Prey selection by Dicyphus hesperus of infected or parasitized greenhouse whitefly

The development of effective biological control programs in which predators are integrated with other natural enemies such as parasitoids and entomopathogens requires an understanding of their interactions. In this study we examined the extent to which the omnivorous mirid bug Dicyphus hesperus, an effective biological control agent of greenhouse whiteflies, accepts prey that are either parasitized by the specialist whitefly parasitoid, Encarsia formosa or infected by the generalist fungal entomopathogen, Beauveria bassiana. In non-choice laboratory experiments, we measured how parasitism and infection of greenhouse whitefly, Trialeurodes vaporariorum, as related to parasitoid age and course of fungal infection, might alter the probability of feeding by second instar or adult female predators. Predation by D. hesperus was similar for parasitized and unparasitized whiteflies, regardless of parasitoid age. However, predators generally avoided feeding on infected whiteflies, particularly when infection was manifested through the presence of oosporein or hyphae on the surface of prey.     

Flavonoids variation in seven tetraploid populations of Dactylis glomerata

133 individual plants representing seven tetraploid populations of Dactytis glomerata were examined for their flavonoid pattern by using HPLC. The chemical results expressed quantitatively as peak heights were submitted to multivariate analysis. This data treatment suggested an obvious discrimination between Eurasian and Mediterranean types, lending support to the taxonomic status of the species. This segregation is based on the distribution of C-glycosylluteolins and methylated derivatives. Moreover among the Mediterranean collections, a Coraica population was conspicuously separated from the other three in regard to the distributionof di-C-glycosylflavones. This chemical approach was compared with an electrophoretic assay concerning seven enzyme systems; their respective conclusions were in good agreement.     

Kinetic characterization and identification of the acylation and glycosylation sites of recombinant human gamma-glutamyltranspeptidase

Gamma-glutamyltranspeptidase (GGT) is a heterodimeric enzyme important for glutathione homeostasis control. It has also been implicated in many physiological disorders, including Parkinson's disease, apoptosis inhibition, and diabetes. In the first step of its ping-pong mechanism it binds glutathione, its in vivo substrate, and releases cysteinylglycine upon formation of an acyl-enzyme intermediate. This intermediate can then react with water to release glutamate as a hydrolysis product or with an amino acid or dipeptide to form a transpeptidation product. Further detailed study of the mechanism underlying these reactions is hindered at least for some GGTs by the low quantities of protein available after a multistep purification from tissue. In the present work the gene for human GGT was cloned into the pPICZalphaA vector and transformed into Pichia pastoris to express as a 68 kDa His-tagged protein. The optimized expression and secretion of this enzyme in 1 L of culture and subsequent purification by immobilized metal affinity chromatography yielded 1.6 mg of purified enzyme having a specific activity of 237 U/mg. Kinetic parameters for the transpeptidation reaction between glutathione and glycylglycine were determined by mass spectrometry, giving a kcat of 13.4 x 10(3) min-1 and apparent KM values of 1.11 mM for glutathione and 8.1 mM for glycylglycine. The GGT-mediated hydrolysis of glutathione was also studied, providing a kcat of 53 min-1 and a KM value of 7.3 microM for glutathione. Incubation of the enzyme with a mechanism-based inhibitor, enzymatic digest, and mass spectrometric analysis provided the first unambiguous identification of Thr381 as the active site nucleophile of human gamma-glutamyltranspeptidase, and confirmed four of the seven N-linked glycosylation sites. These structural and kinetic data are discussed with respect to a homology model generated to facilitate visualization.     

Investigating pre‐analytical requirements for serum and plasma based infrared spectro‐diagnostic

Infrared spectroscopy is a rapid, easy‐to‐operate, label‐free and therefore cost‐effective technique. Many studies performed on biofluids (eg, serum, plasma, urine, sputum, bile and cerebrospinal fluid) have demonstrated its promising application as a clinical diagnostic tool. Given all these characteristics, infrared spectroscopy appears to be an ideal candidate to be implemented into the clinics. However, before considering its translation, a clear effort is needed to standardise protocols for biofluid spectroscopic analysis. To reach this goal, careful investigations to identify and track errors that can occur during the pre‐analytical phase is a crucial step. Here, we report for the first time, results of investigations into pre‐analytical factors that can affect the quality of the spectral data acquired on serum and plasma, such as the impact of long‐term freezing time storage of samples as well as the month‐to‐month reproducibility of the spectroscopic analysis. The spectral data discrimination has revealed to be majorly impacted by a residual water content variation in serum and plasma dried samples.      

Receptor for advanced glycation end-products (RAGE) modulates neutrophil adhesion and migration on glycoxidated extracellular matrix

AGEs (advanced glycation end-products) accumulate in collagen molecules during uraemia and diabetes, two diseases associated with high susceptibility to bacterial infection. Because neutrophils bind to collagen during their locomotion in extravascular tissue towards the infected area we investigated whether glycoxidation of collagen (AGE-collagen) alters neutrophil migration. Type I collagen extracted from rat tail tendons was used for in vitro glycoxidation (AGE-collagen). Neutrophils were obtained from peripheral blood of healthy adult volunteers and were used for the in vitro study of adhesion and migration on AGE- or control collagen. Glycoxidation of collagen increased adhesion of neutrophils to collagen surfaces. Neutrophil adhesion to AGE-collagen was inhibited by a rabbit anti-RAGE (receptor for AGEs) antibody and by PI3K (phosphoinositide 3-kinase) inhibitors. No effect was observed with ERK (extracellular-signal-regulated kinase) or p38 MAPK (mitogen-activated protein kinase) inhibitors. AGE-collagen was able to: (i) induce PI3K activation in neutrophils, and (ii) inhibit chemotaxis and chemokinesis of chemoattractant-stimulated neutrophils. Finally, we found that blocking RAGE with anti-RAGE antibodies or inhibiting PI3K with PI3K inhibitors restored fMLP (N-formylmethionyl-leucyl-phenylalanine)-induced neutrophil migration on AGE-collagen. These results show that RAGE and PI3K modulate adhesion and migration rate of neutrophils on AGE-collagen. Modulation of adhesiveness may account for the change in neutrophil migration rate on AGE-collagen. As neutrophils rely on their ability to move to perform their function as the first line of defence against bacterial invasion, glycoxidation of collagen may participate in the suppression of normal host defence in patients with diabetes and uraemia.     

Kluyveromyces lactis and Saccharomyces cerevisiae, two potent deacidifying and volatile-sulphur-aroma-producing microorganisms of the cheese ecosystem

Cheese flavour is the result of complex biochemical transformations attributed to bacteria and yeasts grown on the curd of smear-ripened cheeses. Volatile sulphur compounds (VSCs) are responsible for the characteristic aromatic notes of several cheeses. In the present study, we have assessed the ability of Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae strains, which are frequently isolated from smear-ripened cheeses, to grow and deacidify a cheese medium and generate VSCs resulting from l-methionine degradation. The Kluyveromyces strains produced a wider variety and higher amounts of VSCs than the S. cerevisiae ones. We have shown that the pathway is likely to be proceeding differently in these two yeast genera. The VSCs are mainly generated through the degradation of 4-methylthio-oxobutyric acid in the Kluyveromyces strains, in contrast to the S. cerevisiae ones which have higher l-methionine demethiolating activity, resulting in a direct conversion of l-methionine to methanethiol. The deacidification activity which is of major importance in the early stages of cheese-ripening was also compared in S. cerevisiae and Kluyveromyces strains.     

Genetic variation and evolutionary history of holly oak: a circum-Mediterranean species-complex [Quercus coccifera L./Q. calliprinos (Webb) Holmboe, Fagaceae]

Holly oak is the only evergreen oak possessing a circum-Mediterranean range; it has two predominant morphological forms, calliprinos and coccifera, described in the eastern and western Mediterranean Basin respectively. The concordance of allozyme and morphotype variation was analysed in the whole holly oak range, and the most plausible historical events responsible for the current geographic pattern of genetic variation were investigated. Individuals from 24 populations were scored for allozyme variation at seven polymorphic loci. Multi-locus genotypes were analysed by using a correspondence analysis (CA) and a Bayesian clustering approach. The relative positions of the populations were obtained from multi-dimensional scaling coupled with UPGMA treatment. A continuous genotype distribution was observed in the CA, and two groups were identified using the Bayesian approach. With a 0.95 threshold, 66 and 69% of the individuals showing the calliprinos and the coccifera morphotypes respectively were assigned to the corresponding genetic groups, which differed by private alleles. As compared to coccifera, the genetic group calliprinos was characterized by higher allelic richness and a strong geographical genetic structure. In agreement with fossil records, the most parsimonious explanation for lack of geographical structure in coccifera is a substantial regression of holly oak in the western Mediterranean Basin during the glaciations and a fast westward post-glacial expansion of coccifera populations, probably from Greece. Two population groups were obtained from the scaling/UPGMA treatment. One included all of the calliprinos populations and a Greek coccifera population, suggesting that the two morphotypes are closely related genetically and constitute two components of the same species.     

Phylogenetic relationships and gene flow between sympatric diploid and tetraploid plants ofDactylis glomerata (Gramineae)

Phylogenetic relationships between sympatric, morphologically indistinguishable diploid and tetraploid plants ofDactylis glomerata L. (Gramineae) in Galicia (Spain) were assessed using allozyme markers for 6 distinct systems. The study exploited recent introduction in Galicia and subsequent hybridization of an alien 4xDactylis subspecies possessing distinct allozymes from those of all the native plants. Opportunities for gene exchanges between the ploidies were estimated from in situ observations of flowering, examination of progenies in 2x/4x natural and experimental crosses, and enzyme analyses. Results show a high genetic similarity between the Galician diploids and tetraploids, which possess peculiar alleles in common. Although the ploidy levels usually have distinct flowering periods, interploidal crosses do occasionally occur. Gene flow is likely much more important from the diploid to the tetraploid level. A good genetic intermixing occurs between the Galician and the alien tetraploid entities which have simultaneous flowering. Autopolyploidization of the diploids followed by various rates of hybridization is proposed as one very probable origin of natural tetraploids inDactylis.     

Involvement of lysine 1047 in type I collagen-mediated activation of polymorphonuclear neutrophils

Oxidative functions of polymorphonuclear neutrophils (PMNs), which play a deciding role in the phagocytosis process, are stimulated by extracellular matrix proteins such as type I collagen. Previous studies have demonstrated the involvement of a DGGRYY sequence located within the alpha(1) chain C-terminal telopeptide in type I collagen-induced PMN activation, but so far the mechanism has not been completely elucidated. We have recently demonstrated that collagen carbamylation (i.e. post-translational binding of cyanate to lysine epsilon-NH(2) groups) impairs PMN oxidative functions, suggesting the potential involvement of lysine residues in this process. The present study was devoted to the identification of lysine residues involved in the collagen-induced activation of PMNs. The inhibition of PMN activation by collagen in the presence of 6-amino-hexanoic acid, a structural analogue of lysine residues, confirmed the involvement of specific lysine residues. Modification of lysine residues by carbamylation demonstrated that only one residue, located within the alpha(1)CB6 collagen peptide, was involved in this mechanism. A recombinant alpha(1)CB6 peptide, designed for the substitution of lysine 1047 by glycine, exhibited decreased activity, demonstrating that the lysine residue at position 1047 within the collagen molecule played a significant role in the mechanism of activation. These results help to understand in more detail the collagen-mediated PMN activation mechanism and confirm the prominent involvement of lysine residues in interactions between extracellular matrix proteins and inflammatory cells.