Slow potential changes due to transport number effects in cells with unstirred membrane invaginations or dendrites

Summary Many neurones are extremely invaginated and possess branching processes, axons and dendrites. In general, they are surrounded by a restricted diffusion space. Many of these cells exhibit large, slow potential changes during the passage of current across their membranes. Whenever currents cross membranes separating aqueous solutions, differences in transport numbers of the major permeant ions give rise to local concentration changes of these ions adjacent to the membranes, which will result in various electrical and osmotic effects. These transport number effects are expected to be enhanced by the presence of membrane invaginations. Dendrites are equivalent to reversed invaginations and there should be significant changes in concentrations of permeant ions within them. In general, the effects of such changes on the electrical response of a cell will be greater when the concentration of a major permeant ion is low. The effects have been modelled in terms of two nondimensional parameters: the invagination transport number parameter and the relative area occupied by the invaginations A. If these two parameters are known, the magnitudes and time course of the slow potential changes can immediately be estimated and the time course converted to real time, if the length of the invaginations (l) and ionic diffusion coefficient (D) within them are also known. Both analytical and numerical solutions have been given and predictions compared. It is shown that in the case of large currents and potentials the analytical solution predictions will underestimate the magnitudes and rates of onset of the voltage responses. The relative magnitude of the transport number effect within the invaginations (or dendrites) and other transport number contributions to slow potential changes have also been assessed and order-of-magnitude values of these are estimated for some biological data.     

Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range

The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin.     

Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence

We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation, extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95% of the probe is specifically attached to sulfhydryl-1 (SH1) on the myosin head. In a solution of labeled subfragment-1 (S-1) at 4 degrees C, absorption anisotropy at 0.1 microseconds after a laser pulse is about 0.27. This anisotropy decays exponentially with a rotational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-1, and with times determined previously by fluorescence and electron paramagnetic resonance. In aqueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range. Furthermore, binding to actin greatly restricts probe motion. Thus the bound eosin is a reliable probe of myosin-head rotational motion in the submicrosecond and microsecond time ranges. Our submicrosecond data for myosin monomers (correlation time 400 ns) also agree with previous results using other techniques, but we also detect a previously unresolvable slower decay component (correlation time 2.6 microseconds), indicating that the faster motions are restricted in amplitude. This restriction is not consistent with the commonly accepted free-swivel model of S-1 attachment in myosin. In synthetic thick filaments of myosin, both fast (700 ns) and slow (5 microseconds) components of anisotropy decay are observed. In contrast to the data for monomers, the anisotropy of filaments has a substantial residual component (26% of the initial anisotropy) that does not decay to zero even at times as long as 50 microseconds, implying significant restriction in overall rotational amplitude. This result is consistent with motion restricted to a cone half-angle of about 50 degrees. The combined results are consistent with a model in which myosin has two principal sites of segmental flexibility, one giving rise to submicrosecond motions (possibly corresponding to the junction between S-1 and S-2) and the other giving rise to microsecond motions (possibly corresponding to the junction between S-2 and light meromyosin).(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormone-responsive cells derived from human dental papilla: Characterization in vitro and in vivo in diffusion chambers

Summary Cells of the dental papilla are capable of odontoblastic, fibroblastic, and endothelial differentiation and formation of dentin and the dental pulp. In the present study dental papilla cells, obtained from human tooth buds (HDP cells), were cultured in vitro through 3 to 7 passages. After exposure to prostaglandin E₂ there was a marked decrease in intracellular cyclic AMP (cAMP) levels as compared to hormone-free controls. Parathyroid hormone and calcitonin had stimulatory effects with 1 and 2 log increases in cAMP, respectively. The HDP cells showed moderate activity of alkaline phosphatase, 1 log higher than that of hamster kidney fibroblasts (BHK 13) and 1 log lower than that of osteoblastic osteosarcoma cells (ROS 17/2). When cultured for 4 or 8 wk in diffusion chambers (DC) implanted in athymic mice, many of the HDP cells underwent odontoblastic morphodifferentiation with very long, single processes extending into the matrix. This matrix contained banded and unbanded collagen fibers. Neither light nor electron microscopy of the DC content revealed mineral deposits. These results suggest that HDP cells have an intrinsic potential for partial odontoblastic differentiation; inductive signals like those originating from odontogenic epithelium are probably essential for the completion of hard tissue formation.     

A moving boundary model of acrosomal elongation

A sperm penetrates an egg by extending a long, actin-filled tube known as the acrosomal process. This simple example of biomotility is one of the most dramatic. In Thyone, a 90 m process can extend in less than 10 s. Experiments have shown that actin monomers stored in the base of the sperm are transported to the growing tip of the acrosomal process where they add to the ends of the existing filaments.The force that drives the elongation of the acrosomal process has not yet been identified although the most frequently discussed candidate is the actin polymerization reaction. Developing what we believe are realistic moving boundary models of diffusion limited actin fiber polymerization, we show that actin filament growth occurs too slowly to drive acrosomal elongation. We thus believe that other forces, such as osmotically driven water flow, must play an important role in causing the elongation. We conjecture that actin polymerization merely follows to give the appropriate shape to the growing structure and to stabilize the structure once water flow ceases.Work partially supported by the United States Department of Energy     

Aminoacyl-tRNA synthetases: inter-site interaction as a possible proofreading mechanism

Smooth muscle cell energetics of taenia caeci during relaxation, activity and maximal contraction were investigated using ³¹P-NMR. In relaxed muscle obtained in calcium-free medium, [ATP], [phosphocreatine] and [sugar phosphate] were 4.4 mM, 7.7 mM and 2.8 mM, respectively. There was only a small difference in the energetics of spontaneously active and maximally contracted muscles, but under both conditions substantial changes occurred as compared with relaxed muscles. The internal pH in relaxed muscle was found to be 7.05, which acidified to 6.5 during contraction. The level of sugar phosphates was found to be not a limiting factor in energetics.     

Diffusion du15N2 dans le sol pendant la mesure de fixation biologique de l'azote

Summary The kinetic of¹⁵N₂ diffusion has been measured in a system similar to that for the estimation of N₂ fixation in plant microorganism associations cultivated in soil. The¹⁵N₂ enrichment of the soil atmosphere reached an homogenous value one hour after injection of¹⁵N₂ and is identical to that obtained by calculation, indicating that no adsorption occurs in the soil particles.

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Diffusion du¹⁵N₂ dans le sol pendant la mesure de fixation biologique de l'azote

Résumé La cinétique de diffusion du¹⁵N₂ est mesurée sur un système identique à ceux pouvant être utilisés pour la mesure de fixation de l'azote chez les associations plantes-microorganismes cultivées sur sol. L'enrichissement homogène de l'atmosphère du sol est obtenu une heure environ après l'injection de¹⁵N₂ et correspond à l'enrichissement calculé, ce qui indique qu'aucune adsorption n'a lieu dans les particules du sol.

     

Physical conditions of propagation media and their influence on the rooting of cuttings

Summary The formation and subsequent growth of roots by cuttings of poinsettia, hydrangea, rose and azalea in various propagation media, Jiffy-7, Jiffy-9 and Grodan under different conditions of aeration was investigated. The interrelationships of the effects of air content of the media, temperature and light intensity on the rooting of poinsettia cuttings was also studied.With low air contents (0 cm moisture tension) in the propagation media the formation and growth of roots was strongly inhibited. The rooting performance of rose appeared to be less affected by the poor aeration. Increasing air content improved rooting but best results were obtained at moisture tensions of 4 to 8 cm. Rooting seems to be better correlated with oxygen diffusion rate (ODR) than with air content.For poinsettia cuttings the optimum temperature for rooting was 24 to 28°C. At low temperatures rooting was delayed while at higher temperatures it was almost completely inhibited. Callus formation increased with temperature but decreased with increasing moisture tension. Conditions which induced large callus formation inhibited root formation.High light intensity during rooting reduced overall rooting performance and the inhibition was most pronounced in conjunction with high moisture tensions.Report No. 255.