Odorant-specific requirements for arrestin function in Drosophila olfaction

The ability to modulate olfactory sensitivity is necessary to detect chemical gradients and discriminate among a multitude of odor stimuli. Desensitization of odorant receptors has been postulated to occur when arrestins prevent the activation of downstream second messengers. A paucity of in vivo data on olfactory desensitization prompts use of Drosophila melanogaster genetics to investigate arrestins' role in regulating olfactory signaling pathways. Physiological analysis of peripheral olfactory sensitivity reveals decreased responsiveness to a host of chemically distinct odorants in flies deficient for arrestin1 (arr1), arrestin2 (arr2), or both. These phenotypes are manifest in odorant- and dose- dependent fashions. Additionally, mutants display altered adaptive properties under a prolonged exposure paradigm. Behaviorally, arr1 mutants are impaired in olfactory-based orientation towards attractive odor sources. As the olfactory deficits vary according to chemical identity and concentration, they indicate that a spectrum of arrestin activity is essential for odor processing depending upon the particular olfactory pathway involved. Arrestin mutant phenotypes are hypothesized to be a consequence of down-regulation of olfactory signaling to avoid cellular excitotoxicity. Importantly, phenotypic rescue of olfactory defects in arr1(1) mutants is achieved through transgenic expression of wild-type arr1. Taken together, these data clearly indicate that arrestins are required in a stimulus-specific manner for wild type olfactory function and add another level of complexity to peripheral odor coding mechanisms that ultimately impact olfactory behavior.     

Incorporation of three endocytosed varicella-zoster virus glycoproteins, gE, gH, and gB, into the virion envelope

The cytoplasmic tails of all three major varicella-zoster virus (VZV) glycoproteins, gE, gH, and gB, harbor functional tyrosine-based endocytosis motifs that mediate internalization. The aim of the present study was to examine whether endocytosis from the plasma membrane is a cellular route by which VZV glycoproteins are delivered to the final envelopment compartment. In this study, we demonstrated that internalization of the glycoproteins occurred in the first 24 h postinfection but was reduced later in infection. Using surface biotinylation of VZV-infected cells followed by a glutathione cleavage assay, we showed that endocytosis was independent of antibody binding to gE, gH, and gB. Subsequently, with this assay, we demonstrated that biotinylated gE, gH, and gB retrieved from the cell surface were incorporated into nascent virus particles isolated after density gradient sedimentation. To confirm and extend this finding, we repeated the above sedimentation step and specifically detected envelopes decorated with Streptavidin-conjugated gold beads on a majority of complete virions through examination by transmission electron microscopy. In addition, a gE-gI complex and a gE-gH complex were found on the virions. Therefore, the above studies established that VZV subsumed a postendocytosis trafficking pathway as one mechanism by which to deliver viral glycoproteins to the site of virion assembly in the cytoplasm. Furthermore, since a recombinant VZV genome lacking only endocytosis-competent gE cannot replicate, these results supported the conclusion that the endocytosis-envelopment pathway is an essential component of the VZV life cycle.     

Ca release induced by cyclic adenosine diphosphoribose (cADPr) in sea urchin egg homogenates: mechanisms of release and heterogeneity of the Ca compartments

A rapid superfusion system measuring the amounts, kinetics, and Ca dependencies of released 45Ca, was used to examine the effects of ryanodine (RY), caffeine (CF), and cyclic ADP ribose (cADPr) on sea urchin egg homogenates. The RY-sensitive compartment had more than twice the Ca release capacity of the CF-sensitive or cADPr-sensitive compartment. cADPr-stimulated 45Ca release required calcium with half-maximal activation at approximately 0.2 to 0.6 microM [Ca2+]. K(1/2) for cADPr activation was approximately 100 nM, and in spite of the Ca requirement for cADPr-stimulated release, the cADPr affinity was not affected by [Ca2+]. Peak 45Ca release rate with cADPr (3 microM) was greater than with CF (20 mM), yet the release amounts were similar and both were [Ca2+]-dependent. When activated with CF and cADPr simultaneously, 45Ca release was large and, no longer [Ca2+]-dependent. Mg competitively inhibited the Ca activation site(s), yet did not inhibit the activation with CF-plus-cADPr. Pre-release of 45Ca by cADPr with low (approximately 0.1 microM) [Ca2+] right-shifted the [Ca2+] dependence of the remaining cADPr-response. These data suggest that (a) only a portion of RY-sensitive compartments empty when stimulated with cADPr or CF, (b) Ca and cADPr act on non-interacting sites, and (c) cADPr-sensitive compartments represent a heterogeneous population with different [Ca2+] dependencies.     

Impacts of biological control and invasive species on a non-target native Hawaiian insect

The potential for classical biological control to cause unintended harm to native species was evaluated in the case of the endemic Hawaiian koa bug, Coleotichus blackburniae White (Hemiptera: Scutelleridae), and parasitoids introduced to Hawaii for control of an agricultural pest, the southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae). Parasitism of C. blackburniae eggs, nymphs and adults by biocontrol agents was quantified across a wide range of habitats and compared to other sources of mortality. Egg mortality due to the biocontrol agent Trissolcus basalis Wollaston (Hymenoptera: Scelionidae) was low (maximum 26%) and confined to elevations below 500 m on a single host plant. Predation, mainly by alien spiders and ants, was the greatest source of egg mortality (maximum 87%). Parasitism of adult C. blackburniae by the biocontrol agent Trichopoda pilipes (F.) (Diptera: Tachinidae) was near zero at 21 of 24 sites surveyed. Three sites with high bug density had higher levels of T. pilipes parasitism, reaching maxima of 70% among adult female bugs, 100% among males and 50% among fifth instars. Male-biased parasitism indicated that T. pilipes is adapted to using male aggregation pheromone for finding C. blackburniae hosts. The relative impacts of biocontrol agents and other sources of mortality were compared using life tables. Invasive species, particularly generalist egg predators, had the greatest impacts on C. blackburniae populations. Effects of intentionally introduced parasitoids were relatively minor, although the tachinid T. pilipes showed potential for large impacts at individual sites. In retrospect, non-target attacks by biological control agents on C. blackburniae were predictable, but the environmental range and magnitude of impacts would have been difficult to foresee.     

Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children. It is thought to result from the transformation of granule cell precursors (GCPs) in the developing cerebellum, but little is known about the early stages of the disease. Here, we identify a pre-neoplastic stage of medulloblastoma in patched heterozygous mice, a model of the human disease. We show that pre-neoplastic cells are present in the majority of patched mutants, although only 16% of these mice develop tumors. Pre-neoplastic cells, like tumor cells, exhibit activation of the Sonic hedgehog pathway and constitutive proliferation. Importantly, they also lack expression of the wild-type patched allele, suggesting that loss of patched is an early event in tumorigenesis. Although pre-neoplastic cells resemble GCPs and tumor cells in many respects, they have a distinct molecular signature. Genes that mark the pre-neoplastic stage include regulators of migration, apoptosis and differentiation, processes crucial for normal development but previously unrecognized for their role in medulloblastoma. The identification and molecular characterization of pre-neoplastic cells provides insight into the early steps in medulloblastoma formation, and may yield important markers for early detection and therapy of this disease.     

The SNARE Ykt6 is released from yeast vacuoles during an early stage of fusion

The farnesylated SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) Ykt6 mediates protein palmitoylation at the yeast vacuole by means of its amino-terminal longin domain. Ykt6 is localized equally to membranes and the cytosol, although it is unclear how this distribution is mediated. We now show that Ykt6 is released efficiently from vacuoles during an early stage of yeast vacuole fusion. This release is dependent on the disassembly of vacuolar SNAREs (priming). In recent literature, it had been demonstrated for mammalian Ykt6 that the membrane-bound form is both palmitoylated and farnesylated at its carboxy-terminal CAAX box, whereas soluble Ykt6 is only farnesylated. In agreement with this, we find that yeast Ykt6 becomes palmitoylated in vitro at its C-terminal CAAX motif. Mutagenesis of the potential palmitoylation site in yeast Ykt6 prevents stable membrane association and is lethal. On the basis of these and other findings, we speculate that Ykt6 is released from membranes by depalmitoylation. Such a mechanism could enable recycling of this lipid-anchored SNARE from the vacuole independent of retrograde transport.     

Selecting cases from nuclear families for case-control association analysis

We examine the efficiency of a number of schemes to select cases from nuclear families for case-control association analysis using the Genetic Analysis Workshop 14 simulated dataset. We show that with this simulated dataset comparing all affected siblings with unrelated controls is considerably more powerful than all of the other approaches considered. We find that the test statistic is increased by almost 3-fold compared to the next best sampling schemes of selecting all affected sibs only from families with affected parents (AF aff), one affected sib with most evidence of allele-sharing from each family (SF), and all affected sibs from families with evidence for linkage (AF L). We consider accounting for biological relatedness of samples in the association analysis to maintain the correct type I error. We also discuss the relative efficiencies of increasing the ratio of unrelated cases to controls, methods to confirm associations and issues to consider when applying our conclusions to other complex disease datasets.     

Human CD69 associates with an N-terminal fragment of calreticulin at the cell surface

CD69 is thought to be a pluripotent signaling molecule expressed on the surface of a number of activated leukocytes including B, T, and NK cells, monocytes, neutrophils, and platelets. While some advances have been made regarding the mechanisms by which CD69 may participate in such diverse functions as cell aggregation, cellular cytotoxicity, and release of cytokines and inflammatory mediators, the most proximal links of signal initiation have not been identified. Our study has identified, by immunoprecipitation and direct protein sequencing (LC/MS/MS), binding of CD69 to an N-terminal protein fragment of calreticulin expressed on the cell surface of human PBMCs. Given the recently identified roles calreticulin plays in cell adhesion and angiogensis, the identification of CD69 binding directly to calreticulin may provide insights into mechanism(s) by which CD69 or other CD69 family members, i.e., LLT1 and AICL participates in such diverse functions.     

Correlated evolution of leaf shape and trichomes in Begonia dregei (Begoniaceae)

Structural features of leaves, including size, shape, and surfaces, vary greatly throughout the plant kingdom. In both functional and phylogenetic analyses of leaves, the various morphological aspects are often considered independently of each other, although it is likely that many combinations of features do not occur at random due to either functional constraint or genetic correlation. The distribution of variation in leaf morphology in the highly variable Begonia dregei species complex was examined in natural populations and in F(2) offspring from a cross between plants from two populations. Leaf shape was quantified using several morphometric measures, and trichomes on leaves were counted and measured. Correlations between leaf shape and the numbers and size of trichomes were examined. There were significant correlations between the shapes of leaves and the presence, number, and size of trichomes among populations and in hybrid plants. Deeply incised leaves had larger numbers of longer trichomes at the sinuses. Higher numbers of trichomes on upper leaf surfaces occurred together with trichomes at the petiole and on the abaxial surface. The potential for independent evolution of leaf shape and trichomes in this group is limited. Hypotheses to explain the correlated development of leaf shape and trichomes are discussed.     

Role for CXCR6 in recruitment of activated CD8+ lymphocytes to inflamed liver

Hepatic infiltration of activated CD8 lymphocytes is a major feature of graft-vs-host disease (GvHD). Chemoattractant cytokines and their receptors are key regulators of lymphocyte trafficking, but the involvement of chemoattractant receptors in the physiologic recruitment of cells into the inflamed liver has not been defined. The present study examines the role of the chemokine receptor CXCR6, which is highly expressed by liver-infiltrating CD8 T cells. Hepatic accumulation of donor CD8, but not donor CD4, lymphocytes was significantly reduced in GvHD induced by transfer of CXCR6(-/-), H-2D(b) lymphocytes into BDF(1), H-2D(bxd) recipients. To determine whether altered recruitment contributes to the reduced accumulation, CXCR6(-/-) or wild-type splenic lymphocytes participating in an active GvHD response were isolated and transferred i.v. into secondary recipients with active GvHD, and the short term (6-h) recruitment of transferred cells to the inflamed liver was assessed. CXCR6(-/-) CD8 (but not CD4) cells displayed a significant (33%) reduction in liver localization, whereas frequencies in blood of CXCR6(-/-) and wild-type CD8 cells were similar. Proliferation and apoptosis of liver-infiltrating donor CD8 cells were unaffected. We conclude that CXCR6 helps mediate the recruitment of activated CD8 lymphocytes in GvHD-induced hepatitis and may be a useful target to treat pathological inflammation in the liver.