Formation of functional alpha3beta4alpha5 human neuronal nicotinic receptors in Xenopus oocytes: a reporter mutation approach.

1. The alpha5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related beta3 subunit, alpha5 forms functional recombinant receptors if expressed together with a pair of typical alpha and beta subunits, but its effect on the properties of the resulting alphabetaalpha5 receptor depends on the alpha and beta subunits chosen and on the expression system. We used a reporter mutation approach to test whether alpha5, like beta3, is incorporated as a single copy in human alpha3beta4alpha5 receptors expressed in oocytes. 2. As previously reported, the main indication of the presence of alpha5 in alpha3beta4alpha5(wt) was an increase in apparent receptor desensitization (compared with alpha3beta4 receptors). If the alpha3beta4alpha5 receptor bore a 9'T mutation in the second transmembrane domain of either alpha3 or beta4, alpha5 incorporation produced a decrease in ACh sensitivity (by 4 fold for alpha3(LT)beta4alpha5 vs. alpha3(LT)beta4 and by 40 fold for alpha3beta4(LT)alpha5 vs. alpha3beta4(LT)). The much greater effect observed in alpha3beta4(LT)alpha5 receptors accords with the hypothesis that alpha5 takes the place of a beta subunit in the receptor. 3. Introducing a 9'T mutation in alpha5 had no effect on the agonist sensitivity of alpha3beta4alpha5 receptors, but reduced apparent desensitisation, as judged by the sag in the current response to high agonist concentrations. 4. Introducing the 9'T mutation in alpha3 or beta4 in the triplet receptor reduced the EC(50) for ACh by a similar extent (7 and 9 fold, respectively), suggesting that alpha3beta4alpha5 receptors contain two copies each of alpha and beta and therefore only one copy of alpha5.

Stoichiometry of human recombinant neuronal nicotinic receptors containing the b3 subunit expressed in Xenopus oocytes.

The neuronal nicotinic subunit beta3 forms functional receptors when co-expressed with both an alpha and a beta subunit, such as alpha3 and beta4. We examined the subunit stoichiometry of these 'triplet' alpha3beta4beta3 receptors by expression in Xenopus oocytes of the alpha3, beta4 and beta3 subunits, either in wild-type form or after insertion of a reporter mutation. The mutation chosen was the substitution of a conserved hydrophobic residue in the second transmembrane domain of the subunits (leucine or valine 9THORN ) with a hydrophilic threonine. In other ion channels within the nicotinic superfamily, this mutation type consistently increases the potency of agonists. In muscle-type nicotinic receptors, the magnitude of this effect is approximately constant for each mutant subunit incorporated. In alpha3beta4beta3 receptors, the ACh EC50 was decreased by approximately 17-fold when this mutation was in alpha3 alone and only by fourfold when beta3 alone was mutated. Mutating beta4 was equivalent to mutating alpha3, suggesting that the 'triplet' receptor contains one copy of beta3 and two copies each of alpha3 and beta4. Mutating beta3 and alpha3 or beta3 and beta4 reduced the ACh EC50 further, to values two- to threefold lower than those seen when only alpha3 or beta4 carried the mutation. In 'pair' alpha3beta4 receptors (known to contain two alpha and three beta subunits), mutating beta4 had a greater effect on the ACh EC50 than mutating alpha3, in agreement with an alpha:beta ratio of 2:3 and a constant and independent effect of each copy of the mutation. Our results suggest that alpha3beta4beta3 neuronal nicotinic receptors contain one copy of beta3 and two copies each of alpha3 and beta4 and confirm that in pair alpha3beta4 receptors the alpha/beta subunits are present in a 2:3 ratio.

Light and suction trap catches of Culicoides midges in southern England.

1. The seasonal flight periods are given for eleven species or species groups of Culicoides midges at Pirbright, Surrey, U.K., from 1979 to 1981 and in 1983. 2. Most species were relatively uniformly distributed both within the study site and between years; only C.clintoni Boorman was limited in its spatial distribution and only C.segnis Campbell and Pelham-Clinton varied greatly in numbers between years. 3. Species fall into one of three patterns of seasonal abundance: spring emergers, summer emergers, or spring to autumn emergers. 4. Only the C.obsoletus group showed a pattern of emergence that could correspond to distinct generations, of one or more species in the group. Three distinct emergences were observed, and possible explanations for these discussed. 5. For all species except C.impunctatus Goetghebuer the catches from suction/light and suction only traps are poorly correlated whereas for C.impunctatus they are highly correlated. This may suggest that this species is not greatly attracted to light.

WHO/FAO Working Team Report: entomology.

A summary of the more important concerns of the Working Team with particular reference to bluetongue (BT) virus (BTV), is as follows: With the exception of Australia, the US, and possibly parts of Africa, there are almost no concrete data that could be used to direct control efforts against the responsible vector(s) of BTV in a specific geographic area. In Australia, a broad plan of attack yielded data that showed that members of the subgenus Avaritia are the primary vector species. It has been clearly shown that Culicoides variipennis (Coq.) is the dominant vector species for most of the US. In the Middle East and certain areas of Africa, isolations from possible vector species indicated that C. imicola K., again probably a species complex and belonging to the subgenus Avaritia, is one of the likely vectors. Except for some subgenera, the taxonomy for much of the Culicoides remains in disarray. The subgenus Oecacta for example still remains a catchall for many species. Although a number of proposals have been made for delineating the species of the C. (Monoculicoides) variipennis species complex, none have been initiated because of the lack of funds. The Working Team strongly feels that the lack of definition of important species complexes is the result of short-term planning that does not consider the fact that efficiency can be increased and a great deal of monies saved by directing control toward the specific species or populations thereof that are responsible for a problem.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of paraffin wax embedding procedures on the ability of the fluorescent antibody test to demonstrate bluetongue virus antigen in insect tissue.

The effect of the reagents used in paraffin wax embedding procedures on the fluorescent antibody technique was investigated with a view to using this test for virus location studies on paraffin wax sections of bluetongue virus infected Culicoides variipennis. Experiments were carried out using tissue smears rather than prepared sections, enabling the effect of individual reagents to be evaluated. Infected smears treated with acetone alone showed bright specific fluorescence, while treatment of infected smears with all other reagents, including low melting point wax and acetone-soluble ester wax, prior to immunofluorescence testing reduced the brightness of specific fluorescence or rendered the test completely ineffective.

Bluetongue virus in a Nigerian dairy cattle herd. 1. Serological studies and correlation of virus activity to vector population.

Newborn calves were bled at monthly intervals and examined for serum antibodies to blue tongue virus (BTV). Maternal immunity persisted for 3 months and it was possible to calculate decay rates for virus neutralizing antibody. Calves were subclinically infected with BTV within a few months of becoming susceptible and neutralization tests were used to deduce the serotype responsible. A profile of virus activity was built up over a 12 month period. Frequent light trap catches were used to examine the population dynamics of suspected Culicoides vector species. Two species, imicola and schultzei were present throughout the wet and dry seasons and survival were sufficiently long to account for virus transmission at any time of the year.