共查询到20条相似文献,搜索用时 31 毫秒
1.
E.A. Dukhanina T.I. Lukyanova A.S. Dukhanin S.G. Georgieva 《Cell cycle (Georgetown, Tex.)》2018,17(4):479-485
S100A4 is a Ca2+-binding protein that performs an important role in metastasis. It is also known for its antitumor functions. S100A4 is expressed by a specialized subset of CD4+CD25+ lymphocytes and is present on those cell's membranes along with peptidoglycan recognition proteins (PGRPs). There, by interacting with major heat shock protein Hsp70, S100A4 plays an important cytotoxic role. The resulting stably formed complex of PGRPs, S100A4 and Hsp70 is required for the identification and binding between a lymphocyte and a target cell. Here, we investigated the S100A4 functions in CD4+CD25+PGRPs+S100A4+ lymphocyte cytotoxicity against target cells. We demonstrated that those lymphocytes do not form a stable complex with the tumor target cells that themselves have S1004A on their surface. That observation can be explained by our finding that S100A4 precludes the formation of a stable complex between PGRPs, S100A4 (on the lymphocytes’ surface), and Hsp70 (on the target cells’ surface). The decrease in S100A4 level in CD4+CD25+PGRPs+S100A4+ lymphocytes inhibits their cytotoxic activity, while the addition of S100A4 in the medium restores it. Thus, the resistance of target cells to CD4+CD25+PGRPs+ S100A4+ lymphocyte cytotoxicity depends on their S100A4 expression level and can be countered by S100A4 antibodies. 相似文献
2.
Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis. 相似文献
3.
An alternative culture system has been developed based on a conventional tissue culture plate (3.5 cm diameter) which is changed into a closed perfusion chamber. The system can easily be scaled up from one to several chambers. The shape and the size of the area of cell growth may be designed to individual experimental demands. The whole culture chamber is optically accessible, so cell growth and morphology can be evaluated by light microscopy. Furthermore the cellular physiology can be characterised by any fluorimetric assay using a bottom type fluorescence reader. A peristaltic pump sustains a constant medium flow through the chamber thus creating true homeostasis. The use of HPLC-valves and connectors allows the switching between different media or assay solutions. Thus it is possible to perform in situ assays also measuring transient effects. A protocol for vitality tests using calcein-AM is worked out for an adherent cell line and for a suspension cell line. The lower detection limits are 7 × 102 cells cm-2 for the adherent cells and 5 × 104 cells mL-1 for the suspension cells. The upper limits are 1–2 × 105 cells cm-2 respectively 8 × 106 cells mL-1. 相似文献
4.
Hyun Jin Kim Soichiro Yamaguchi Qin Li Insuk So Shmuel Muallem 《The Journal of biological chemistry》2010,285(22):16513-16520
TRPML3 is a H+-regulated Ca2+ channel that shuttles between intracellular compartments and the plasma membrane. The A419P mutation causes the varitint-waddler phenotype as a result of gain-of-function (GOF). The mechanism by which A419P leads to GOF is not known. Here, we show that the TRPML3 pore is dynamic when conducting Ca2+ to change its conductance and permeability, which appears to be mediated by trapping Ca2+ within the pore. The pore properties can be restored by strong depolarization or by conducting Na+ through the pore. The A419P mutation results in expanded channel pore with altered permeability that limits modulation of the pore by Ca2+. This effect is specific for the A419P mutation and is not reproduced by other GOF mutations, including A419G, H283A, and proline mutations in the fifth transmembrane domain. These findings describe a novel mode of a transient receptor potential channel behavior and suggest that pore expansion by the A419P mutation may contribute to the varitint-waddler phenotype. 相似文献
5.
A versatile method for the determination of the ionization of guanosine is described. Suitably derivatized alkyl-guanosines are partitioned between organic solvents and aqueous buffer solutions at various pH values. Ionization is revealed by a change in partition coefficient with pH. The method is ideally suited for application to micro samples since any quantitative method can be used to determine the partition coefficient. A procedure for distinguishing N1 or O6, N2, and C8 alkylation of guanosine is described. 相似文献
6.
V. A. Kvitsinskii E. V. Mudretskaya S. N. Pavlov S. A. Perevoznikov 《Plasma Physics Reports》2002,28(1):78-81
A series of experiments with a fully ionized turbulent lithium plasma is described. Discharges with a heat flux density onto the wall of 1–3 kW/cm2 and an electron density of ~1015 cm?3 are obtained. The energy can be transferred to the wall by both Li+ and Li++ ions. The measurements show that the photon flux corresponding to the main resonant transition of lithium atoms is a factor of 104–105 less than it could be if all the ions arriving at the wall recombined there. A mechanism is proposed for energy transfer onto the wall via the recombination of Li++ ions to Li+ ions in the cold wall region of the discharge and the subsequent energy emission by Li+ ions. 相似文献
7.
D B Kohn M J Weber P L Carl J A Katzenellenbogen P K Chakravarty 《Analytical biochemistry》1979,97(2):269-276
A peptidyl derivative of [3H]aniline, Gly-Gly-Arg-[3H]anilide, can be used as a substrate in a convenient and sensitive assay procedure for trypsin, urokinase, and plasminogen activator from transformed cells. The extent of hydrolysis can be determined simply by selective extraction of the product [3H]aniline into an organic phase containing a scintillant. (The uncleaved peptide is not appreciably soluble in this phase and is not counted.) The reaction is of comparable sensitivity to fluorimetric assays, but has the advantage that no cleanup of the biological sample is required, since it is far less subject to interference from fluorescence quenching. Other peptidyl anilides should be useful for assaying proteolytic enzymes with widely varying specificities. 相似文献
8.
A Single-File Model for Potassium Transport in Squid Giant Axon: Simulation of Potassium Currents at Normal Ionic Concentrations
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H. -H. Kohler 《Biophysical journal》1977,19(2):125-140
A physical model for potassium transport in squid giant axon is proposed. The model is designed to explain the empirical data given by the Hodgkin-Huxley model and related experiments. It is assumed that K+ moves across the axon membrane by single-file diffusion through narrow pores. In the model a pore has three negatively charged sites that can be occupied alternatively by K+ or by a gating particle, GP++, coming from the external surface. GP++ is considered to be part of the membrane rather than a diffusible component of the surrounding solutions. A high activation barrier for GP++ is supposed at the inner membrane border so that it cannot change over to the internal surface. Therefore potassium diffusion can be blocked by GP++ penetrating into the pores. This mechanism controls the dynamic behaviour of the model. The time-dependent probabilities of the pore states are described by a system of differential equations. The rate constants in these equations depend on the ionic concentrations, the membrane voltage, and the electrostatic interaction between ions in a single pore. Detailed computational tests for normal composition of external and internal solutions show that the model agrees remarkably well with the stationary and dynamic behaviour of the Hodgkin-Huxley model. However, the hyperpolarization delay is not reproduced. A structural modification, concerning this delay and the way in which GP++ is attached to the membrane, is proposed, and the qualitative behavior of the model at varied external and internal concentrations is discussed. 相似文献
9.
Doris Taylor Richard Baker Paul Hochstein 《Biochemical and biophysical research communications》1977,76(2):205-211
Incubation of red cells at 37° with the ionophore A23187 results in a loss of ATP that is dependent on the concentrations of A23187 and Ca2+ in the medium. ATP hydrolysis is greatest at micromolar concentrations of Ca2+ and decreases as Ca2+ in the medium is raised to millimolar levels. The ATP depletion is due to stimulation of calcium ATPase by A23187-mediated Ca2+ influx into the cell. The biphasic nature of Ca2+-stimulated ATP depletion in whole cells reflects the activity of Ca2+-ATPase in membrane preparations at varying Ca2+ concentrations. The ionophore can be removed by washing the cells with plasma or bovine serum albumin-containing medium and the ATP levels restored to normal by reincubating with 5 mM adenosine for 1 hr. 相似文献
10.
The A-repeat region of the lncRNA Xist is critical for X inactivation and harbors several N6-methyladenosine (m6A) modifications. How the m6A modification affects the conformation of the conserved AUCG tetraloop hairpin of the A-repeats and how it can be recognized by the YTHDC1 reader protein is unknown. Here, we report the NMR solution structure of the (m6A)UCG hairpin, which reveals that the m6A base extends 5′ stacking of the A-form helical stem, resembling the unmethylated AUCG tetraloop. A crystal structure of YTHDC1 bound to the (m6A)UCG tetraloop shows that the (m6A)UC nucleotides are recognized by the YTH domain of YTHDC1 in a single-stranded conformation. The m6A base inserts into the aromatic cage and the U and C bases interact with a flanking charged surface region, resembling the recognition of single-stranded m6A RNA ligands. Notably, NMR and fluorescence quenching experiments show that the binding requires local unfolding of the upper stem region of the (m6A)UCG hairpin. Our data show that m6A can be readily accommodated in hairpin loop regions, but recognition by YTH readers requires local unfolding of flanking stem regions. This suggests how m6A modifications may regulate lncRNA function by modulating RNA structure. 相似文献
11.
Rapid indexing of the sunblotch disease of avocados using a complementary DNA probe to avocado sunblotch viroid 总被引:1,自引:0,他引:1
PETER PALUKAITIS ‡ REW G. RAKOWSKI DONALD McE. ALEXANDER† ROBERT H. SYMONS 《The Annals of applied biology》1981,98(3):439-449
A routine procedure has been established for the sensitive and specific detection of avocado sunblotch viroid in partially purified nucleic acid extracts of avocado leaves by hybridisation analysis with 32P-complementary DNA prepared against the purified viroid. Avocado sunblotch viroid was shown to be present in 12 avocado trees that had indexed positive in a biological test for sunblotch disease but was absent from 10 trees that indexed negative. The complete correlation between sunblotch disease and the presence of viroid indicates that the complementary DNA hybridisation assay procedure can be used for the indexing of sunblotch disease. The overall procedure of leaf extraction and hybridisation analysis can be completed in 5 days and is to be compared with up to 2 yr required for indexing by biological methods. The level of avocado sunblotch viroid in partially purified nucleic acid extracts of a number of different sources of sunblotch infected avocado leaves was found to vary 10 000-fold from 0.2% to 2 × 10-5% by weight. The lower limit of detectability of the viroid by the hybridisation assay is considered to be about 10-5% by weight; this is at least 103 times more sensitive than the detection of the viroid by polyacrylamide gel electrophoresis of the leaf nucleic acid extracts followed by staining. 相似文献
12.
There are three types of myeloid leukemic cells. One type (Fc+C3+D+) can be induced by a protein in serum from mice injected with bacterial endotoxin to form rosettes for Fc and C3 receptors, migrate in agar, attach to the surface of a Petri dish and differentiate to mature macrophages and granulocytes. A second type (Fc+C3+D−) can be induced by this protein to form Fc and C3 rosettes, but not to migrate, attach or form mature cells and the third type of cell (Fc−C3−D−), could not even be induced to form rosettes. Fc+C3+D+, Fc+C3+D− and Fc−C3−D− cells before induction, showed 50%, 5% and 0% cells with a concanavalin A (ConA)-induced cap, respectively. Treatment with vinblastine or colchicine, but not with lumicolchicine, increased the frequency of cap formation to 100% in Fc+C3+D+, 95% in Fc+C3+D− but only to 50% in Fc−C3−D− cells. Of the properties that can be induced, the induction of C3 rosettes, cell migration and cell attachment can be determined 24 h after induction. The increased ability to form a cap produced by vinblastine, did not change the inducibility of cells for these properties. The results indicate that although free surface receptors for ConA and receptors anchored to tubulin can form a cap on myeloid leukemic cells, there are also receptors that may be anchored to structures other than tubulin, that did not form a cap. It is suggested that the ability of myeloid leukemic cells to differentiate is associated with the frequency of ConA surface receptors that are free or have specific types of anchorage. 相似文献
13.
A low-affinity Ca2+-ATPase from erythrocyte membranes has been purified by agarose suspension electrophoresis and polyacrylamide gel electrophoresis in the absence of detergents. For maximal activity a calcium concentration above 10 mM is required. The activity is independent of magnesium. The value for ATP is about 60 μM. The enzyme appears in two forms (A and B) with similar amino acid composition. The specific activity of A is higher than that of B. Gel electrophoresis in SDS of A gives a pattern consisting of two bands. B gives the same pattern; the only difference between the patterns is the ratio of the amounts of protein in the bands. The apparent molecular weight of the proteins in the two SDS bands has been estimated at 23 000 and 21 000, respectively. The results obtained can be explained by assuming that the two proteins corresponding to the two bands obtained in SDS electrophoresis have a similar structure and can associate to complexes A and B. We have also shown that electrophoretic and chromatographic supporting media can induce aggregation of (membrane) proteins. Artificial complexes can thus be formed and cause misinterpretation of the data obtained. This may be the reason why some authors have speculated that Ca2+-ATPase is active only in complex with other proteins such as spectrin and actin. 相似文献
14.
Susan Rittenhouse-Simmons Daniel Deykin 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,543(4):409-422
Thrombin-induced release of arachidonic acid from human platelet phosphatidylcholine is found to be more than 90% impaired by incubation of platelets with 1 mM dibutyryl cyclic adenosine monophosphate (Bt2 cyclic AMP) or with 0.6 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist. Incorporation of arachidonic acid into platelet phospholipids is not enhanced by Bt2 cyclic AMP. The addition of external Ca2+ to thrombin-treated platelets incubated with Bt2 cyclic AMP or TMB-8 does not counteract the observed inhibition. However, when divalent cation ionophore A23187 is employed as an activating agent, much less inhibition is produced by Bt2 cyclic AMP or TMB-8. The inhibition which does result can be overcome by added Ca2+. Inhibition of arachidonic acid liberation by Bt2 cyclic AMP, but not by TMB-8, can be overcome by high concentrations of A23187. When Mg2+ is substituted for Ca2+, ionophore-induced release of arachidonic acid from phosphatidylcholine of inhibitor-free controls is depressed and inhibition by Bt2 cyclic AMP is slightly enhanced. The phospholipase A2 activity of platelet lysates is increased by the presence of added Ca2+, however, the addition of either A23187 or Bt2 cyclic AMP is without effect on this activity. We suggest that Bt2 cyclic AMP may promote a compartmentalization of Ca2+, thereby inhibiting phospholipase A activity. The compartmentalization may be overcome by ionophore. By contrast, TMB-8 may immobilize platelet Ca2+ stores in situ or restrict access of Ca2+ to phospholipase A in a manner not susceptible to reversal by high concentrations of ionophore. 相似文献
15.
We report the results of thermodynamic and kinetic studies on the gelation of mixtures of sickle cell (S) deoxyhemoglobin with normal human adult (A) and fetal (F) deoxyhemoglobins. The delay time of thermally induced gelation was monitored by the increase in turbidity. At the completion of gelation the solubility was determined by sedimenting the polymers and measuring the supernatant concentration spectrophotometrically. Addition of hemoglobins A or F, at mole fractions from 0 to 0.6, resulted in large increases in both the solubility and the delay time. For a 50:50 mixture of deoxyhemoglobin F with deoxyhemoglobin S, the solubility increased by a factor of 1.8 and the delay time by a factor of 107 relative to pure deoxyhemoglobin S at the same total concentration, while for a 50:50 mixture of deoxyhemoglobins A and S the solubility increased by a factor of 1.4 and the delay time by a factor of 104. The relative delay times were independent of both temperature and total hemoglobin concentration. The data have been analyzed according to theoretical models which treat the effects of temperature, concentration, non-ideality and solution composition on the thermodynamics and kinetics of gelation. The increased solubility in mixtures with deoxyhemoglobin F is fully explained by a model in which only deoxyhemoglobin S molecules polymerize. The effect of fetal hemoglobin (α2γ2) and hybrid α2γβS molecules is to increase the solution non-ideality through the contribution of their excluded volume. The smaller increase in the solubility observed in comparable mixtures with deoxyhemoglobin A requires that the hybrid α2βAβS molecules copolymerize with the deoxyhemoglobin S. The kinetic results for the mixtures can be quantitatively accounted for using a nucleation model in which the equilibrium properties of the polymer are used to describe the critical nucleus. The very large increases in delay time observed for the mixtures can be explained by assuming that only α2β2S molecules participate in the formation of a nucleus containing about 25 monomers. As in the thermodynamic analysis, the smaller effect of adding deoxyhemoglobin A can be attributed to the contribution of the hybrid molecules in forming the critical nucleus. Thus the difference between the polymerization properties of mixtures of deoxyhemoglobin S with deoxyhemoglobins A and F can be attributed solely to the copolymerization of the α2βAβS hybrid molecule and the absence of any significant copolymerization of the α2γβS hybrid. 相似文献
16.
AimThe feasibility of using 230 MeV proton cyclotrons in proton therapy centers as a spallation neutron source for Boron Neutron Capture Therapy (BNCT) was investigated.BackgroundBNCT is based on the neutron irradiation of a 10B-containing compound located selectively in tumor cells. Among various types of neutron generators, the spallation neutron source is a unique way to generate high-energy and high-flux neutrons.Materials and MethodsNeutron beam was generated by a proton accelerator via spallation reactions and then the produced neutron beam was shaped to be appropriate for BNCT. The proposed Beam Shaping Assembly (BSA) consists of different moderators, a reflector, a collimator, as well as thermal and gamma filters. In addition, the simulated Snyder head phantom was utilized to evaluate the dose distribution in tumor and normal tissue due to the irradiation by the designed beam. MCNPX2.6 Monte Carlo code was used to optimize BSA as well as evaluate dose evaluation.ResultsA BSA was designed. With the BSA configuration and a beam current of 104 nA, epithermal neutron flux of 3.94 × 106 [n/cm2] can be achieved, which is very low. Provided that we use the beam current of 5.75 μA, epithermal neutron flux of 2.18 × 108 [n/cm2] can be obtained and the maximum dose of 38.2 Gy-eq can be delivered to tumor tissue at 1.4 cm from the phantom surface.ConclusionsResults for 230 MeV protons show that with proposed BSA, proton beam current about 5.75 μA is required for this purpose. 相似文献
17.
This report describes several modifications of the original radioenzymatic assay for serotonin (4) which increase the sensitivity of the assay 10-fold as well as enhance its reliability. Serotonin is converted to [3H]melatonin, in two steps. First, serotonin is acetylated to N-acetylserotonin by acetic anhydride. The N-acetylserotonin is then incubated with hydroxyindole-O-methyltransferase and S-[methyl-3H]adenosyl methionine and is converted to [3H]melatonin. The radioactive melatonin is extracted with toluene-isoamyl alcohol (7:3), dried, reconstituted, isolated by one-dimensional, silica gel, thin-layer chromatography, and counted in a liquid scintillation counter. The assay is specific and sensitive to approximately 5 pg of serotonin and thus can be used to measure serotonin levels in single brain nuclei or microliter quantities of biological fluids. The assay can be easily adapted for the direct measurement of N-acetylserotonin. A large number of samples can be assayed in a single working day. 相似文献
18.
James S. McKenzie Adrian J. Charlton James A. Donarski Alan D. MacNicoll Julie C. Wilson 《Metabolomics : Official journal of the Metabolomic Society》2010,6(4):574-582
A modified Lorentzian distribution function is used to model peaks in two-dimensional (2D) 1H–13C heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra. The model fit is used to determine
accurate chemical shifts from genuine signals in complex metabolite mixtures such as blood. The algorithm can be used to extract
features from a set of spectra from different samples for exploratory metabolomics. First a reference spectrum is created
in which the peak intensities are given by the median value over all samples at each point in the 2D spectra so that 1H–13C correlations in any spectra are accounted for. The mathematical model provides a footprint for each peak in the reference
spectrum, which can be used to bin the 1H–13C correlations in each HSQC spectrum. The binned intensities are then used as variables in multivariate analyses and those
found to be discriminatory are rapidly identified by cross referencing the chemical shifts of the bins with a database of
13C and 1H chemical shift correlations from known metabolites. 相似文献
19.
Z. He N. Petesch K.-P. Voges W. Röben K.D. Philipson 《The Journal of membrane biology》1997,156(2):149-156
The Na+-Ca2+ exchanger plays an important role in cardiac contractility by moving Ca2+ across the plasma membrane during excitation-contraction coupling. A 20 amino acid peptide, XIP, synthesized to mimic a region
of the exchanger, inhibits exchange activity. We identify here amino acid residues important for inhibitory function. Effects
of modified peptides on Na+-Ca2+ exchange activity were determined. Exchange activity was assessed as 45Ca2+ uptake into Na+-loaded cardiac sarcolemmal vesicles. We find that the entire length of XIP is important for maximal potency, though the major
inhibitory components are between residues 5 and 16. Basic and aromatic residues are most important for the inhibitory function
of XIP. Substitutions of arginine 12 and arginine 14 with alanine or glutamine dramatically decrease the potency of XIP, suggesting
that these residues play a key role in possible charge-charge interactions. Substitutions of other basic residues with alanines
or glutamines had less effect on the potency of XIP. All aromatic residues participate in binding with the exchanger, probably
via hydrophobic interactions as indicated by tryptophan fluorescence. A tyrosine is required at position 6 for maximal inhibition
and phenylalanine 5 and tyrosine 8 can only be replaced by other aromatic residues. Tyrosine 10 and tyrosine 13 can be replaced
with other bulky residues. A specific conformation of XIP, with structural constrains provided by all parts of the molecule,
is required for optimal inhibitory function.
Received: 19 September 1996/Revised: 20 November 1996 相似文献
20.
A method is described by which nonionic detergents of the alkyl-phenol class can be labeled with 125I. The detergent is labeled to a high specific activity, which provides a sensitive method for the detection of detergent molecules bound to proteins, and, in addition, may provide a method by which proteins may be labeled indirectly. 相似文献