Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

La structure logique de la contre-épreuve expérimentale

Résumé Claude Bernard a réservé à la contre-épreuve un rôle fondamental dans le raisonnement expérimental. Il a soutenu que la seule preuve qu'un phénomène joue le rôle de cause par rapport à un autre, c'est qu'en supprimant le premier on fait cesser le second. Cependant, la contre-épreuve expérimentale n'en reste pas moins une expérimentation; la structure logique des deux, preuve et contre-épreuve, est la même. Or, si la preuve exige la contre-épreuve, la dernière à son tour exige la première comme sa contre-épreuve. On arrive ainsi à une suite infinie d'expérimentations dont chaque membre se rapporte à l'autre comme soit à sa preuve, soit à sa contre-épreuve. Cependant, ce travail expérimental, qui s'arrête dans sa réalité à un moment choisi, il est vrai, plus ou moins arbitrairement, se poursuit dans la pensée de l'expérimentateur passant ainsi des résultats individuels à leur généralisation. Au sein du raisonnement expérimental il y a donc un raisonnement analogique et c'est le sentiment du déterminisme (Claude Bernard) et la pensée causale qu'il implique plutôt que le nombre des preuves et contre-épreuves qui surgissent comme les seuls critères de la vérité expérimentale.

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Summary Claude Bernard ascribed to the counter-proof a fundamental role in his experimental method and the reasoning on which the method rests. He saw the only proof of the causal nature of a given phenomenon in the fact that the effect fails to appear after elimination of the alleged cause. However, the experimental counter-proof still remains an experiment; the logical structure of both proof and counter-proof, is the same; it is true that the proof calls for a counter-proof; but the latter in its turn calls for the proof as for its counter-proof. One thus arives at an infinite chain of experiments, each link referring to each other as to its proof or counter-proof. This experimental work, nevertheless, comes to its end, though at a moment still chosen more or less arbitrarily; the work continues however in the thought of the experimenter, thus passing from individual results to generalization. There is embodied in the experimental method and in experimental thought a reasoning by analogy. The sense of determinism (sentiment du déterminisme:Claude Bernard) and the causal thought on which this sense rests rather than the number of proofs and counter-proofs emerge as the ultimate criteria of experimental truth.

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Zusammenfassung Claude Bernard hat dem experimentellen Gegenbeweis (oder Kontrollversuch) eine hervorragende Stellung in seiner experimentellen Methode und dem ihr zugrundeliegenden Denkvorgang eingeräumt. Seiner Überzeugung nach ist der einzige Beweis der ursächlichen Natur eines Faktors darin zu suchen, dass die Wirkung ausbleibt, wenn man den als Ursache angeschuldigten Faktor ausschaltet. Indessen ist auch der experimentelle Gegenbeweis nichts anderes und nicht mehr als ein Experiment. Beide, Experiment und Gegenexperiment, haben die gleiche logische Struktur. Wenn demnach jedes Experiment den Gegenbeweis verlangt, so bedarf auch der Gegenbeweis seinerseits des Beweises als seines Gegenbeweises. So gelangt man schliesslich zu einer unendlichen Kette von Experimenten, deren jedes Glied sich zu jedem anderen wie zu seinem Beweis oder Gegenbeweis verhält. Diese experimentelle Arbeit wird aber in einem mehr oder weniger willkürlich gewählten Augenblick abgebrochen; sie wird indessen in Gedanken vom Experimentalforscher fortgesetzt, der auf diese Weise schliesslich von den Einzelresultaten zu ihrer Verallgemeinerung schreitet. Innerhalb der experimentellen Methode trifft man also auf ein Denken nach Analogie. DerBernard-sche Kausal sinn (le sentiment du déterminisme) und das diesem zurgrundeliegende kausale Denken, nicht aber die Anzahl der experimentellen Beweise und Gegenbeweise sind es, welche die letzten Kriterien der experimentellen Wahrheit enthalten.

     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

ADP ribosylation of Arg41 of Rap by ExoS inhibits the ability of Rap to interact with its guanine nucleotide exchange factor, C3G

ExoS is a bifunctional type III cytotoxin that is secreted by Pseudomonas aeruginosa. The N-terminal domain comprises a RhoGAP activity, while the C-terminal domain comprises a ADP-ribosyltransferase activity. Previous studies showed that ExoS ADP ribosylated Ras at Arg41 which interfered with the ability of Ras to interact with its guanine nucleotide exchange factor. Rap and Ras share considerable primary amino acid homology, including Arg41. In this study, we report that ExoS ADP ribosylates Rap1b at Arg41 and that ADP ribosylation of Arg41 inhibits the ability of C3G to stimulate guanine nucleotide exchange. The mechanism responsible for this inhibition is one in which ADP-ribosylated Rap binds inefficiently to C3G, relative to wild type Rap. This identifies a second member of the Ras GTPase subfamily that can be ADP ribosylated by ExoS and indicates that ExoS can inhibit both Ras and Rap signaling pathways in eukaryotic cells.