共查询到20条相似文献,搜索用时 15 毫秒
1.
In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment "ball-on-stick" model with a "ball" 1.66 microm in diameter and a "stick" 41 microm in length and 0.14 microm wide. Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 microm in diameter and a "stick" 53 microm in length and 0.17 microm wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 Vo, where Vo is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: Lpg = 0.30 x 10(-15) m(3)/Ns (0.0017 microm/min-atm) and ELp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.27 x 10(-15) m(3)/Ns (0.0015 microm/min-atm) and ELp[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: Lpg = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp[cpa] = 37.6 kJ/mole (9.0 kcal/mole). The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70 degrees C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C. gigas. 相似文献
2.
Subzero water transport characteristics of boar spermatozoa confirm observed optimal cooling rates 总被引:1,自引:0,他引:1
Incomplete understanding of the water transport parameters (reference membrane permeability, L(pg), and activation energy, E(Lp)) during freezing in the presence of extracellular ice and cryoprotective agents (CPAs) is one of the main limiting factors in reconciling the difference between the numerically predicted value and the experimentally determined optimal rates of freezing in boar (and in general mammalian) gametes. In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the water transport during freezing of boar spermatozoa. Water transport data during freezing of boar sperm cell suspensions were obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 6% (v/v) glycerol. Using previously published values, the boar sperm cell was modeled as a cylinder of length 80.1 microm and a radius of 0.31 microm with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L(pg) and E(Lp)) were determined. The "combined-best-fit" parameters at 5 and 20 degrees C/min for boar spermatozoa in the presence of extracellular ice are: L(pg) = 3.6 x 10(-15) m(3)/N. s (0.02 microm/min-atm) and E(Lp) = 122.5 kJ/mole (29.3 kcal/mole) (R(2) = 0.99); and the corresponding parameters in the presence of extracellular ice and glycerol are: L(pg)[cpa] = 0.90 x 10(-15) m(3)/N. s (0.005 microm/min-atm) and E(Lp)[cpa] = 75.7 kJ/mole (18.1 kcal/mole) (R(2) = 0.99). The water transport parameters obtained in the present study are significantly different from previously published parameters for boar and other mammalian spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The theoretically predicted optimal rates of freezing using the new parameters ( approximately 30 degrees C/min) are in close agreement with previously published but experimentally determined optimal cooling rates. This analysis reconciles a long-standing difference between theoretically predicted and experimentally determined optimal cooling rates for boar spermatozoa. 相似文献
3.
A novel hydrogel was obtained by reticulation of chitosan with dextrin enzymatically linked to vinyl acrylate (dextrin-VA), without cross-linking agents. The hydrogel had a solid-like behaviour with G′ (storage modulus) >> G″ (loss modulus). Glucose diffusion coefficients of 3.9 × 10−6 ± 1.3 × 10−6 cm2/s and 2.9 × 10−6 ± 0.5 × 10−6 cm2/s were obtained for different substitution degrees of the dextrin-VA (20% and 70% respectively). SEM observation revealed a porous structure, with pores ranging from 50 μm to 150 μm. 相似文献
4.
Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents. 总被引:10,自引:0,他引:10
Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp)[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp)[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions. 相似文献
5.
Hernandez VH Bortolozzi M Pertegato V Beltramello M Giarin M Zaccolo M Pantano S Mammano F 《Nature methods》2007,4(4):353-358
Gap junction channels assembled from connexin protein subunits mediate intercellular transfer of ions and metabolites. Impaired channel function is implicated in several hereditary human diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP(3)) through connexin channels is associated with peripheral neuropathies and deafness, respectively. Here we present a method to estimate the permeability of single gap junction channels to second messengers. Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fluorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP(3). The unitary permeabilities to cAMP (47 x 10(-3) +/- 15 x 10(-3) microm(3)/s) and InsP(3) (60 x 10(-3) +/- 12 x 10(-3) microm(3)/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 +/- 3 x 10(-3) microm(3)/s), an exogenous tracer. This method permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signaling pathways. 相似文献
6.
Membrane insertion and lateral diffusion of fluorescence-labelled cytochrome c oxidase subunit IV signal peptide in charged and uncharged phospholipid bilayers. 总被引:5,自引:0,他引:5
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The synthetic 25-residue signal peptide of cytochrome c oxidase subunit IV was labelled with the fluorophor 7-nitrobenz-2-oxa-1,3-diazole (NBD) at its single cysteine residue. Addition of small unilamellar vesicles of 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) to the labelled peptide resulted in a shift of the NBD excitation and emission spectra to shorter wavelengths. Binding of the peptide to the vesicles was measured by the increase in the fluorescence emission yield. A surface partition constant of (3.9 +/- 0.5) x 10(3) M-1 was derived from these titrations. When the membrane contained, in addition to POPC, negatively charged 1-palmitoyl 2-oleoyl phosphatidylglycerol (POPG), the NBD fluorescence spectra were further shifted to shorter wavelengths and exhibited increased quantum yields. The apparent partition constants were increased to 10(4)-10(5) M-1 for vesicles with 20 or 100 mol% POPG. Lateral diffusion of the peptide was measured by fluorescence recovery after photobleaching in multibilayers of POPC, POPG, POPC/POPG (4:1) and 1,2-dimyristoyl phosphatidylcholine. The lateral diffusion coefficients of the peptide in bilayers of POPC (8 x 10(-8) cm2/s at 21 degrees C) were 1.5-1.6-fold greater than those of NBD-labelled phospholipids (5 x 10(-8) cm2/s at 21 degrees C), but 1.5-1.8-fold smaller (3 x 10(-8) cm2/s in 20% POPG and at 21 degrees C) than the lipid diffusion coefficients in the negatively charged bilayers. It is concluded that the signal peptide associates with phospholipid bilayers in two different forms, which depend on the lipid charge. The experiments with POPC bilayers are well explained by a model in which the peptide partitions into the region of the phospholipid head-groups and diffuses along the membrane/water interface. If POPG is present in the membrane, electrostatic attractions between the basic residues of the peptide and the acidic lipid head-groups result in a deeper penetration of the bilayer. For this case, two models that are both consistent with the experimental data are discussed, in which the peptide either forms an oligomer of three to six partially helical membrane-spanning monomers, or inserts into the bilayer with its amphiphilic helical segment aligned parallel to the plane of the membrane and located near the head-group and outer hydrocarbon region of the bilayer. 相似文献
7.
cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling. 相似文献
8.
Transducin is a heterotrimer formed by a fatty acylated alpha-subunit and a farnesylated betagamma-subunit. The role of these two covalent modifications and of adjacent hydrophobic and charged amino acid residues in reversible anchoring at disk model membranes is investigated at different pH values, salt concentrations, and lipid packing densities using the monolayer expansion technique and CD spectroscopy. The heterotrimer only binds if the acetylated alpha-subunit is transformed into its surface-active form by divalent cations. In the presence of salts the alpha(GDP)-subunit, the betagamma-complex, and the heterotrimer bind to POPC monolayers at 30 mN/m, estimated to mimic the lateral packing density of disk membranes, with apparent binding constants of Kapp = (1.1 +/- 0.3) x 10(6) M-1 (reflecting the penetration of the fatty acyl chain together with approximately three adjacent hydrophobic amino acid residues), Kapp = (3.5 +/- 0.5) x 10(6) M-1 (reflecting the penetration of the farnesyl chain), and Kapp = (1.6 +/- 0.3) x 10(6) M-1 (reflecting a major contribution of the alpha(GDP)-subunit with only a minor contribution from the betagamma-complex). The apparent binding constant of the alpha(GTP)-subunit is distinctly smaller than that of the alpha(GDP)-subunit. Binding to negatively charged POPC/POPG (75/25 mole/mole) monolayers is reinforced by 2-3 cationic residues for the betagamma-complex. The alpha-subunit shows no electrostatic attraction and the heterotrimer shows even a slight electrostatic repulsion which becomes the dominating force in the absence of salts. 相似文献
9.
Sperm cryopreservation of live-bearing fishes, such as those of the genus Xiphophorus is only beginning to be studied, although these fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. To explore optimization of techniques for sperm cryopreservation of these fishes, this study measured the volumetric shrinkage response during freezing of sperm cells of Xiphophorus helleri by use of a shape-independent differential scanning calorimeter (DSC) technique. Volumetric shrinkage during freezing of X. helleri sperm cell suspensions was obtained in the presence of extracellular ice at a cooling rate of 20 degrees C/min in three different media: (1) Hanks' balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol; and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of 33.3 microm in length and 0.59 microm in diameter with an osmotically inactive cell volume (V(b)) of 0.6V(o), where V(o) is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) of the Xiphophorus helleri sperm cell membrane were determined. The best-fit membrane permeability parameters at 20 degrees C/min in the absence of CPAs were: L(pg)=0.776 x 10(-15)m3/Ns (0.0046 microm/min atm), and E(Lp)=50.1 kJ/mol (11.97 kcal/mol) (R2=0.997). The corresponding parameters in the presence of 14% glycerol were L(pg)[cpa]=1.063 x 10(-15)m3/Ns (0.0063 microm/min atm), and E(Lp)[cpa]=83.81 kJ/mol (20.04 kcal/mol) (R2=0.997). The parameters in the presence of 10% DMSO were L(pg)[cpa]=1.4 x 10(-15)m3/Ns (0.0083 microm/min atm), and E(Lp)[cpa]=90.96 kJ/mol (21.75 kcal/mol) (R2=0.996). Parameters obtained in this study suggested that the optimal rate of cooling for X. helleri sperm cells in the presence of CPAs ranged from 20 to 35 degrees C/min and were in close agreement with recently published, empirically determined optimal cooling rates. 相似文献
10.
Rappolt M Vidal MF Kriechbaum M Steinhart M Amenitsch H Bernstorff S Laggner P 《European biophysics journal : EBJ》2003,31(8):575-585
We have studied the structural, dynamic and mechanical properties of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine (POPC)/cholesterol binary mixtures by small-angle X-ray scattering. Our investigations were concentrated on the biologically most relevant pressure-temperature-cholesterol regime, i.e. the liquid crystalline phase and its phase boundary to the lamellar gel phase within a cholesterol concentration up to 25 mol%. From the dependence of the transition pressure we derived a value of 19 kJ/mol for the transition enthalpy Delta H(m) of POPC in excess water. With increasing cholesterol concentration, Delta H(m) drops to about 7 kJ/mol at 20 mol% cholesterol. Time-resolved pressure-scan (p-scan) and temperature-jump (T-jump) experiments reveal that at low cholesterol content (<5-8 mol%) the fluidity and also the bilayer compressibility increase remarkably. In contrast, at concentrations between 5 and 25 mol% cholesterol the bilayer becomes again more rigid and the lipid bilayer spacing increases about 2 A. Theses changes are attributed to the onset of phase separation between liquid disordered and liquid ordered phases. The fluid-fluid miscibility gap for this mono-unsaturated lecithin species is strongly enlarged compared with saturated lecithins. 相似文献
11.
We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on the membranes of living nerve cells. The binding of the benzodiazepine Ro 7-1986/602 (N-des-diethyl-fluorazepam), labeled with the fluorophore Alexa 532, to the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9 +/- 1.9) nm and the rate constant for ligand-receptor dissociation kdisS = (1.28 +/- 0.08) x 10(-3) s(-1) show that there is a specific and high affinity interaction between the dye-labeled ligand (Ro-Alexa) and the receptor site. The binding was saturated at approx. 100 nM and displacement of 10 nM Ro-Alexa, with a 1,000-fold excess of midazolam, showed a non-specific binding of 7-10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [D(bound1) = (1.32 +/- 0.26) microm2/s; D(bound2) = (2.63 +/- 0.63) x 10(-2) microm2/s] are related to binding sites, which shows a mono-exponential decay in a time-dependent dissociation of the ligand-receptor complex. 相似文献
12.
The fluorescence decay kinetics of 1-methylpyrene in small unilamellar l-alpha-dimyristoylphosphatidylcholine vesicles above the phase transition temperature has been studied as a function of concentration and temperature. When the 1-methylpyrene/phospholipid ratio equals 1:2000 no excimer is observed and the fluorescence decay is monoexponential. When this ratio is equal to or higher than 1 200, excimer is observed and the monomer and excimer decays can be adequately described by two exponential terms. The deviation of the monomer decays from monoexponentiality cannot be described by a model where the diffusion-controlled excimer formation is time dependent. The observed decays are compatible with the excimer formation scheme which is valid in an isotropic medium. The activation energy of excimer formation is found to be 29-9 +/-1.4 kJ mol . The (apparent) excimer formation constant and the excimer lifetime at different temperatures have been determined. The diffusion coefficient associated with the excimer formation process varies between 2 x 10(-10) m(2)/s at 70 degrees C to 4 x 10(-11) m(2)/s at 25 degrees C. 相似文献
13.
The binding behavior of a fluorescently labeled muscimol derivative to the GABA(A) receptor was analyzed at rat hippocampal neurons by fluorescence correlation spectroscopy. After muscimol had been labeled with the fluorophore Alexa Fluor 532, specific binding constants for binding of the dye-labeled ligand (Mu-Alexa) to the GABA(A) receptor were determined. We found a high specific binding affinity of Mu-Alexa with a K(D) value of 3.4 +/- 0.5 nM and a rate constant of ligand-receptor dissociation (k(diss)) of (5.37 +/- 0.95) x 10(-2) s(-1). A rate constant of ligand-receptor association (k(ass)) of (1.57 +/- 0.28) x 10(7) L mol(-1) s(-1) was calculated. The following diffusion coefficients were observed: D(free) = 233 +/- 20 microm(2)/s (n = 66) for free diffusing Mu-Alexa, D(bound1) = 2.8 +/- 0.9 microm(2)/s (n = 64) for the lateral mobility, and D(bound2) = 0.14 +/- 0.05 microm(2)/s (n = 56) for the hindered mobility of the GABA(A) receptor-ligand complex in the cell membrane. Saturation of Mu-Alexa binding was observed at a concentration of 50 nM. A maximum number of binding sites [B(max) = 18.4 +/- -0.4 nM (n = 5)] was found. Similar K(i) values of 4.5 +/- 1.0 nM for nonlabeled muscimol and 8.8 +/- 1.8 nM for Mu-Alexa were found by RRAs using [(3)H]muscimol as a radioligand. A concentration-dependent increase in the level of specific Mu-Alexa binding was demonstrated by the positive cooperative activity of co-incubated midazolam, which was selectively found in GABA(A) receptor-ligand complexes with hindered mobility. 相似文献
14.
Forder JR Bui JD Buckley DL Blackband SJ 《American journal of physiology. Heart and circulatory physiology》2001,281(3):H1280-H1285
Myocardial tissue slices were isolated from the left ventricular free wall (7 slices) and left ventricular papillary muscle (3 slices) of New Zealand White male rabbits (n = 4) and were subsequently superfused with a modified St. Thomas' Hospital cardioplegic solution at 19 degrees C. The diffusion-weighted images were obtained with a 600-MHz nuclear magnetic resonance spectrometer using diffusion gradient b-values that ranged from 166 to 6,408 s/mm(2); the apparent diffusion coefficient of water in the tissues were subsequently calculated. All of the tissue samples that were studied exhibited nonmonoexponential diffusion. Data from seven slices were mathematically fitted by a biexponential expression with a fast diffusion component of 0.72 +/- 0.07 x 10(-3) mm(2)/s, and a slow diffusion component of 0.060 +/- 0.033 x 10(-3) mm(2)/s. The fast component dominated the calculated apparent diffusion coefficient of the tissue, composed of 82 +/- 3% of the overall diffusion-dependent signal decay. Thus myocardial tissue exhibits characteristics consistent with multiple compartments of diffusion. This work has important implications for myocardial diffusion tensor imaging, as well as the changes in diffusion that have been reported following myocardial ischemia. 相似文献
15.
The agonist-induced dynamic regulation of the beta(2)-adrenergic receptor (beta(2)-AR) on living cells was examined by means of fluorescence correlation spectroscopy (FCS) using a fluorescence-labeled arterenol derivative (Alexa-NA) in hippocampal neurons and in alveolar epithelial type II cell line A549. Alexa-NA specifically bound to the beta(2)-AR of neurons with a K(D) value of 1.29 +/- 0.31 nM and of A549 cells with a K(D) of 5.98 +/- 1.62 nM. The receptor density equaled 4.5 +/- 0.9 microm(-2) in neurons (rho(N)) and 19.9 +/- 2.0 microm(-2) in A549 cells (rho(A549)). Kinetic experiments revealed comparable on-rate constants in both cell types (k(on) = 0.49 +/- 0.03 s(-1) nM(-1) in neurons and k(on) = 0.12 +/- 0.02 s(-1) nM(-1) in A549 cells). In addition to the free ligand diffusing with a D(free) of (2.11 +/- 0.04) x 10(-6) cm(2)/s, in both cell types receptor-ligand complexes with two distinct diffusion coefficients, D(bound1) (fast lateral mobility) and D(bound2) (hindered mobility), were observed [D(bound1) = (5.23 +/- 0.64) x 10(-8) cm(2)/s and D(bound2) = (6.05 +/- 0.23) x 10(-10) cm(2)/s for neurons, and D(bound1) = (2.88 +/- 1.72) x 10(-8) cm(2)/s and D(bound2) = (1.01 +/- 0.46) x 10(-9) cm(2)/s for A549 cells]. Fast lateral mobility of the receptor-ligand complex was detected immediately after addition of the ligand, whereas hindered mobility (D(bound2)) was observed after a delay of 5 min in neurons (up to 38% of total binding) and of 15-20 min in A549 cells (up to 40% of total binding). Thus, the receptor-ligand complexes with low mobility were formed during receptor regulation. Consistently, stimulation of receptor internalization using the adenylate cyclase activator forskolin shifted the ratio of receptor-ligand complexes toward D(bound2). Intracellular FCS measurements and immunocytochemical studies confirmed the appearance of endocytosed receptor-ligand complexes in the cytoplasm subjacent to the plasma membrane after stimulation with the agonist terbutaline (1 microM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin alpha-hederin. 相似文献
16.
Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP) experiments, we have developed a finite element (FE) method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4-8 folds faster (0.93+/-0.06 microm(2)/sec) than those anchored on different compartments in plasma membrane (at lipid raft: 0.11+/-0.01 microm(2)/sec and outside: 0.18+/-0.02 microm(2)/sec). The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained relatively stationary upon epidermal growth factor (EGF) stimulation. This result suggests that EGF induced a Src activation at lipid rafts with well-coordinated spatiotemporal patterns. Our FE-based method also provides an integrated platform of image analysis for studying molecular mobility and reconstructing the spatiotemporal activation maps of signaling molecules in live cells. 相似文献
17.
As part of a programme of comparative measurements of diffusional water permeability (Pd) the red blood cells (RBC) from Little Penguin (Eudyptula minor) were studied. The cell dimensions were measured with light and electron microscopy, and by a newly described non-invasive technique, NMR q-space analysis. In view of its relative novelty for cell biologists, an overview of this technique is presented. The RBC revealed an ellipsoidal shape that is characteristic of avian RBC, with axis lengths ("diameters") estimated to be: a=16.0 microm; b=9.6 microm; c=5.0 microm. The values of P(d)were: 2.0 x 10(-3)cm s(-1)at 5 degrees C, 3.3 x 10(-3)cm s(-1)at 10 degrees C, 4.6 x 10(-3)cm s(-1)at 15 degrees C and approximately 5.4 x 10(-3)cm s(-1)at 20, 25, 30, 37 and 42 degrees C.There was a lack of inhibition of water permeability by p-chloromercuribenzensulfonate (PCMBS), the well-known inhibitor of RBC aquaporin. It was notable that in the temperature range 5-20 degrees C the NMR parameters, and hence the permeability, varied linearly as is found for other species, but at temperatures higher than 20 degrees C there was no temperature-dependence of Pd. Consequently, there was an obvious break at approximately 20 degrees C in the Arrhenius plot, of the mean residence life time of water inside the cells, 1/Te, versus temperature. For temperatures less than 20 degrees C the activation energy E(a,d) was 45.6 +/- 6.6 kJ/mol. For temperatures higher than 25 degrees C E(a,d) was zero. The lack of inhibition of water permeability by PCMBS and the very high value of E(a,d) for diffusive water exchange suggests that the water permeation occurs primarily via the membrane bilayer per se, i.e., there is no aquaporin in Little Penguin RBC. The discontinuity at approximately 20 degrees C in the Arrhenius plot is an interesting finding, not seen before in other species, and we suggest that it reflects a phase transition in the membrane lipids. 相似文献
18.
The use of cryosurgery in the treatment of uterine fibroids is emerging as a possible treatment modality. The two known mechanisms of direct cell injury during the tissue freezing process are linked to intracellular ice formation and cellular dehydration. These processes have not been quantified within uterine fibroid tumor tissue. This study reports the use of a combination of freeze-substitution microscopy and differential scanning calorimetry (DSC) to quantify freeze-induced dehydration within uterine fibroid tumor tissue. Stereological analysis of histological tumor sections was used to obtain the initial cellular volume (V(o)) or the Krogh model dimensions (deltaX, the distance between the microvascular channels = 15.5 microm, r(vo), the initial radius of the extracellular space = 4.8 micro m, and L, the axial length of the Krogh cylinder = 19.1 microm), the interstitial volume ( approximately 23%), and the vascular volume ( approximately 7%) of the fibroid tumor tissue. A Boyle-van't Hoff plot was then constructed by examining freeze-substituted micrographs of "equilibrium"-cooled tissue slices to obtain the osmotically inactive cell volume, V(b) = 0.47V(o). The high interstitial volume precludes the use of freeze-substitution microscopy data to quantify freeze-induced dehydration. Therefore, a DSC technique, which does not suffer from this artifact, was used to obtain the water transport data. A model of water transport was fit to the calorimetric data at 5 and 20 degrees C/min to obtain the "combined best fit" membrane permeability parameters of the embedded fibroid tumor cells, assuming either a Krogh cylinder geometry, L(pg) = 0.92 x 10(-13) m(3)/Ns (0.55 microm/min atm) and E(Lp) = 129.3 kJ/mol (30.9 kcal/mol), or a spherical cell geometry (cell diameter = 18.3 microm), L(pg) = 0.45 x 10(-13) m(3)/Ns (0.27 microm/min atm) and E(Lp) = 110.5 kJ/mol (26.4 kcal/mol). In addition, numerical simulations were performed to generate conservative estimates, in the absence of ice nucleation between -5 and -30 degrees C, of intracellular ice volume in the tumor tissue at various cooling rates typical of those experienced during cryosurgery (< or =100 degrees C/min). With this assumption, the Krogh model simulations showed that the fibroid tumor tissue cells cooled at rates < or = 50 degrees C/min are essentially dehydrated; however, at rates >50 degrees C/min the amount of water trapped within the tissue cells increases rapidly with increasing cooling rate, suggesting the formation of intracellular ice. 相似文献
19.
K De Bock W Derave M Ramaekers E A Richter P Hespel 《Journal of applied physiology》2007,102(1):183-188
The effect of carbohydrate intake before and during exercise on muscle glycogen content was investigated. According to a randomized crossover study design, eight young healthy volunteers (n = 8) participated in two experimental sessions with an interval of 3 wk. In each session subjects performed 2 h of constant-load bicycle exercise ( approximately 75% maximal oxygen uptake). On one occasion (CHO), they received carbohydrates before ( approximately 150 g) and during (1 g.kg body weight(-1).h(-1)) exercise. On the other occasion they exercised after an overnight fast (F). Fiber type-specific relative glycogen content was determined by periodic acid Schiff staining combined with immunofluorescence in needle biopsies from the vastus lateralis muscle before and immediately after exercise. Preexercise glycogen content was higher in type IIa fibers [9.1 +/- 1 x 10(-2) optical density (OD)/microm(2)] than in type I fibers (8.0 +/- 1 x 10(-2) OD/microm(2); P < 0.0001). Type IIa fiber glycogen content decreased during F from 9.6 +/- 1 x 10(-2) OD/microm(2) to 4.5 +/- 1 x 10(-2) OD/microm(2) (P = 0.001), but it did not significantly change during CHO (P = 0.29). Conversely, in type I fibers during CHO and F the exercise bout decreased glycogen content to the same degree. We conclude that the combination of carbohydrate intake both before and during moderate- to high-intensity endurance exercise results in glycogen sparing in type IIa muscle fibers. 相似文献
20.
Intramolecular excimer formation in pyrene-labeled phosphatidylcholine was used as a tool to determine thermodynamic characteristics of the lamellar to hexagonal phase transitions in a binary lipid system dilinoleoylphosphatidylethanolamine (DLPE)/palmitoyloleoylphosphatidylcholine (POPC). Upon an L alpha/HII phase transition, the activation energy Ea for excimer formation increased from 5.6 +/- 0.2 kcal/mol to 6.3 +/- 0.2 kcal/mol, while the activation entropy delta S decreased from -40.0 +/- 0.8 cal/K.mol to -38.4 +/- 0.8 cal/K.mol. The results are consistent with the idea of molecular splaying of the acyl chains in the hexagonal phase. It is estimated that the molecular area at the terminal carbon of the lipid acyl chains increases by a factor of 2.2 upon the L alpha HII transition in DLPE/POPC. 相似文献