Cutting edge: characterization of allorestricted and peptide-selective alloreactive T cells using HLA-tetramer selection

The vast majority of alloreactive T cells recognize foreign MHC molecules in a peptide-dependent manner. A subpopulation of these peptide-dependent alloreactive T cells is peptide-specific and contains T cells that are of interest for tumor immunotherapy. Allorestricted T cells (i.e., peptide-specific and alloreactive) specific for tumor-associated Ags can be raised in vitro. However, it is technically difficult to distinguish between peptide-specific and peptide-nonspecific alloreactive T cells by functional assays in vitro. Here we show for the first time that allorestricted T cells specifically bind HLA-peptide tetrameric complexes, as nominal Ag-specific T cells would do. In consequence, fluorescent HLA-peptide tetrameric complexes can be used for sorting and cloning of allorestricted CTLs specific for a peptide of interest. We also show by the mean of HLA-peptide tetramers the existence of peptide-selective alloreactive T cells that recognize a conformation on the foreign-MHC brought about by some but not all peptides bound.     

Could the egg itself be the source of the oviposition deterrent marker in the ectoparasitoid Dinarmus basalis?

Females of Dinarmus basalis, a solitary ectoparasitoid, strongly avoid superparasitising hosts bearing eggs which are 20 or more hours old. Our objective was to determine the mechanism by which the factors inducing host discrimination and such an oviposition deterring effect act, and the origin of the deterrent. This study showed that the information is carried by the parasitised host. It is acquired by a transfer of molecules from the wasp egg onto the host and it requires contact between egg and host for at least 4 h. The factors transferred do not, as commonly reported in other species, come from the female's genital apparatus but from the live egg itself. Consequently, the D. basalis egg directly participates in the success of parasitism by releasing factors which elicit the oviposition host deterring effect. The originality and the ecological importance of these results are discussed.     

Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France

A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.     

Purification, cloning, and sequencing of a 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1

A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22 degrees C, compared to 30 and 37 degrees C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K(m) for 3,5-dicholorophenol was 49.3 +/- 3.1 microM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.     

Trophic interactions in a high arctic snow goose colony

We examined the role of trophic interactions in structuringa high arctic tundra community characterized by a large breedingcolony of greater snow geese (Chen caerulescens atlantica).According to the exploitation ecosystem hypothesis of Oksanenet al. (1981), food chains are controlled by top-down interactions.However, because the arctic primary productivity is low, herbivorepopulations are too small to support functional predator populationsand these communities should thus be dominated by the plant/herbivore trophic-level interaction. Since 1990, we have beenmonitoring annual abundance and productivity of geese, the impactof goose grazing, predator abundance (mostly arctic foxes, Alopexlagopus) and the abundance of lemmings, the other significantherbivore in this community, on Bylot Island, Nunavut, Canada.Goose grazing consistently removed a significant proportionof the standing crop (     

Palaeoecology of triassic stem turtles sheds new light on turtle origins

Competing hypotheses of early turtle evolution contrast sharply in implying very different ecological settings-aquatic versus terrestrial-for the origin of turtles. We investigate the palaeoecology of extinct turtles by first demonstrating that the forelimbs of extant turtles faithfully reflect habitat preferences, with short-handed turtles being terrestrial and long-handed turtles being aquatic. We apply this metric to the two successive outgroups to all living turtles with forelimbs preserved, Proganochelys quenstedti and Palaeochersis talampayensis, to discover that these earliest turtle outgroups were decidedly terrestrial. We then plot the observed distribution of aquatic versus terrestrial habits among living turtles onto their hypothesized phylogenies. Both lines of evidence indicate that although the common ancestor of all living turtles was aquatic, the earliest turtles clearly lived in a terrestrial environment. Additional anatomical and sedimentological evidence favours these conclusions. The freshwater aquatic habitat preference so characteristic of living turtles cannot, consequently, be taken as positive evidence for an aquatic origin of turtles, but must rather be considered a convergence relative to other aquatic amniotes, including the marine sauropterygians to which turtles have sometimes been allied.     

Higher-accuracy method for measuring minichromosome stability in Saccharomyces cerevisiae

The minichromosome maintenance assay isfrequently used to characterize mutations genetically that affect the initiation of DNA replication or to decipherfunctional components in autonomously replicating sequences. The assay determines minichromosome loss by measuring the percentage of plasmid-containing cells in cultures after a period of growth in nonselective medium. Here we analyze data acquisition errors that contribute to the low accuracy of the routine versions of the assay. We propose modifications that eliminate errors in the acquisition of two variables and significantly improve the accuracy of the assay.     

Effects of a hypocaloric,low-carbohydrate diet on weight loss,blood lipids,blood pressure,glucose tolerance,and body composition in free-living overweight women

The purpose of the current study was to examine the effects of a very low-carbohydrate diet on weight loss and biochemical parameters in overweight women. Twenty women completed an 8-week trial that reduced their daily carbohydrate intake from 232 to 71 g (p < 0.05) and reduced energy by 2,644 kJ/day (8,384 to 5,740 kJ, p < 0.001). The average weight loss was 5.0 kg (p < 0.0001), with a net decrease in body mass index of 1.82 kg/m2, a loss of 3.4% body fat (4 kg, p < 0.0001), and a loss of 1.0 kg lean mass (p < 0.05). There were no significant changes in fasting blood glucose, fasting serum insulin, oral glucose tolerance, free or total insulin-like growth factor-1, or total IGFBP-3. Systolicblood pressure decreased by an average of 9.0 mmHg (1 mmHg = 133.322 Pa) (p < 0.01) and diastolic blood pressure decreased by 7 mmHg (p < 0.05). Total cholesterol decreased 1.2 mM (p < 0.001), all of which was accounted for by a decrease in low-density lipoprotein cholesterol (p < 0.001) with no change in high-density lipoprotein cholesterol (baseline, 1.17 mM; week 8, 1.22 mM). Total triacylglycerol decreased 0.6 mM (p < 0.01), and the ratio of triacylglycerol/HDL also significantly decreased (baseline, 1.40; week 8, 0.87; p < 0.001). Serum beta-hydroxybutyrate concentrations rose significantly by week 2 and declined thereafter but remained significantly higher than baseline values for the duration of the intervention. Therefore, carbohydrate restriction to 70 g or less with concomitant energy restriction, without changes in protein or fat consumption, promotes weight loss, and improvements in body composition, blood pressure, and blood lipids without compromising glucose tolerance in moderately overweight women.