Current noise parameters derived from voltage noise and impedance in embryonic heart cell aggregates.

We have recorded membrane impedance and voltage noise in the pacemaker range of potentials (-70 to -59 mV) from spheroidal aggregates of 7-d embryonic chick ventricle cells made quiescent by exposure to tetrodotoxin in medium containing 4.5 mM K+. The input capacitance is proportional to aggregate volume and therefore to total membrane area. The specific membrane capacitance is 1.24 microF/cm2. The input resistance at constant potential is inversely proportional to aggregate volume and therefore to total membrane area. The specific membrane resistance in 18 k omega . cm2 at -70 mV and increases to 81 k omega . cm2 at -59 mV. The RC time constant is 22 ms at -70 mV and increases to 146 ms at -59 mV. The aggregate transmembrane small-signal impedance can be represented by a parallel RC circuit itself in parallel with an inductive branch consisting of a resistor (rL) and an inductor (L) in series. The time constant of the inductive branch (L/rL) is 340 ms, and is only weakly dependent on potential. Correlation functions of aggregate voltage noise and the impedance data were modeled by a population of channels with simple open-close kinetics. The time constant of a channel (tau s) derived from the noise analysis is 300 ms. The low frequency limit of the pacemaker current noise (SI[0]), derived from the voltage noise and impedance, increases from 10(-20) A2/Hz . cm2 at -67 mV to 10(-19) A2/Hz . cm2 at -61 mV.     

Cellular interaction between mouse pancreatic alpha-cell and beta-cell lines: possible contact-dependent inhibition of insulin secretion

The endocrine cells in the pancreatic islet have cellular communication between the heterotypic cells as well as the homotypic cells. The present study was conducted to elucidate the cellular interaction between pancreatic alpha cells and beta cells utilizing differentiated mouse cell lines (i.e., alphaTC clone 6 and betaTC cells). Co-culture of these two cell lines on a gyratory shaker generated numerous cellular aggregates of homogenous size within 48 h. Immunohistochemical staining for insulin and glucagon demonstrated that betaTC cells were located in the central core of each aggregate, while alphaTC cells formed a mantle layer surrounding the betaTC cells. This segregation was observed regardless of the ratios of the two cell types employed. Although glucagon at concentrations of 10(-8) M or higher stimulated insulin secretion from betaTC cells in both monolayer and aggregates, an increase in the ratio of alphaTC/betaTC cells in aggregate cultures was accompanied by a decrease in secreted insulin and a rise in intracellular insulin content of the betaTC component. The inhibitory effect of alphaTC cells on betaTC insulin secretion was not limited to aggregate culture, since insulin secretion from betaTC cells was also suppressed, and intracellular insulin content increased, by co-culture of alphaTC with betaTC cells in monolayer. On the other hand, the secreted and intracellular insulin of betaTC cells was not affected by alphaTC cells in a Transwell co-culture system in which direct cell-to-cell contacts were prevented by a semipermeable membrane that permitted chemical communication via medium metabolites. These data suggest that the insulin secretion from betaTC cells may be inhibited possibly as a result of the contact with alphaTC cells.     

Hydraulic Permeability of Immobilized Bacterial Cell Aggregates

A dense aggregate of cells was retained in a reactor by a supported porous membrane. A continuous flow of nutrient medium was maintained through the cell aggregate and membrane. The hydraulic resistance of the cell aggregate was monitored throughout experiments with either growing or chemically cross-linked cells, under conditions of varying flow rates. Digital image analysis was used to characterize the sizes, separations, and orientations of several thousand individual cells in electron micrographs of chemically cross-linked cell aggregates. Two nonlinear phenomena were observed. First, the hydraulic resistance varied in direct relation to and reversibly with flow rate. Second, in constant flow-rate experiments the hydraulic resistance increased with time at a faster rate than could be attributed to cell growth. Both of these phenomena were dependent upon and could be explained by the ability of cells to move with respect to one another, under the influences of Brownian motion and of convection. Such relative motion could allow changes in net alignment of cells in the direction of flow and in the volume fraction of cells in the aggregate. This explanation is consistent with image analysis data. The observed sensitivity of hydraulic resistance to flow rate was inconsistent with a model that assumed elastic deformation of individual cells, and no evidence of cell deformation was found in electron micrographs.     

Direct measurements of the compressive properties of single proteoglycan aggregates

Proteoglycan aggregate is a major component of the extracellular matrix in articular cartilage and is considered to be responsible for the resistance to compression of this tissue. The reduced stiffness of articular cartilage due to the loss of proteoglycan aggregate has been reported in osteoarthritis. In order to understand the mechanical properties of extracellular matrix in articular cartilage at molecular level, the compressive properties of 36 single molecules of proteoglycan aggregate were directly measured using a laser tweezers/interferometer system. The proteoglycan aggregates showed resistance when compressed to approximately 30% of their contour length. The stiffness of proteoglycan aggregates increased non-linearly from 2.6+/-3.8 pN/microm (compressed to 30-35% of their contour length) to 115.5+/-30.9 pN/microm (compressed to 2.5-5% of their contour length).     

Isolation and characterization of the hemichrome-stabilized membrane protein aggregates from sickle erythrocytes. Major site of autologous antibody binding

Because the interaction of denatured hemoglobins (i.e. hemichromes) with the red cell membrane has been associated with several abnormalities commonly observed in hemichrome-containing erythrocytes, we have undertaken to isolate and characterize the hemichrome-rich membrane protein aggregates from sickle cells. The aggregates were isolated by two procedures: one at low ionic strength by centrifugation of detergent-solubilized spectrin-depleted inside-out vesicles, and the other at physiological ionic strength by detergent solubilization of whole cells followed by cytoskeletal disruption and centrifugation. The extensively washed aggregates obtained by both methods yielded similar results. These insoluble complexes were found to be highly cross-linked by predominantly intermolecular disulfide bonds; however, other nonreducible covalent linkages were also observed. Both in the presence and absence of reducing agents, the aggregate disintegrated when the hemichromes were removed by high ionic strength, suggesting that the aggregate depended heavily on the cohesive properties of the hemichromes for stability. Protein assays demonstrated that the aggregates comprised approximately 1.3% of the total membrane protein, roughly two-thirds of which appeared to be globin chains. Other major components identified in the aggregate were band 3, ankyrin, bands 4.1, 4.9, and 5, glycophorins A and B, and autologous IgG. Quantitative analysis of the IgG content demonstrated that three-fourths of the surface-bound IgG on washed sickle cells was clustered at these aggregate sites, representing an enrichment of approximately 250-fold over nonaggregated regions of the membrane. Since clustered cell surface IgG is thought to trigger removal of erythrocytes from circulation, the hemichrome-induced membrane reorganization at these aggregate sites may be an important cause of the greatly shortened life span of sickle cells.     

Embryonic myocardial cell aggregates: volume and pulsation rate

Spontaneously beating aggregates of myocardial cells from whole heart, atria, and apical portions of the ventricles were prepared by trypsin-dissociation and gyratory reaggregation of 4-, 7-, and 14-day-old chick embryo tissue. Pulsation rate and volume of aggregates were determined. The pulsation rate for a given volume aggregate decreased as the age of the donor embryo increased. Atrial aggregates of a given size beat faster than ventricular aggregates of the same size. However, in all cases the pulsation rate varied inversely with the aggregate volume. These results are not in agreement with the pacemaker concept as generally accepted, which predicts that a mass of heart cells would take on the pulsation rate of the fastest cell or cells within it. Differential composition of large and small aggregates was ruled out as a determining factor in the inverse rate-volume relationship. We suggest that (a) limited diffusion in large aggregates compared to small aggregates or (b) the larger total membrane capacitance of the electrically coupled cells of larger aggregates compared with that of smaller aggregates, plays a major role in setting the pulsation rate.     

Establishment of a peritubular myoid-like cell line and interactions between established testicular cell lines in culture

Established cell lines and primary cultures derived from somatic cells of the testis have been used to study cell-cell interactions. Primary cultures of Sertoli cells or Sertoli-derived cell lines from the mouse (TM4) and rat (TR-ST) will aggregate when plated on monolayers of primary cultures of peritubular myoid cells or a rat (TR-M) cell line which has many properties of peritubular myoid cells. Time-lapse cinematography and scanning and transmission electron microscopy reveal that Sertoli cells formed aggregates after 1 day in coculture, display surface activity and move on the monolayer. When these aggregates touch one another, they rapidly combine. By the 4th day of culture, spherical aggregates are composed of 50 to 200 cells. They do not display surface activity or movement on the myoid monolayer. On the 5th and 6th day of culture most spherical aggregates have flattened to form dome-shaped aggregates in close association with the monolayer. Cells in the aggregates are characterized by long microvilli and some ruffles. In large aggregates, cells sometimes form close associations within the aggregates although junctions are seldom observed. Sertoli-derived cell lines will not aggregate on monolayers of Leydig-derived (TM3) or testicular endothelial-derived (TR-1) cell lines. Neither TM3 nor TR-1 cells will aggregate when plated on myoid monolayers. The TR-M cells produced an extensive extracellular matrix beneath the cells which contains collagen, an amorphous globular material resembling elastin and a fibrous noncollagenous component. Sertoli cells plated on this matrix will not aggregate. Thus the aggregation of Sertoli cells on myoid cell monolayers is cell type, but not species dependent and not determined solely by extracellular matrix components produced by TR-M cells.     

Membrane currents in retinal bipolar cells of the axolotl

By whole-cell patch-clamping bipolar cells isolated from enzymatically dissociated retinae, we have studied the nonsynaptic ionic currents that may play a role in shaping the bipolar cell light response and in determining the level of voltage noise in these cells. Between -30 and -70 mV, the membrane current of isolated bipolar cells is time independent, and the input resistance is 1-2 G omega. Depolarization past -30 mV activates an outward current (in less than 100 ms), which then inactivates slowly (approximately 1 s). Inactivation of this current is removed by hyperpolarization over the range -20 to -80 mV. This current is carried largely by K ions. It is not activated by internal Ca2+. The membrane current of isolated bipolar cells is noisy, and the variance of this noise has a minimum between -40 and -60 mV. At its minimum, the standard deviation of the voltage noise produced by nonsynaptic membrane currents is at least 100 microV. The membrane currents of depolarizing bipolar cells in slices of retina were investigated by whole-cell patch-clamping. Their membrane properties were similar to those of isolated bipolar cells, but with a larger membrane capacitance and a smaller input resistance. Their membrane current noise also showed a minimum near -40 to -60 mV. The time-dependent potassium current in axolotl bipolar cells is not significantly activated in the physiological potential range and can therefore play little role in shaping the bipolar cells' voltage response to light. Differences in the waveform of the light response of bipolar cells and photoreceptors must be ascribed to shaping by the synapses between these cells. The noise minimum in the bipolar membrane current is near the dark potential of these cells, and this may be advantageous for the detection of weak signals by the bipolar cells.     

Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye

The electrical sheet resistance between living cells grown on planar electronic contacts of semiconductors or metals is a crucial parameter for bioelectronic devices. It determines the strength of electrical signal transduction from cells to chips and from chips to cells. We measured the sheet resistance by applying AC voltage to oxidized silicon chips and by imaging the voltage change across the attached cell membrane with a fluorescent voltage-sensitive dye. The phase map of voltage change was fitted with a planar core-coat conductor model using the sheet resistance as a free parameter. For nerve cells from rat brain on polylysine as well as for HEK293 cells and MDCK cells on fibronectin we find a similar sheet resistance of 10 MOmega. Taking into account the independently measured distance of 50 nm between chip and membrane for these cells, we obtain a specific resistance of 50 Omegacm that is indistinguishable from bulk electrolyte. On the other hand, the sheet resistance for erythrocytes on polylysine is far higher, at approximately 1.5 GOmega. Considering the distance of 10 nm, the specific resistance in the narrow cleft is enhanced to 1500 Omegacm. We find this novel optical method to be a convenient tool to optimize the interface between cells and chips for bioelectronic devices.     

Fluctuations in interbeat interval in rhythmic heart-cell clusters. Role of membrane voltage noise.

Small clusters of ventricular cells prepared from 7-d chick heart maintain spontaneous, stationary, rhythmic beating in culture for many hours. For clusters containing I-125 cells, mean interbeat interval (IBI) is 0.45 +/- 0.08 s and is independent of cell number (N), whereas, the coefficient of variation of IBI (C) is proportional to N-1/2. Because membrane voltage noise in such clusters would also be expected to vary as N-1/2, we propose a model relating fluctuation in IBI (sigma IBI) to voltage noise (sigma v). A simplified model consisting of random voltage fluctuations superimposed upon a linear pacemaker depolarization of slope a is used to analyze the N-dependent shape of the IBI histogram. Values of sigma v derived from the relation sigma IBI = sigma v/a, or calculated from the skewness of the measured IBI histograms, both agree well with those extrapolated from steady-state noise recorded from resting heart-cell aggregates.     

Effects of hyperthermia on spectrin expression patterns of murine lymphocytes

In this study the influence of whole-body hyperthermia on the distribution of spectrin in murine lymphocytes isolated from various lymphoid tissues is examined. Lymphocytes normally vary in terms of the pattern of spectrin distribution within the cell. In certain populations of lymphocytes, spectrin is distributed into a dense submembranous aggregate that can be easily identified by immunofluorescence microscopy. In these lymphocytes, little or no spectrin is seen at the plasma membrane region in the rest of the cell. Other lymphocytes have no such cytoplasmic aggregates, and the protein is seen at the region of the plasma membrane. Following whole-body hyperthermia (40.5 degrees C for 90 min) there is a 100% increase in cells exhibiting polar spectrin aggregates in the spleen, while lymphocytes from the thymus show no alteration in the number of cells showing such aggregates. The increase in the percentage of splenic cells that express aggregated spectrin is a result of increases occurring in both T- and B-cell subsets. This increase gradually returns to control levels by 48 h post-heating. During recovery to control levels this phenomenon is resistant to additional changes when a second heat treatment is applied. The effects described above are not observed when the experiments are performed in vitro; therefore, it is likely that the in vivo heat-induced alteration in the splenic lymphocyte population reflects the physiological response of lymphocytes to stimuli during a natural fever. The role that spectrin may play in the modulation of lymphocyte membrane properties is discussed.     

Changes in animal cell natural aggregates in suspended batch cultures

Some anchorage-dependent animal cells can form natural aggregates in stirred tanks. Baby hamster kidney (BHK) natural aggregates are described and characterized. Total cell concentration and viability could be obtained after aggregate mechanical aissociation, with negligible cell lysis and no change in cell membrane permeability. During a normal batch run, aggregates were formed immediately after inoculation, a few spherical aggregates increasing size during the initial growth phase. At the end of the growth phase, an increase in aggregate concentration was observed, without a considerable increase in aggregate diameter. At the end of the batch run, 160 h after inoculation, aggregates disintegrated into smaller, non-spherical units, following a sharp viability decrease. Cell concentrations of 1. 2 · 10⁶ cells/ml were obtained, with 60% of the total cells being in aggregates; the cell concentration in aggregates achieved 5 · 10⁸ cells/ml, with a porosity of 55%. Viability was consistently in the range 85–90%, both for aggregate and suspended cells.     

Physiological studies of chloramine resistance developed by Klebsiella pneumoniae under low-nutrient growth conditions.

This study investigated the physiological mechanisms of resistance to chloramines developed by Klebsiella pneumoniae grown in a nutrient-limited environment. Growth under these conditions resulted in cells that were smaller than cells grown under high-nutrient conditions and extensively aggregated. Cellular aggregates ranged from 10 to more than 10,000 cells per aggregate, with a mean population aggregate size of 90 cells. This aggregation may have been facilitated by the presence of extracellular polymer material. By using glucose as a reference of capsule content, it was determined that growth under low-nutrient conditions produced cells with 8 x 10(-14) to 41 x 10(-14) g of carbohydrate per cell, with a mean +/- standard deviation of 27 x 10(-14) +/- 16 x 10(-14) g of carbohydrate per cell. In comparison, growth under high-nutrient conditions resulted in 2.7 x 10(-14) to 5.9 x 10(-14) g of carbohydrate per cell, with a mean and standard deviation of 4.3 x 10(-14) +/- 1.2 x 10(-14) g of carbohydrate per cell. Cell wall and cell membrane lipids also varied with growth conditions. The ratio of saturated to unsaturated fatty acids in cells grown under low-nutrient conditions was approximately five times greater than that in cells grown under high-nutrient conditions, suggesting possible differences in membrane permeability. An analysis of sulfhydryl (-SH) groups revealed no quantitative difference with respect to growth conditions. However, upon exposure to chloramines, only 33% of the -SH groups of cells grown under low-nutrient conditions were oxidized, compared with 80% oxidization of -SH groups in cells grown under high-nutrient conditions. The reduced effectiveness of chloramine oxidization of -SH groups in cells grown under low-nutrient conditions may be due to restricted penetration of chloramines into the cells, conformational changes of enzymes, or a combination of both factors. The results of this study suggest that chloramine resistance developed under low-nutrient growth conditions may be a function of multiple physiological factors, including cellular aggregation and protection of sulfhydryl groups within the cell.     

Physiological studies of chloramine resistance developed by Klebsiella pneumoniae under low-nutrient growth conditions.

This study investigated the physiological mechanisms of resistance to chloramines developed by Klebsiella pneumoniae grown in a nutrient-limited environment. Growth under these conditions resulted in cells that were smaller than cells grown under high-nutrient conditions and extensively aggregated. Cellular aggregates ranged from 10 to more than 10,000 cells per aggregate, with a mean population aggregate size of 90 cells. This aggregation may have been facilitated by the presence of extracellular polymer material. By using glucose as a reference of capsule content, it was determined that growth under low-nutrient conditions produced cells with 8 x 10(-14) to 41 x 10(-14) g of carbohydrate per cell, with a mean +/- standard deviation of 27 x 10(-14) +/- 16 x 10(-14) g of carbohydrate per cell. In comparison, growth under high-nutrient conditions resulted in 2.7 x 10(-14) to 5.9 x 10(-14) g of carbohydrate per cell, with a mean and standard deviation of 4.3 x 10(-14) +/- 1.2 x 10(-14) g of carbohydrate per cell. Cell wall and cell membrane lipids also varied with growth conditions. The ratio of saturated to unsaturated fatty acids in cells grown under low-nutrient conditions was approximately five times greater than that in cells grown under high-nutrient conditions, suggesting possible differences in membrane permeability. An analysis of sulfhydryl (-SH) groups revealed no quantitative difference with respect to growth conditions. However, upon exposure to chloramines, only 33% of the -SH groups of cells grown under low-nutrient conditions were oxidized, compared with 80% oxidization of -SH groups in cells grown under high-nutrient conditions. The reduced effectiveness of chloramine oxidization of -SH groups in cells grown under low-nutrient conditions may be due to restricted penetration of chloramines into the cells, conformational changes of enzymes, or a combination of both factors. The results of this study suggest that chloramine resistance developed under low-nutrient growth conditions may be a function of multiple physiological factors, including cellular aggregation and protection of sulfhydryl groups within the cell.     

Supercharging accelerates T-tubule membrane potential changes in voltage clamped frog skeletal muscle fibers.

In voltage-clamp studies of single frog skeletal muscle fibers stained with the potentiometric indicator 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl] vinyl]pyridinium betaine (di-8 ANEPPS), fluorescence transients were recorded in response to both supercharging and step command pulses. Several illumination paradigms were utilized to study global and localized regions of the transverse tubule system (T-system). The rising phases of transients obtained from global illumination regions showed distinct accelerations when supercharging pulses were applied (95% of steady-state fluorescence achieved in 1.5 ms with supercharging pulses versus 14.6 ms with step pulses). When local transients were recorded at the edge of the muscle fiber, their kinetics resembled those of the applied waveform, but a similar relationship was not observed in transients from regions near the edge chosen to minimize the surface membrane contribution. We developed a model of the T-system capable of simulating membrane potential changes as a function of time and distance along the T-system cable and the associated fluorescence changes in regions corresponding to the experimental illumination strategies. A critical parameter was the access resistance term, for which values of 110-150 Omega.cm2 were adequate to fit the data. The results suggest that the primary mechanism through which supercharging pulses boost the kinetics of T-system voltage changes most likely involves their compensating the voltage attenuation across the access resistance at the mouth of the T-tubule.     

Hydrodynamic effects on BHK cells grown as suspended natural aggregates

Baby hamster kidney (BHK) cell aggregates grown in stirred vessels with different working volumes and impeller sizes were characterized. Using batch cultures, the range of agitation rates studied (25-100 rpm) led to aggregates with maximum sizes of 150 mum. Necrotic centers were not observed and cell specific productivity was independent of aggregate size. High cell viability was found for both single and adherent cells without an increase in cell death when agitation rate was increased. The increase in agitation rate affected aggregates by reducing their size and increasing their concentration and cell concentration in aggregates, while increasing the fraction of free cells in suspension. The experimental relationship between aggregate size and power dissipation rate per unit of mass was close to -1/4, suggesting a correlation with a critical turbulence microscale; this was independent of vessel scale and impeller geometry over the range investigated. Viscous stresses in the viscous dissipation subrange (below Kolmogoroff eddies) appear to be responsible for aggregate breakage. Under intense agitation BHK cells grown in the absence of microcarriers existed as aggregates without cell damage, whereas cells grown on the surface of microcarriers were largely reduced. This is a clear advantage for scaleup purposes if aggregates are used as a natural immobilization system in stirred vessels. (c) 1995 John Wiley & Sons, Inc.     

F9 embryonal carcinoma cells can differentiate into endoderm-like cells

The mouse teratocarcinoma cell line, F9, has been used in many laboratories as the epitome of the “nullipotent” embryonal carcinoma cell line. However, careful inspection of F9 cultures reveals the presence of small numbers of cells which possess several properties of endoderm, particularly parietal endoderm, and which can be shown to derive from the embryonal carcinoma component. Furthermore, tumors of F9 cells include isolated patches of endoderm-like cells surrounded by a thick secretion resembling Reichert's membrane. The proportion of endoderm-like cells in F9 cultures can be increased to varying degrees by causing the cells to form aggregates and/or maintaining them at high density for several days, although the endoderm-like cells produced in these ways contribute very little to the formation of subcutaneous tumors from the resultant mixed cultures. Differentiated cell types other than endoderm are rarely observed in F9 monolayer or aggregate cultures, even after several weeks. Cloning studies support the view that most, if not all, F9 cells can differentiate, albeit at very low incidence.     

Model of the mass transport to the surface of animal cells cultured in a rotating bioreactor operated in micro gravity

A mathematical model is used to investigate the transport of dissolved oxygen from the bulk fluid to the surface of aggregates of animal cells cultured in a rotating bioreactor. These aggregates move through different regions of the bioreactor with a local flow field and concentration distribution that vary with time. The time variation of the Sherwood number and the surface concentration for a range of parameters typical of a cell science experiment executed in the Rotating Wall Perfused Vessel (RWPV) bioreactor in space are investigated. The Reynolds numbers experienced by the aggregate are generally low (Re < 1) and the Peclet numbers range from O(1) to O(100). Comparison of the results from the numerical solution of the mathematical model with those from a quasi-steady model, using a steady-state correlation for mass transport on a sphere, indicate that the quasi-steady assumption is not a good model to compute the instantaneous Sherwood number. This indicates a significant history effect in the Sherwood number response to the variations of acceleration of the aggregates in the bioreactor. A high resistance to the mass transport from the bulk fluid to the surface of the aggregate exists for the bioreactor operated in micro gravity. The difference between the surface concentration and the free stream concentration was as high as 30% for aggregates larger than 3 mm. Diffusion reduces the variations of the free stream concentration resulting in a nearly constant value for the concentration at the surface of the aggregates.     

The factors that promote the development of symmetry properties in aggregates from dissociated echinoid embryos

Summary Embryos of Hemicentrotus pulcherrimus at the 16 cell, 400 cell or mesenchyme blastula stage of development were dissociated into single cells. The cells were reaggregated, and the development of individual aggregates was monitored. Only aggregates from 16 cell embryos developed into pluteus-like larvae with radial or bilateral symmetry. When embryos at these three developmental stages were incompletely dissociated so that there were mixtures of single cells and groups of undissociated cells, the percentage of aggregates from 16 cell embryos that developed in a pluteus-like manner was greater than in aggregates from completely dissociated 16 cell embryos. Also a small percentage of aggregates from 400 cell embryos now developed into pluteus-like larvae. In both of these experiments small aggregates tend to develop in a more normal manner than larger aggregates.In order to test the role of undissociated cells in promoting pluteus-like development in aggregates from incompletely dissociated blastula stage embryos, pieces of intact animal, lateral, or vegetal blastula wall were grafted to aggregates formed from completely dissociated embryos. While each kind of graft improved the ability of the aggregate to develop in a pluteus-like manner, grafts of vegetal blastula wall were most effective. In an aggregate, a graft differentiates according to its presumptive fate and influences the cells of the aggregate to differentiate in an appropriate manner. The ability of the graft to influence the development of the other cells in the aggregate depends on the developmental stage of the cells that make up the aggregate and the size of the aggregate.     

On the effect of prestin on the electrical breakdown of cell membranes

The voltage-dependent activity of prestin, the outer hair cell (OHC) motor protein essential for its electromotility, enhances the mammalian inner ear's auditory sensitivity. We investigated the effect of prestin's activity on the plasma membrane's (PM) susceptibility to electroporation (EP) via cell-attached patch-clamping. Guinea pig OHCs, TSA201 cells, and prestin-transfected TSA cells were subjected to incremental 50 mus and/or 50 ms voltage pulse trains, or ramps, at rates from 10 V/s to 1 kV/s, to a maximum transmembrane potential of +/-1000 mV. EP was determined by an increase in capacitance to whole-cell levels. OHCs were probed at the prestin-rich lateral PM or prestin-devoid basal portion; TSA cells were patched at random points. OHCs were consistently electroporated with 50 ms pulses, with significant resistance to depolarizing pulses. Although EP rarely occurred with 50 mus pulses, prior stimulation with this protocol had a significant effect on the sensitivity to EP with 50 ms pulses, regardless of polarity or PM domain. Consistent with these results, resistance to EP with depolarizing 10-V/s ramps was also found. Our findings with TSA cells were comparable, showing resistance to EP with both depolarizing 50-ms pulses and 10 V/s ramps. We conclude prestin significantly affects susceptibility to EP, possibly via known biophysical influences on specific membrane capacitance and/or membrane stiffness.