Measurement of erythrocyte membrane elasticity by flicker eigenmode decomposition.

We have studied the flickering of erythrocytes at wavelengths comparable to the cell dimension. To do this we have analyzed the edge fluctuations of the cell to a resolution of 5 nm by combining phase contrast microscopy with fast image processing. By measuring the edge excitations simultaneously at four orthogonal positions around the cell, the eigenmodes of equal azimuthal mode numbers m = 0,1,2 could be separated. From a continuous time sequence of 100 s of video frames taken at 40 ms time intervals, we determined the time-auto correlation function for the modes m = 0,1,2 and calculated their mean square amplitudes <delta n2m> as well as their decay times tau m. To explain the results we also present the theoretically calculated energy eigenmodes of an erythrocyte, accounting for the constraint that the cell is in contact with the substrate along an annular ring, which agreed well with the experimental findings. We found that the softest mode is a "hindered translational" mode with m = 1 of the adhered cell, which is almost insensitive to the shear elastic modulus. Comparison of the calculated and measured amplitudes yielded an average value for the bending stiffness of kc = 4 x 10(-19) J, which is much larger than the value obtained by flicker analysis at short wavelengths (kc = 2.3 x 10(-20) J). It would, however, agree well with the value expected from the red cell membrane area compressibility modulus of K = 4.5 x 10(-1)N/m, which corresponds to a lipid bilayer containing approximately 50 mol % of cholesterol. In contradiction to our theoretical expectations we found that the flicker eigenmodes seemed not to be influenced by the membrane shear elasticity, which will be discussed in terms of an unusual coupling between the lipid bilayer and the cytoskeleton.     

Mechanism of electroporative dye uptake by mouse B cells.

The color change of electroporated intact immunoglobulin G receptor (Fc gammaR-) mouse B cells (line IIA1.6) after direct electroporative transfer of the dye SERVA blue G (Mr 854) into the cell interior is shown to be dominantly due to diffusion of the dye after the electric field pulse. Hence the dye transport is described by Fick's first law, where, as a novelty, time-integrated flow coefficients are introduced. The chemical-kinetic analysis uses three different pore states (P) in the reaction cascade (C <==> P1 <==> P2 <==> P3), to model the sigmoid kinetics of pore formation as well as the biphasic pore resealing. The rate coefficient for pore formation k(p) is dependent on the external electric field strength E and pulse duration tE. At E = 2.1 kV cm(-1) and tE = 200 micros, k(p) = (2.4 +/- 0.2) x 10(3) s(-1) at T = 293 K; the respective (field-dependent) flow coefficient and permeability coefficient are k(f)0 = (1.0 +/- 0.1) x 10(-2) s(-1) and P0 = 2 cm s(-1), respectively. The maximum value of the fractional surface area of the dye-conductive pores is 0.035 +/- 0.003%, and the maximum pore number is Np = (1.5 +/- 0.1) x 10(5) per average cell. The diffusion coefficient for SERVA blue G, D = 10(-6) cm2 s(-1), is slightly smaller than that of free dye diffusion, indicating transient interaction of the dye with the pore lipids during translocation. The mean radii of the three pore states are r(P1) = 0.7 +/- 0.1 nm, r(P2) = 1.0 +/- 0.1 nm, and r(P3) = 1.2 +/- 0.1 nm, respectively. The resealing rate coefficients are k(-2) = (4.0 +/- 0.5) x 10(-2) s(-1) and k(-3) = (4.5 +/- 0.5) x 10)(-3) s(-1), independent of E. At zero field, the equilibrium constant of the pore states (P) relative to closed membrane states (C) is K(p)0 = [(P)]/[C] = 0.02 +/- 0.002, indicating 2.0 +/- 0.2% water associated with the lipid membrane. Finally, the results of SERVA blue G cell coloring and the new analytical framework may also serve as a guideline for the optimization of the electroporative delivery of drugs that are similar in structure to SERVA blue G, for instance, bleomycin, which has been used successfully in the new discipline of electrochemotherapy.     

A molecular scheme for the reaction between acetylcholine and nicotinic channels.

In outside-out patches of mouse-muscle membrane, embryonic-like channels were activated by pulses of acetylcholine (ACh). On increasing the ACh concentration, the rate of desensitization, 1/tau d, increased linearly with the peak open probability, indicating desensitization from the open state. Desensitization had only one time constant tau d at each ACh concentration. Recovery from desensitization was only approximately 10 times slower than desensitization, whereas the probability of steady-state channel opening, declined to < 0.01 with > 10(-6) M ACh. The peak probability of opening in > 10(-4) M ACh pulse was close to 1. A linear reaction scheme was not compatible with these results. The scheme had to be expanded resulting in a circular scheme with two additional ACh binding steps to desensitized channel states. The approximate rate constants of all reaction steps in the circular scheme could be determined using computer simulations. The model predicted that clusters of channel opening had the average duration tau d at the respective ACh concentration. In cell-attached patches on intact muscle fibers, similar average cluster durations were observed at the respective ACh concentration. This indicates that tau d in the intact muscle fibers has similar values as in outside-out patches.     

cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block

Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- >> SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10- fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.     

Electron paramagnetic resonance of the excited triplet state of metal-free and metal-substituted cytochrome c.

The photoactivated metastable triplate states of the porphyrin (free-base, i.e., metal-free) zinc and tin derivatives of horse cytochrome c were investigated using electron paramagnetic resonance. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range 3.8-150 K are discussed in terms of porphyrin-protein interactions. The zero-field splitting parameters D for the free-base, Zn and Sn derivatives are 465 x 10(-4), 342 x 10(-4) and 353 x 10(-4) cm-1, respectively, and are temperature invariant over the temperature ranges studied. AN E value at 4 K of 73 x 10(-4) cm-1 was obtained for Zn cytochrome c, larger than any previously found for Zn porphyrins derivatives of hemeproteins, showing that the heme site of cytochrome c imposes an asymmetric field. Though the E value for Zn cytochrome c is large, the geometry of the site appears quite constrained, as indicated by a spectral line shape showing a single species. Intersystem crossing occurred predominantly to the T2 > zero-field spin sublevel. EPR line shape changes with respect to temperature of Zn cyt c are interpreted in terms of vibronic coupling, and a maximum Jahn-Teller crystal-field splitting of approximately 180 cm-1 is obtained. Sn cytochrome c in comparison with the Zn protein exhibits a photoactivated triplet line shape that is less well resolved in the X-Y region. The magnitude of E value is approximately 60 x 10(-4) cm-1 at 4 K; its value rapidly tends toward zero with increasing temperature, from which a value for the Jahn-Teller crystal-field splitting of > or = 40 cm-1 is estimated. In contrast to those for the metal cytochromes, the magnitude of E value for the free-base derivative was essentially zero at all temperatures studied. This finding is discussed as a consequence of an excited-state tautomerization process that occurs even at 4 K.     

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.     

PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies

High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.     

Monte Carlo simulations of supercoiling free energies for unknotted and trefoil knotted DNAs.

A new Monte Carlo (MC) algorithm is proposed for simulating inextensible circular chains with finite twisting and bending rigidity. This new algorithm samples the relevant Riemann volume elements in a uniform manner, when the constraining potential vanishes. Simulations are performed for filaments comprising 170 subunits, each containing approximately 28 bp, which corresponds to a DNA length of 4770 bp. The bending rigidity is chosen to yield a persistence length, P = 500 A, and the intersubunit potential is taken to be a hard-cylinder potential with diameter d = 50 A. This value of d yields the same second virial coefficient as the electrostatic potential obtained by numerical solution of the Poisson-Boltzmann equation for 150 mM salt. Simulations are performed for unknotted circles and also for trefoil knotted circles using two different values of the torsional rigidity, C = (2.0 and 3.0) x 10(-19) dyne cm2. In the case of unknotted circles, the simulated supercoiling free energy varies practically quadratically with linking difference delta l. The simulated twist energy parameter ET, its slope dET/dT, and the mean reduced writhe <w>/delta l for C = 3 x 10(-19) dyne cm2 all agree well with recent simulations for unknotted circles using the polygon-folding algorithm with identical P, d, and C. The simulated ET vs. delta l data for C = 2.0 x 10(-19) dyne cm2 agree rather well with recent experimental data for p30 delta DNA (4752 bp), for which the torsional rigidity, C = 2.07 x 10(-19) dyne cm2, was independently measured. The experimental data for p30 delta are enormously more likely to have arisen from C = 2.0 x 10(-19) than from C = 3.0 x 10(-19) dyne cm2. Serious problems with the reported experimental assessments of ET for pBR322 and their comparison with simulated data are noted. In the case of a trefoil knotted DNA, the simulated value, (ET)tre, exceeds that of the unknotted DNA, (ET)unk, by approximately equal to 1.40-fold at magnitude of delta l = 1.0, but declines to a plateau about 1.09-fold larger than (ET)unk when magnitude of delta l > or = 15. Although the predicted ratio, (ET)tre/(ET)unk approximately equal to 1.40, agrees fairly well with recent experimental measurements on a 5600-bp DNA, the individual measured ET values, like some of those reported for pBR322, are so large that they cannot be simulated using P = 500 A, d = 50 A, and any previous experimental estimate of C.     

Hydrophobic ions amplify the capacitive currents used to measure exocytotic fusion.

The detection of exocytotic fusion in patch-clamped secretory cells depends on measuring an increase in the cell membrane capacitance as new membrane is added to the plasma membrane. However, in the majority of secretory cells, secretory vesicles are too small (< 200 nm in diameter) to cause a detectable signal. We have found that incubations of normal mouse mast cells with the hydrophobic anion dipicrylamine (DPA), increases cell membrane capacitance by about three times. The large capacitive current induced by DPA was voltage-dependent, having a maximum value at -10 mV. The DPA-induced charge movement could be described by a single barrier model in which the DPA molecules move between two stable states in the bulk lipid matrix of the membrane. More importantly, the DPA treatment produced a sevenfold increase in the size of the capacitance steps observed upon the exocytotic fusion of single secretory granules. A similar amplification of DPA on the secretory vesicle capacitance was observed in a cell with larger (< or = 5 microns in diameter) or with smaller secretory granules (< 250 nm in diameter). Additionally, the increased granule membrane capacitance enlarged the transient capacitive discharge measured upon formation of a fusion pore in normal mast cell granules. Our results indicate that hydrophobic ions provide an important tool for high resolution studies of membrane capacitance.     

Bidirectional transepithelial water transport: measurement and governing mechanisms.

In the search for the mechanisms whereby water is transported across biological membranes, we hypothesized that in the airways, the hydration of the periciliary fluid layer is regulated by luminal-to-basolateral water transport coupled to active transepithelial sodium transport. The luminal-to-basolateral (JWL-->B) and the basolateral-to-luminal (JWB-->L) transepithelial water fluxes across ovine tracheal epithelia were measured simultaneously. The JWL-->B (6.1 microliter/min/cm2) was larger than JWB-->L (4.5 microliter/min/cm2, p < 0.05, n = 30). The corresponding water diffusional permeabilities were PdL-->B = 1.0 x 10(-4) cm/s and PdB-->L = 7.5 x 10(-5) cm/s. The activation energy (Ea) of JWL-->B (11.6 kcal/mol) was larger than the Ea of JWB-->L (6.5 kcal/mol, p < 0.05, n = 5). Acetylstrophanthidin (100 microM basolateral) reduced JWL-->B from 6.1 to 4.4 microliter/min/cm2 (p < 0. 05, n = 5) and abolished the PD. Amiloride (10 microM luminal) reduced JWL-->B from 5.7 to 3.7 microliter/min/cm2 (p < 0.05, n = 5) and reduced PD by 44%. Neither of these agents significantly changed JWB-->L. These data indicate that in tracheal epithelia under homeostatic conditions, JWB-->L was dominated by diffusion (Ea = 4.6 kcal/mol), whereas approximately 30% of JWL-->B was coupled to the active Na+,K+-ATPase pump (Ea = 27 kcal/mol).     

The reduction in electroporation voltages by the addition of a surfactant to planar lipid bilayers.

The effects of a nonionic surfactant, octaethyleneglycol mono n-dodecyl ether (C12E8), on the electroporation of planar bilayer lipid membranes made of the synthetic lipid 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 100-450 mV) rectangular voltage pulses were used to electroporate the bilayers, followed by a prolonged, low-amplitude ( approximately 65 mV) voltage clamp to monitor the ensuing changes in transmembrane conductance. The electroporation thresholds of the membranes were found for rectangular voltage pulses of given durations. The strength-duration relationship was determined over a range from 10 micros to 10 s. The addition of C12E8 at concentrations of 0.1, 1, and 10 microM to the bath surrounding the membranes decreased the electroporation threshold monotonically with concentration for all durations (p < 0.0001). The decrease from control values ranged from 10% to 40%, depending on surfactant concentration and pulse duration. For a 10-micros pulse, the transmembrane conductance 150 micros after electroporation (G150) increased monotonically with the surfactant concentration (p = 0.007 for 10 microM C12E8). These findings suggest that C12E8 incorporates into POPC bilayers, allowing electroporation at lower intensities and/or shorter durations, and demonstrate that surfactants can be used to manipulate the electroporation threshold of lipid bilayers.     

Osmotic gradient-induced water permeation across the sarcolemma of rabbit ventricular myocytes

The mechanism of water permeation across the sarcolemma was characterized by examining the kinetics and temperature dependence of osmotic swelling and shrinkage of rabbit ventricular myocytes. The magnitude of swelling and the kinetics of swelling and shrinkage were temperature dependent, but the magnitude of shrinkage was very similar at 6 degrees, 22 degrees, and 37 degrees C. Membrane hydraulic conductivity, Lp, was approximately 1.2 x 10(-10) liter.N-1.s-1 at 22 degrees C, corresponding to an osmotic permeability coefficient, Pf, of 16 microns.s-1, and was independent of the direction of water flux, the magnitude of the imposed osmotic gradient (35-165 mosm/liter), and the initial cell volume. This value of Lp represents an upper limit because the membrane was assumed to be a smooth surface. Based on capacitive membrane area, Lp was 0.7 to 0.9 x 10(-10) liter.N-1.s-1. Nevertheless, estimates of Lp in ventricle are 15 to 25 times lower than those in human erythrocytes and are in the range of values reported for protein- free lipid bilayers and biological membranes without functioning water channels (aquaporin). Evaluation of the effect of unstirred layers showed that in the worst case they decrease Lp by < or = 2.3%. Analysis of the temperature dependence of Lp indicated that its apparent Arrhenius activation energy, Ea', was 11.7 +/- 0.9 kcal/mol between 6 degrees and 22 degrees C and 9.2 +/- 0.9 kcal/mol between 22 degrees and 37 degrees C. These values are significantly greater than that typically found for water flow through water-filled pores, approximately 4 kcal/mol, and are in the range reported for artificial and natural membranes without functioning water channels. Taken together, these data strongly argue that the vast majority of osmotic water flux in ventricular myocytes penetrates the lipid bilayer itself rather than passing through water-filled pores.     

Cell fission and formation of mini cell bodies by high frequency alternating electric field.

We report the use of high frequency alternating electric fields (AC) to induce deformation of sea urchin eggs, leading to budding of membrane vesicles or fission of cells. Several mini cell bodies can be prepared from a single egg by carefully manipulating the frequency and amplitude of the AC field and the ratio between the interelectrode spacing and the cell diameter, alpha. alpha values between 2.2 and 3.5 have been found to be optimal for inducing fission of sea urchin eggs. In a typical experiment, a sea urchin egg (diameter = 75 microns), suspended in a low ionic medium (conductance < 2 mS/m), was located under the microscope between two platinum wire electrodes, separated by a distance of approximately 200 microns. A medium strength AC field (< 100 V/cm at 2 MHz) was applied to attract the egg to one of the two electrodes via dielectrophoresis. This process took place in a few seconds. The voltage was then slowly increased to approximately 1000 V/cm over approximately 30 s. The cell elongated and separated into two fragments, the larger one containing the nucleus. When the field was turned off, the mother cell and the daughter vesicle retracted to form spherical mini cell bodies that appear to be stable as assessed by the absence of swelling for the duration of the experiment (approximately 15 min). This indicates that membranes of these mini cell bodies were not leaky to ions and small molecules. This procedure could be repeated a few times to make several mini cell bodies from a single egg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphology and development of rhabdovirus-like particles inCuscuta odorata (Convolvulaceae) simultaneous infected with virus and mycoplasma-like organisms

Summary Electron microscopic examination ofCuscuta odorata, used for transmission trials, revealed mycoplasma-like organisms (MLO) as well as rhabdovirus-like particles, unknown toCuscuta. The virus infection is confined to certain phloem-parenchyma cells and a 1–2 cell thick layer of parenchyma cells with thickened walls surrounding the central cylinder. Virus particles, mostly bacilliform, could be detected mainly in the nucleus but also in the cytoplasm. They reach a length of 350–400 nm and a diameter of approximately 75 nm. Virus assembly takes place exclusively in the nucleus. Virus maturation occurs in membrane bound areas within the nucleus, which have no connection with the perinuclear space. Formation of nucleocapsids is always associated with a nuclear viroplasm. Envelopment of virus particles occurs in these membrane bound areas. Budding into the perinuclear space does not occur. Virus infection leads to degeneration and finally to death of the protoplast.Abbreviations cy cytoplasm - m membrane stacks - mt mitochondria - my mycoplasma-like organisms - nc nucleocapsid - ncp nucleocapsid particles - nf nuclear filaments - np nucleoplasm - nu nucleus - nvp nuclear viroplasm - oc obliterated cells - p plastid - pc passage cells - ph phloem - ps perinuclear space - spc strand of parenchymatous cells - v virus particle - x xylem     

Modeling electroporation in a single cell. I. Effects Of field strength and rest potential.

This study develops a model for a single cell electroporated by an external electric field and uses it to investigate the effects of shock strength and rest potential on the transmembrane potential V(m) and pore density N around the cell. As compared to the induced potential predicted by resistive-capacitive theory, the model of electroporation predicts a smaller magnitude of V(m) throughout the cell. Both V(m) and N are symmetric about the equator with the same value at both poles of the cell. Larger shocks do not increase the maximum magnitude of V(m) because more pores form to shunt the excess stimulus current across the membrane. In addition, the value of the rest potential does not affect V(m) around the cell because the electroporation current is several orders of magnitude larger than the ionic current that supports the rest potential. Once the field is removed, the shock-induced V(m) discharges within 2 micros, but the pores persist in the membrane for several seconds. Complete resealing to preshock conditions requires approximately 20 s. These results agree qualitatively and quantitatively with the experimental data reported by Kinosita and coworkers for unfertilized sea urchin eggs exposed to large electric fields.     

Electrofusion between heterogeneous-sized mammalian cells in a pellet: potential applications in drug delivery and hybridoma formation.

High-efficiency electrofusion between cells of different sizes was achieved by application of fusing electric pulses to cells in centrifuged pellets. Larger target cells (Chinese hamster ovary or L1210 cells) were stacked among smaller human erythrocytes or erythrocyte ghosts by sequential centrifugation at 700 g to form five-tier pellets in a specially designed centrifugation-electrofusion chamber. The membranes of erythrocytes and ghost were labeled with fluorescent membrane dye (1,1' dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine (Dil)), and the contents of ghosts were loaded with water-soluble fluorescent dye (42-kDa fluorescein isothiocyanate dextran (FITC-dextran)), to monitor heterogeneous cell fusion. Fusion efficiency was assayed by the extent of either membrane dye mixing or contents (FITC-dextran) mixing with target cells. Four rectangular electric pulses at 300 V and 80 microseconds each were found to give the optimal fusion results of approximately 80% heterogeneous fusion by the content-mixing assay and approximately 95% by the membrane-dye-mixing assay. Cell viability remained greater than 80% after electrofusion. Because of the electric breakdown of cell membranes at the beginning of the pulse, the pellet resistance and hence the partial voltage across the pellet reduced rapidly during the remaining pulse time. This voltage redistribution favored the survival of fused cells. The limited colloidal-osmotic swelling of cells in pellets enhanced cell-cell contact and increased the pellet resistance after each pulse. As a result, the partial voltage across the pellet was restored when the next pulse was applied. This redistribution of pulse voltage in the pellet system permitted the breakdown of cell membranes at a lower applied voltage threshold than that required for electrofusion of cells in suspension or in dielectrophoretic cell chains. The cell viability and soluble dye retention within cells (FITC-dextran) remained at the same high levels for 3 h when the cells were incubated in respective culture media with serum at 37 degrees C. Viability and dye retention decreased significantly within 30 min when cells were incubated in phosphate-buffered saline without serum. The pellet technique was applied to form hybridomas by fusion of larger SP2/0 murine myelomas with smaller naive mouse lymphocytes. An optimum of 173 +/- 70 hypoxanthine aminopterin thymidine (HAT)-selected clones of the hybridomas was obtained from 40,000 SP2/0 cells and 1.5 x 10(6) lymphocytes used in each trial. This high-efficiency fusion technique may be adapted to mediate drug and gene transfer to target cells ex vivo as well as to form hybrid cells with limited cell sources.     

Sequence composition effects on the stabilities of triple helix formation by oligonucleotides containing N7-deoxyguanosine.

A nonnatural nucleoside, 7-(2-deoxy-beta-D-erythro-pento-furanosyl)-guanine (d7G), mimics protonated cytosine and specifically binds GC base pairs within a pyrimidine - purine - pyrimidine triple helix. The differences in association constants (KT) determined by quantitative footprint titration experiments at neutral pH reveal dramatic sequence composition effects on the energetics of triple helix formation by oligonucleotides containing d7G. Purine tracts of sequence composition 5'-d(AAAAAGAGAGAGAGA)-3' are bound by oligonucleotide 5'-d(TTTTT7GT7GT7GT7GT7GT)-3' three orders of magnitude less strongly than by 5'-d(TTTTTmCTmCTmCTmCTmCT)-3' (KT = 1.5 x 10(6) M(-1) and KT > or = 3 x 10(9) M(-1) respectively). Conversely, purine tracts of sequence composition 5'-d(AAAAGAAAAGGGGGGA)-3' are bound by oligonucleotide 5'-d(TTTTmCTTTT7G7G7G7G7G7GT)-3' five orders of magnitude more strongly than by 5'-d(TTTTmCTTTTmCmCmCmCmCT)-3' (KT > or = 3 x 10(9) M(-1) and KT < 5 x 10(4) M(-1) respectively). The complementary nature of d7G and mC expands the repertoire of G-rich sequences which may be targeted by triple helix formation.     

Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.     

Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF)

Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2–3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations.