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.     

A Bacillus subtilis plasmid that can be packaged as single-stranded DNA in Escherichia coli: use for oligodeoxynucleotide-directed mutagenesis

A Bacillus subtilis/Escherichia coli shuttle vector was modified to contain the origin of DNA replication of the E. coli filamentous phage f1, in both orientations. Upon superinfection with and f1-related phage of an E. coli strain containing either of the modified vectors, the single-stranded (ss) form of the plasmid was packaged in virions and released to the culture medium. Each of these ss DNAs has been purified from the virions and used as a template for oligodeoxynucleotide-directed mutagenesis. The resulting mutations were demonstrated by DNA sequencing. The capacity of these vectors to be isolated as phage ss DNA from E. coli and to replicate as plasmids in B. subtilis makes them convenient substrates for the production of oligodeoxynucleotide-directed mutations for studies in B. subtilis.     

Catabolite repression of the synthesis of inducible polygalacturonase and pectinesterase byAspergillus niger sp.

Several recent reports have described large numbers of monoclonal antibodies that cross-react with toxins A and B ofClostridium difficile; this suggests that the toxins share major epitopes. Our results show that monoclonal antibodies (MAb) against other antigens bind nonspecifically to both toxins. Therefore, we believe that the cross-reacting MAb bind by this manner and not by a true immune reaction.     

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.      

Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.

Activation of the cell surface receptor Fas/APO-1 (CD95) induces apoptosis in lymphocytes and regulates immune responses. The cytoplasmic membrane protein Bcl-2 inhibits lymphocyte killing by diverse cytotoxic agents, but we found it provided little protection against Fas/APO-1-transduced apoptosis in B lymphoid cell lines, thymocytes and activated T cells. In contrast, the cowpox virus protease inhibitor CrmA blocked Fas/APO-1-transduced apoptosis, but did not affect cell death induced by gamma-radiation or serum deprivation. Signalling through Fas/APO-1 did not down-regulate Bcl-2 or induce its antagonists Bax and Bcl-xS. In Fas/APO-1-deficient lpr mice, Bcl-2 transgenes markedly augmented the survival of antigen-activated T cells and the abnormal accumulation of lymphocytes (although they did not interfere with deletion of auto-reactive cells in the thymus). These data raise the possibility that Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.     

Uptake, release and metabolism of docosahexaenoic acid (DHA, c22:6 omega 3) in human platelets and neutrophils

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.