation on the 2,2,-azobis(2-amidinopropane dihydrochloride) (AAPH)-induced autoxidation of glycerol trioleate (GtH) in the micelles of cetyl trimethyl ammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Triton X-100, the addition of vitamin C accelerates the autoxidation of GtH even in the absence of the free radical initiator, AAPH. The initiating rate, Ri, of vitamin C (VC)-induced autoxidation of GtH is related to the micelle charge, i.e. Ri,=,14.4,,10,6 [VC] s,1 in SDS (anionic micelle), Ri,=,1961,,10,6 [VC] s,1 in Triton X-100 (neutral micelle) and Ri is a maximum in CTAB (cationic micelle) when the vitamin C concentration is ,300,M. Thus, vitamin C can initiate autoxidation of GtH in micelles, especially in the neutral one. Moreover, the attempt to explore whether ,-tocopherol (TocH) could rectify vitamin C-induced autoxidation of GtH leads us to find that the rate constant of TocH reacting with the anionic radical of vitamin C (VC.,), k,inh, is ,103M,1,s,1, which is less than that of the ,-tocopherol radical (Toc.) with vitamin C (kinh,=,,105,M,1,s,1). Thus, the equilibrium constant of the reaction Toc.+VC,,TocH+VC., is prone strongly to the regeneration of Toc. by vitamin C rather than the reverse reaction. Copyright 2006 John Wiley & Sons, Ltd. [source]

    Fabrication of poly(aniline- co -pyrrole) hollow nanospheres with Triton X-100 micelles as templates

    Chuanqiang Zhou
    Abstract A one-step route has been reported for the fabrication of poly(aniline- co -pyrrole) (PACP) copolymer hollow nanospheres via the oxidation polymerization of a mixture of aniline and pyrrole in the presence of Triton X-100. It was found that the variations in polymerization conditions, such as the concentrations of Triton X-100 and comonomers, and [pyrrole]/[aniline] molar ratios, could change the size and uniformity of copolymer hollow nanospheres. The result of DLS has attested the presence of the spherical Triton X-100 micelles swelled by the comonomers in reaction system, and such micelles might play template for the formation of hollow nanospheres, followed by developing a possible formation mechanism. The chemical structures and crystallinity of products were characterized by FTIR, UV,visible, 1H NMR spectra, and XRD patterns, respectively, to prove the copolymer chemical structures of hollow nanospheres. The thermal-stability and solubility of PACP were improved compared with homopolymers (polyaniline and pyrrole). 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 3563,3572, 2008 [source]

    Breakdown kinetics of aggregates from poly(ethylene glycol- bl -propylene sulfide) di- and triblock copolymers induced by a non-ionic surfactant

    Simona Cerritelli
    Abstract We explored the effects of addition of the nonionic surfactant Triton X-100 on the stability of aggregates of poly(ethylene glycol- bl -propylene sulfide) di- and triblock copolymers. Fluorescence spectra of pyrene, used as a probe molecule, elucidated the various stages of transformation from pure copolymeric micelles to surfactant-rich micelles. Turbidity measurements yielded insight into the mechanism of the interaction, the hydrophobicity of the copolymer driving the process. Triton X-100 tends to strongly interact with highly hydrophobic copolymers by inserting into the core of the micellar aggregates. On the other hand, Triton X-100 tends to interact with the corona of micelles formed by less hydrophobic copolymers which, for this reason, are more stable upon addition of this destabilizing agent. Kinetic data give evidence that only monomers, not micelles of surfactant, interact with the copolymer micelles. 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2477,2487, 2008 [source]

    Surfactant-Induced Amorphous Aggregation of Tobacco Mosaic Virus Coat Protein: A Physical Methods Approach

    Yuliy V. Panyukov
    Abstract The interactions of non-ionic surfactant Triton X-100 and the coat protein of tobacco mosaic virus, which is an established model for both ordered and non-ordered protein aggregation, were studied using turbidimetry, differential scanning calorimetry, isothermal titration calorimetry, and dynamic light scattering. It was found that at the critical aggregation concentration (equal to critical micelle concentration) of 138,,10,6M, Triton X-100 induces partial denaturation of tobacco mosaic virus coat protein molecules followed by protein amorphous aggregation. Protein aggregation has profound ionic strength dependence and proceeds due to hydrophobic sticking of surfactant-protein complexes (start aggregates) with initial radii of 46 nm. It has been suggested that the anionic surfactant sodium dodecyl sulfate forms mixed micelles with Triton X-100 and therefore reverses protein amorphous aggregation with release of protein molecules from the amorphous aggregates. A stoichiometric ratio of 5 was found for Triton X-100-sodium dodecyl sulfate interactions. [source]

    Characterization of RAT, an autolysis regulator in Staphylococcus aureus

    S. S. Ingavale
    Summary In trying to identify genetic loci involved in the regulation of cap5 genes in Staphylococcus aureus, we isolated a transposon mutant that exhibited a growth defect, enhanced autolysis and increased sensitivity to Triton X-100 and penicillin, attributable in part to increased murein hydrolase activity. Analysis of the chromosomal sequence flanking the transposon insertion site revealed that the gene disrupted in the mutant encodes an open reading frame of 147 amino acids. We named this gene rat, which stands for regulator of autolytic activity. Sequence analysis indicated that Rat is homologous to the MarR and, to a lesser extent, the SarA protein families. Mutations in rat resulted in decreased expression of known autolytic regulators lytSR, lrgAB and arlRS. Gel shift studies indicated that Rat binds to the lytRS and arlRS promoters, thus confirming Rat as a DNA-binding protein to these known repressors of autolytic activity. As anticipated, rat appears to be a negative regulator of autolysin genes including lytM and lytN. These data suggest that the rat gene product is an important regulator of autolytic activity in S. aureus. [source]

    Automutanolysin disrupts clinical isolates of cariogenic streptococci in biofilms and planktonic cells

    P. Thanyasrisung
    Introduction:, Dental caries remains one of the most common chronic infectious diseases throughout the world. The formation of dental plaque is one of the caries risk factors. As a consequence, the removal of plaque may reduce the incidence of caries development. We identified an autolysin produced by Streptococcus mutans named auto-mutanolysin (Aml). Aml selectively lyses S. mutans and Streptococcus sobrinus. The specificity towards these cariogenic bacteria suggests that Aml may be used to prevent dental caries. Here, with the aim towards therapeutic application, we investigated the lytic activity of Aml against clinical isolates of S. mutans and S. sobrinus using planktonic cells and biofilms. Methods:, Planktonic cell suspensions and biofilms of clinically isolated streptococci were treated with Aml in the absence or the presence of Triton X-100. The lytic activity of Aml was monitored as the change in turbidity. The disruption of biofilms was evaluated by detecting the released DNA by polymerase chain reaction and observing the alteration of optical density of treated biofilms. Results:, Triton X-100 enhances the lytic ability of Aml. Using planktonic cells, Aml had various lysis levels against clinical strains. Repeated Aml treatment showed disruption of the biofilm using the representative clinical strains. Conclusion:, Our study demonstrates that Aml has an ability to lyse planktonic and biofilm cells of clinically isolated mutans streptococci in the presence of Triton X-100. These results suggest the possibility of using Aml as an alternative or additional approach for caries prevention. [source]