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.      

Increased Carotid Intima-Media Thickness and Reduced Distensibility in Human Class III Obesity: Independent and Differential Influences of Adiposity and Blood Pressure on the Vasculature

Carotid intima-media-thickness (cIMT) and carotid distensibility (distensibility), structural and functional properties of carotid arteries respectively, are early markers, as well as strong predictors of cardiovascular disease (CVD). The characteristic of these two parameters in individuals with BMI>40.0 kg/m² (Class III obesity), however, are largely unknown. The present study was designed to document cIMT and distensibility in this population and to relate these to other factors with established association with CVD in obesity. The study included 96 subjects (65 with BMI>40.0 kg/m² and 31, age- and gender-matched, with BMI of 18.5 to 30.0 kg/m²). cIMT and distensibility were measured by non-invasive high resolution ultrasonography, circulatory CD133⁺/KDR⁺ angiogenic cells and endothelial microparticles (EMP) by flow cytometry, and plasma levels of adipokines, growth factors and cytokines by Luminex immunoassay kits. The study results demonstrated increased cIMT (0.62±0.11 mm vs. 0.54±0.08 mm, P = 0.0002) and reduced distensibility (22.52±10.79 10⁻³kpa⁻¹ vs. 29.91±12.37 10⁻³kpa⁻¹, P<0.05) in individuals with BMI>40.0 kg/m². Both cIMT and distensibility were significantly associated with traditional CVD risk factors, adiposity/adipokines and inflammatory markers but had no association with circulating angiogenic cells. We also demonstrated, for the first time, elevated plasma EMP levels in individuals with BMI>40.0 kg/m². In conclusion, cIMT is increased and distensibility reduced in Class III obesity with the changes predominantly related to conventional CVD risk factors present in this condition, demonstrating that both cIMT and distensibility remain as CVD markers in Class III obesity.     

Development of sero-diagnostic and molecular tools for the control of important tick-borne pathogens of cattle in Africa

Tick-borne diseases (TBDs) are a major economic constraint to livestock production in sub-Saharan Africa. ILRI is focussing on developing a range of products, such as vaccines, diagnostics and decision support services to underpin improved control programmes against these diseases. We have developed three highly sensitive and specific enzyme linked immuno-assays (ELISAs), which allow precise diagnosis of Theileria parva, Babesia bigemina and Anaplasma marginale. These tests have been standardised and validated using defined experimental and field infection sera. Parasite specific recombinant antigens and monoclonal antibodies against bovine immunoglobulins as secondary antibodies have played an important role in in enhancing the sensitivity and specificity of the assays. They have been further evaluated in on-farm longitudinal sero-epidemiological studies to define infection dynamics and disease risks in various farming systems in Kenya and Uganda. In addition, DNA-based tests for differentiation of Theileria species and characterisation of Theileria parva stocks have been developed. These tests have been derived through physical mapping and sequencing of key elements of the T. parva genome, which include repetitive and telomeric regions, minisatellite sequences, antigen genes and a number of random DNA sequences. These tools are currently being deployed in conjunction with field immunisation programmes to determine the biological impact of introducing live vaccines of T. parva on population dynamics.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.