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ABSTRACTIn a search for tools to distinguish antigenic variants ofEhrlichia ruminantium, we sequenced the major antigenic protein genes (map1genes) of 21 different isolates and found that the sequence polymorphisms were too great to permit the design of probes which could be used as markers for immunogenicity. Phylogenetic comparison of the 21 deduced MAP1 sequences plus another 9 sequences which had been previously published did not reveal any geographic clustering among the isolates. Maximum likelihood analysis of codon and amino acid changes over the phylogeny provided no statistical evidence that the gene is under positive selection pressure, suggesting that it may not be important for the evasion of host immune responses.

1. The electrical properties of squid giant axons were examined by means of admittance bridges at frequencies from 0.5 to 300 kHz. A simple equivalent circuit was used to estimate the membrane capacity. 2. The calculated membrane capacities decreased monotonically over the whole frequency range. 3. At 100 kHz and higher frequencies the membrane capacity was independent of potential. 4. At frequencies greater than 20 kHz, exposure of the axons to saturated or 0.9 saturated solutions of n‐pentane (275‐306 micrometer) reduced the capacity per unit area by 0.1‐0.15 micro F cm‐2. 5. At 1 kHz the effect of the saturated pentane solutions depended on the membrane potential. In axons having potentials between ‐60 and zero mV the pentane solutions lowered the capacity, whereas for potentials between ‐160 and ‐60 mV they produced little or no change. 6. Saturated solutions of n‐hexane, n‐heptane and n‐octane exhibited qualitatively similar, but quantitatively smaller influences on the membrane capacity, the changes declining as the chain length increased. 7. Under voltage clamp, the peak inward and steady‐state outward currents were partially suppressed by the hydrocarbons. Saturated solutions of n‐pentane usually reduced the former (reversibly) by 60‐80% and the latter by 20‐40%. Solutions of n‐hexane, n‐heptane and n‐octane appeared to have successively less effect. Except in deteriorating axons, none of the hydrocarbons produced any consistent changes in the passive membrane resistance, the resting potential or in the reversal potential of the transient inward current. 8. Both the changes in the clamp currents and in the membrane capacity were largely, though not usually completely, reversible. In the hydrocarbon solution the axons deteriorated more rapidly than normal. 9. The responses of axons of Doryteuthis plei to the hydrocarbons were very similar to those of Loligo forbesi with the exception that for the former all observed changes were some five times faster. 10. The time courses of the peak inward and steady‐state outward currents on exposure of the axons to n‐pentane resembled the time course of the change in membrane capacity at 100 kHz. 11. The simplest interpretation of the high frequency capacity results is suggested to be that, as for lipid bilayers, the membranes become thicker through adsorption of the hydrocarbon.

1. Sodium and potassium currents have been recorded in intracellularly perfused squid giant axons before, during and after exposure to solutions of n‐pentane in artificial sea water. 2. The currents were fitted with equations similar to those proposed by Hodgkin & Huxley (1952) and the changes in the parameters of these equations in the presence of pentane were calculated. 3. In the range of membrane potential ‐40 to 40 mV, the time constants for activation (tau m) and inactivation (tau h) of the Na current, and for activation (tau n) of the K current were all reduced by the pentane. 4. The curve of the steady‐state inactivation parameter (h infinity) for the Na current against membrane potential was shifted by the pentane in a hyperpolarizing direction (at h infinity = 0.5 this shift was approx. ‐15 mV in 275 microM‐pentane) and the slope at all potentials was reduced. 5. The curve of the steady‐state activation parameter (m infinity) for the Na current against membrane potential was also shifted by the pentane in a hyperpolarizing direction (in 153 microM‐pentane, 10 mV at m infinity = 0.5). 6. The maximum Na and K conductances gNa and gK were lowered by the pentane, though not usually completely reversibly. 7. The changes in position and slope of the steady‐state inactivation curve have been tentatively accounted for in terms of an increase in membrane thickness.

1. The anaesthetic effects of aqueous solutions of the n‐alkanes pentane to nonane on the propagated action potential of squid axons have been investigated for a range of axon diameters. 2. By the use of small axons (approx. 200 microns diameter) to minimize effects due to long diffusion times and alkane depletion it was found that n‐pentane and n‐hexane caused a rapid reversible inhibition of the impulse, while higher homologues had progressively less effect, n‐nonane being apparently inert. 3. The rate of action potential decline due to the n‐alkanes was found to be strongly dependent on axon diameter. For n‐hexane, n‐heptane and n‐octane the rate of decline was inversely proportional to the square of the axon diameter. 4. The mechanisms which may underly the increased sensitivity of small axons to impulse blockade by n‐alkanes are discussed. A quantitative comparison is made between the effects of n‐hexane, n‐heptane and n‐octane on the action potential. It is argued that this supports the idea of a real decline in anaesthetic potency on ascending the homologous series, rather than an effect due to long diffusion times and solution depletion.

The effects of the n‐alkanes propane to hexane, cyclopropane, cyclopentane and cyclohexane and carbon tetrachloride on the ionic currents and electrical capacity of the squid giant axon membrane have been examined. Both the peak inward and steady‐state outward currents were reduced reversibly by each substance, though propane at 1 atm had very little effect. The membrane capacity at 100 kHz was reduced by all substances except propane at 1 atm. Na currents were recorded in intracellularly perfused axons before and during exposure to the hydrocarbons and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the curves of the steady‐state activation and inactivation parameters (m infinity and h infinity) against membrane potential, changes in the peak heights of the activation and inactivation time constants (tau m and tau h) and reductions in the maximum Na conductance (gNa) have been tabulated. The effects of the various hydrocarbons and carbon tetrachloride on the parameters of the Hodgkin‐Huxley equations suggest that the suppression of the Na current by these substances originates from several different phenomena. The underlying physico‐chemical events are considered in the light of the observed capacity changes and of information on artificial pore‐containing membranes.

The effects of several n‐alkanols and n‐alkyl oxyethylene alcohols, methyl octanoate, glycerol 1‐monooctanoate and dioctanoyl phosphatidylcholine on the ionic currents and electrical capacity of the squid giant axon membrane have been examined. The peak inward current in voltage‐clamped axons was reduced reversibly by each substance. For n‐pentanol to n‐decanol the concentrations required to suppress the peak inward current by 50% were determined. From these data, it was estimated that the standard free energy per CH2 for adsorption to the site of action was ‐3.04 kJ mole‐1, as compared with ‐3.11 kJ mole‐1 for adsorption into phospholipid bilayers or an n‐alkane/aqueous solution interface. The membrane capacity at 100 kHz was not greatly by any of the test substances at concentrations which reduced the inward current by 50%. Na currents under voltage clamp were recorded in intracellularly perfused axons before, during and sometimes after exposure to the test substances and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the curves of the steady‐state activation and inactivation parameters (m infinity and h infinity) against membrane potential, changes in the peak heights of the activation and inactivation time constants (tau m and tau h) and reductions in the maximum Na conductance (gNa) have been tabulated. All of the test substances shifted the voltage dependence of the steady‐state activation in the depolarizing direction and lowered the peak time constants for both activation and inactivation. The origins of these effects, and of the differences in the present results from those of the hydrocarbons (Haydon & Urban, 1983), have been discussed in terms of the physico‐chemical properties of the two groups of substances and with reference to their effects on artificial membranes.