Testing the efficiency of three 15N-labeled nitrogen compounds for indirect labeling of grasshoppers via plants in the field

In field studies of plant–insect herbivore interactions it is often difficult to establish which herbivore has fed on a particular plant. We investigated the suitability of three different ¹⁵N‐labeled nitrogen compounds (ammonium, nitrate, and glycine) for indirect marking of three grasshopper species [Omocestus viridulus (L.), Chorthippus parallelus (Zett.), and Chorthippus biguttulus (L.) (Orthoptera: Acrididae)] through labeling their food plants in the field. In two short‐term experiments grassland plots of 1 m² were separately labeled with either one of the different nitrogen compounds. Grasshoppers were caged on three food‐plant species [Dactylis glomerata L., Holcus lanatus L. (Poaceae), and Trifolium repens L. (Fabaceae)] present in these plots for 72 h. Significantly enriched δ¹⁵N values in grasshoppers were found in all plant/grasshopper combinations. Enrichment in grasshoppers was positively correlated with the enrichment of plants and labeling with nitrate resulted in highest ¹⁵N enrichment. In a long‐term experiment, individuals of C. biguttulus were placed in a cage covering an area of 1 m² for 37 days, with sampling of grasshoppers at regular intervals. δ¹⁵N values of the grasshopper and a common food plant, D. glomerata, increased steadily over time, up to 40‐fold by the end of the experiment. Our results demonstrate that ¹⁵N‐labeling of plants is an appropriate tool for the investigation of insect–plant interactions under natural conditions.     

Signaling through CD5 Activates a Pathway Involving Phosphatidylinositol 3-Kinase, Vav, and Rac1 in Human Mature T Lymphocytes

CD5 acts as a coreceptor on T lymphocytes and plays an important role in T-cell signaling and T-cell–B-cell interactions. Costimulation of T lymphocytes with anti-CD5 antibodies results in an increase of the intracellular Ca²⁺ levels, and subsequently in the activation of Ca²⁺/calmodulin-dependent (CaM) kinase type IV. In the present study, we have characterized the initial signaling pathway induced by anti-CD5 costimulation. The activation of phosphatidylinositol (PI) 3-kinase through tyrosine phosphorylation of its p85 subunit is a proximal event in the CD5-signaling pathway and leads to the activation of the lipid kinase activity of the p110 subunit. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit the CD5-induced response as assessed in interleukin-2 (IL-2) secretion experiments. The expression of an inactivated Rac1 mutant (Rac1 · N17) in T lymphocytes transfected with an IL-2 promoter-driven reporter construct also abrogates the response to CD5 costimulation, while the expression of a constitutively active Rac1 mutant (Rac1-V12) completely replaces the CD5 costimulatory signal. The Rac1-specific guanine nucleotide exchange factor Vav is heavily phosphorylated on tyrosine residues upon CD5 costimulation, which is a prerequisite for its activation. A role for Vav in the CD5-induced signaling pathway is further supported by the findings that the expression of a dominant negative Vav mutant (Vav-C) completely abolishes the response to CD5 costimulation while the expression of a constitutively active Vav mutant [Vav(Δ1–65)] makes the CD5 costimulation signal superfluous. Wortmannin is unable to block the Vav(Δ1–65)- or Rac1 · V12-induced signals, indicating that both Vav and Rac1 function downstream from PI 3-kinase. Vav and Rac1 both act upstream from the CD5-induced activation of CaM kinase IV, since KN-62, an inhibitor of CaM kinases, and a dominant negative CaM kinase IV mutant block the Vav(Δ1–65)-and Rac1 · V12-mediated signals. We propose a model for the CD5-induced signaling pathway in which the PI 3-kinase lipid products, together with tyrosine phosphorylation, activate Vav, resulting in the activation of Rac1 by the Vav-mediated exchange of GDP for GTP.     

Expression of glycosphingolipids in serum-free primary cultures of mouse kidney cells: male-female differences and androgen sensitivity

The expression of neutral glycosphingolipids was examined in primary kidney cell cultures derived from adult male and female beige mutant mice (C57BL/6J;bg ^j/bg ^j) with enrichment for proximal tubule cells during preparation of explants and using defined serum-free medium for the culture conditions. Cell proliferated for 7 daysin vitro to provide confluent or nearly confluent monolayers of epithelial-type growth indicative of proximal tubule cells. The malevs female differences in neutral glycosphingolipids seen in the kidneyin vivo were retained in these 7 day cultures. Cultures derived from males contained galacto- and digalactosylceramides whereas those from females did not express these types of glycolipids. Also, male cells had higher ratios of sphingosine: phytosphingosine containing species in Nfa (non-hydroxy fatty acid) globotriaosylceramide and in glucosylceramide than females. The shift in sphingosine: phytosphingosine to male ratios in Nfa globotriaosylceramide and in glucosylceramide could be stimulated in female kidney cells by treatment with 10^–5 M testosterone or 5-dihydrotestosterone. The male-specific expression of neutral glycosphingolipids, then, appears to be stable character of male-type differentiation in mouse kidney that is passed on during proliferation in culture. Female kidney cells retain an ability to respond to androgens with specific changes in neutral glycosphingolipid expression during 7 days of growthin vitro in serum-free conditions, but do not respond with the induction of the male-specific glycolipids galacto-and digalactosylceramides as seenin vivo.     

Pulsed-field gel electrophoresis-fingerprinting, genome size estimation and rrn loci number of Rhizobium galegae

I. HUBER AND S. SELENSKA-POBELL. 1994. The genomes of several Rhizobium galegae (of.) strains, which effectively nodulate Galega officinalis host, were analysed by pulsed-field gel electrophoresis (PFGE). Individual PFGE-fingerprints were obtained for every particular strain when the rarely cutting restriction endonucleases Spe I and Asn I were applied. In hybridization experiments, where a DNA fragment carrying the rrnB ribosomal RNA operon of Escherichia coli was used as a probe, the number of the resulting strain-specific Spe I and Asn I bands was reduced to three for all of the strains studied. This suggests that in Rh. galegae (of.) there are at least three rrn loci. On the basis of the lengths of the Spe I fragments, separated by PFGE, the genome size of five Rh. galegae (of.) strains was estimated to be 5852 ± 198 kbp.     

Central vasopressin V(1a) and V(1b) receptors modulate the cardiovascular response to air-jet stress in conscious rats.

This study investigates the contribution of central vasopressin receptors in the modulation of systolic arterial pressure (SAP) and heart rate (HR) response to air-jet stress in conscious Wistar rats equipped with a femoral arterial catheter and intracerebroventricular cannula using novel non-peptide and selective vasopressin V(1a) (SR49059) and V(1b) (SSR149415) antagonists. The effects of stress on SAP and HR were evaluated by measuring the maximal response to stress, the latency of the maximal response, the duration of the recovery period, and the increase in the low frequency (LF) short-term variability component. Stress induced a parallel and almost immediate increase in both SAP and HR, followed by enhanced LF SAP variability in the recovery period. Pretreatment of rats with V(1a) antagonist did not affect the maximal increase or the latency of SAP and HR response to acute stress, but shortened the recovery period of SAP and HR and prevented the increase in LF SAP. The V(1b) antagonist reduced the maximal increase in SAP without affecting HR and their latencies, shortened the recovery period of SAP and inhibited the increase in LF SAP variability. These results indicate that both central V(1a) and V(1b) receptors mediate cardiovascular changes induced by air-jet stress in conscious rats.     

Contrasting metacommunity structure and beta diversity in an aquatic‐floodplain system

Habitat connectivity and dispersal interact to structure metacommunities, but few studies have examined these patterns jointly for organisms across the aquatic–terrestrial ecotone. We assessed metacommunity structure and beta diversity patterns of instream benthic invertebrates, riparian carabid beetles (Order: Coleoptera; Family: Carabidae) and riparian spiders (Order: Araneae) at fifteen sites in a river‐floodplain system. Sampling took place over a three‐year period (2010–2012) in the Rhine‐Main‐Observatory LTER site on the Kinzig River, central Germany. This allowed disentangling the combined influence, and temporal variability, of habitat connectivity (i.e. between aquatic and terrestrial) and dispersal ability (i.e. between spiders and beetles, and aerial and aquatic dispersing invertebrates) on the dominant paradigms structuring these metacommunities. We found mostly consistent differences in the manner that metacommunities were structured between groups, with lower levels of variability explained for beetles compared to the other groups. Beetles were consistently structured more by turnover than nestedness components, with greater beta diversity than expected by chance and a minor spatial compared to environmental signal emerging with variance partitioning. Conversely, spiders and benthic invertebrates had lower beta diversity and greater nestedness than null expectation, and a clearer spatial signal controlling metacommunity structure. Our results suggest varying levels of mass effects and species sorting shape river‐floodplain metacommunities, depending on habitat connectivity and dispersal ability. That is, greater connectivity and lower fragmentation along the river compared to the terrestrial zone promoted mass effects, and differences in overall dispersal ability and mode (i.e. active and passive) for instream and riparian communities shifted paradigms between mass effects and species sorting.     

A systems biology approach reveals major metabolic changes in the thermoacidophilic archaeon Sulfolobus solfataricus in response to the carbon source L‐fucose versus D‐glucose

Archaea are characterised by a complex metabolism with many unique enzymes that differ from their bacterial and eukaryotic counterparts. The thermoacidophilic archaeon Sulfolobus solfataricus is known for its metabolic versatility and is able to utilize a great variety of different carbon sources. However, the underlying degradation pathways and their regulation are often unknown. In this work, the growth on different carbon sources was analysed, using an integrated systems biology approach. The comparison of growth on L‐fucose and D‐glucose allows first insights into the genome‐wide changes in response to the two carbon sources and revealed a new pathway for L‐fucose degradation in S. solfataricus. During growth on L‐fucose major changes in the central carbon metabolic network, as well as an increased activity of the glyoxylate bypass and the 3‐hydroxypropionate/4‐hydroxybutyrate cycle were observed. Within the newly discovered pathway for L‐fucose degradation the following key reactions were identified: (i) L‐fucose oxidation to L‐fuconate via a dehydrogenase, (ii) dehydration to 2‐keto‐3‐deoxy‐L‐fuconate via dehydratase, (iii) 2‐keto‐3‐deoxy‐L‐fuconate cleavage to pyruvate and L‐lactaldehyde via aldolase and (iv) L‐lactaldehyde conversion to L‐lactate via aldehyde dehydrogenase. This pathway as well as L‐fucose transport shows interesting overlaps to the D‐arabinose pathway, representing another example for pathway promiscuity in Sulfolobus species.     

The nucleotide‐dependent interaction of FlaH and FlaI is essential for assembly and function of the archaellum motor

The motor of the membrane‐anchored archaeal motility structure, the archaellum, contains FlaX, FlaI and FlaH. FlaX forms a 30 nm ring structure that acts as a scaffold protein and was shown to interact with the bifunctional ATPase FlaI and FlaH. However, the structure and function of FlaH has been enigmatic. Here we present structural and functional analyses of isolated FlaH and archaellum motor subcomplexes. The FlaH crystal structure reveals a RecA/Rad51 family fold with an ATP bound on a conserved and exposed surface, which presumably forms an oligomerization interface. FlaH does not hydrolyze ATP in vitro, but ATP binding to FlaH is essential for its interaction with FlaI and for archaellum assembly. FlaH interacts with the C‐terminus of FlaX, which was earlier shown to be essential for FlaX ring formation and to mediate interaction with FlaI. Electron microscopy reveals that FlaH assembles as a second ring inside the FlaX ring in vitro. Collectively these data reveal central structural mechanisms for FlaH interactions in mediating archaellar assembly: FlaH binding within the FlaX ring and nucleotide‐regulated FlaH binding to FlaI form the archaellar basal body core.