The rockefeller university

Chromosome-dependent spindle assembly requires the chromosomal recruitment and activation of Aurora B, the kinase subunit of the chromosomal passenger complex (CPC). It remains unclear how the chromosome-activated kinase spatially transmits signals to organize the micron-scale spindle. Here we reveal that the CPC must detect two structures, chromosomes and microtubules, to support spindle assembly in Xenopus egg extracts. While Aurora B is enriched on chromosomes in metaphase, we establish that a fraction of Aurora B is targeted to the metaphase spindle and phosphorylates microtubule-bound substrates. We demonstrate that chromosomally activated Aurora B must be targeted to microtubules to drive spindle assembly. Moreover, although the CPC-microtubule interaction can activate Aurora B, which further promotes microtubule assembly, this positive feedback is not initiated without chromosomes. We propose that the dual detection of chromosomes and microtubules by the CPC is a critical step in assembling spindles around and only around chromosomes.

A self-cleaving elastin-like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP alpha subunit was tagged with a self-cleaving ELP-intein tag and coexpressed with the beta, beta', and omega subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP-intein purification. Thus, this simple ELP-based method should be applicable for the nonchromatographic purification of other recombinant, in vivo-assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large-scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.

As conventional transcriptional factors that are activated in diverse signaling pathways, nuclear receptors play important roles in many physiological processes that include energy homeostasis. The MED1 subunit of the Mediator coactivator complex plays a broad role in nuclear receptor-mediated transcription by anchoring the Mediator complex to diverse promoter-bound nuclear receptors. Given the significant role of skeletal muscle, in part through the action of nuclear receptors, in glucose and fatty acid metabolism, we generated skeletal muscle-specific Med1 knockout mice. Importantly, these mice show enhanced insulin sensitivity and improved glucose tolerance as well as resistance to high-fat diet-induced obesity. Furthermore, the white muscle of these mice exhibits increased mitochondrial density and expression of genes specific to type I and type IIA fibers, indicating a fast-to-slow fiber switch, as well as markedly increased expression of the brown adipose tissue-specific UCP-1 and Cidea genes that are involved in respiratory uncoupling. These dramatic results implicate MED1 as a powerful suppressor in skeletal muscle of genetic programs implicated in energy expenditure and raise the significant possibility of therapeutical approaches for metabolic syndromes and muscle diseases through modulation of MED1-nuclear receptor interactions.

Background: The hearing of tetrapods including humans is enhanced by an active process that amplifies the mechanical inputs associated with sound, sharpens frequency selectivity, and compresses the range of responsiveness. The most striking manifestation of the active process is spontaneous otoacoustic emission, the unprovoked emergence of sound from an ear. Hair cells, the sensory receptors of the inner ear, are known to provide the energy for such emissions; it is unclear, though, how ensembles of such cells collude to power observable emissions. Methodology and Principal Findings: We have measured and modeled spontaneous otoacoustic emissions from the ear of the tokay gecko, a convenient experimental subject that produces robust emissions. Using a van der Pol formulation to represent each cluster of hair cells within a tonotopic array, we have examined the factors that influence the cooperative interaction between oscillators. Conclusions and Significance: A model that includes viscous interactions between adjacent hair cells fails to produce emissions similar to those observed experimentally. In contrast, elastic coupling yields realistic results, especially if the oscillators near the ends of the array are weakened so as to minimize boundary effects. Introducing stochastic irregularity in the strength of oscillators stabilizes peaks in the spectrum of modeled emissions, further increasing the similarity to the responses of actual ears. Finally, and again in agreement with experimental findings, the inclusion of a pure-tone external stimulus repels the spectral peaks of spontaneous emissions. Our results suggest that elastic coupling between oscillators of slightly differing strength explains several properties of the spontaneous otoacoustic emissions in the gecko.

A theory is put forward that emphasises differences in neuronal responses to fluctuations in steroid hormone levels compared to constant hormone levels. We propose that neuronal functions that regulate gonadotrophin release from the anterior pituitary tend to be more sensitive to rapid increases in the levels of oestrogens than they are to constant oestrogen levels. By contrast, neurones that control certain behavioral functions are affected just as well by constant oestrogen levels as by positively accelerating levels of oestrogen. In addition to providing examples of data from recent experiments that examine actions of the long-term effects of oestrogen on mouse behaviour, we illustrate the behavioural effects of microinjections of adeno-associated viral vectors of small interfering RNA directed against the mRNA for oestrogen receptor-alpha (ER alpha). This manipulation provides for a long-term loss of ER alpha function in a neuranatomically specific manner. The theoretical distinction between temporal features of oestrogen sensitivity of neuroendocrine versus behavioural function is not absolute, but is intended to stimulate new experimentation that examines temporal features of oestrogen administration.

The PMTs of the CMS Hadron Forward calorimeter were found to generate a large size signal when their windows were traversed by energetic charged particles. This signal, which is due to. Cerenkov light production at the PMT window, could interfere with the calorimeter signal and mislead the measurements. In order to find a viable solution to this problem, the response of four different types of PMTs to muons traversing their windows at different orientations is measured at the H2 beam-line at CERN. Certain kinds of PMTs with thinner windows show significantly lower response to direct muon incidence. For the four anode PMT, a simple and powerful algorithm to identify such events and recover the PMT signal using the signals of the quadrants without window hits is also presented. For the measurement of PMT responses to. Cerenkov light, the Hadron Forward calorimeter signal was mimicked by two different setups in electron beams and the PMT performances were compared with each other. Superior performance of particular PMTs was observed.

Structural data of integrin alpha IIb beta 3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a "bent," low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that "clasping" the alpha IIb head domain to the beta 3 tail decreased fibrinogen binding. To study the role of alpha IIb extension about the genu, we introduced a disulfide "clamp" between the alpha IIb thigh and calf-1 domains. Clamped alpha IIb beta 3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn(2+) or by coexpressing the clamped alpha IIb with a beta 3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (M(r) 7,500) or alpha IIb beta 3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAbAP5 in the presence of kistrin. Collectively, our studies support a role for alpha IIb extension about the genu in the binding of ligands of 340,000 and 900,000 M(r) with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with alpha IIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in beta 3 that affect the intrinsic affinity of the binding pocket for ligand.

This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x.

The endocannabinoid system is a neuroactive lipid signaling system that functions to gate synaptic transmitter release. Accumulating evidence has demonstrated that this system is responsive to modulation by both stress and glucocorticoids within the hypothalamus and limbic structures; however, the nature of this regulation is more complex than initially assumed. The aim of the current review is to summarize the research to date which examines the effects of acute stress and glucocorticoid administration on endocannabinoid signaling in limbic-hypothalamic-pituitary-adrenal (LHPA) axis, and in turn the role endocannabinoid signaling plays in the neurobehavioural responses to acute stress and glucocorticoid administration. The majority of research suggests that acute stress produces a mobilization of the endocannabinoid 2-arachidonoylglycerol (2-AG) while concurrently reducing the tissue content of the other endocannabinoid ligand anandamide. Genetic and pharmacological studies demonstrate that the reduction in anandamide signaling may be involved in the initiation of HPA axis activation and the generation of changes in emotional behaviour, while the increase in 2-AG signaling may be involved in terminating the stress response, limiting neuronal activation and contributing to changes in motivated behaviours. Collectively, these studies reveal a complex interplay between endocannabinoids and the HPA axis, and further identify endocannabinoid signaling as a critical regulator of the stress response. (C) 2009 Elsevier Inc. All rights reserved.

The MLL1 gene is a frequent target for recurrent chromosomal translocations, resulting in transformation of hematopoietic precursors into leukemia stem cells. Here, we report on structure-function studies that elucidate molecular events in MLL1 binding of histone H3K4me3/2 marks and recruitment of the cyclophilin CyP33. CyP33 contains a PPIase and a RRM domain and regulates MLL1 function through HDAC recruitment. We find that the PPIase domain of CyP33 regulates the conformation of MLL1 through proline isomerization within the PHD3-Bromo linker, thereby disrupting the PHD3-Bromo interface and facilitating binding of the MLL1-PHD3 domain to the CyP33-RRM domain. H3K4me3/2 and CyP33-RRM target different surfaces of MLL1-PHD3 and can bind simultaneously to form a ternary complex. Furthermore, the MLL1-CyP33 interaction is required for repression of HOXA9 and HOXC8 genes in vivo. Our results highlight the role of PHD3-Bromo cassette as a regulatory platform, orchestrating MLL1 binding of H3K4me3/2 marks and cyclophilin-mediated repression through HDAC recruitment.