Phenotypic effects in Xenopus and zebrafish suggest that one-eyed pinhead functions as antagonist of BMP signalling

Zebrafish one-eyed pinhead (oep) is essential for embryonic axis and dorsal midline formation by promoting Nodal signalling and is thought to act as a permissive factor. Here we describe that oep elicits profound phenotypic effects when overexpressed in Xenopus and zebrafish. In Xenopus, wild-type oep inhibits mesoderm induction, disrupts axis formation and neuralizes animal caps. A secreted Oep dorsoanteriorizes and neuralizes Xenopus embryos indicative of BMP inhibition. In zebrafish, misexpression of smad1 in oep mutant embryos also reveals an interaction of oep with BMP signalling. Furthermore, the phenotypic effect of nodal overexpression can be rescued by coexpression of oep both in Xenopus and zebrafish. Taken together, our results support an interaction between oep and nodal but they suggest also (1) that the role of oep in Nodal signalling may include negative as well as positive regulation, (2) that oep is able to function in an active fashion and (3) that oep exerts a regulatory effect on the BMP signalling pathway.     

Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome

We present extensive explicit solvent molecular dynamics analysis of three RNA three-way junctions (3WJs) from the large ribosomal subunit: the 3WJ formed by Helices 90–92 (H90–H92) of 23S rRNA; the 3WJ formed by H42–H44 organizing the GTPase associated center (GAC) of 23S rRNA; and the 3WJ of 5S rRNA. H92 near the peptidyl transferase center binds the 3′-CCA end of amino-acylated tRNA. The GAC binds protein factors and stimulates GTP hydrolysis driving protein synthesis. The 5S rRNA binds the central protuberance and A-site finger (ASF) involved in bridges with the 30S subunit. The simulations reveal that all three 3WJs possess significant anisotropic hinge-like flexibility between their stacked stems and dynamics within the compact regions of their adjacent stems. The A-site 3WJ dynamics may facilitate accommodation of tRNA, while the 5S 3WJ flexibility appears to be essential for coordinated movements of ASF and 5S rRNA. The GAC 3WJ may support large-scale dynamics of the L7/L12-stalk region. The simulations reveal that H42–H44 rRNA segments are not fully relaxed and in the X-ray structures they are bent towards the large subunit. The bending may be related to L10 binding and is distributed between the 3WJ and the H42–H97 contact.     

Evidence that L-glutamate can act as an exogenous signal to modulate root growth and branching in Arabidopsis thaliana

The roots of many plant species are known to use inorganic nitrogen, in the form of , as a cue to initiate localized root proliferation within nutrient-rich patches of soil. We report here that, at micromolar concentrations and in a genotype-dependent manner, exogenous l-glutamate is also able to elicit complex changes in Arabidopsis root development. l-Glutamate is perceived specifically at the primary root tip and inhibits mitotic activity in the root apical meristem, but does not interfere with lateral root initiation or outgrowth. Only some time after emergence do lateral roots acquire l-glutamate sensitivity, indicating that their ability to respond to l-glutamate is developmentally regulated. Comparisons between different Arabidopsis ecotypes revealed a remarkable degree of natural variation in l-glutamate sensitivity, with C24 being the most sensitive. The aux1-7 auxin transport mutant had reduced l-glutamate sensitivity, suggesting a possible interaction between l-glutamate and auxin signaling. Surprisingly, two loss-of-function mutants at the AXR1 locus (axr1-3 and axr1-12) were hypersensitive to l-glutamate. A pharmacological approach, using agonists and antagonists of mammalian ionotropic glutamate receptors, was unable to provide evidence of a role for their plant homologs in sensing exogenous glutamate. We discuss the mechanism of l-glutamate sensing and the possible ecological significance of the observed l-glutamate-elicited changes in root architecture.     

Multiyear dual nitrate isotope signatures suggest that N‐saturated subtropical forested catchments can act as robust N sinks

In forests of the humid subtropics of China, chronically elevated nitrogen (N) deposition, predominantly as ammonium (NH₄⁺), causes significant nitrate (NO₃^?) leaching from well‐drained acid forest soils on hill slopes (HS), whereas significant retention of NO₃^? occurs in near‐stream environments (groundwater discharge zones, GDZ). To aid our understanding of N transformations on the catchment level, we studied spatial and temporal variabilities of concentration and natural abundance (δ¹⁵N and δ¹⁸O) of nitrate (NO₃^?) in soil pore water along a hydrological continuum in the N‐saturated Tieshanping (TSP) catchment, southwest China. Our data show that effective removal of atmogenic NH₄⁺ and production of NO₃^? in soils on HS were associated with a significant decrease in δ¹⁵N‐NO₃^?, suggesting efficient nitrification despite low soil pH. The concentration of NO₃^? declined sharply along the hydrological flow path in the GDZ. This decline was associated with a significant increase in both δ¹⁵N and δ¹⁸O of residual NO₃^?, providing evidence that the GDZ acts as an N sink due to denitrification. The observed apparent ¹⁵N enrichment factor (ε) of NO₃^? of about ?5‰ in the GDZ is similar to values previously reported for efficient denitrification in riparian and groundwater systems. Episode studies in the summers of 2009, 2010 and 2013 revealed that the spatial pattern of δ¹⁵N and δ¹⁸O‐NO₃^? in soil water was remarkably similar from year to year. The importance of denitrification as a major N sink was also seen at the catchment scale, as largest δ¹⁵N‐NO₃^? values in stream water were observed at lowest discharge, confirming the importance of the relatively small GDZ for N removal under base flow conditions. This study, explicitly recognizing hydrologically connected landscape elements, reveals an overlooked but robust N sink in N‐saturated, subtropical forests with important implications for regional N budgets.     

Evidence to suggest that the spontaneous release of acetylcholine from rat hippocampal tissue is carrier-mediated

The effect ofl- andd-stereoisomers of 2-(4-phenylpiperidino) cyclohexanol (AH 5183) on the spontaneous release of acetylcholine (Ach) from rat hippocampal tissue was studied.l-AH 5183 was approximately 100 times more potent than wasd-AH 5183 in reducing spontaneous ACh release. Spontaneous ACh release was also temperature dependent. These results may suggest that the spontaneous release of ACh from brain tissue is carrier-mediated.     

Diverse inhibitors of intracellular signalling act as adenosine receptor antagonists.

Inhibitors of intracellular signalling events, including enzyme inhibitors, are often used to investigate signal transduction pathways. We examined whether some inhibitors that act on the ATP site of enzymes are also potent adenosine receptor antagonists. Competitive radioligand binding assays in membranes or brain sections show that genistein, chelerythrine, and SQ22536 [9-(tetrahydro-2'-furyl) adenine] block A(1), A(2A), and A(3) adenosine receptors in concentrations of these drugs commonly used to examine cellular signalling (K(i) of [(3)H]-DPCPX (1,3-dipropyl-8-cyclopentylxanthine) competition mean (95% confidence interval): 2.6 (1.5-4.8) microM, 5.7 (2.1-15.8) microM, 59.4 (17.3-203.8) microM; K(i) of [(3)H]-SCH58261 [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine] competition: 15.3 (8.1-28.8) microM, 37.6 (10.3-137.4) microM, 16.7 (11.5-24.3) microM for genistein, chelerythrine, and SQ22536, respectively). Given that adenosine receptors are present on most cells, that adenosine is often present, and that adenosine receptors interact functionally with several signalling pathways, these results may be of significance also when studying signalling via other receptors.     

Evidence that endo-1,4-beta-glucanases act on cellulose in suspension-cultured poplar cells

Suspension-cultured poplar (Populus alba) cells produce two distinct endo-1,4-beta-glucanases, one of which is released in the extracellular culture medium and the other localized in their walls. Two cDNA clones, PopCel1 and PopCel2, isolated from a poplar cDNA library, encode the extracellular and the wall-bound endo-1, 4-beta-glucanases, respectively, based upon deduced amino acid sequences. The products of these two genes contained domains conserved in endo-1,4-beta-glucanase (family 9) and showed 91.5% amino acid identity. The levels of both PopCel1 and PopCel2 mRNAs increased during the lag phase of growth and decreased rapidly during the linear phase. After the levels had decreased, they were again increased by addition of sucrose to the culture medium and further enhanced by the addition of 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of sucrose. The accumulation of the mRNAs was correlated with the solubilization of cello-oligosaccharides. Cello-oligosaccharides and xyloglucan were also solubilized from the wall preparations of poplar cells incubated with enzyme preparations from the extracellular culture medium and walls. An antibody against both PopCel proteins reduced the production of cello-oligosaccharides by the extracellular enzyme by 90% and that by the wall-bound enzyme by 55%, and also prevented xyloglucan solubilization. The results show that the accumulation of poplar endo-1,4-beta-glucanases is regulated indirectly by auxin in the presence of sucrose and can act on cellulose in suspension-cultured poplar cells.     

Intracranial dialysis and microinfusion studies suggest that morphine may act in the ventromedial hypothalamus to inhibit female rat sexual behavior.

The present investigation examined the neural sites and mechanisms of opiate inhibition of female sexual behavior. Systemic administration of morphine (10 mg/kg) significantly reduced ovarian steroid-induced estrous behavior in female rats. This behavioral inhibition was prevented when the opiate receptor antagonist naloxone (5 mg/kg) was administered 30 min prior to morphine. Bilateral infusion of morphine directly into the ventromedial hypothalamus (VMH) also inhibited hormone-dependent estrous behavior for at least 2 hr. Furthermore, naloxone infusion into the VMH 20 min before behavior testing reduced the inhibitory effects of systemically administered morphine on lordosis. These results suggest that morphine may inhibit female sexual behavior by acting directly on the VMH, the primary site at which ovarian steroids facilitate this behavior. In a separate experiment we used in vivo brain microdialysis to test the hypothesis that morphine inhibits lordosis by interfering with norepinephrine (NE) neurotransmission in the VMH. In control rats, the onset of mating was associated with increased NE release in the VMH. Morphine-treated animals displayed neither behavioral estrus nor elevated NE release from the VMH when tested with stimulus males. These data are consistent with the hypothesis that morphine suppresses NE release in the VMH. Nevertheless, mechanisms other than or in addition to attenuation of hypothalamic NE release may contribute to the inhibitory effects of morphine on lordosis.     

Characterization of Aplysia enticin and temptin, two novel water-borne protein pheromones that act in concert with attractin to stimulate mate attraction

Mate attraction in Aplysia involves a long-distance water-borne signal (attractin) that is released during egg laying. Other pheromones are predicted to be released during egg laying that act in concert with albumen gland attractin to stimulate attraction, but their identities are unknown. To identify other candidate water-borne pheromones, we employed differential library screening of an albumen gland cDNA library, Northern blot analysis, purification, characterization, cloning, and expression of albumen gland proteins, matrix-assisted laser desorption ionization mass spectrometry, pheromone secretion assays, behavioral bioassays, immunolocalization studies, and comparative genomics. Four genes, Alb-23, Alb-24, Alb-69, and Alb-172, were highly expressed in Aplysia californica albumen glands and encoded novel proteins. The products of the Alb-24 ("enticin") and Alb-172 ("temptin") precursors were soluble and highly abundant in albumen gland extracts, whereas Alb-23 and Alb-69 were membrane-associated proteins. A comparative analysis showed that the predicted Aplysia brasiliana enticin and temptin proteins were 90 and 91% identical, respectively, to their A. californica homologs. T-maze attraction bioassay studies have previously demonstrated that egg cordons alone are attractive to Aplysia but that attractin alone is not. In the present study, however, the combination of attractin, enticin, and temptin was found to be significantly attractive to potential mates and doubled the number of animals attracted to this stimulus compared with control animals. The combined data strongly suggest that enticin and temptin are novel candidate water-borne protein pheromones that act in concert with attractin to attract Aplysia to form and maintain egglaying and mating aggregations.     

A multivariate approach to human mate preferences

Human mate choice is complicated, with various individual differences and contextual factors influencing preferences for numerous traits. However, focused studies on human mate choice often do not capture this multivariate complexity. Here, we consider multiple factors simultaneously to demonstrate the advantages of a multivariate approach to human mate preferences. Participants (N = 689) rated the attractiveness of opposite-sex online dating profiles that were independently manipulated on facial attractiveness, perceived facial masculinity/femininity, and intelligence. Participants were also randomly instructed to either consider short- or long-term relationships. Using fitness surfaces analyses, we assess the linear and nonlinear effects and interactions of the profiles' facial attractiveness, perceived facial masculinity/femininity, and perceived intelligence on participants' attractiveness ratings. Using hierarchical linear modeling, we were also able to consider the independent contribution of participants' individual differences on their revealed preferences for the manipulated traits. These individual differences included participants' age, socioeconomic status, education, disgust (moral, sexual, and pathogen), sociosexual orientation, personality variables, masculinity, and mate value. Together, our results illuminate various previously undetectable phenomena, including nonlinear preference functions and interactions with individual differences. More broadly, the study illustrates the value of considering both individual variation and population-level measures when addressing questions of sexual selection, and demonstrates the utility of multivariate approaches to complement focused studies.     

Prephenate dehydratase from the aphid endosymbiont (Buchnera) displays changes in the regulatory domain that suggest its desensitization to inhibition by phenylalanine

Buchnera aphidicola, the prokaryotic endosymbiont of aphids, complements dietary deficiencies with the synthesis and provision of several essential amino acids. We have cloned and sequenced a region of the genome of B. aphidicola isolated from Acyrthosiphon pisum which includes the two-domain aroQ/pheA gene. This gene encodes the bifunctional chorismate mutase-prephenate dehydratase protein, which plays a central role in L-phenylalanine biosynthesis. Two changes involved in the overproduction of this amino acid have been detected. First, the absence of an attenuator region suggests a constitutive expression of this gene. Second, the regulatory domain of the Buchnera prephenate dehydratase shows changes in the ESRP sequence, which is involved in the allosteric binding of phenylalanine and is strongly conserved in prephenate dehydratase proteins from practically all known organisms. These changes suggest the desensitization of the enzyme to inhibition by phenylalanine and would permit the bacterial endosymbiont to overproduce phenylalanine.     

Evidence from rhesus macaques suggests that male coloration plays a role in female primate mate choice

Male animals of many species use conspicuous coloration to attract mates. Among mammals, primates possess the most brilliant secondary sexual coloration. However, whether colour plays a part in primate female mate choice remains unknown. Adult male rhesus macaques undergo a hormonally regulated increased reddening of facial and anogenital skin during their mating season. We experimentally investigated whether red male facial coloration is preferred by simultaneously presenting female rhesus macaques (n = 6) with computer-manipulated pale and red versions of 24 different male faces. The duration and direction of gaze were measured to discern visual preferences. Females exhibited preferences for the red versions of male faces. It is proposed that male coloration might provide a cue to male quality.     

Evidence that forskolin binds to the glucose transporter of human erythrocytes

Binding of [4-3H]cytochalasin B and [12-3H]forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of [12-3H]forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of [12-3H]forskolin and [4-3H]cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of [4-3H]cytochalasin B or [12-3H]forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either [4-3H]cytochalasin B or [12-3H]forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.     

Identification of functionally important domains of human cytomegalovirus gO that act after trimer binding to receptors

Human cytomegalovirus (HCMV) entry involves trimer (gH/gL/gO) that interacts with PDGFRα in fibroblasts. Entry into epithelial and endothelial cells requires trimer, which binds unidentified receptors, and pentamer (gH/gL/UL128-131), which binds neuropilin-2. To identify functionally important domains in trimer, we screened an overlapping 20-mer gO peptide library and identified two sets of peptides: 19/20 (a.a. 235–267) and 32/33 (a.a. 404–436) that could block virus entry. Soluble trimer containing wild type gO blocked HCMV entry, whereas soluble trimers with the 19/20 or 32/33 sequences mutated did not block entry. Interestingly, the mutant trimers retained the capacity to bind to cellular receptors including PDGFRα. Peptide 19/20 and 32/33 sequences formed a lobe extending from the surface of gO and an adjacent concave structure, respectively. Neither of these sets of sequences contacted PDGFRα. Instead, our data support a model in which the 19/20 and 32/33 trimer sequences function downstream of receptor binding, e.g. trafficking of HCMV into endosomes or binding to gB for entry fusion. We also screened for peptides that bound antibodies (Abs) in human sera, observing that peptides 20 and 26 bound Abs. These peptides engendered neutralizing Abs (NAbs) after immunization of rabbits and could pull out NAbs from human sera. Peptides 20 and 26 sequences represent the first NAb epitopes identified in trimer. These studies describe two important surfaces on gO defined by: i) peptides 19/20 and 32/33, which apparently act downstream of receptor binding and ii) peptide 26 that interacts with PDGFRα. Both these surfaces are targets of NAbs.     

Evidence that calcium acts as an intracellular messenger for adrenergic responses in human erythrocytes

Adrenergic stimulation of membrane protein phosphorylation has been studied in human erythrocytes. The adrenergic enhancement in phosphorylation of band 2 could be mimicked by the calcium-specific ionophore A23187 in the presence of 10 micron extracellular calcium. Experiments with the potassium ionophore, valinomycin, showed that potassium efflux was not the primary effector of the response. Trifluoperazine, an inhibitor of the Ca2+-dependent regulatory protein, calmodulin, inhibited phosphorylation stimulation by either norepinephrine or the calcium ionophore. The norepinephrine response was observed in the absence of extracellular calcium, implicating Ca2+ released from cellular bound pools in mediating the response.     

Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA

DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m(5)C MTases) of both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence motifs that are conserved among m(5)C MTases, including the consensus S:-adenosyl-L-methionine-binding motifs and the active site ProCys dipeptide. DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans. The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-L-homocysteine (AdoHcy) has been determined at 1.8 A resolution. The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HHAI, a confirmed bacterial m(5)C MTase, and the smaller target recognition domains of DNMT2 and M.HHAI are also closely related in overall structure. The small domain of DNMT2 contains three short helices that are not present in M.HHAI. DNMT2 binds AdoHcy in the same conformation as confirmed m(5)C MTases and, while DNMT2 shares all sequence and structural features with m(5)C MTases, it has failed to demonstrate detectable transmethylase activity. We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant complex in vitro. While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif.     

Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction

Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome-microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.