Nuclear import of N-terminal FAK by activation of the FcepsilonRI receptor in RBL-2H3 cells

As FAK integrates membrane receptor signalling, yet is also found in the nucleus, we investigated whether nuclear FAK is regulated by membrane receptor activation. Activation of the mast cell FcepsilonRI receptor leads to the release and synthesis of inflammatory mediators as well as increased proliferation and survival. Using RBL-2H3 basophilic leukaemia cells, FAK and the FcepsilonRI receptor were co-localised following cross-linking of IgE with antigen. This also resulted in a significant increase in the nucleus of several N-terminal FAK fragments, the largest of which included the kinase domain but not the focal adhesion targeting domain. This was confirmed using cells that stably expressed recombinant EGFP-FAK. Furthermore, treatment of EGFP-FAK expressing cells with Leptomycin B, an inhibitor of nuclear export, resulted in increased nuclear localisation of EGFP-FAK. Therefore, FAK can shuttle between the nuclear and cytoplasmic compartments and the cellular distribution of N-terminal FAK is regulated by membrane receptor activation.     

First derivatives of myo-inositol 1,4,6-trisphosphate modified at positions 2 and 3: structural analogues of D-myo-inositol 1,4,5-trisphosphate

Novel, structurally modified potential mimics of the second messenger D-myo-inositol 1,4,5-trisphosphate, based on the biologically active regioisomer D-myo-inositol 1,4,6-trisphosphate, were synthesised. DL-5-O-Benzyl-1,4,6-tri-O-p-methoxybenzyl-myo-inositol was the key intermediate for the preparation of the following compounds: DL-3-deoxy-, DL-3-deoxy-2-O-methyl-, DL-3-O-(2-hydroxyethyl)-, DL-3-O-(3-hydroxypropyl)- and DL-3-O-(4-hydroxybutyl)-myo-inositol 1,4,6-trisphosphate. DL-1,4,6-Tri-O -allyl-5-O-benzyl-myo-inositol was used to prepare DL-2-O-methyl-myo-inositol 1,4,6-trisphosphate. Deoxy-compounds were prepared by reduction of the corresponding tosylated intermediate using Super Hydride. The hydroxyalkyl groups were introduced at the C-3 of myo-inositol using the corresponding benzyl protected hydroxy alkyl bromide via the cis-2,3-O-dibutylstannylene acetal. Methylation and benzylation at C-2 was accomplished using methyl iodide and benzyl bromide, respectively, in the presence of sodium hydride. Deblocking of p-methoxybenzyl groups was accomplished with TFA in dichloromethane and the allyl groups were removed by isomerisation to the cis-prop-1-enyl derivative, which was hydrolysed under acidic conditions to give the corresponding 1,4,6-triol. The 1,4,6-triols were phosphitylated with the P(III) reagent bis(benzyloxy)(diisopropylamino)phosphine in the presence of 1H-tetrazole then oxidised with 3-chloroperoxybenzoic acid followed by deblocking by hydrogenolysis to give DL-2-O-methyl-, DL-3-O-deoxy-, DL-3-O-deoxy-2-O-methyl-, DL-3-O-(2-hydroxyethyl)-, DL-3-O-(3-hydroxypropyl)- and DL-3-O-(4-hydroxybutyl)-myo-inositol 1,4,6-trisphosphate, respectively.     

Natural selection on unpalatable species imposed by state-dependent foraging behaviour

Müllerian mimicry is typically thought to arise as a consequence of defended prey species adopting a similar way of signalling their unprofitability, thereby reducing the costs of predator education. Here we consider subsequent selection on the morphology of prey species, in the potentially lengthy period of time when predators are generally aware of the noxious qualities of their prey (and so no further learning is involved). Using a pair of stochastic dynamic programming equations which describe both the toxin burdens of a predator and its energy level, we identified the optimal state-dependent rules that maximize a predator's long-term survivorship, and examined the implications of this behaviour for the evolution of prey morphologies. When palatable prey are in short supply then those prey species which contain relatively low doses of toxins become profitable to consume by hungry predators. Under these conditions, a weakly defended prey could gain selective advantage in the post educational period by resembling a prey species which contained a higher dose of the same or different toxins, although the precise nature of the ecological relationship between model and mimic could either be mutualistic or parasitic depending on how mimic density increases when favoured by selection. Our work formally demonstrates that one does not always need to invoke educational effects to explain why two or more unpalatable species have evolved a similar appearance, or to explain why mimetic similarity among distasteful species is maintained over time. When two species contain high levels of different toxins then they may gain mutual advantage by resembling one another, not only by educating the predator as to their common unprofitability (classical Müllerian mimicry), but also by increasing predator uncertainty as to the specific kind of toxin a prey item contains.     

Disruption of the actin cytoskeleton can complement the ability of Nef to enhance human immunodeficiency virus type 1 infectivity

The human immunodeficiency virus (HIV) protein Nef has been shown to increase the infectivity of HIV at an early point during infection. Since Nef is known to interact with proteins involved in actin cytoskeleton rearrangements, we tested the possibility that Nef may enhance HIV infectivity via a mechanism that involves the actin cytoskeleton. We find that disruption of the actin cytoskeleton complements the Nef infectivity defect. The ability of disruption of the actin cytoskeleton to complement the Nef defect was specific to envelopes that fuse at the cell surface, including a variety of HIV envelopes and the murine leukemia virus amphotropic envelope. In contrast, the infectivity of HIV virions pseudotyped to enter cells via endocytosis, which is known to complement the HIV Nef infectivity defect and can naturally penetrate the cortical actin barrier, was not altered by actin cytoskeleton disruption. The results presented here suggest that Nef functions to allow the HIV genome to penetrate the cortical actin network, a known barrier for intracellular parasitic organisms.     

Regulation of R7 and R8 differentiation by the spalt genes

Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of the developing ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt was previously proposed to promote R7 and R8 terminal differentiation. Here we show that spalt is required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta0.5, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specification or differentiation in the larval disc but promotes terminal differentiation during pupation. We show that spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. We demonstrate that spalt and senseless are part of a genetic network, which regulates rhodopsin 6 and rhodopsin 1. Taken together, our results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8 differentiation during pupal stages, including the regulation of rhodopsin expression.     

Structure-function analysis of the Drosophila retinal determination protein Dachshund

Dachshund (Dac) is a highly conserved nuclear protein that is distantly related to the Ski/Sno family of corepressor proteins. In Drosophila, Dac is necessary and sufficient for eye development and, along with Eyeless (Ey), Sine oculis (So), and Eyes absent (Eya), forms the core of the retinal determination (RD) network. In vivo and in vitro experiments suggest that members of the RD network function together in one or more complexes to regulate the expression of downstream targets. For example, Dac and Eya synergize in vivo to induce ectopic eye formation and they physically interact through conserved domains. Dac contains two highly conserved domains, named DD1 and DD2, but no function has been assigned to either of them in an in vivo context. We performed structure-function studies to understand the relationship between the conserved domains of Dac and the rest of the protein and to determine the function of each domain during development. We show that only DD1 is essential for Dac function and while DD2 facilitates DD1, it is not absolutely essential in spite of more than 500 million years of conservation. Moreover, the physical interaction between Eya and DD2 is not required for the genetic synergy between the two proteins. Finally, we show that DD1 also plays a central role for nuclear localization of Dac.     

Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR

Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site.     

Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential

Due to the acidic nature of the stomach, enteric organisms must withstand extreme acid stress for colonization and pathogenesis. Escherichia coli contains several acid resistance systems that protect cells to pH 2. One acid resistance system, acid resistance system 2 (AR2), requires extracellular glutamate, while another (AR3) requires extracellular arginine. Little is known about how these systems protect cells from acid stress. AR2 and AR3 are thought to consume intracellular protons through amino acid decarboxylation. Antiport mechanisms then exchange decarboxylation products for new amino acid substrates. This form of proton consumption could maintain an internal pH (pHi) conducive to cell survival. The model was tested by estimating the pHi and transmembrane potential (DeltaPsi) of cells acid stressed at pH 2.5. During acid challenge, glutamate- and arginine-dependent systems elevated pHi from 3.6 to 4.2 and 4.7, respectively. However, when pHi was manipulated to 4.0 in the presence or absence of glutamate, only cultures challenged in the presence of glutamate survived, indicating that a physiological parameter aside from pHi was also important. Measurements of DeltaPsi indicated that amino acid-dependent acid resistance systems help convert membrane potential from an inside negative to inside positive charge, an established acidophile strategy used to survive extreme acidic environments. Thus, reversing DeltaPsi may be a more important acid resistance strategy than maintaining a specific pHi value.     

Insect antifeedant compounds from Nothofagus dombeyi and N. pumilio

A bioassay-guided purification of the extracts of Nothofagus dombeyi and N. pumilio leaves yielded several triterpenes and flavonoids including 2-O-acetylmaslinic acid, 3-O-acetyl 20,24,25-trihydroxydammarane, and 3,20,24,25-tetrahydroxydammarane as new natural products. All the isolated compounds were assessed for antifeeding activity against the 5th instar larvae of Ctenopsteustis obliquana. 12-Hydroxyoleanolic lactone and pectolinarigenin from N. dombeyi and dihydrooroxylin A from N. pumilio, showed significant antifeeding activity.     

Delineation of Cohen syndrome following a large-scale genotype-phenotype screen

Cohen syndrome is an autosomal recessive condition associated with developmental delay, facial dysmorphism, pigmentary retinopathy, and neutropenia. The pleiotropic phenotype, combined with insufficient clinical data, often leads to an erroneous diagnosis and has led to confusion in the literature. Here, we report the results of a comprehensive genotype-phenotype study on the largest cohort of patients with Cohen syndrome assembled to date. We found 22 different COH1 mutations, of which 19 are novel, in probands identified by our diagnostic criteria. In addition, we identified another three novel mutations in patients with incomplete clinical data. By contrast, no COH1 mutations were found in patients with a provisional diagnosis of Cohen syndrome who did not fulfill the diagnostic criteria ("Cohen-like" syndrome). This study provides a molecular confirmation of the clinical phenotype associated with Cohen syndrome and provides a basis for laboratory screening that will be valuable in its diagnosis.