A Functional Screen Provides Evidence for a Conserved, Regulatory, Juxtamembrane Phosphorylation Site in Guanylyl Cyclase A and B

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ∼20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation – processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200–1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor 32PO4 co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC50 properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders

Community ecology of the Middle Miocene primates of La Venta, Colombia: the relationship between ecological diversity, divergence time, and phylogenetic richness

It has been suggested that the degree of ecological diversity that characterizes a primate community correlates positively with both its phylogenetic richness and the time since the members of that community diverged (Fleagle and Reed in Primate communities. Cambridge University Press, New York, pp 92–115, 1999). It is therefore questionable whether or not a community with a relatively recent divergence time but high phylogenetic richness would be as ecologically variable as a community with similar phylogenetic richness but a more distant divergence time. To address this question, the ecological diversity of a fossil primate community from La Venta, Colombia, a Middle Miocene platyrrhine community with phylogenetic diversity comparable with extant platyrrhine communities but a relatively short time since divergence, was compared with that of modern Neotropical primate communities. Shearing quotients and molar lengths, which together are reliable indicators of diet, for both fossil and extant species were plotted against each other to describe the dietary “ecospace” occupied by each community. Community diversity was calculated as the area of the minimum convex polygon encompassing all community members. The diversity of the fossil community was then compared with that of extant communities to test whether the fossil community was less diverse than extant communities while taking phylogenetic richness into account. Results indicate that the La Ventan community was not significantly less ecologically diverse than modern communities, supporting the idea that ecological diversification occurred along with phylogenetic diversification early in platyrrhine evolution

Structural Analysis of a Repetitive Protein Sequence Motif in Strepsirrhine Primate Amelogenin

Strepsirrhines are members of a primate suborder that has a distinctive set of features associated with the development of the dentition. Amelogenin (AMEL), the better known of the enamel matrix proteins, forms 90% of the secreted organic matrix during amelogenesis. Although AMEL has been sequenced in numerous mammalian lineages, the only reported strepsirrhine AMEL sequences are those of the ring-tailed lemur and galago, which contain a set of additional proline-rich tandem repeats absent in all other primates species analyzed to date, but present in some non-primate mammals. Here, we first determined that these repeats are present in AMEL from three additional lemur species and thus are likely to be widespread throughout this group. To evaluate the functional relevance of these repeats in strepsirrhines, we engineered a mutated murine amelogenin sequence containing a similar proline-rich sequence to that of Lemur catta. In the monomeric form, the MQP insertions had no influence on the secondary structure or refolding properties, whereas in the assembled form, the insertions increased the hydrodynamic radii. We speculate that increased AMEL nanosphere size may influence enamel formation in strepsirrhine primates

An Essential Role for DYF-11/MIP-T3 in Assembling Functional Intraflagellar Transport Complexes

MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia 1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. elegans MIP-T3 ortholog DYF-11 is an intraflagellar transport (IFT) protein that plays a critical role in assembling functional kinesin motor-IFT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the IFT machinery, including Kinesin-II, as well as IFT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that DYF-11 functions as a novel component of IFT subcomplex B. Consistent with an evolutionarily conserved cilia-associated role, mammalian MIP-T3 localizes to basal bodies and cilia, and zebrafish mipt3 functions synergistically with the Bardet-Biedl syndrome protein Bbs4 to ensure proper gastrulation, a key cilium- and basal body-dependent developmental process. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis and further highlight the emerging role of this organelle in vertebrate development

Emerging evidence of a link between the polycystins and the mTOR pathways

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease characterized by the formation of renal cysts. This disease can be caused by mutations in two genes, PKD1 and PKD2, which encode polycystin-1 (PC-1) and -2 (PC-2), respectively

A systematic review of mental disorder, suicide, and deliberate self harm in lesbian, gay and bisexual people

Background: Lesbian, gay and bisexual (LGB) people may be at higher risk of mental disorders than heterosexual people.Method: We conducted a systematic review and meta-analysis of the prevalence of mental disorder, substance misuse, suicide, suicidal ideation and deliberate self harm in LGB people. We searched Medline, Embase, PsycInfo, Cinahl, the Cochrane Library Database, the Web of Knowledge, the Applied Social Sciences Index and Abstracts, the International Bibliography of the Social Sciences, Sociological Abstracts, the Campbell Collaboration and grey literature databases for articles published January 1966 to April 2005. We also used Google and Google Scholar and contacted authors where necessary. We searched all terms related to homosexual, lesbian and bisexual people and all terms related to mental disorders, suicide, and deliberate self harm. We included papers on population based studies which contained concurrent heterosexual comparison groups and valid definition of sexual orientation and mental health outcomes.Results: Of 13706 papers identified, 476 were initially selected and 28 (25 studies) met inclusion criteria. Only one study met all our four quality criteria and seven met three of these criteria. Data was extracted on 214,344 heterosexual and 11,971 non heterosexual people. Meta-analyses revealed a two fold excess in suicide attempts in lesbian, gay and bisexual people [ pooled risk ratio for lifetime risk 2.47 (CI 1.87, 3.28)]. The risk for depression and anxiety disorders (over a period of 12 months or a lifetime) on meta-analyses were at least 1.5 times higher in lesbian, gay and bisexual people (RR range 1.54-2.58) and alcohol and other substance dependence over 12 months was also 1.5 times higher (RR range 1.51-4.00). Results were similar in both sexes but meta analyses revealed that lesbian and bisexual women were particularly at risk of substance dependence (alcohol 12 months: RR 4.00, CI 2.85, 5.61; drug dependence: RR 3.50, CI 1.87, 6.53; any substance use disorder RR 3.42, CI 1.97-5.92), while lifetime prevalence of suicide attempt was especially high in gay and bisexual men (RR 4.28, CI 2.32, 7.88).Conclusion: LGB people are at higher risk of mental disorder, suicidal ideation, substance misuse, and deliberate self harm than heterosexual people

Phylogenetic Analysis of Seven WRKY Genes across the Palm Subtribe Attaleinae (Arecaceae) Identifies Syagrus as Sister Group of the Coconut

BACKGROUND:The Cocoseae is one of 13 tribes of Arecaceae subfam. Arecoideae, and contains a number of palms with significant economic importance, including the monotypic and pantropical Cocos nucifera L., the coconut, the origins of which have been one of the "abominable mysteries" of palm systematics for decades. Previous studies with predominantly plastid genes weakly supported American ancestry for the coconut but ambiguous sister relationships. In this paper, we use multiple single copy nuclear loci to address the phylogeny of the Cocoseae subtribe Attaleinae, and resolve the closest extant relative of the coconut. METHODOLOGY/PRINCIPAL FINDINGS:We present the results of combined analysis of DNA sequences of seven WRKY transcription factor loci across 72 samples of Arecaceae tribe Cocoseae subtribe Attaleinae, representing all genera classified within the subtribe, and three outgroup taxa with maximum parsimony, maximum likelihood, and Bayesian approaches, producing highly congruent and well-resolved trees that robustly identify the genus Syagrus as sister to Cocos and resolve novel and well-supported relationships among the other genera of the Attaleinae. We also address incongruence among the gene trees with gene tree reconciliation analysis, and assign estimated ages to the nodes of our tree. CONCLUSIONS/SIGNIFICANCE:This study represents the as yet most extensive phylogenetic analyses of Cocoseae subtribe Attaleinae. We present a well-resolved and supported phylogeny of the subtribe that robustly indicates a sister relationship between Cocos and Syagrus. This is not only of biogeographic interest, but will also open fruitful avenues of inquiry regarding evolution of functional genes useful for crop improvement. Establishment of two major clades of American Attaleinae occurred in the Oligocene (ca. 37 MYBP) in Eastern Brazil. The divergence of Cocos from Syagrus is estimated at 35 MYBP. The biogeographic and morphological congruence that we see for clades resolved in the Attaleinae suggests that WRKY loci are informative markers for investigating the phylogenetic relationships of the palm family