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.     

Lymphokine-mediated activation of a T cell-dependent IgA antipolysaccharide response

Immune responses to bacterial polysaccharides are important to host immunity at mucosal surfaces. We previously showed that BALB/c mice produce substantial T cell-dependent IgA responses to alpha (1,3) glucan determinants on the bacterial capsular polysaccharide dextran B1355. The data in this study demonstrate that the requirement for T cells for the activation of the IgA anti-alpha (1,3) dextran B1355 response can be replaced by T cell-derived nonantigen specific helper factors that appear to act during the late stages of B cell differentiation. Supernatants from the activated T cell lines cr-15 and (DL)C.C3.11.75, which contain interferon and late-acting T cell replacing factor activity, supported terminal differentiation of dextran-stimulated B cells to IgA anti-alpha (1,3) glucan antibody-forming cells and substantially increased IgM anti-alpha (1,3) glucan responses in culture. Although supernatants with interleukin 2 activity did not support optimal antigen-driven plaque-forming cell responses, they synergized with supernatants having interferon and T cell replacing factor activity in the production of IgA and IgM anti-alpha (1,3) glucan responses and IgM anti-SRBC responses. Supernatants from the T cell lines B6.11 and (DL)A.4 contained B cell growth factor activity but did not support activation of IgA anti-alpha (1,3) glucan PFC. These studies suggest that interferon and/or T cell replacing factor play an important role in the antigen-driven differentiation of B cells of the IgA and IgM isotypes to antibody-forming cells.     

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.      

An assay for beta-adrenergic receptors in isolated human fat cells

The beta-adrenergic receptors have been characterized in isolated human adipocytes using a potent beta-adrenergic antagonist (-)-[3H]dihydroalprenolol. Binding of (-)-[3H]dihydroalprenolol to isolated fat cells was stereospecific and saturable, the maximum number of binding sites calculated being 7.8 +/- 2.2 pmol of bound ligand/10(7) cells, corresponding to 450,000 binding sites/cell. The dissociation constant was estimated to be 2.7 +/- 1.1 nM. The results with competition-inhibition experiments using beta-adrenergic agonists and antagonists indicated that the binding sites in isolated adipocytes were predominantly of the beta1-subtype; about 80% of the receptors were of this type. With the present method, specific beta-adrenergic receptor number and affinity in isolated human adipocytes could be determined in about 1 g of human adipose tissue.     

Relationship between the tissue level of cyclic AMP and the fat cell size of human adipose tissue.

The relationship between mean fat cell size, maximal tissue cyclic AMP concentration, and glycerol release was investigated in human subcutaneous adipose tissue incubated in vitro with or without isoprenaline or noradrenaline added at maximal effective concentrations. Basal and stimulated glycerol release and cyclic AMP concentration were each related to the fat cell size. Whether or not the phosphodiesterase inhibitor theophylline was present in the incubation system, basal and noradrenaline-induced cyclic AMP levels were significantly correlated with the fat cell size. The noradrenaline-induced cyclic AMP levels resulted in twice as rapid glycerol release as could be expected from the basal ratio between glycerol release and cyclic AMP. Furthermore, both basal and noradrenaline-induced glycerol release in relation to the cyclic AMP levels were more rapid in enlarge fat cells. It is concluded that basal and catecholamine-induced production of cyclic AMP is related to the fat cell size and that a quantitative relationship exists between rate of lipolysis and maximal tissue levels of cyclic AMP in human adipose tissue. Basal and noradrenaline-induced lipolysis are probably regulated by different mechanisms and the lipolytic sensitivity to cyclic AMP seems increased in large fat cells.