Novel alkoxybenzamide inhibitors of poly(ADP-ribose) polymerase

We have previously described poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors based on a substituted benzyl-phthalazinone scaffold. As an alternative chemical template, a novel series of alkoxybenzamides were developed with restricted conformation through intramolecular hydrogen bond formation; the compounds exhibit low nM enzyme and cellular activity as PARP-1 inhibitors.     

Sun-Compass Orientation in Mediterranean Fish Larvae

Mortality is very high during the pelagic larval phase of fishes but the factors that determine recruitment success remain unclear and hard to predict. Because of their bipartite life history, larvae of coastal species have to head back to the shore at the end of their pelagic episode, to settle. These settlement-stage larvae are known to display strong sensory and motile abilities, but most work has been focused on tropical, insular environments and on the influence of coast-related cues on orientation. In this study we quantified the in situ orientation behavior of settlement-stage larvae in a temperate region, with a continuous coast and a dominant along-shore current, and inspected both coast-dependent and independent cues. We tested six species: one Pomacentridae, Chromis chromis, and five Sparidae, Boops boops, Diplodus annularis, Oblada melanura, Spicara smaris and Spondyliosoma cantharus. Over 85% of larvae were highly capable of keeping a bearing, which is comparable to the orientation abilities of tropical species. Sun-related cues influenced the precision of bearing-keeping at individual level. Three species, out of the four tested in sufficient numbers, oriented significantly relative to the sun position. These are the first in situ observations demonstrating the use of a sun compass for orientation by wild-caught settlement-stage larvae. This mechanism has potential for large-scale orientation of fish larvae globally.     

DTNBP1, a schizophrenia susceptibility gene, affects kinetics of transmitter release

Schizophrenia is one of the most debilitating neuropsychiatric disorders, affecting 0.5-1.0% of the population worldwide. Its pathology, attributed to defects in synaptic transmission, remains elusive. The dystrobrevin-binding protein 1 (DTNBP1) gene, which encodes a coiled-coil protein, dysbindin, is a major susceptibility gene for schizophrenia. Our previous results have demonstrated that the sandy (sdy) mouse harbors a spontaneously occurring deletion in the DTNBP1 gene and expresses no dysbindin protein (Li, W., Q. Zhang, N. Oiso, E.K. Novak, R. Gautam, E.P. O'Brien, C.L. Tinsley, D.J. Blake, R.A. Spritz, N.G. Copeland, et al. 2003. Nat. Genet. 35:84-89). Here, using amperometry, whole-cell patch clamping, and electron microscopy techniques, we discovered specific defects in neurosecretion and vesicular morphology in neuroendocrine cells and hippocampal synapses at the single vesicle level in sdy mice. These defects include larger vesicle size, slower quantal vesicle release, lower release probability, and smaller total population of the readily releasable vesicle pool. These findings suggest that dysbindin functions to regulate exocytosis and vesicle biogenesis in endocrine cells and neurons. Our work also suggests a possible mechanism in the pathogenesis of schizophrenia at the synaptic level.     

Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11

A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.     

Steric factors moderate conformational fluidity and contribute to the high proton sensitivity of Root effect hemoglobins

The structural basis of the extreme pH dependence of oxygen binding to Root effect Hbs is a long-standing puzzle in the field of protein chemistry. A previously unappreciated role of steric factors in the Root effect was revealed by a comparison of pH effects on oxygenation and oxidation processes in human Hb relative to Spot (Leiostomus xanthurus) and Carp (Cyprinodon carpio) Hbs. The Root effect confers five-fold increased pH sensitivity to oxygenation of Spot and Carp Hbs relative to Hb A(0) in the absence of anionic effectors, and even larger relative elevations of pH sensitivity of oxygenation in the presence of 0.2M phosphate. Remarkably, the Root effect was not evident in the oxidation of the Root effect Hbs. This finding rules out pH-dependent alterations in the thermodynamic properties of the heme iron, measured in the anaerobic oxidation reaction, as the basis of the Root effect. The alternative explanation supported by these results is that the elevated pH sensitivity of oxygenation of Root effect Hbs is attributable to globin-dependent steric effects that alter oxygen affinity by constraining conformational fluidity, but which have little influence on electron exchange via the heme edge. This elegant mode of allosteric control can regulate oxygen affinity within a given quaternary state, in addition to modifying the T-R equilibrium. Evolution of Hb sequences that result in proton-linked steric barriers to heme oxygenation could provide a general mechanism to account for the appearance of the Root effect in the structurally diverse Hbs of many species.     

In Vivo Delta Opioid Receptor Internalization Controls Behavioral Effects of Agonists

Background

GPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal.

Methods and Findings

In this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking.

Conclusions

Together our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs.     

Mechanisms governing B cell developmental defects in invariant chain-deficient mice

Invariant chain (Ii)-deficient mice exhibit profound B cell defects that have remained poorly understood, because they could not be simply explained by impaired Ag presentation. We found that Ii deficiency induced cell autonomous defects of two distinct B cell lineages. The life span of mature follicular (FO) B cells was reduced, accounting for their markedly decreased frequency, whereas, in contrast, marginal zone (MZ) B cells accumulated. Other Ii-expressing lineages such as B1 B cells and dendritic cells were unaffected. Surprisingly, the life span of FO B cells was fully corrected in Ii/I-Abeta doubly deficient mice, revealing that Ii-free I-Abeta chains alter FO B cell survival. In contrast, the accumulation of MZ B cells was controlled by a separate mechanism independent of I-Abeta. Interestingly, in Ii-deficient mice lacking FO B cells, the MZ B cells invaded the FO zone, suggesting that intact follicules contribute to the retention of B cells in the MZ. These findings reveal unexpected consequences of Ii deficiency on the development and organization of B cell follicles.     

Mining online genomic resources in Anolis carolinensis facilitates rapid and inexpensive development of cross-species microsatellite markers for the Anolis lizard genus

Online sequence databases can provide valuable resources for the development of cross-species genetic markers. In particular, mining expressed tag sequences (EST) for microsatellites and developing conserved cross-species microsatellite markers can provide a rapid and relatively inexpensive method to develop new markers for a range of species. Here, we adopt this approach to develop cross-species microsatellite markers in Anolis lizards, which is a model genus in evolutionary biology and ecology. Using EST sequences from Anolis carolinensis, we identified 127 microsatellites that satisfied our criteria, and tested 49 of these in five species of Anolis (carolinensis, distichus, apletophallus, porcatus and sagrei). We identified between 8 and 25 new variable genetic markers for five Anolis species. These markers will be a valuable resource for studies of population genetics, comparative mapping, mating systems, behavioural ecology and adaptive radiations in this diverse lineage.     

Australian Dust Storm Associated with Extensive Aspergillus sydowii Fungal “Bloom” in Coastal Waters

A massive central Australian dust storm in September 2009 was associated with abundant fungal spores (150,000/m³) and hyphae in coastal waters between Brisbane (27°S) and Sydney (34°S). These spores were successfully germinated from formalin-preserved samples, and using molecular sequencing of three different genes (the large subunit rRNA gene [LSU], internal transcribed spacer [ITS[, and beta-tubulin gene), they were conclusively identified as Aspergillus sydowii, an organism circumstantially associated with gorgonian coral fan disease in the Caribbean. Surprisingly, no human health or marine ecosystem impacts were associated with this Australian dust storm event. Australian fungal cultures were nontoxic to fish gills and caused a minor reduction in the motility of Alexandrium or Chattonella algal cultures but had their greatest impacts on Symbiodinium dinoflagellate coral symbiont motility, with hyphae being more detrimental than spores. While we have not yet seen any soft coral disease outbreaks on the Australian Great Barrier Reef similar to those observed in the Caribbean and while this particular fungal population was non- or weakly pathogenic, our observations raise the possibility of future marine ecosystem pathogen impacts from similar dust storms harboring more pathogenic strains.