The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering.

Agrin derived from Torpedo electric organ induces the clustering of acetylcholine receptors (AChRs) on cultured myotubes. As a first step toward characterizing the plasma membrane receptor for agrin, we have examined agrin binding to cultured myotubes. Agrin binding is saturable as measured by radioimmunoassay and, like agrin-induced AChR clustering, requires extracellular calcium. Immunofluorescence shows that on myotubes incubated with agrin at 4 degrees C, agrin binds in a uniform, finely punctate pattern that correlates poorly with the distribution of AChRs. Myotubes stimulated with agrin at 37 degrees C for greater than or equal to 2 hr show a coclustering of agrin binding sites and AChRs. By contrast, if anti-AChR antibodies are used either to cluster or to internalize AChRs, the distribution and number of agrin binding sites remain unchanged. The aggregation and calcium dependence of the putative agrin receptor may represent important control points in postsynaptic differentiation.     

Altitudinal patterns for latitudinally varying traits and polymorphic markers in Drosophila melanogaster from eastern Australia

Altitudinal changes in traits and genetic markers can complement the studies on latitudinal patterns and provide evidence of natural selection because of climatic factors. In Drosophila melanogaster, latitudinal variation is well known but altitudinal patterns have rarely been investigated. Here, we examine five traits and five genetic markers on chromosome 3R in D. melanogaster collected at high and low altitudes from five latitudes along the eastern coast of Australia. Significant altitudinal differentiation was observed for cold tolerance, development time, ovariole number in unmated females, and the microsatellite marker DMU25686. Differences tended to match latitudinal patterns, in that trait values at high altitudes were also found at high latitudes, suggesting that factors linked to temperature are likely selective agents. Cold tolerance was closely associated with average temperature and other climatic factors, but no significant associations were detected for the other traits. Genes around DMU25686 represent good candidates for climatic adaptation.     

Is there genetic structure in populations of Helicoverpa armigera from Australia?

Recent studies suggest that populations of the pest moth Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) may be genetically differentiated over short distances and time periods within Queensland, Australia. To test for genetic structure in another region of Australia, we characterized population differentiation in Victorian samples of H. armigera using eight microsatellite loci. We found no evidence of genetic structure among samples from different locations or among samples collected at different times. Moreover, Victorian samples were not differentiated from other samples of H. armigera from Queensland and New Zealand. All samples showed substantial deviations from Hardy–Weinberg equilibrium, suggesting a high frequency of null alleles typically found in microsatellites of Lepidoptera. These results indicate that populations of H. armigera are not strongly structured among regions in south‐eastern Australia.     

Thermal tolerance trade-offs associated with the right arm of chromosome 3 and marked by the hsr-omega gene in Drosophila melanogaster

Drosophila melanogaster occurs in diverse climatic regions and shows opposing clinal changes in resistance to heat and resistance to cold along a 3000 km latitudinal transect on the eastern coast of Australia. We report here on variation at a polymorphic 8 bp-indel site in the heat shock hsr-omega gene that maps to the right arm of chromosome 3. The frequency of the genetic element marked by the L form of the gene was strongly and positively associated with latitude along this transect, and latitudinal differences in L frequency were robustly associated with latitudinal differences in maximum temperature for the hottest month. On a genetic background mixed for genes from each end of the cline a set of 10 lines was derived, five of which were fixed for the L marker, the absence of In(3R)P and 12 kb of repeats at a second polymorphic site at the 3' end of hsr-omega, and five that were fixed for the S marker, In(3R)P and 15 kb of hsr-omega repeats. For two different measures of heat tolerance S lines outperformed L lines, and for two different measures of cold tolerance L lines outperformed S lines. These data suggest that an element on the right arm of chromosome 3, possibly In(3R)P, confers heat resistance but carries the trade-off of also conferring susceptibility to cold. This element occurs at high frequency near the equator. The alternate element on the other hand, at high frequency at temperate latitudes, confers cold resistance at the cost of heat susceptibility.     

HERITABLE VARIATION IN ETHANOL TOLERANCE AND ITS ASSOCIATION WITH BIOCHEMICAL TRAITS IN DROSOPHILA MELANOGASTER

To help elucidate mechanisms of larval ethanol tolerance seven isochromosomal lines of Drosophila melanogaster with different second chromosomes were fed a growth-limiting concentration of ethanol (4.5% v/v) and examined for associations between growth traits and biochemical characteristics that had previously been implicated in the determination of tolerance variation. Repeated measures of survival and development time over four generations verified the inherited nature of these traits. Significant variation among the lines were evident for flux from ethanol into lipid, for activity levels of alcohol dehydrogenase and glycerol-3-phosphate oxidase (GPO), and for levels of long chain and unsaturated fatty acids. A high degree of positive association occurred among the variables. A partial correlation analysis controlling for performance of the lines on ethanol-free medium revealed a strong association between the degree of long chain fatty acid content and line survival when ethanol was fed. The correlation between GPO activity and survival in an ethanol environment appeared to depend on the association of GPO activity with long chain fatty acid content. The positive correlations of flux from ethanol into lipid with many of the other variables suggested that the ADH pathway influenced the level of ethanol tolerance. These associations are all consistent with the hypothesis that the lipid content of body tissues, especially the levels of long chain and unsaturated fatty acids in cell membranes, may have an important influence on both spatial and interspecific variation in the ethanol tolerance of larvae.     

Structure, function, and motility of vacuoles in filamentous fungi

Current information on the structure and function of motile tubular vacuoles in Pisolithus tinctorius and other fungi is reviewed. The use of fluorochromes to label the vacuole lumen is evaluated and observations on the structure and motility of vacuoles in P. tinctorius are differentiated from possible artifacts. The styryl dyes FM4-64 and MDY-64, used in yeast to demonstrate endocytosis, show little or no labeling of internal membranes in undamaged P. tinctorius cells. This agrees with our data showing that other probes for endocytosis such as Lucifer yellow CH are not taken up by hyphal tip cells. Overall, the observations do not support endocytosis in hyphal tips. It has been suggested that tubular vacuole systems carry out longitudinal transport, and evidence in favor of this hypothesis is evaluated. New data are presented to show that many of the large vacuoles in subapical cells are attached to the plasma membrane and are relatively immobile, while video sequences show movement of fluorochrome in pulses along a series of several large vacuoles, all interconnected via tubules. Tubular vacuoles from thick sections of hyphae processed under anhydrous conditions are shown by X-ray microanalysis to contain relatively high levels of P and K, as seen previously in the larger vacuoles. These results provide further evidence for a role of the tubular vacuoles in longitudinal transport of P. Copyright 1998 Academic Press.     

Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.      

Effects of chronic modification of dietary fat and carbohydrate on the insulin, corticosterone and metabolic responses of rats fed acutely with glucose, fructose or ethanol

1. Male rats were fed for 14 days on powdered diets containing (by weight) 53% of starch, or on diets in which 20g of starch per 100g of diet was replaced by lard or corn oil. They were then fed acutely by stomach tube with a single dose of glucose, fructose or ethanol of equivalent energy contents, or with 0.15m-NaCl. The serum concentrations of corticosterone, insulin, glucose, glycerol, triacylglycerol and cholesterol were measured up to 6h after this treatment. 2. Feeding saline (0.9% NaCl) acutely to the rats maintained on the three powdered diets produced a small transient increase in circulating corticosterone that was similar to that in rats maintained on the normal 41B pelleted diet. 3. Feeding glucose acutely to the rats on the powdered diets produced peak concentrations of corticosterone that were 2–3-fold higher than those seen in rats maintained on the 41B diet. The duration of this response increased in the order starch diet<lard diet<corn-oil diet. This abnormal corticosterone response to glucose feeding appeared to be responsible for an increased activity in phosphatidate phosphohydrolase in the livers of rats fed the starch and lard diets of 2.9- and 4.9-fold respectively. The latter increase was similar to that produced by ethanol, whereas glucose did not increase the phosphohydrolase activity in the liver of rats maintained on the 41B diet. 4. Feeding fructose acutely produced even more marked increases than glucose in the concentrations of circulating corticosterone in rats given the powdered diets, but unlike glucose did not increase circulating insulin. The duration of the corticosterone response again increased in the order starch diet<lard diet<corn-oil diet. The concentrations of circulating glucose were increased by fructose feeding in rats maintained on these diets, but they were not altered in the rats maintained on the 41B pellets. A prolonged increase in serum corticosterone concentrations was also observed when fructose was fed to rats maintained on pelleted diets enriched with corn oil or beef tallow rather than with starch or sucrose. However, these effects were less marked than those seen with rats fed on the powdered diets. 5. These results are discussed in relation to the mechanism whereby high dietary fat exaggerates the effects of ethanol, fructose and sorbitol in stimulating triacylglycerol synthesis in the liver.