Overlapping motifs (PTAP and PPEY) within the Ebola virus VP40 protein function independently as late budding domains: involvement of host proteins TSG101 and VPS-4

The VP40 protein of Ebola virus can bud from mammalian cells in the form of lipid-bound, virus-like particles (VLPs), and late budding domains (L-domains) are conserved motifs (PTAP, PPxY, or YxxL; where "x" is any amino acid) that facilitate the budding of VP40-containing VLPs. VP40 is unique in that potential overlapping L-domains with the sequences PTAP and PPEY are present at amino acids 7 to 13 of VP40 (PTAPPEY). L-domains are thought to function by interacting with specific cellular proteins, such as the ubiquitin ligase Nedd4, and a component of the vacuolar protein sorting (vps) pathway, tsg101. Mutational analysis of the PTAPPEY sequence of VP40 was performed to understand further the contribution of each individual motif in promoting VP40 budding. In addition, the contribution of tsg101 and a second member of the vps pathway, vps4, in facilitating budding was addressed. Our results indicate that (i) both the PTAP and PPEY motifs contribute to efficient budding of VP40-containing VLPs; (ii) PTAP and PPEY can function as L-domains when separated and moved from the N terminus (amino acid position 7) to the C terminus (amino acid position 316) of full-length VP40; (iii) A VP40-PTAP/tsg101 interaction recruits tsg101 into budding VLPs; (iv) a VP40-PTAP/tsg101 interaction recruits VP40 into lipid raft microdomains; and (v) a dominant-negative mutant of vps4 (E228Q), but not wild-type vps4, significantly inhibited the budding of Ebola virus (Zaire). These results provide important insights into the complex interplay between viral and host proteins during the late stages of Ebola virus budding.     

Physiological characteristics of the tympanic organ in noctuoid moths

Summary The tympanic organ ofSpodoptera frugiperda, Mocis latipes, Erebus odorata (Noctuidae) andMaenas jussiae (Arctiidae) was stimulated with acoustic stimuli of 20 kHz, 45 ms and 5 s duration, and intensities ranging from 30 to 100 dB. The electric activity of the auditory receptors was recorded at the tympanic nerve with a stainless steel hook electrode. In all of these moth species there is an intensity range (ca. 20 dB) in which the response of each auditory receptor (A₁ and A₂ cells) to 45 ms pulses varies in a linear relation to the logarithm of stimulus intensity. For intensities higher than this value, depending on the species and the cell analysed, the spike discharge may continue to increase, may saturate or may diminish (Fig. 2). InE. odorata andM. latipes the A₁-cell response shows a decrease for stimulus intensities higher than 30 dB above the threshold. In the former species there is a statistically significant linear relation between the A₂-cell response and the decrease of the A₁-cell response, but this is not the case inM. latipes (Fig. 3). The similarity of the responses ofE. odorata to those described inEmpyreuma pugione (Coro and Pérez 1984) suggest that also in this noctuid species one may assume that the A₂ cell inhibits the A₁ receptor. In all of these moth species there is a maximum firing rate of the auditory cells at the beginning of the response to pure tones of 5 s and an exponential decrease of their discharge frequency with the course of time (Fig. 5). The analysed species differ in the adaptation rates of their auditory receptors. In all of these species the A₂ cell adapts more rapidly than the A₁ cell. In most of these species the stimulus intensity influences the adaptation rate of the auditory receptors (Fig. 7). These results are compared with data obtained by other authors, and it is concluded that there are more interspecific differences in the physiological characteristics of the auditory receptors in noctuoid species than those reported so far.Abbreviation AP action potential     

Microbes from apiarian sources: Bacillus spp. in frass of the greater wax moth

Frass from the greater wax moth, Galleria mellonella, obtained from feral colonies of honey bees, Apis mellifera; from managed honey bee colonies; and from a laboratory culture of the wax moth was sampled for aerobic Gram-positive spore-forming rods. One hundred eighty-five strains belonging to the genus Bacillus were isolated, and most were identified. One hundred and three of the isolates were from frass from the wax moth culture, 61 were from frass from managed honey bee colonies, and 21 were from frass from feral honey bee colonies. The species most frequently isolated varied with the source. Fifty-eight isolates from frass from managed honey bee colonies were B. cereus which was isolated from this source only, but B. sphaericus was the most frequent isolate from frass from the wax moth culture. Bacillus megaterium and organisms belonging to the B. alvei-B. thiaminolyticus spectrum were the most frequent isolates from frass from feral honey bee colonies. Most strains isolated produced caprylate esterase-lipase, leucine aminopeptidase, and phosphoamidase. The numbers of isolates, the species, and the enzymatic activity of the strains varied with the source of the frass. In fact, the complete microbial complement varied with the source. These results are discussed in relation to possible roles of Bacillus spp. in the nutrition of the wax moth as well as the microbial ecology of the honey bee colony.     

Improving the long‐term storage of a mammalian biosensor cell line via genetic engineering

The unique properties of mammalian cells make them valuable for a variety of applications in medicine, industry, and diagnostics. However, the utility of such cells is restricted due to the difficulty in storing them non‐frozen for an extended time and still maintaining their stability and responsiveness. In order to extend the active life span of a mammalian biosensor cell line at room and refrigerated temperatures, we have over expressed genes that are reported to provide protection from apoptosis, stress, or oxidation. We demonstrated that over expression of genes from the extremophile, Artemia franciscana, as well as GADD45β, extends room‐temperature storage of fully active cells 3.5‐fold, while over production of several anti‐apoptotic proteins extended 4°C storage 2‐ to 3‐fold. Methodologies like these that improve the stability of mammalian‐cell‐based technologies in the absence of freezers may enable widespread use of these tools in applications that have been considered impractical based solely on limited storage characteristics. Biotechnol. Bioeng. 2010; 106: 474–481. © 2010 Wiley Periodicals, Inc.     

Sex differences in antipredator tail-waving displays of the diurnal yellow-headed gecko Gonatodes albogularis from tropical forests of Colombia

Many vertebrate species show display behaviors when predators are in their vicinity. Some of these displays may inform the predator of the improbability of capturing the prey (i.e., pursuit-deterrent displays) and are potentially advantageous to both predator and prey. Here we present data on a tail display performed by Gonatodes albogularis, a diurnal tropical gecko. We performed transect surveys in three habitats near Bogotá in Colombia. Geckos detected during transects were approached by the observer in a standardized way, and details of their tail-waving displays were recorded. In control recordings animals were watched from a distant site without approaching them. Results showed sexual differences in tail-waving display: when approached by the observer, males performed this behavior more frequently than females. We found no significant differences between males and females in flight-initiation distances and height above the substratum when they were initially located. Results also showed that males displayed more frequently when approached than when the simulated predator remained stationary. We interpret these results as evidence that the display functions as a pursuit-deterrent signal to potential predators. However, as some tail displays were performed in the presence of conspecifics, the display may also have a social function.     

Natural variation in life history and aging phenotypes is associated with mitochondrial DNA deletion frequency in Caenorhabditis briggsae

Background  

Mutations that impair mitochondrial functioning are associated with a variety of metabolic and age-related disorders. A barrier to rigorous tests of the role of mitochondrial dysfunction in aging processes has been the lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Toward the goal of developing such a model system, we studied natural variation in life history, metabolic, and aging phenotypes as it relates to levels of a naturally-occurring heteroplasmic mitochondrial ND5 deletion recently discovered to segregate among wild populations of the soil nematode, Caenorhabditis briggsae. The normal product of ND5 is a central component of the mitochondrial electron transport chain and integral to cellular energy metabolism.     

Monounsaturated fatty acyl-coenzyme A is predictive of atherosclerosis in human apoB-100 transgenic, LDLr-/- mice

ACAT2, the enzyme responsible for the formation of cholesteryl esters incorporated into apolipoprotein B-containing lipoproteins by the small intestine and liver, forms predominantly cholesteryl oleate from acyl-CoA and free cholesterol. The accumulation of cholesteryl oleate in plasma lipoproteins has been found to be predictive of atherosclerosis. Accordingly, a method was developed in which fatty acyl-CoA subspecies could be extracted from mouse liver and quantified. Analyses were performed on liver tissue from mice fed one of four diets enriched with one particular type of dietary fatty acid: saturated, monounsaturated, n-3 polyunsaturated, or n-6 polyunsaturated. We found that the hepatic fatty acyl-CoA pools reflected the fatty acid composition of the diet fed. The highest percentage of fatty acyl-CoAs across all diet groups was in monoacyl-CoAs, and values were 36% and 46% for the n-3 and n-6 polyunsaturated diet groups and 55% and 62% in the saturated and monounsaturated diet groups, respectively. The percentage of hepatic acyl-CoA as oleoyl-CoA was also highly correlated to liver cholesteryl ester, plasma cholesterol, LDL molecular weight, and atherosclerosis extent. These data suggest that replacing monounsaturated with polyunsaturated fat can benefit coronary heart disease by reducing the availability of oleoyl-CoA in the substrate pool of hepatic ACAT2, thereby reducing cholesteryl oleate secretion and accumulation in plasma lipoproteins.     

Use of Real-Time Quantitative PCR for the Analysis of φLC3 Prophage Stability in Lactococci

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.     

Quantitative profiling of the detergent-resistant membrane proteome of iota-b toxin induced vero cells

Enzyme-mediated 18O/16O differential labeling of proteome samples often suffers from incomplete exchange of the carboxy-terminus oxygen atoms, resulting in ambiguity in the measurable abundance differences. In this study, an 18O/16O labeling strategy was optimized for and applied to the solution-based comparative analysis of the detergent-resistant membrane proteome (DRMP) of untreated and Iota-b (Ib)-induced Vero cells. Solubilization and tryptic digestion of the DRMP was conducted in a buffer containing 60% methanol. Unfortunately, the activity of trypsin is attenuated at this methanol concentration hampering the ability to obtain complete oxygen atom turnover. Therefore, the incorporation of the 18O atoms was decoupled from the protein digestion step by carrying out the trypsin-mediated heavy atom incorporation in a buffer containing 20% methanol; a concentration at which trypsin activity is enhanced compared to purely aqueous conditions. After isotopic labeling, the samples were combined, fractionated by strong cation exchange and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. In total, over 1400 unique peptides, corresponding to almost 600 proteins, were identified and quantitated, including all known caveolar and lipid raft marker proteins. The quantitative profiling of Ib-induced DRMP from Vero cells revealed several proteins with altered expression levels suggesting their possible role in Ib binding/uptake.