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Biomedical subjects

L F Jaffe

Publications and source records attributed to L F Jaffe.

17 recordsLinked to original sources

Strong electrical currents leave the primitive streak of chick embryos.

The electrical fields above chick embryos were explored with a vibrating probe. These fields indicate that steady currents with exit densities of the order of 100 microamperes per square centimeter leave the whole streak and return elsewhere through the epiblast. The epicenter of these strong exit currents lies near Hensen's node. They are probably pumped into the intraembryonic space by the epiblast and then leak out of the streak because it is a zone of junctional disruption.

Animals

Role of subdermal current shunts in the failure of frogs to regenerate.

Large, uniform, skin-driven currents (20-40 muamp/cm2) leave the ends of limb stumps of post-metamorphic frogs (Rana pipiens) from about the first through the tenth day after amputation. However, right after amputation, while currents of comparable density may leave the periphery of the cut surface, current densities are greatly depressed in the center of this surface. We suggest that this depression is brought about by shunting through the subdermal lymph space (characteristic of anurans but not urodeles); continues in covert form after formation of a wound epithelium; and helps explain the ability of small, imposed currents to initiate frog limb regeneration.

Action Potentials

Neurites grow faster towards the cathode than the anode in a steady field.

We explanted fragments of embryonic chick dorsal root ganglia on to polylysine coated glass and cultured them in a medium containing one unit of nerve growth factor plus enough methylcellulose to give viscosities from 0.01-3,000 poise. We allowed them to grow out in the absence of a field, and then selected explants with halos of neurites which were relatively dense, relatively symmetrical, and practically free of glial cells. These selected explants were then exposed to electrical fields of up to 140 mV/mm for some hours. In media with viscosities of one poise or less, the field some times dragged the central cell mass of an explant towards the anode. However, in cases where the central cell mass did not move, fields of 70-140 mV/mm induced that sector of each neurite halo which faced the cathode to grow out several times faster than the one facing the anode.

Animals

Reduction of sodium dependent stump currents disturbs urodele limb regeneration.

We have asked the question whether the natural electric currents which leave urodele limb stumps are in any way needed for their regeneration. As an initial test, we have greatly reduced such currents in the tiger salamander, Ambystoma tigrinum, by applying 0.5 mM amiloride to the stump skin or by immersion of the animals in sodium depleted media. We have also reduced such currents in the red spotted newt, Notophthalmus viridescens, by such immersion. Limb regeneration in half of the amiloride-treated animals was either entirely blocked or grossly deficient, while the others regenerated normally. Limb regeneration in sodium depleted media was consistently inhibited for some weeks but then recovered. These results are consistent with the hypothesis that stump currents are in some way needed for normal regeneration.

Ambystoma

Electrophoresis along cell membranes.

Bioelectric fields may segregate charged components floating in the plasma membranes of cells by a process of electrophoresis along the membrane. Molecules in cell membranes may be sorted to different portions of the cell surface by such electrical gradients. We present here a theory to support this hypothesis.

Cell Membrane

Bioelectricity and regeneration: large currents leave the stumps of regenerating newt limbs.

Electrical currents near regenerating newt limbs were measured with a recently developed vibrating probe. Steady currents with local surface densities of 10 to 100 muA/cm2 or more leave the end of the stump during the first 5-10 days after amputation and are balanced by currents with densities of only 1-3 muA/cm2 that enter the intact skin around the stump. They are immediately dependent upon the entry of sodium ions into this skin and are therefore inferred to be skin-driven. The outward currents are comparable in direction, density, duration, and position to artificially imposed currents previously found sufficient to induce significant regeneration of amputated adult frog limbs. This comparison suggests that the endogenous stump currents play some causal role in initiating regeneration.

Amiloride

Free calcium increases explosively in activating medaka eggs.

We have used the calcium-specific light-emitting protein aequorin to follow changes in free calcium concentration during fertilization and cleavage of eggs from medaka, a fresh-water fish. Aequorin-injected medaka eggs show a very low resting glow before they are fertilized, indicating a low calcium concentration in the resting state. Upon activation by sperm, the calcium-mediated light emission increases to a level some 10,000 times the resting level with a 1 to 2 sec time constant for an e-fold increase, and then slowly retruns to the resting level. Upon activation by the ionophore A23187, the early rise in luminescence is much slower, but once a threshold has been reached the subsequent rise becomes as rapid as the normal sperm-induced response. We infer that the explosive rise in calcium involves calcium-stimulated calcium release, and that a sperm normally triggers this rise by somehow inducing a more modest and localized rise in calcium.

Aequorin

Relations between ameboid movement and membrane-controlled electrical currents.

We have studied the pattern of electrical currents through amebas (mainly Chaos chaos) with an ultrasensitive extracellular vibrating probe. Amebas drive both steady currents and current pulses through themselves. Relatively steady current with an average surface density of 0.1-0.2 muA/cm2 enters the rear quarter of an ameba and leaves its pseudopods. Streaming reversals are preceded by changes in this current pattern and the region with the largest new inward current becomes the new tail. Ion substitution studies suggest that some of the steady inward current is carried by calcium ions. Characteristic stimulated pulses of current sometimes follow the close approach of the vibrating probe to the side of an advancing pseudopod. Such a pulse enters the cytoplasm through a small patch of membrane near the probe (and seems to leave through the adjacent membrane), is usually followed by hyaline cap and then by pseudopod initiation, is calcium dependent, lasts about 5-10 s, and has a peak density of about 0.4 muA/cm2. Spontaneous pulses of similar shape and duration may enter or leave any part of an animal. They are much less localized, tend to have higher peak densities, and occur in physiological salt solutions at about 0.2-4 times per minute. Retraction of a pseudopod is always accompanied or preceded by a spontaneous pulse which leaves its sides.

Amoeba

A simple, selective method for freeze-fracturing spherical cells.

A simple and selective method for freeze-fracturing spherical cells is described. The cells are loaded into the holes of a thin nickel screen. A metal hat is applied to the cell monolayer and the whole assembly, hat-cells-screen, is frozen and then fractured by ripping the hat off. The fractured face on the screen is replicated. By varying the size of the screen holes and by applying the hat to either side of the screen, this method can selectively expose the E face (or the outer half of plasma membrane), the P face (or the inner half of the plasma membrane), or the cytoplasm of the cells. It also provides a means to produce fractures at a preselected area on the cell, if the cells can be loaded onto the screen in an oriented fashion.

Cell Membrane

Polarizing fucoid eggs drive a calcium current through themselves.

Calcium ions enter the prospective growth pole of polarizing Pelvetia eggs faster than the opposite pole and leave this antipode faster than the growth pole. The calcium current is greatest when first measured at 6 hours after fertilization and decreases as the time of final commitment to growth in a particular direction approaches.

Biological Transport, Active

The pulse current pattern generated by developing fucoid eggs.

Using a newly developed extracellular vibrating electrode, we have made the first study of the spatial distribution of the growth currents around a single developing egg. This pattern was studied during the current pulses wihic traverse two-celled Pelvetia embryos. These pulses can be stimulated to occur with a periodicity of 70 min by mild acidification of the dea water medium. Current enters only at the growing rhizoid's tip while leaving both the base of the rhizoid cell and the whole outer membrane of the thallus cell. The field in front of the rhizoid cell falls off as the inverse cube of the distance from the rhizoid cell's center in the manner of a dipole field. The total inward and outward currents are equal, agreeing with theory. The current density at the rhizoid cell's base is twice that at the top of the thallus cell and this probably represents a change in the outer membrane's properties. There are no significant differences in the durrent density over the thallus cell. These results suggest a model in which the pulse current leaks in through newly opened channels in the growing tip and leaks out elsewhere due to the resultant fall in the membrane potential.

Electrodes

Large electrical currents traverse growing pollen tubes.

Using a newly developed vibrating electrode, we have explored the electric fields around lily pollen germinating in vitro. From these field measurements, we infer that each weeted pollen drives a steady current of a few hundred picoamperes through itself. Considered as a flow of positive ions, this current enters an ungerminated grain's prospective growth site and leaves it opposite end. After a grain germinates and forms a tube, this current enters most of the growing tube and leaves the whole grain. The current densities over both of these extended surface regions are relatively uniform, and the boundary zone, near the tube's base, is relatively narrow. This current continues as long as the tube grows, and even continues when elongation, as well as cytoplasmic streaming, are blocked by 1 mug/ml of cytochalasin B. After a otherwise indistinguishable minority of tubes have grown to lengths of a millimeter or more, their current comes to include an endless train of discrete and characteristic current pulses as well as a steady component. These pulses are about 30s long, never overlap, recur every 60-100s, and seem to enter a region more restricted to be growing tip than the steady current's sink. In most ways, the current through growing lily pollen resembles that known to flow through focoid eggs.

Cytochalasin B