Diapause incidence in the two-spotted spider mite increases due to predator presence, not due to selective predation

We recently reported evidence for increased diapause incidence in the spider mite Tetranychus urticae in presence of the predatory mite Typhlodromus pyri. This effect may arise from (1) selective predation on non-diapause spider mites, (2) predator-induced diapause in spider mites, or (3) both. Using a different strain of T. urticae, we first recovered increased diapause incidence in association with predators. Then, we tested for selective feeding in two-choice experiments with equal numbers of non-diapause and diapause spider mites. We found that the predatory mite had a significant preference for the latter. This indicates that increased diapause incidence in association with predatory mites is not due to selective predation. Therefore, predator-mediated physiological induction of diapause seems a more likely explanation. The cues leading to induction appear to relate to the predators, not their effects, since predation simulated by spider-mite removal or puncturing did not significantly affect diapause incidence. Why spider mites benefit from this response, remains an open question.This revised version was published online in May 2005 with a corrected cover date.     

Interleukin-10 regulates arterial pressure in early primate pregnancy

OBJECTIVES: In pregnancy, the placental contribution of cytokines to maternal immunosuppression has been established, however their role in normal maternal blood pressure regulation has not been identified. We investigate the contribution of interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) to the vasodilation of early pregnancy in non-human primates. We also sequenced the IL-10 baboon gene and compared it with humans. METHODS: The effect of four different treatments, administered sequentially (semi-random-design) on resting 18h, night time, or hourly mean arterial pressure (MAP) and heart rate (HR) were measured using telemetry. An anti-human IL-10 monoclonal antibody (MAb, 1mg, n=7), anti-TNF-alpha antibody (n=3), a combination of anti-IL-10 and anti-TNF-alpha antibodies (n=5) or saline (n=3) control were administered intravenously to baboons in early pregnancy. Plasma and placental IL-10 concentration was measured before and after injection in all animals. RESULTS: Anti-human IL-10 MAb caused a significant increase in MAP of 2.6+/-0.5mmHg over the 18-h period (p<0.05). Administration of TNF-alpha alone or in combination with IL-10 did not alter MAP. There was 97% sequence homology of IL-10 cDNA between humans and baboons. CONCLUSIONS: IL-10 was shown to regulate the vasodilation of early pregnancy in Papio hamadryas. This partial role of IL-10 in the early BP response of primate pregnancy may be relevant to pathophysiological states of human pregnancy such as preeclampsia.     

Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.     

A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex

The structure of the scFv fragment FITC-E2, obtained from a naive phage antibody scFv library derived from human donors, was determined at 2.1 A resolution in the free form and at 3.0 A in the complexed form. The wild-type (wt) scFv binds fluorescein with a K(D) of 0.75 nM. The free scFv readily crystallizes by compacting its 18 amino acid-long CDR-H3, partially occluding the binding site and further blocking access by binding to the "bottom" of a neighboring scFv molecule with a cluster of exposed aromatic residues within CDR-H3. Only upon mutating one of the residues involved in this dominant crystal contact, an exposed tryptophan in the middle of CDR-H3, crystals of the complex could be obtained. A series of alanine mutants within the putative antigen binding site, covering a range of binding affinities, were used to relate macroscopic thermodynamic and kinetic binding parameters to single-molecule disruption forces measured by AFM. The effects of the mutations on the binding properties, particularly on the fraction of binding-competent molecules within the population, cannot be fully explained by changes in the strength of local interactions. The significant conformational change of CDR-H3 between the free and the liganded form illustrates the plasticity of the binding site. An accompanying study in this issue by Curcio and colleagues presents the molecular dynamics simulation of the forced unbinding experiments and explores possible effects of the mutations on the unbinding pathway of the hapten.     

Gene expression and digit homology in the chicken embryo wing

The bird wing is of special interest to students of homology and avian evolution. Fossil and developmental data give conflicting indications of digit homology if a pentadactyl "archetype" is assumed. Morphological signs of a vestigial digit I are seen in bird embryos, but no digit-like structure develops in wild-type embryos. To examine the developmental mechanisms of digit loss, we studied the expression of the high-mobility group box containing Sox9 gene, and bone morphogenetic protein receptor 1b (bmpR-1b)-markers for precondensation and prechondrogenic cells, respectively. We find an elongated domain of Sox9 expression, but no bmpR-1b expression, anterior to digit II. We interpret this as a digit I domain that reaches precondensation, but not condensation or precartilage stages. It develops late, when the tissue in which it is lodged is being remodeled. We consider these findings in the light of previous Hoxd-11 misexpression studies. Together, they suggest that there is a digit I vestige in the wing that can be rescued and undergo development if posterior patterning cues are enhanced. We observed Sox9 expression in the elusive "element X" that is sometimes stated to represent a sixth digit. Indeed, incongruity between digit domains and identities in theropods disappears if birds and other archosaurs are considered primitively polydactyl. Our study provides the first gene expression evidence for at least five digital domains in the chick wing. The failure of the first to develop may be plausibly linked to attenuation of posterior signals.     

Systematic comparison of surface coatings for protein microarrays

To process large numbers of samples in parallel is one potential of protein microarrays for research and diagnostics. However, the application of protein arrays is currently hampered by the lack of comprehensive technological knowledge about the suitability of 2-D and 3-D slide surface coatings. We have performed a systematic study to analyze how both surface types perform in combination with different fluorescent dyes to generate significant and reproducible data. In total, we analyzed more than 100 slides containing 1152 spots each. Slides were probed against different monoclonal antibodies (mAbs) and recombinant fusion proteins. We found two surface coatings to be most suitable for protein and antibody (Ab) immobilization. These were further subjected to quantitative analyses by evaluating intraslide and slide-to-slide reproducibilities, and the linear range of target detection. In summary, we demonstrate that only suitable combinations of surface and fluorescent dyes allow the generation of highly reproducible data.     

Alternative pre-mRNA processing regulates cell-type specific expression of the <Emphasis Type="Italic">IL4l1</Emphasis> and <Emphasis Type="Italic">NUP62</Emphasis> genes

Background  

Given the complexity of higher organisms, the number of genes encoded by their genomes is surprisingly small. Tissue specific regulation of expression and splicing are major factors enhancing the number of the encoded products. Commonly these mechanisms are intragenic and affect only one gene.     

The p21-activated protein kinase-related kinase Cla4 is a coincidence detector of signaling by Cdc42 and phosphatidylinositol 4-phosphate

Signal transduction pathways that co-regulate a given biological process often are organized into networks by molecules that act as coincidence detectors. Phosphoinositides and the Rho-type GTPase Cdc42 regulate overlapping processes in all eukaryotic cells. However, the coincidence detectors that link these pathways into networks remain unknown. Here we show that the p21-activated protein kinase-related kinase Cla4 of yeast integrates signaling by Cdc42 and phosphatidylinositol 4-phosphate (PI4P). We found that the Cla4 pleckstrin homology (PH) domain binds in vitro to several phosphoinositide species. To determine which phosphoinositides regulate Cla4 in vivo, we analyzed phosphatidylinositol kinase mutants (stt4, mss4, and pik1). This indicated that the plasma membrane pool of PI4P, but not phosphatidylinositol 4,5-bisphosphate or the Golgi pool of PI4P, is required for localization of Cla4 to sites of polarized growth. A combination of the Cdc42-binding and PH domains of Cla4 was necessary and sufficient for localization to sites of polarized growth. Point mutations affecting either domain impaired the ability of Cla4 to regulate cell morphogenesis and the mitotic exit network (localization of Lte1). Therefore, Cla4 must retain the ability to bind both Cdc42 and phosphoinositides, the hallmark of a coincidence detector. PI4P may recruit Cla4 to the plasma membrane where Cdc42 activates its kinase activity and refines its localization to cortical sites of polarized growth. In mammalian cells, the myotonic dystrophy-related Cdc42-binding kinase possesses p21-binding and PH domains, suggesting that this kinase may be a coincidence detector of signaling by Cdc42 and phosphoinositides.     

Subunits of a yeast oligomeric G protein-coupled receptor are activated independently by agonist but function in concert to activate G protein heterotrimers

G protein-coupled receptors (GPCRs) form dimeric or oligomeric complexes in vivo. However, the function of oligomerization in receptor-mediated G protein activation is unclear. Previous studies of the yeast alpha-factor receptor (STE2 gene product) have indicated that oligomerization promotes signaling. Here we have addressed the mechanism by which oligomerization facilitates G protein signaling by examining the ability of ligand binding- and G protein coupling-defective alpha-factor receptors to form complexes in vivo and to correct their signaling defects when co-expressed (trans complementation). Newly and previously identified receptor mutants indicated that ligand binding involves the exofacial end of transmembrane domain (TM) 4, whereas G protein coupling involves ic1, ic3, the C-terminal tail, and the intracellular ends of TM2 and TM3. Mutant receptors bearing substitutions in these domains formed homo-oligomeric or hetero-oligomeric complexes in vivo, as indicated by results of fluorescence resonance energy transfer experiments. Co-expression of ligand binding- and G protein coupling-defective mutant receptors did not significantly improve signaling. In contrast, co-expression of ic1 and ic3 mutations in trans but not in cis significantly increased signaling efficiency. Therefore, we suggest that subunits of the alpha-factor receptor: 1) are activated independently rather than cooperatively by agonist, and 2) function in a concerted fashion to promote G protein activation, possibly by contacting different subunits or regions of the G protein heterotrimer.