Efficacy of modified hive entrances and a bottom screen device for controlling Aethina tumida (Coleoptera: Nitidulidae) infestations in Apis mellifera (Hymenoptera: Apidae) colonies

This study was designed to test whether hive entrances reduced with polyvinyl chloride pipe reduce the ingress of Aethina tumida Murray into Apis mellifera L. colonies and whether screen-mesh bottom boards alleviate side effects associated with restricted entrances. Forty-eight colonies distributed equally between two locations each received one of six experimental treatments: 1) conventional solid bottom board and open entrance, 2) ventilated bottom board and open entrance, 3) conventional bottom and 1.9-cm-i.d. pipe entrance, 4) conventional bottom and 3.8-cm pipe entrance, 5) screen bottom and 1.9-cm pipe entrance, and 6) screen bottom and 3.8-cm pipe entrance. Results were inconsistent between apiaries. In apiary 1, colonies with 3.8-cm pipe entrances had fewer A. tuzmida than colonies with open entrances, but this benefit was not apparent in apiary 2. Pipe entrances tended to reduce colony and brood production in both apiaries, and these losses were only partly mitigated with the addition of screened bottom boards. Pipe entrances had no measurable liability concerning colony thermoregulation. There were significantly fewer frames of adult A. mellifera in colonies with 3.8- or 1.9-cm pipe entrances compared with open entrances but more in colonies with screens. There were more frames of pollen in colonies with open or 3.8-cm pipe entrances than 1.9-cm entrances. We conclude that the efficacy of reduced hive entrances in reducing ingress of A. tumida remains uncertain due to observed differences between apiaries. Furthermore, there were side effects associated with restricted entrances that could be only partly mitigated with screened bottom boards.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

Microhabitat distribution of protostelids in a Tropical Wet Forest in Costa Rica

A microhabitat study of protostelids was carried out in a Tropical Wet Forest at the La Selva Biological Station in Costa Rica. Nine species were recorded from sterile wheat straws placed out and then re-collected over a period of six weeks from two different litter microhabitats in an area of primary forest. All nine species were present on straws placed in the aerial litter microhabitat, but only six species were present on straws placed in the forest floor litter microhabitat. Total colonies, percent of straws colonized, and mean number of species per straw increased significantly over time. One species (Schizoplasmodiopsis pseudoendospora) typical of temperate litter was the overwhelming dominant on the forest floor litter, while Echinostelium bisporum, a species rare in temperate litter microhabitats, was the single most abundant species in the aerial litter microhabitat. Both of these species had significantly increased frequencies over time. Two species abundant in temperate aerial litter microhabitats and one species abundant in temperate forest floor litter were rare at La Selva. Our data conform to those obtained in an earlier study carried out in tropical forests in the mountains of Puerto Rico and provide additional support towards developing a model of microhabitat distribution of protostelids in terrestrial ecosystems.     

Stability of diacylglycerol acyltransferase in dehydrated bovine muscle tissue

Meaningful estimates of diacylglycerol acyltransferase (EC 2.3.1.20) activity in different tissue samples require effective, unbiased methods of sample storage. Samples of the pars costalis diaphragmatis muscle (skirt muscle of the diaphragm) were obtained from 18- to 20-month-old cattle and assayed for microsomal protein content and diacylglycerol acyltransferase activity after having been stored under various conditions as dissected tissue or microsomes prepared from dissected tissue. There was relative enrichment of diacylglycerol acyltransferase specific activity (p<0.05) when samples prepared from the pars costalis diaphragmatis muscle were dehydrated and stored for 2 weeks, as compared to the control condition (in which the microsome fraction was prepared from fresh pars costalis diaphragmatis muscle and assayed immediately). The results suggested that dehydration was an effective method of storage for bovine muscle samples destined for estimation of the microsomal diacylglycerol acyltransferase activity. The dehydration approach for preparing samples for analysis of diacylglycerol acyltransferase activity might also prove useful to investigators who are interested in obtaining reliable estimates of the activity of other enzymes in tissue samples.     

Purification and preliminary characterization of brain aspartoacylase

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) in the brain to produce acetate and L-aspartate. An aspartoacylase deficiency, with concomitant accumulation of NAA, is responsible for Canavan disease, a lethal autosomal recessive disorder. To examine the mechanism of this enzyme the genes encoding murine and human aspartoacylase were cloned and expressed in Escherichia coli. A significant portion of the enzyme is expressed as soluble protein, with the remainder found as inclusion bodies. A convenient enzyme-coupled continuous spectrophotometric assay has been developed for measuring aspartoacylase activity. Kinetic parameters were determined with the human enzyme for NAA and for selected N-acyl analogs that demonstrate relaxed substrate specificity with regard to the nature of the acyl group. The clinically relevant E285A mutant reveals an altered enzyme with poor stability and barely detectable activity, while a more conservative E285D substitution leads to only fivefold lower activity than native aspartoacylase.     

Interaction of a peptide from the receptor-binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: changes in backbone dynamics induced by binding

The C-terminal receptor-binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N NMR relaxation experiments of the (15)N-labeled recombinant PAK pilin peptide in complex with an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus, were performed in order to probe for changes in the mobilities and dynamics of the peptide backbone as a result of antibody binding. The major results of these studies are as follows: binding of Fab leads to the preferential ordering of the first turn over the second turn in each isomer, binding of Fab partially stabilizes peptide backbone regions undergoing slow (microsecond to millisecond) exchange-related motions, and binding of Fab leads to a greater loss in backbone conformational entropy at pH 7.2 versus pH 4.5. The biological implications of these results will be discussed in relation to the role that fast and slow backbone motions play in PAK pilin peptide immunogenicity and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.     

Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy

Total internal reflection excitation used in combination with fluorescence correlation spectroscopy (TIR-FCS) is a method for characterizing the dynamic behavior and absolute concentrations of fluorescent molecules near or at the interface of a planar substrate and a solution. In this work, we demonstrate for the first time the use of TIR-FCS for examining the interaction kinetics of fluorescent ligands in solution which specifically and reversibly associate with receptors in substrate-supported planar membranes. Fluorescence fluctuation autocorrelation functions were obtained for a fluorescently labeled IgG reversibly associating with the mouse receptor FcgammaRII, which was purified and reconstituted into substrate-supported planar membranes. Data were obtained as a function of the IgG solution concentration, the Fc receptor surface density, the observation area size, and the incident intensity. Best fits of the autocorrelation functions to appropriate theoretical forms gave measures of the average surface density of bound IgG, the local solution concentration of IgG, the kinetic rate constant for surface dissociation, and the rate of diffusion through the depth of the evanescent field. The average number of observed fluorescent molecules, both in solution and bound to the surface, scaled with the solution concentration of IgG, observation area size, and Fc receptor surface density as expected. The dissociation rate constant and rate of diffusion through the evanescent field agree with previous results, and all measured parameters were independent of the incident intensity.     

Cryptic repeated genomic recombination during speciation in Gossypium gossypioides

The Mexican cotton Gossypium gossypioides is a perplexing entity, with conflicting morphological, cytogenetic, and molecular evidence of its phylogenetic affinity to other American cottons. We reevaluated the evolutionary history of this enigmatic species using 16.4 kb of DNA sequence. Phylogenetic analyses show that chloroplast DNA (7.3 kb), nuclear ribosomal internal transcribed spacers (ITS; 0.69 kb), and unique nuclear genes (8.4 kb) yield conflicting resolutions for G. gossypioides. Eight low-copy nuclear genes provide a nearly unanimous resolution of G. gossypioides as the basalmost American diploid cotton, whereas cpDNA sequences resolve G. gossypioides deeply nested within the American diploid clade sister to Peruvian G. raimondii, and ITS places G. gossypioides in an African (rather than an American) clade. These data, in conjunction with previous evidence from the repetitive fraction of the genome, implicate a complex history for G. gossypioides possibly involving temporally separated introgression events from genetically divergent cottons that are presently restricted to different hemispheres. Based on repetitive nuclear DNA, it appears that G. gossypioides experienced nuclear introgression from an African species shortly after divergence from the remainder of the American assemblage. More recently, hybridization with a Mexican species may have resulted in cpDNA introgression, and possibly a second round of cryptic nuclear introgression. Gossypium gossypioides provides a striking example of the previously unsuspected chimeric nature of some plant genomes and the resulting phylogenetic complexity produced by multiple historical reticulation events.     

OmpF enhances the ability of BtuB to protect susceptible Escherichia coli cells from colicin E9 cytotoxicity

The outer membrane (OM) vitamin B(12) receptor, BtuB, is the primary receptor for E group colicin adsorption to Escherichia coli. Cell death by this family of toxins requires the OM porin OmpF but its role remains elusive. We show that OmpF enhances the ability of purified BtuB to protect bacteria against the endonuclease colicin E9, demonstrating either that the two OM proteins form the functional receptor or that OmpF is recruited for subsequent translocation of the bacteriocin. While stable binary colicin E9-BtuB complexes could be readily shown in vitro, OmpF-containing complexes could not be detected, implying that OmpF association with the BtuB-colicin complex, while necessary, must be weak and/or transient in nature.     

Social correlates of stress in adult males of the great gerbil,Rhombomys opimus,in years of high and low population densities

The great gerbil, Rhombomys opimus, is the most social species in the Gerbillinae. The social structure consists of family groups that occupy isolated systems of burrows consisting of one breeding male, from one to seven females, and juveniles. During a year of peak density and one of density decline, we studied the influence of group size, group composition, local density, and distance to the nearest groups on fecal corticosterone and testosterone concentrations in breeding males. We also examined the relationship of hormone concentrations to the survival of males during the summer drought between the spring and the fall. We found that males differed in concentrations of steroid hormones. Concentrations of testosterone were lower whereas those of corticosterone tended to be higher in a year of high population densities compared with higher testosterone and lower corticosterone in a year with a lower density. This finding suggests that stress may be greater in higher densities because of increased social contact. Stepwise regression analysis revealed a positive and significant influence of the number of adult females in a family group on concentrations of fecal corticosterone and testosterone in adult males. Concentrations of corticosterone were also significantly higher in males that disappeared from family groups between the spring and the fall compared with males still alive in family groups in the fall. There was no change in concentrations of testosterone. These results suggest that social interactions within large family groups may be an important source of stress for adult males.