Beta turn propensity and a model polymer scaling exponent identify intrinsically disordered phase-separating proteins

The complex cellular milieu can spontaneously demix, or phase separate, in a process controlled in part by intrinsically disordered (ID) proteins. A protein''s propensity to phase separate is thought to be driven by a preference for protein–protein over protein–solvent interactions. The hydrodynamic size of monomeric proteins, as quantified by the polymer scaling exponent (v), is driven by a similar balance. We hypothesized that mean v, as predicted by protein sequence, would be smaller for proteins with a strong propensity to phase separate. To test this hypothesis, we analyzed protein databases containing subsets of proteins that are folded, disordered, or disordered and known to spontaneously phase separate. We find that the phase-separating disordered proteins, on average, had lower calculated values of v compared with their non-phase-separating counterparts. Moreover, these proteins had a higher sequence-predicted propensity for β-turns. Using a simple, surface area-based model, we propose a physical mechanism for this difference: transient β-turn structures reduce the desolvation penalty of forming a protein-rich phase and increase exposure of atoms involved in π/sp² valence electron interactions. By this mechanism, β-turns could act as energetically favored nucleation points, which may explain the increased propensity for turns in ID regions (IDRs) utilized biologically for phase separation. Phase-separating IDRs, non-phase-separating IDRs, and folded regions could be distinguished by combining v and β-turn propensity. Finally, we propose a new algorithm, ParSe (partition sequence), for predicting phase-separating protein regions, and which is able to accurately identify folded, disordered, and phase-separating protein regions based on the primary sequence.     

A phylogenetic analysis of the Orchidaceae: evidence from rbcL nucleotide

Cladistic parsimony analyses of rbcL nucleotide sequence data from 171 taxa representing nearly all tribes and subtribes of Orchidaceae are presented here. These analyses divide the family into five primary monophyletic clades: apostasioid, cypripedioid, vanilloid, orchidoid, and epidendroid orchids, arranged in that order. These clades, with the exception of the vanilloids, essentially correspond to currently recognized subfamilies. A distinct subfamily, based upon tribe Vanilleae, is supported for Vanilla and its allies. The general tree topology is, for the most part, congruent with previously published hypotheses of intrafamilial relationships; however, there is no evidence supporting the previously recognized subfamilies Spiranthoideae, Neottioideae, or Vandoideae. Subfamily Spiranthoideae is embedded within a single clade containing members of Orchidoideae and sister to tribe Diurideae. Genera representing tribe Tropideae are placed within the epidendroid clade. Most traditional subtribal units are supported within each clade, but few tribes, as currently circumscribed, are monophyletic. Although powerful in assessing monophyly of clades within the family, in this case rbcL fails to provide strong support for the interrelationships of the subfamilies (i.e., along the spine of the tree). The cladograms presented here should serve as a standard to which future morphological and molecular studies can be compared.     

Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes

Proteomics is a valuable tool for establishing and comparing the protein content of defined tissues, cell types, or subcellular structures. Its use in non-model species is currently limited because the identification of peptides critically depends on sequence databases. In this study, we explored the potential of a preliminary cDNA database for the non-model species Pisum sativum created by a small number of massively parallel pyrosequencing (MPSS) runs for its use in proteomics and compared it to comprehensive cDNA databases from Medicago truncatula and Arabidopsis thaliana created by Sanger sequencing. Each database was used to identify proteins from a pea leaf chloroplast envelope preparation. It is shown that the pea database identified more proteins with higher accuracy, although the sequence quality was low and the sequence contigs were short compared to databases from model species. Although the number of identified proteins in non-species-specific databases could potentially be increased by lowering the threshold for successful protein identifications, this strategy markedly increases the number of wrongly identified proteins. The identification rate with non-species-specific databases correlated with spectral abundance but not with the predicted membrane helix content, and strong conservation is necessary but not sufficient for protein identification with a non-species-specific database. It is concluded that massively parallel sequencing of cDNAs substantially increases the power of proteomics in non-model species.     

Biochemical and mass spectrometric characterization of human N-acylethanolamine-hydrolyzing Acid amidase inhibition

The mechanism of inactivation of human enzyme N-acylethanolamine-hydrolyzing acid amidase (hNAAA), with selected inhibitors identified in a novel fluorescent based assay developed for characterization of both reversible and irreversible inhibitors, was investigated kinetically and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 1-Isothiocyanatopentadecane (AM9023) was found to be a potent, selective and reversible hNAAA inhibitor, while two others, 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-Benzyloxycarbonyl-L-serine β-lactone (N-Cbz-serine β-lactone), inhibited hNAAA in a covalent and irreversible manner. MS analysis of the hNAAA/covalent inhibitor complexes identified modification only of the N-terminal cysteine (Cys126) of the β-subunit, confirming a suggested mechanism of hNAAA inactivation by the β-lactone containing inhibitors. These experiments provide direct evidence of the key role of Cys126 in hNAAA inactivation by different classes of covalent inhibitors, confirming the essential role of cysteine for catalysis and inhibition in this cysteine N-terminal nucleophile hydrolase enzyme. They also provide a methodology for the rapid screening and characterization of large libraries of compounds as potential inhibitors of NAAA, and subsequent characterization or their mechanism through MALDI-TOF MS based bottom up-proteomics.     

Gonadal development in hermaphrodite mice

Summary The genetic background and morphology of spontaneous mosaic hermaphrodites of mice have been described. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8. 1978. This work was supported by Grant HD 04083 from the National Institute of Child Health and Human Development.     

Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.      

The influence of age on fecal steroid hormone levels in male Budongo Forest chimpanzees (Pan troglodytes schweinfurthii)

Potential interactions between age and endocrinological functioning have been understudied in wild ape populations. Therefore, we examined the relationship between age and the secretion of androgens and glucocorticoids in 15 juvenile, subadult, and adult male chimpanzees (Pan troglodytes schweinfurthii) free ranging in the Budongo Forest of Uganda. One hundred and nine fecal samples were opportunistically collected, between 07:30 and 13:30 hr, during the wet season. Fecal samples were preserved, by oven drying, and steroid content extracted before radioimmunoassay for dehydroepiandrosterone-sulfate (DHEA-S), testosterone (TEST), cortisol (CORT), and corticosterone (CCT). Employing indexes of age as predictive factors, linear mixed-effects modeling and non-parametric statistical comparisons of fecal steroid levels were conducted. Age was observed to significantly influence the production of both glucocorticoids and androgens in male Budongo Forest chimpanzees. Basically, whereas TEST and CORT increased, DHEA-S and CCT levels slightly declined as animals matured.     

Population structure of Sclerotinia sclerotiorum in an Australian canola field at flowering and stem-infection stages of the disease cycle.

Populations of the ascomycete pathogen Sclerotinia sclerotiorum sampled from a canola field were analysed using microsatellite markers. Fifty isolates were collected from ascospore-infested canola petals and, later in the season, another 55 isolates were obtained from stem lesions; these isolates were used to compare inoculum and disease-causing populations. Fifty-five unique haplotypes were identified, with gene diversity ranging from 0.40 to 0.71. Genotypic diversity was higher in the inoculum population than it had been in the previous year, but analysis of molecular variance (AMOVA) showed that less than 10% of the variation was attributable to differences between the 2 years. Genotypic disequilibrium measures were consistent with the occurrence of both clonal reproduction and out-crossing. There was no significant population subdivision between the ascospore and stem-lesion populations, as measured with fixation indices (R(ST) = 0.015, p = 0.90) and AMOVA, suggesting that there are no genetically defined subgroups of isolates more likely to proceed from petal colonization to cause stem infection. This might be because S. sclerotiorum possesses wide-ranging pathogenicity mechanisms that account for the lack of host specificity observed to date.