The rockefeller university

The inherent instability of heptahelical G protein-coupled receptors (GPCRs) during purification and reconstitution is a primary impediment to biophysical studies and to obtaining high-resolution crystal structures. New approaches to stabilizing receptors during purification and screening reconstitution procedures are needed. Here we report the development of a novel homogeneous time-resolved fluorescence assay (HTRF) to quantify properly folded CC-chemokine receptor 5 (CCR5). The assay permits high-throughput thermal stability measurements of femtomole quantities of CCR5 in detergent and in engineered nanoscale apolipoprotein-bound bilayer (NABB) particles. We show that recombinantly expressed CCR5 can be incorporated into NABB particles in high yield, resulting in greater thermal stability compared with that of CCR5 in a detergent solution. We also demonstrate that binding of CCR5 to the HIV-1 cellular entry inhibitors maraviroc, AD101, CMPD 167, and vicriviroc dramatically increases receptor stability. The HTRF assay technology reported here is applicable to other membrane proteins and could greatly facilitate structural studies of GPCRs.

Amplification in the mammalian inner ear is thought to result from a nonlinear active process known as the cochlear amplifier. Although there is much evidence that outer hair cells (OHCs) play a central role in the cochlear amplifier, the mechanism of amplification remains uncertain. In non-mammalian ears hair bundles can perform mechanical work and account for the active process in vitro, yet in the mammalian cochlea membrane-based electromotility is required for amplification in vivo. A key issue is how OHCs conduct mechanical power amplification at high frequencies. We present a physical model of a segment of the mammalian cochlea that can amplify the power of external signals. In this representation both electromotility and active hair-bundle motility are required for mechanical power gain at high frequencies. We demonstrate how the endocochlear potential, the OHC resting potential, Ca2+ gradients, and ATP-fueled myosin motors serve as the energy sources underlying mechanical power gain in the cochlear amplifier.

Mature microRNAs (miRNAs) are single-stranded RNA molecules of 20-23 nucleotide (nt) length that control gene expression in many cellular processes. These molecules typically reduce the stability of mRNAs, including those of genes that mediate processes in tumorigenesis, such as inflammation, cell cycle regulation, stress response, differentiation, apoptosis and invasion. miRNA targeting is mostly achieved through specific base-pairing interactions between the 5' end ('seed' region) of the miRNA and sites within coding and untranslated regions (UTRs) of mRNAs; target sites in the 3' UTR lead to more effective mRNA destabilization. Since miRNAs frequently target hundreds of mRNAs, miRNA regulatory pathways are complex. To provide a critical overview of miRNA dysregulation in cancer, we first discuss the methods currently available for studying the role of miRNAs in cancer and then review miRNA genomic organization, biogenesis and mechanism of target recognition, examining how these processes are altered in tumorigenesis. Given the critical role miRNAs play in tumorigenesis processes and their disease-specific expression, they hold potential as therapeutic targets and novel biomarkers. Copyright. (C) 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time-and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule.

The mechanosensory hair cells of many auditory receptor organs are tuned by an electrical resonance that increases their responses to stimulation over a narrow band of frequencies. The small oscillations of membrane potential characteristic of this phenomenon have previously been detectable only through intracellular electrode measurements, which are laborious and preclude analysis at the level of an entire sensory organ. We used a voltage-sensitive dye to image hair-cell electrical resonance in an intact preparation of the bullfrog's sacculus, a receptor organ sensitive to low-frequency seismic and auditory stimuli. Imaging revealed distinct populations of hair cells whose resonant response varied with the frequency of transepithelial electrical stimulation. Most of the hair cells in the saccular epithelium in vitro were electrically tuned to stimulation at 25-50 Hz. The frequency dependence of the fluorescence signal was sensitive to pharmacological blockade of large-conductance Ca2+-sensitive K+ channels and to enzymatic digestion. At an elevated concentration of Ca2+, we observed transient fluorescence signals that probably represented action potentials. The stroboscopic imaging and analysis techniques described here present a general approach for studying subthreshold oscillations in electrically excitable cells.

The year 2010 marked the centennial of the Rockefeller University Hospital, one of the great philanthropic achievements of 20th-century science. For 100 years, the Hospital played a central role in the development and growth of medical science by enabling physician-scientists to make intensive study of human biology and disease. With ingenuity and devotion, they greatly enriched clinical medicine as well as basic biological science. This account emphasizes the founding and first half-century of the Hospital as it became a germinal center for clinical investigation. The second half of the century saw rapid change in medicine and health care with vexing problems, many yet unsolved. This history should serve as a call to arms for maintaining the linkage of science and medicine, supporting patient-oriented research as a basic discipline of medicine.

Polar lipids must flip-flop rapidly across biological membranes to sustain cellular life [1, 2], but flipping is energetically costly [3] and its intrinsic rate is low. To overcome this problem, cells have membrane proteins that function as lipid transporters (flippases) to accelerate flipping to a physiologically relevant rate. Flippases that operate at the plasma membrane of eukaryotes, coupling ATP hydrolysis to unidirectional lipid flipping, have been defined at a molecular level [2]. On the other hand, ATP-independent bidirectional flippases that translocate lipids in biogenic compartments, e.g., the endoplasmic reticulum, and specialized membranes, e.g., photoreceptor discs [4, 5], have not been identified even though their activity has been recognized for more than 30 years [1]. Here, we demonstrate that opsin is the ATP-independent phospholipid flippase of photoreceptor discs. We show that reconstitution of opsin into large unilamellar vesicles promotes rapid (tau < 10 s) flipping of phospholipid probes across the vesicle membrane. This is the first molecular identification of an ATP-independent phospholipid flippase in any system. It reveals an unexpected activity for opsin and, in conjunction with recently available structural information on this G protein-coupled receptor [6, 7], significantly advances our understanding of the mechanism of ATP-independent lipid flip-flop.

The ultimate goal of hepatitis C virus (HCV) treatment is the eradication of the virus. Ongoing research continues to add to knowledge of the HCV life cycle, revealing new potential viral and host targets for the development of therapy. Understanding of HCV was initially hampered by the inability to achieve viral replication in cell culture. Advances such as the HCV replicon and complete cell culture systems, however, have permitted rapid growth in knowledge and accelerated testing of candidate antiviral agents. Among potential targets are viral entry factors, including scavenger receptor type B1 (SR-B1) and CD81, as well as neutralizing antibodies against the viral glycoproteins. Popular targets related to translation and replication are the NS3/4A protease (inhibited by telaprevir and boceprevir) and the NS5B polymerase, as well as the NS2/3 autoprotease, the NS3 helicase, and nonenzymatic targets such as NS4B and NS5A proteins. Host targets are also available, including microRNAs and cyclophilins. This article summarizes a presentation by Charles M. Rice, PhD, at the IAS-USA live continuing medical education course, Management of Hepatitis C Virus in the New Era: Small Molecules Bring Big Changes, held in New York City in April 2011.

Pathologic rheumatoid factor (RF) levels are hallmarks of several human diseases. Production of monoclonal RF in vitro is essential for studies of the antigenic specificities of RF, as well as for a dissection of the mechanisms of aberrant RF+ B cell activation. We have expanded upon previous methods to develop a flow cytometry-based method to efficiently clone monoclonal antibodies (mAbs) from humans with expansions of RF-like, immunoglobulin heavy chain variable region (IgVH) 1-69 gene segment-containing B cells. The cloned variable regions are expressed as IgM and produced during culture at concentrations between 5 and 20 mu g/ml. Using this system, we show that clonal Igs from patients with HCV-related mixed cryoglobulinemia, when expressed as IgM, have RF activity. We anticipate that this system will be useful for the cloning and expression of mAbs partially encoded by VH1-69 and for determination of the reactivity patterns of polyspecific, low-affinity IgMs of human pathogenic importance. (C) 2010 Elsevier B.V. All rights reserved.

In the last 20 years, it has become clear that developmental genes and their regulators, noncoding RNAs including microRNAs and long-noncoding RNAs, within signaling pathways play a critical role in the pathogenesis of cancer. Many of these pathways were first identified in genetic screens in Drosophila and other lower organisms. Mammalian orthologs were subsequently identified and genes within the pathways cloned and found to regulate cell growth. Genes and pathways expressed during embryonic development, including the Notch, Wnt/beta-Catenin, TGF-beta/BMP, Shh/Patched, and Hippo pathways are mutated, lost, or aberrantly regulated in a wide variety of human cancers, including skin, breast, blood, and brain cancers, including medulloblastoma. These biochemical pathways affect cell fate determination, axis formation, and patterning during development and regulate tissue homeostasis and regeneration in adults. Medulloblastoma, the most common malignant nervous system tumor in childhood, are thought to arise from disruptions in cerebellar development [reviewed by Marino, S. (2005)]. Defining the extracellular cues and intracellular signaling pathways that control cerebellar neurogenesis, especially granule cell progenitor (GCP) proliferation and differentiation has been useful for developing models to unravel the mechanisms underlying medulloblastoma formation and growth. In this chapter, we will review the development of the cerebellar cortex, highlighting signaling pathways of potential relevance to tumorigenesis.