Reproductive mode of grape phylloxera (Daktulosphaira vitifoliae, Homoptera: Phylloxeridae) in Europe: molecular evidence for predominantly asexual populations and a lack of gene flow between them.

The genetic structure of European grape phylloxera populations, Daktulosphaira vitifoliae (Homoptera: Phylloxeridae), was analyzed using 6 polymorphic microsatellite markers. Genetic diversity data of 6 populations originating from northern and southern European viticultural regions was assessed for geographic differences, and the structure of 2 additional populations was examined in more detail, focusing on specific host plant and habitat characteristics. To test for "signatures" of clonal reproduction, different population genetic measures were applied to the data obtained from these populations. A total of 195 multilocus genotypes were detected in 360 individuals tested. Significant deviations from Hardy-Weinberg equilibrium, negative FIS values (from -0.148 to -0.658 per population), and the presence of multicopy genotypes revealed that the current major reproductive mode at each of the locations tested was asexual. The high genotypic diversity detected within and among populations, however, together with the occurrence of unique D. vitifoliae genotypes, indicates sexual recombination events took place, probably prior to the multiple introductions into Europe. The absence of overlapping genotypes between the sampling sites suggests low migration rates among the populations studied and implies that the main mode of insect dispersal is through infested plant material carried by human agency. The specific features of European D. vitifoliae habitats are illustrated to discuss the role of habitat and life cycle in the genetic structure of this globally important pest aphid species.     

The crystal structure of the apoenzyme of the iron-sulphur cluster-free hydrogenase

The iron-sulphur cluster-free hydrogenase (Hmd, EC 1.12.98.2) from methanogenic archaea is a novel type of hydrogenase that tightly binds an iron-containing cofactor. The iron is coordinated by two CO molecules, one sulphur and a pyridone derivative, which is linked via a phosphodiester bond to a guanosine base. We report here on the crystal structure of the Hmd apoenzyme from Methanocaldococcus jannaschii at 1.75 A and from Methanopyrus kandleri at 2.4 A resolution. Homodimeric Hmd reveals a unique architecture composed of one central and two identical peripheral globular units. The central unit is composed of the intertwined C-terminal segments of both subunits, forming a novel intersubunit fold. The two peripheral units consist of the N-terminal domain of each subunit. The Rossmann fold-like structure of the N-terminal domain contains a mononucleotide-binding site, which could harbour the GMP moiety of the cofactor. Another binding site for the iron-containing cofactor is most probably Cys176, which is located at the bottom of a deep intersubunit cleft and which has been shown to be essential for enzyme activity. Adjacent to the iron of the cofactor modelled as a ligand to Cys176, an extended U-shaped extra electron density, interpreted as a polyethyleneglycol fragment, suggests a binding site for the substrate methenyltetrahydromethanopterin.     

Epidermal fatty acid-binding protein is increased in rat lungs following in vivo treatment with keratinocyte growth factor

Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with recombinant human keratinocyte growth factor (Palifermin) via intratracheal instillation and analyzed the expression of epidermal fatty acid-binding protein mRNA and protein by quantitative RT-PCR, immunoblotting as well as immunohistochemistry. Keratinocyte growth factor-treatment in vivo leads to an increased expression of epidermal fatty acid-binding protein mRNA and protein in the total lung. Epidermal fatty acid-binding protein mRNA expression per alveolar epithelial type II cell remains constant as shown in isolated type II cells. Epidermal fatty acid-binding protein immunoreactivity is seen in most if not all hyperplastic alveolar epithelial type II cells, and is mainly localized to the cytoplasm. The increase in epidermal fatty acid-binding protein gene expression associated with type II cell hyperplasia might contribute to the molecular mechanisms mediating lung protection by keratinocyte growth factor.     

Magnetic particles for the separation and purification of nucleic acids

Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and work-consuming process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products, and not suited for automation and up-scaling. During the last few years, specifically functionalised magnetic particles were developed. Together with an appropriate buffer system, they allow for the quick and efficient purification directly after their extraction from crude cell extracts. Centrifugation steps were avoided. In addition, the new approach provided for an easy automation of the entire process and the isolation of nucleic acids from larger sample volumes. This review describes traditional methods and methods based on magnetic particles for nucleic acid purification. The synthesis of a variety of magnetic particles is presented in more detail. Various suppliers of magnetic particles for nucleic acid separation as well as suppliers offering particle-based kits for a variety of different sample materials are listed. Furthermore, commercially available manual magnetic separators and automated systems for magnetic particle handling and liquid handling are mentioned.     

Eutrophication-induced changes in benthic algae affect the behaviour and fitness of the marine amphipod Gammarus locusta

This study, conducted in mesocosms, natural field sites, and in laboratory aquaria, showed that eutrophication altered the nutrient status and dominance patterns among marine macroalgae, which in turn, stimulated gammaridean density. Gammaridean abundance correlated positively with both nutrient addition and the amount of green algae (also stimulated by nutrient enrichment). Path analysis indicated that the direct effect of nutrients on gammaridean density was of less importance than the indirect effect through increased production of green algae. In cage colonisation experiments, either in the field or in a control mesocosm kept under ambient nutrient conditions, more gammarids colonised nutrient enriched algae (E-algae) than algae with ambient nutrient levels (A-algae). Gammarus locusta generally grew faster on nutrient enriched algal specimens and when reared on green rather than on brown algae (fucoids). The nutrient status of periphytic algae did not affect gammaridean growth significantly, but the number of egg-carrying females (and thus egg production) was significantly higher among gammarids reared on E-periphyton. The gammaridean habitat preference order (red > green > brown > periphyton) was almost the reverse of their growth rate in feeding assays (periphyton > green > brown). This implies that macroalgae may be more important as a habitat than as a food source for these animals, which then have to become mobile in search of optimal food items. In this process, algal nutrient content was important as the gammarids in our study actively chose high quality nutrient-rich food, which, in addition, increased their fitness. Stimulated growth rates and egg production may ultimately lead to population increase, which, combined with the preference for high nutrient food items may dampen the initial effect of nutrient enrichment (i.e. blooms of green macroalgae) in shallow coastal waters.     

Synthesis of Staphylococcus aureus lipoteichoic acid derivatives for determining the minimal structural requirements for cytokine induction

For the investigation of the minimal structural requirements for cytokine induction, Staphylococcus aureus lipoteichoic acid derivatives with two, three, four, and five glycerophosphate backbone moieties, carrying each a d-alanyl residue, were needed. Based on two different glycerophosphate building blocks and 6b-O-phosphitylated gentiobiosyl diacylglycerol the desired target molecules (compounds 1-4) could be readily obtained and provided for biological studies.     

Evolution of multi-enzyme complexes: the case of tryptophan synthase

The prototypical tryptophan synthase is a stable heterotetrameric alpha-betabeta-alpha complex. The constituting TrpA and TrpB1 subunits, which are encoded by neighboring genes in the trp operon, activate each other in a bi-directional manner. Recently, a novel class of TrpB2 proteins has been identified, whose members contain additional amino acids that might sterically prevent complex formation with TrpA. To test this hypothesis, we characterized the TrpA and TrpB proteins from Sulfolobus solfataricus. This hyperthermophilic archaeon does not contain a TrpB1 protein but instead contains two TrpB2 homologues that are encoded within (TrpB2i) and outside (TrpB2o) the trp operon. We find that TrpB2i and TrpA form a weak and transient complex during catalysis, with a uni-directional activation of TrpA by TrpB2i. In contrast, TrpB2o and TrpA do not form a detectable complex. These results suggest a model for the evolution of the tryptophan synthase in which TrpB2o, TrpB2i, and TrpB1 reflect the stepwise increase of TrpB affinity for TrpA and the refinement of functional subunit interaction, concomitant with the co-localization of the encoding genes in the trp operon.     

Spectroscopic and biochemical studies on protein variants of quinaldine 4-oxidase: Role of E736 in catalysis and effects of serine ligands on the FeSI and FeSII clusters

Quinaldine 4-oxidase (Qox), which catalyzes the hydroxylation of quinaldine to 1H-4-oxoquinaldine, is a heterotrimeric (LMS)2 molybdo-iron/sulfur flavoprotein belonging to the xanthine oxidase family. Variants of Qox were generated by site-directed mutagenesis. Replacement in the large subunit at E736, which is presumed to be located close to the molybdenum, by aspartate (QoxLE736D) resulted in a marked decrease in kcat app for quinaldine, while Km app was largely unaffected. Although a minor reduction of the glutamine substituted variant QoxLE736Q by quinaldine occurred, its activity was below detection, indicating that the carboxylate group of E736 is crucial for catalysis. Replacement of cysteine ligands C40, C45, or C60 (FeSII) and of the C120 or C154 ligands to FeSI in the small subunit of Qox by serine led to decreased iron contents of the protein preparations. Substitutions C40S and C45S (Fe1 of FeSII) suppressed the characteristic FeSII EPR signals and significantly reduced catalytic activity. In QoxSC154S (Fe1 of FeSI), the g-factor components of FeSI were drastically changed. In contrast, Qox proteins with substitutions of C48 and C60 (Fe2 of FeSII), and of the C120 ligand at Fe2 of FeSI, retained considerable activity and showed less pronounced changes in their EPR parameters. Taken together, the properties of the Qox variants suggest that Fe1 of both FeSI and FeSII are the reducible iron sites, whereas the Fe2 ions remain in the ferric state. The location of the reducible iron sites of FeSI and FeSII appears to be conserved in enzymes of the xanthine oxidase family.