Comparison of a lipophilic cation and microelectrodes to measure membrane potentials of the giant-celled algae,Chara australis (Charophyta) andGriffithsia monilis (Rhodophyta)

Summary The vacuolar equilibrium potential of the lipophilic cation TPMP⁺ (triphenyl methyl phosphonium) in the giant algaeChara australis andGriffithsia monilis was directly measured. The TPMP⁺ equilibrium potential was approximately 100mV less negative than the measured vacuolar electrical potential. Thus TPMP⁺ does not act as a probe of the vacuolar electrical potential and appears to be extruded against an electrochemical gradient. Measurement of the plasmalemma equilibrium potential of TPMP⁺ showed that extrusion of TPMP⁺ apparently occurred at both the tonoplast and plasmalemma inChara and at the plasmalemma inGriffithsia. It is concluded that TPMP⁺ cannot be used as a membrane potential probe inChara orGriffithsia.     

Binding of radioactively labeled saxitoxin to the squid giant axon

Summary The binding of saxitoxin, a specific inhibitor of the sodium conductance in excitable membranes, has been measured in giant axons from the squid,Loligo pealei. Binding was studied by labeling saxitoxin with tritium, using a solvent-exchange technique, and measuring the toxin uptake by liquid scintillation counting. Total toxin binding is the sum of a saturable, hyperbolic binding component, with a dissociation constant at 2–4°C of 4.3±1.7nm (meanse), and a linear, nonsaturable component. The density of saturable binding sites is 166±20.4 m^–2. From this density and published values of the maximum sodium conductance, the conductance per toxin site is estimated to be about 7 pS, assuming sequential activation and inactivation processes (F. Bezanilla & C.M. Armstrong, 1977,J. Gen. Physiol. 70: 549). This single site conductance value of 7 pS is in close agreement with estimates of the conductance of one open sodium channel from measurements of gating currents and of noise on squid giant axons, and is consistent with the hypothesis that one saxitoxin molecule binds to one sodium channel.     

The composition of intracellular granules from the metal-accumulating cells of the common garden snail (Helix aspersa).

Certain cells in the hepatopancreas of the common garden snail (Helix aspersa) contain intracellular granules that are sites of metal-ion accumulation. These granules have been extracted and investigated by u.v. and i.r. spectroscopy, atomic-absorption spectroscopy, X-ray microanalysis, thermogravimetric analysis, enzymic assay and microanalysis. The deposits contain about 18% (w/w) water, 5% (w/w) organic matter and 76% (w/w) inorganic material of which the main components are Ca2+, Mg2+ and P2O7(4)-. The possible origin of these granules is discussed, as is their role in detoxifying heavy-metal ions.     

Tumor necrosis factor induction of endothelial cell surface antigens is independent of protein kinase C activation or inactivation. Studies with phorbol myristate acetate and staurosporine.

We have investigated whether TNF-induced changes in human endothelial cell (EC) surface Ag expression are mediated by protein kinase C (PKC). This suggestion arose from the observations that PMA, a potent PKC activator, can mimic TNF by inducing expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1 (ICAM-1), and class I MHC molecules on human EC. However, in contrast to the actions of PMA, TNF neither causes membrane translocation of PKC nor induces the phosphorylation of the myristoylated alanine-rich C kinase substrate, two measures of PKC activation. Moreover, the PKC inhibitor staurosporine can block PMA-induced endothelial leukocyte adhesion molecule 1 expression at 4 h, but does not inhibit the actions of TNF. At 24 h, staurosporine itself induces intercellular adhesion molecule 1 and class I MHC, and acts additively with TNF. Twenty four hour treatment with PMA causes loss of PKC. We propose that at 24 h, staurosporine and PMA share a mechanism of action, namely diminution of PKC activity. However, 24 h treatment with TNF does not reduce the amount of PKC nor does it prevent activation of PKC by PMA. We conclude that TNF effects in EC are not mediated by PKC activation or inactivation.     

High frequency transformation of Streptomyces niveus protoplasts by plasmid DNA.

A procedure has been developed for transforming protoplasts of the novobiocin producing strain Streptomyces niveus at high frequency. This required the isolation of strains LH13 and LH20 defective in DNA restriction from the wild type (ATCC 19793) which is transformed at very low frequencies. The LH13 and LH20 derivatives were obtained by curing pIJ702 DNA from the few S. niveus transformed protoplasts obtained by transformation of the wild type with high concentrations of pIJ702 DNA. Protoplasts of S. niveus strains LH13 and LH20 produced about 10(6) transformants/micrograms DNA with modified pIJ702 DNA derived by replication in S. niveus. Unmodified DNA (derived from replication in S: lividans) from a series of pIJ101, SCP2 and pSN2-based derivatives, gave transformation frequencies in the range of 10(2)-10(3) transformants/micrograms DNA. Optimal conditions for the formation and transformation of S. niveus protoplasts are described.     

A comparison of rate and uniformity of embryo development in Meishan and European white pigs.

A comparison was made of the rate and uniformity of development of embryos recovered from Meishan and European white sows. The time of ovulation was estimated to be 34.3 and 49.0 h after the onset of oestrus in large white and Meishan sows, respectively. Embryos were recovered from a total of 38 Meishan and 37 European pigs between 18 and 219 h after the estimated time of ovulation. Embryos recovered after 18-59 or 44-82 h were classified into one of 11 stages (from early fertilization to early blastocyst), and the maximum blastocyst diameter was measured for embryos recovered 140-219 h after ovulation. There was no evidence of a difference between the genotypes in the stage or size of embryos at these times or of large differences between the genotypes in the extent of variation in embryo stage within females, although a minority of European white females had very variable embryos. As the differences between the embryos of the Meishan and the European white were small, it seems unlikely that greater uniformity of Meishan embryo development is a major cause of the higher prenatal survival in that breed.     

The initial heat production in garfish olfactory nerve fibres.

A study has been made of the temperature changes associated with the passage of a single impulse in the non-myelinated fibres of the garfish olfactory nerve: and the time course of these temperature changes has been compared with the time course of the electrical events during the action potential. As in other non-myelinated nerves studied the observed temperature changes result from a biphasic initial heat production consisting of a transient evolution of heat (the positive heat) followed by a rapid heat reabsorption (referred to as the negative heat). There is no evidence of any additional phases of initial heat production. At 0 degrees C the measured positive initial heat is 224 mucal/g impulse (937 muJ/g impulse); and the corresponding negative initial heat is 230 mucal/g impulse (962 muJ/g impulse). The residual initial heat is very small, being about -6 mucal/g impulse (-25 muJ/g impulse). In the range 0-10 degrees C there is no significant effect of temperature on the magnitude of either the positive or the negative phases of heat production. The experimental thermal records were analysed to determine the true time course of the temperature changes in the nerve undistorted by the recording system. The time course of the temperature changes does not fit with that of the transmembrane voltage change as represented by the monophasic compound action potential recorded externally from the same point on the nerve. A better fit is obtained if the temperature changes are compared with the square of the voltage change in accordance with the view that the heat derives almost wholly from free energy changes and entropy changes in the membrane capacity. The best fit is obtained if it is assumed that the membrane potential does not discharge to zero during the action potential but that at the peak of the action potential the charge (and hence the p.d.) across the membrane capacity retains about 24% of its resting value.     

Ionic properties of the acetylcholine receptor in cultured rat myotubes

The acetylcholine reversal potential (Er) of cultured rat myotubes is -3mV. When activated, the receptor is permeable to K+ and Na+, but not to Cl- ions. Measurement of Er in Tris+-substituted, Na-free medium also indicated a permeability to Tris+ ions. Unlike adult frog muscle the magnitude of Er was insensitive to change in external Ca++ (up to 30 mM) or to changes in external pH (between 6.4 and 8.9). The equivalent circuit equation describing the electrical circuit composed of two parallel ionic batteries (EK and ENa) and their respective conductances (gK and gNa), which has been generally useful in describing the Er of adult rat and frog muscle, could also be applied to rat myotubes when Er was measured over a wide range of external Na+ concentrations. The equivalent circuit equation could not be applied to myotubes bathed in media of different external K+ concentrations. In this case, the Er was more closely described by the Goldman constant field equation. Under certain circumstances, it is known that the receptor in adult rat and frog muscle can be induced to reversibly shift from behavior described by the equivalent circuit equation to that described by the Goldman equation. Attempts to similarly manipulate the responses of cultured rat myotubes were unsussessful. These trials included a reduction in temperature (15 degress C), partial alpha-bungarotoxin blodkade, and activation of responses with the cholinergic agonist, decamethonium.