Proteomic analysis of the androgen receptor via MS-compatible purification of biotinylated protein on streptavidin resin

The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high-temperature elution in urea-containing buffers, we show that mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde cross-linked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen-activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.     

A smaller Macadamia from a more vagile tribe: inference of phylogenetic relationships, divergence times, and diaspore evolution in Macadamia and relatives (tribe Macadamieae; Proteaceae)

Tribe Macadamieae (91 spp., 16 genera; Proteaceae) is widespread across the southern hemisphere on all major fragments of Gondwana except New Zealand and India. Macadamia is cultivated outside its natural range as a "nut" crop (notably in Hawaii, where it is the principal orchard crop). We sampled seven DNA regions and 53 morphological characters from the tribe to infer its phylogeny and address the common assumption that the distribution of the extant diversity of the tribe arose by the rafting of ancestors on Gondwanan fragments. Macadamia proves to be paraphyletic with respect to the African genus Brabejum, the South American genus Panopsis, and the Australian species Orites megacarpus. We erect two new generic names, Nothorites and Lasjia, to produce monophyly at that rank. The earliest disjunctions in the tribe are inferred to be the result of long-distance dispersal out of Australia (with one possible exception), rather than vicariance. Evolution of tardy fruit dehiscence is correlated with these dispersals, and the onset of the Antarctic Circumpolar Current (ACC) precedes them. We suggest that the ancestors of extant diversity arrived on their respective continents via the ACC, and we recognize that this is a mechanism precluded, rather than facilitated, by Gondwana's terrestrial continuity.     

The conformational properties of methyl alpha-(2,8)-di/trisialosides and their N-acyl analogues: implications for anti-Neisseria meningitidis B vaccine design

The conformational properties of di- and trisaccharide fragments of the polysialic acid O-antigen capsular polysaccharide (CPS) of Neisseria meningitidis B (NmB) have been investigated by a combination of solution phase NMR spectroscopy and explicit-solvent molecular dynamics (MD) simulations. Simulations employing 100 ns of conventional MD, as well as 160 ns of replica exchange MD (REMD), with the GLYCAM06 force field were shown to be in agreement with experimental NMR scalar J-coupling and NOE values. The presence of conformational families has been determined by monitoring interglycosidic torsion angles, by comparing structural superimpositions, as well as via a Bayesian statistical analysis of the torsional data. Attempts to augment the immunogenicity of NmB CPS often involve chemical modifications of the N-acetyl moiety. Here the effects of these chemical group modifications on the conformational properties of the trisialoside have been probed via REMD simulations of the N-glycolyl, N-propionyl, N-propyl and N-butanoyl analogues. Although there were conformational families unique to each non-native analogue, the chemical modifications resulted in largely equivalent overall conformational phase-spaces compared to the native trisialoside. On the basis of the conformational distributions, these shared conformational properties suggest that a recurrent global conformational epitope may be present in both the native and chemically modified CPS fragments. Explanations are therefore provided for monoclonal antibody cross-reactivity, in terms of recognition of a shared global CPS conformation, as well as for lack of cross-reactivity, in terms of fine structural differences associated with the N-acyl groups, which may be dominant in highly matured antibody responses.     

Immunostimulatory action of AC II--an ayurvedic formulation useful in HIV

Immunostimulatory activity of AC II, a registered ayurvedic preparation prepared at Amala Ayurvedic Research Centre for treating HIV and AIDS is reported. AC II administration could significantly enhance the mitogen-induced proliferation of lymphocytes of spleen cells. It was also found to increase cell-mediated immune responses in normal and tumor-bearing control animals. Oral administration of AC II significantly enhanced Natural Killer cell activity in normal and tumor-bearing animals on the 7th day, which was observed earlier than the tumor-bearing control animals and normal animals. Antibody dependent cellular cytotoxicity (ADCC) was also increased in AC II treated normal and tumor-bearing animals. An early enhancement of antibody-dependent complement-mediated cytotoxicity was also observed by the administration of AC II in normal as well as tumor-bearing animals. Treatment with AC II elevated the levels of IL-2, TNF-alpha and IFN-gamma in normal mice. Administration of AC II was also found to increase the cytotoxic T lymphocyte production in EL4 treated mice. These studies support the use of this immune stimulatory preparation in HIV patients.     

The population genetics of using homing endonuclease genes in vector and pest management

Homing endonuclease genes (HEGs) encode proteins that in the heterozygous state cause double-strand breaks in the homologous chromosome at the precise position opposite the HEG. If the double-strand break is repaired using the homologous chromosome, the HEG becomes homozygous, and this represents a powerful genetic drive mechanism that might be used as a tool in managing vector or pest populations. HEGs may be used to decrease population fitness to drive down population densities (possibly causing local extinction) or, in disease vectors, to knock out a gene required for pathogen transmission. The relative advantages of HEGs that target viability or fecundity, that are active in one sex or both, and whose target is expressed before or after homing are explored. The conditions under which escape mutants arise are also analyzed. A different strategy is to place HEGs on the Y chromosome that cause one or more breaks on the X chromosome and so disrupt sex ratio. This strategy can cause severe sex-ratio biases with efficiencies that depend on the details of sperm competition and zygote mortality. This strategy is probably less susceptible to escape mutants, especially when multiple X shredders are used.     

Novel function of PERK as a mediator of force-induced apoptosis

Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response.     

Clutch size in the tropical scincid lizard Emoia sanfordi, a species endemic to the Vanuatu Archipelago

The majority of species in the scincid genus Emoia (Squamata: Scincidae) have a fixed clutch size of two eggs per clutch and produce between two and four clutches per year. One lineage within Emoia, the Emoia samoensis species group, consists of 13 species occurring in Melanesia and the islands of the southwestern Pacific Ocean, and exhibits variation in clutch size, with previously reported clutch sizes of two to five eggs. Little is known about reproduction in several members of this lineage including Emoia sanfordi, a large-bodied lizard endemic to the archipelago of Vanuatu in the South Pacific. We analyzed reproduction and clutch size in E. sanfordi females and discovered that there is a substantial amount of intraspecific variation, with clutch size ranging from two to seven eggs, with a modal clutch size of five eggs. Females were reproductively active throughout the study period of June through October and appear to be laying multiple clutches. The variation in clutch size seen in E. sanfordi is congruent with the variation previously reported within other closely related species.     

Dual fluorescent protein reporters for studying cell behaviors in vivo

Fluorescent proteins (FPs) are useful tools for visualizing live cells and their behaviors. Protein domains that mediate subcellular localization have been fused to FPs to highlight cellular structures. FPs fused with histone H2B incorporate into chromatin allowing visualization of nuclear events. FPs fused to a glycosylphosphatidylinositol anchor signal sequence label the plasma membrane, highlighting cellular shape. Thus, a reporter gene containing both types of FP fusions would allow for effective monitoring of cell shape, movement, mitotic stage, apoptosis, and other cellular activities. Here, we report a binary color‐coding system using four differently colored FP reporters that generates 16 distinct color codes to label the nuclei and plasma membranes of live cells in culture and in transgenic mice. As an initial test of this system in vivo, the promoter of the human Ubiquitin C (UBC) gene was used to widely express one of the color‐code reporters. Widespread expression of the reporter was attained in embryos; however, both male and female transgenic mice were infertile. In contrast, the promoter of the mouse Oct4/Pou5f1 gene linked to two different color‐code reporters specifically labeled blastocysts, primordial germ cells, and postnatal germ cells, and these mice were fertile. Time‐lapse movies of fluorescently‐labeled primordial germs cells demonstrate the utility of the color‐code system to visualize cell behaviors. This set of new FP reporters should be a useful tool for labeling distinct cell populations and studying their behaviors in complex tissues in vivo. genesis 47:708–717, 2009. © 2009 Wiley‐Liss, Inc.