The Galpha protein controls a pH-dependent signal path to the induction of phytoalexin biosynthesis in Eschscholzia californica

The function of a Galpha protein in the elicitation of phytoalexin (benzophenanthridine) biosynthesis was characterized in cultured cells of California poppy (Eschscholzia californica). Both the decrease of Galpha content via antisense transformation and the expression of recombinant anti-Galpha single-chain antibodies strongly impaired the induction of alkaloid biosynthesis by low elicitor concentrations. All transgenic cell types were deficient in two elicitor-triggered early signal events: activation of phospholipase A2 (PLA2) and efflux of vacuolar protons. The lacking H+ efflux could be restored (1) by adding lysophosphatidylcholine (LPC), a product of PLA2 activity, to vacuoles in situ and (2) by exposing intact cells to isotonic, near-neutral HEPES buffers. The latter treatment induced alkaloid biosynthesis in the absence of elicitor and in Galpha-deficient cells. We conclude that Galpha mediates the stimulation of PLA2 by low elicitor concentrations and that the resulting peak of LPC initiates a transient efflux of vacuolar protons. In this way, an acidic peak of the cytoplasmic pH is generated that causes the expression of enzymes of phytoalexin production independent of the hypersensitive response.     

Tracking genes of ecological relevance using a genome scan in two independent regional population samples of Arabis alpina

Understanding the genetic basis of adaptation in response to environmental variation is fundamental as adaptation plays a key role in the extension of ecological niches to marginal habitats and in ecological speciation. Based on the assumption that some genomic markers are correlated to environmental variables, we aimed to detect loci of ecological relevance in the alpine plant Arabis alpina L. sampled in two regions, the French (99 locations) and the Swiss (109 locations) Alps. We used an unusually large genome scan [825 amplified fragment length polymorphism loci (AFLPs)] and four environmental variables related to temperature, precipitation and topography. We detected linkage disequilibrium among only 3.5% of the considered AFLP loci. A population structure analysis identified no admixture in the study regions, and the French and Swiss Alps were differentiated and therefore could be considered as two independent regions. We applied generalized estimating equations (GEE) to detect ecologically relevant loci separately in the French and Swiss Alps. We identified 78 loci of ecological relevance (9%), which were mainly related to mean annual minimum temperature. Only four of these loci were common across the French and Swiss Alps. Finally, we discuss that the genomic characterization of these ecologically relevant loci, as identified in this study, opens up new perspectives for studying functional ecology in A. alpina, its relatives and other alpine plant species.     

Crystal structure of full length topoisomerase I from Thermotoga maritima

DNA topoisomerases are a family of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. Bacterial and archeal type IA topoisomerases, including topoisomerase I, topoisomerase III, and reverse gyrase, are crucial in regulation of DNA supercoiling and maintenance of genetic stability. The crystal structure of full length topoisomerase I from Thermotoga maritima was determined at 1.7A resolution and represents an intact and fully active bacterial topoisomerase I. It reveals the torus-like structure of the conserved transesterification core domain comprising domains I-IV and a tightly associated C-terminal zinc ribbon domain (domain V) packing against domain IV of the core domain. The previously established zinc-independence of the functional activity of T.maritima topoisomerase I is further supported by its crystal structure as no zinc ion is bound to domain V. However, the structural integrity is preserved by the formation of two disulfide bridges between the four Zn-binding cysteine residues. A functional role of domain V in DNA binding and recognition is suggested and discussed in the light of the structure and previous biochemical findings. In addition, implications for bacterial topoisomerases I are provided.     

Phenon cluster analysis as a method to investigate epidemiological relatedness between sources of Campylobacter jejuni

AIMS: To develop a method for assessing the relative epidemiological significance of possible infection sources for human campylobacteriosis. METHODS AND RESULTS: Using fluorescent amplified fragment length polymorphism (AFLP), 243 apparently epidemiologically unrelated Campylobacter jejuni isolates were genotyped (77 human, 46 cattle, 49 pet and 71 poultry isolates). In total 136 different phena were identified, of which 48 were clusters grouping at least two isolates. Isolates from different sources were frequently clustered together, underlining the high degree of source mixing and the lack of host specificity of C. jejuni. The phena were classified into different phenon types according to the sources of the isolates they contained. The occurrence of these phenon types was analysed using an area-proportional Euler diagram to describe epidemiological relatedness among C. jejuni isolates. Group separation statistics revealed that 43% of analysed human isolates expressed maximum similarity to other human isolates, 9% to cattle isolates, 21% to pet isolates and 27% to poultry isolates; these results were in accordance with the pattern observed in the phenon cluster analysis. CONCLUSIONS: Based on the grouping of strains into molecular similarity clusters, ecological patterns between sources can be investigated. Significance and IMPACT OF THE STUDY: This approach is a new methodological contribution to establish the relative epidemiological significance of concurrent infection sources.     

High energy phosphate transfer by NDPK B/Gβγcomplexes - an alternative signaling pathway involved in the regulation of basal cAMP production

The activation of heterotrimeric G proteins induced by G protein coupled receptors (GPCR) is generally believed to occur by a GDP/GTP exchange at the G protein α -subunit. Nevertheless, nucleoside diphosphate kinase (NDPK) and the β-subunit of G proteins (Gβ) participate in G protein activation by phosphate transfer reactions leading to the formation of GTP from GDP. Recent work elucidated the role of these reactions. Apparently, the NDPK isoform B (NDPK B) forms a complex with β; γ; dimers in which NDPK B acts as a histidine kinase phosphorylating G#x03B2; at His266. Out of this high energetic phosphoamidate bond the phosphate can be transferred specifically onto GDP. The formed GTP binds to the G protein α -subunit and thus activates the respective G protein. Evidence is presented, that this process occurs independent of the classical GPCR-induced GTP/GTP exchange und thus contributes, e.g. to the regulation of basal cAMP synthesis in cells.     

Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit

Transient expression of foreign genes in plant tissues is a valuable tool for plant biotechnology. To shorten the time for gene functional analysis in fruits, we developed a transient methodology that could be applied to tomato (Solanum lycopersicum cv Micro Tom) fruits. It was found that injection of Agrobacterium cultures through the fruit stylar apex resulted in complete fruit infiltration. This infiltration method, named fruit agroinjection, rendered high levels of 35S Cauliflower mosaic virus-driven beta-glucuronidase and yellow fluorescence protein transient expression in the fruit, with higher expression levels around the placenta and moderate levels in the pericarp. Usefulness of fruit agroinjection was assayed in three case studies: (1) the heat shock regulation of an Arabidopsis (Arabidopsis thaliana) promoter, (2) the production of recombinant IgA antibodies as an example of molecular farming, and (3) the virus-induced gene silencing of the carotene biosynthesis pathway. In all three instances, this technology was shown to be efficient as a tool for fast transgene expression in fruits.     

Site-specific photocrosslinking to probe interactions of Arf1 with proteins involved in budding of COPI vesicles

ADP-ribosylation factor 1 (Arf1) plays an important role in early and intra-Golgi protein trafficking. During this process, Arf1 interacts with many different proteins and other molecules that regulate its state of activation or are involved in its intracellular function. To determine which of these proteins interact directly with Arf1 during coat protein type I (COPI) vesicle biogenesis, we probed the molecular environment of Arf1 by use of site-specific photocrosslinking. This method was first used successfully in the field of protein trafficking to study the mechanisms involved in protein translocation across the endoplasmic reticulum during protein synthesis. In such a hydrophobic environment, crosslink yields of up to 30% have been observed. We have now applied this method to study the mechanism of vesicle budding from the cytosolic face of the Golgi apparatus, an aqueous environment. Although the crosslink yield is significantly lower under these conditions, due to predominant reaction of the photolabile probes with water, a specific interaction of Arf1 with subunits of coatomer, the major coat protein of COPI vesicles, could readily be identified.     

Angiotensin II modulates VEGF-driven angiogenesis by opposing effects of type 1 and type 2 receptor stimulation in the microvascular endothelium

Vascular endothelial growth factor (VEGF) is a main stimulator of pathological vessel formation. Nevertheless, increasing evidence suggests that Angiotensin II (Ang II) can play an augmentory role in this process. We thus analyzed the contribution of the two Ang II receptor types, AT(1)R and AT(2)R, in a mouse model of VEGF-driven angiogenesis, i.e. oxygen-induced proliferative retinopathy. Application of the AT(1)R antagonist telmisartan but not the AT(2)R antagonist PD123,319 largely attenuated the pathological response. A direct effect of Ang II on endothelial cells (EC) was analyzed by assessing angiogenic responses in primary bovine retinal and immortalized rat microvascular EC. Selective stimulation of the AT(1)R by Ang II in the presence of PD123,319 revealed a pro-angiogenic activity which further increased VEGF-driven EC sprouting and migration. In contrast, selective stimulation of the AT(2)R by either CGP42112A or Ang II in the presence of telmisartan inhibited the VEGF-driven angiogenic response. Using specific inhibitors (pertussis toxin, RGS proteins, kinase inhibitors) we identified G(12/13) and G(i) dependent signaling pathways as the mediators of the AT(1)R-induced angiogenesis and the AT(2)R-induced inhibition, respectively. As AT(1)R and AT(2)R stimulation displays opposing effects on the activity of the monomeric GTPase RhoA and pro-angiogenic responses to Ang II and VEGF requires activation of Rho-dependent kinase (ROCK), we conclude that the opposing effects of the Ang II receptors on VEGF-driven angiogenesis converge on the regulation of activity of RhoA-ROCK-dependent EC migration.     

DHTKD1 Mutations Cause 2-Aminoadipic and 2-Oxoadipic Aciduria

Abnormalities in metabolite profiles are valuable indicators of underlying pathologic conditions at the molecular level. However, their interpretation relies on detailed knowledge of the pathways, enzymes, and genes involved. Identification and characterization of their physiological function are therefore crucial for our understanding of human disease: they can provide guidance for therapeutic intervention and help us to identify suitable biomarkers for monitoring associated disorders. We studied two individuals with 2-aminoadipic and 2-oxoadipic aciduria, a metabolic condition that is still unresolved at the molecular level. This disorder has been associated with varying neurological symptoms. Exome sequencing of a single affected individual revealed compound heterozygosity for an initiating methionine mutation (c.1A>G) and a missense mutation (c.2185G>A [p.Gly729Arg]) in DHTKD1. This gene codes for dehydrogenase E1 and transketolase domain-containing protein 1, which is part of a 2-oxoglutarate-dehydrogenase-complex-like protein. Sequence analysis of a second individual identified the same missense mutation together with a nonsense mutation (c.1228C>T [p.Arg410^∗]) in DHTKD1. Increased levels of 2-oxoadipate in individual-derived fibroblasts normalized upon lentiviral expression of the wild-type DHTKD1 mRNA. Moreover, investigation of L-lysine metabolism showed an accumulation of deuterium-labeled 2-oxoadipate only in noncomplemented cells, demonstrating that DHTKD1 codes for the enzyme mediating the last unresolved step in the L-lysine-degradation pathway. All together, our results establish mutations in DHTKD1 as a cause of human 2-aminoadipic and 2-oxoadipic aciduria via impaired turnover of decarboxylation 2-oxoadipate to glutaryl-CoA.     

N-cadherin specifies first asymmetry in developing neurons

The precise polarization and orientation of developing neurons is essential for the correct wiring of the brain. In pyramidal excitatory neurons, polarization begins with the sprouting of opposite neurites, which later define directed migration and axo-dendritic domains. We here show that endogenous N-cadherin concentrates at one pole of the newborn neuron, from where the first neurite subsequently emerges. Ectopic N-cadherin is sufficient to favour the place of appearance of the first neurite. The Golgi and centrosome move towards this newly formed morphological pole in a second step, which is regulated by PI3K and the actin/microtubule cytoskeleton. Moreover, loss of function experiments in vivo showed that developing neurons with a non-functional N-cadherin misorient their cell axis. These results show that polarization of N-cadherin in the immediate post-mitotic stage is an early and crucial mechanism in neuronal polarity.