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.      

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.      

Natural killer cells in mice and man and their possible biological significance

Conclusions Despite active and extensive research on human and murine NK cells in the last few years a number of basic questions remain to be answered. These include the nature of the antigen(s) on target cells to which NK cells react, the nature of the receptors on NK cells allowing the recognition, the mechanism of NK cell cytotoxicity, the exact relationship between NK cell activities in vivo and in vitro, genetic control of NK cells, involvement of NK cells in the field of bone marrow transplantation, and finally their involvement in immunosurveillance of malignancies. Although the etiology and biologic significance of NK cells remain obscure, there is suggestive evidence that these cells may be in volved in resistance to and immunosurveillance of malignancies, since there is experimental evidence for a correlation between in vivo and in vitro NK cell activities. The fact that athymic mice do not have a higher incidence of malignancies than conventional mice and express delayed appearance and higher resistance to virally and chemically induced tumors (Stutman, 1975) is consistent with a protective role of the NK cell effector mechanism. In addition, their spontaneous occurrence without any necessity for priming and their ability to recognize and kill malignant cells makes NK cells almost perfect candidates for the part of immunosurveillors. The similarity between bone marrow effector cells and NK cells also seems unlikely to be fortuitous. In summary, this interesting cell requires further attention.     

Segregation analyses of 1,476 population-based Australian families affected by prostate cancer

Segregation analyses aim to detect genetic factors that have a major effect on an individual's risk of disease and to describe them in terms of mode of inheritance, age-specific cumulative risk (penetrance), and allele frequency. We conducted single- and two-locus segregation analyses of data from 1,476 men with prostate cancer diagnosed at age <70 years and ascertained through population registries in Melbourne, Sydney, and Perth, Australia, and from their brothers, fathers, and both maternal and paternal lineal uncles. Estimation and model selection were based on asymptotic likelihood theory and were performed through use of the software MENDEL. All two-locus models gave better fits than did single-locus models, even if lineal uncles were excluded or if we censored data (age and disease status) for relatives at 1992, when prostate-specific-antigen testing started to have a major impact on the incidence of prostate cancer in Australia. Among the genetic models that we considered, the best-fitting ones included a dominantly inherited increased risk that was greater, in multiplicative terms, at younger ages, as well as a recessively inherited or X-linked increased risk that was greater, in multiplicative terms, at older ages. The recessive and X-linked effects were strongly confounded, and it was not possible to fit them together. Penetrance to age 80 years was approximately 70% (95% confidence interval [CI] 57%-85%) for the dominant effect and virtually 100% for the recessive and X-linked effects. Approximately 1/30 (95% CI 1/80-1/12) men would carry the dominant risk, and 1/140 (95% CI 1/220-1/90) would carry the recessive risk or 1/200 (95% CI 1/380-1/100) would carry the X-linked risk. Within discussed limitations, these analyses confirm the genetic heterogeneity, of prostate cancer susceptibility, that is becoming evident from linkage analyses, and they may aid future efforts in gene discovery.