The rockefeller university

Oligomeric beta-amyloid (A beta) has recently been linked to synaptic plasticity deficits, which play a major role in progressive cognitive decline in Alzheimer's disease (AD). Here we present evidence that chronic oral administration of carvedilol, a nonselective beta-adrenergic receptor blocker, significantly attenuates brain oligomeric beta-amyloid content and cognitive deterioration in 2 independent AD mouse models. We found that carvedilol treatment significantly improved neuronal transmission, and that this improvement was associated with the maintenance of number of the less stable "learning" thin spines in the brains of AD mice. Our novel observation that carvedilol interferes with the neuropathologic, biochemical, and electrophysiological mechanisms underlying cognitive deterioration in AD supports the potential development of carvedilol as a treatment for AD. (C) 2011 Elsevier Inc. All rights reserved.

Antibodies against the CD4 binding site (CD4bs) on the HIV-1 spike protein gp120 can show exceptional potency and breadth. We determined structures of NIH45-46, a more potent clonal variant of VRC01, alone and bound to gp120. Comparisons with VRC01-gp120 revealed that a four-residue insertion in heavy chain complementarity-determining region 3 (CDRH3) contributed to increased interaction between NIH45-46 and the gp120 inner domain, which correlated with enhanced neutralization. We used structure-based design to create NIH45-46(G54W), a single substitution in CDRH2 that increases contact with the gp120 bridging sheet and improves breadth and potency, critical properties for potential clinical use, by an order of magnitude. Together with the NIH45-46-gp120 structure, these results indicate that gp120 inner domain and bridging sheet residues should be included in immunogens to elicit CD4bs antibodies.

Activated transcription of the bacteriophage T4 late genes, which is coupled to concurrent DNA replication, is accomplished by an initiation complex containing the host RNA polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the DNA-mounted sliding-clamp processivity factor of the phage T4 replisome (gp45, the activator). We have determined the 3.0 angstrom-resolution X-ray crystal structure of gp33 complexed with its RNA polymerase binding determinant, the beta-flap domain. Like domain 4 of the promoter specificity sigma factor (sigma(4)), gp33 interacts with RNA polymerase primarily by clamping onto the helix at the tip of the beta-flap domain. Nevertheless, gp33 and sigma(4) are not structurally related. The gp33/beta-flap structure, combined with biochemical, biophysical, and structural information, allows us to generate a structural model of the T4 late promoter initiation complex. The model predicts protein/protein interactions within the complex that explain the presence of conserved patches of surface-exposed residues on gp33, and provides a structural framework for interpreting and designing future experiments to functionally characterize the complex.

Study Objective.: To better understand the precise role of sensory corpuscles within the female external genitalia. Design: After IRB approval, waste tissue samples were obtained from 10 normal girls (aged 2-9 years) who underwent surgery for labial fusion. Immunocytochemistry against protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), vasoactive intestinal peptide (VIP), 5-hydroxytryptamine transporter (5HTT), 5-hydroxytryptamine receptor 1A (5HT1A), Neuronal Peptide Y (NPY), neuronal nitric oxide synthase (nNOS), and estrogen receptors (ER) alpha and beta was performed. Results: Pacinian-like corpuscles were identified in epithelium of labia minora of prepubertal girls. A central structure composed of an axon surrounded by a central core, outer core, external capsule, surrounded by encapsulated stroma, and a subsidiary innervation in the outer aspect of the corpuscle stroma stained for PGP 9.5 in the outer core and layers of the external capsule, NSE positive cells in layers of the outer core, 5HTT in stroma of the corpuscle and cells located in layers of the outer core, 5HT1A in cells of outer core, NPY in stroma of the corpuscle, and nNOS in external core and external capsule of the central structure. ER alpha was present in stroma, external core, and external capsule, and ER beta in stroma of the corpuscle with subsidiary innervation in the stroma positive to PGP 9.5, VIP, and NPY. Conclusion: PGP 9.5, NSE, ER alpha, nNOS, and 5HTT immunoreaction detected in the outer core and external capsule could indicate these areas may play an important role in the functional aspects of the Pacinian-like corpuscle.

We developed an assay for quantifying the reward value of nutrient and used it to analyze the effects of metabolic state and leptin. In this assay, mice chose between two sippers, one of which dispensed water and was coupled to optogenetic activation of dopaminergic (DA) neurons and the other of which dispensed natural or artificial sweeteners. This assay measured the reward value of sweeteners relative to lick-induced optogenetic activation of DA neurons. Mice preferred optogenetic stimulation of DA neurons to sucralose, but not to sucrose. However, the mice preferred sucralose plus optogenetic stimulation versus sucrose. We found that food restriction increased the value of sucrose relative to sucralose plus optogenetic stimulation, and that leptin decreased it. Our data suggest that leptin suppresses the ability of sucrose to drive taste-independent DA neuronal activation and provide new insights into the mechanism of leptin's effects on food intake.

Eukaryotic DNA is packaged in nucleosomes. How does this sequestration affect the ability of transcription regulators to access their sites? We cite evidence against the idea that nucleosome positioning is determined primarily by the intrinsic propensities of DNA sequences to form nucleosomes - such that, for example, regulatory sites would be 'nucleosome-free'. Instead, studies in yeast show that nucleosome positioning is primarily determined by specific DNA-binding proteins. Where nucleosomes would otherwise compete with regulatory protein binding (a modest but potentially biologically important effect), this obstacle can be relieved by at least two strategies for exposing regulatory sites. In contrast to their lack of effect on nucleosome positioning, DNA sequence differences do directly affect both the efficiencies with which nucleosomes form in regions flanking regulatory sites before induction, and the extent of their removal upon induction. These nucleosomes, evidently, inhibit basal transcription but are poised to be removed quickly upon command.

Chromatin reorganization is governed by multiple post-translational modifications of chromosomal proteins and DNA(1,2). These histone modifications are reversible, dynamic events that can regulate DNA-driven cellular processes(3,4). However, the molecular mechanisms that coordinate histone modification patterns remain largely unknown. In metazoans, reversible protein modification by O-linked N-acetylglucosamine (GlcNAc) is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA)(5,6). However, the significance of GlcNAcylation in chromatin reorganization remains elusive. Here we report that histone H2B is GlcNAcylated at residue S112 by OGT in vitro and in living cells. Histone GlcNAcylation fluctuated in response to extracellular glucose through the hexosamine biosynthesis pathway (HBP)(5,6). H2B S112 GlcNAcylation promotes K120 monoubiquitination, in which the GlcNAc moiety can serve as an anchor for a histone H2B ubiquitin ligase. H2B S112 GlcNAc was localized to euchromatic areas on fly polytene chromosomes. In a genome-wide analysis, H2B S112 GlcNAcylation sites were observed widely distributed over chromosomes including transcribed gene loci, with some sites co-localizing with H2B K120 monoubiquitination. These findings suggest that H2B S112 GlcNAcylation is a histone modification that facilitates H2BK120 monoubiquitination, presumably for transcriptional activation.

Glucagon levels are elevated in diabetes and some liver diseases. Increased glucagon secretion leads to abnormal stimulation of glucagon receptors (GRs) and consequent elevated glucose production in the liver. Blocking glucagon receptor signaling has been proposed as a potential treatment option for diabetes and other conditions associated with hyperglycemia. Elucidating mechanisms of GR desensitization and downregulation may help identify new drug targets besides GR itself. The present study explores the mechanisms of GR internalization and the role of PKC alpha. GPCR kinases (GRKs) and beta-arrestins therein. We have reported previously that PKCa mediates GR phosphorylation and desensitization. While the PKC agonist, PMA, did not affect GR internalization when tested alone, it increased glucagon-mediated GR internalization by 25-40% in GR-expressing HEK-293 cells (HEK-GR cells). In both primary hepatocytes and HEK-GR cells, glucagon treatment recruited PKCa to the plasma membrane where it colocalized with GR. We also observed that overexpression of GRK2, GRK3, or GRK5 enhanced GR internalization. In addition, we found that GR utilizes both clathrin- and caveolin-mediated endocytosis in HEK-GR cells. Glucagon triggered translocation of both beta-arrestin1 and beta-arrestin2 from the cytosol to the perimembrane region, and overexpression of beta-arrestin1 and beta-arrestin2 increased GR internalization. Furthermore, both beta-arrestin1 and beta-arrestin2 colocalized with GR and with Cav-1, suggesting the possible involvement of these arrestins in GR internalization. (C) 2011 Elsevier Inc. All rights reserved.

Hepatitis C virus (HCV) replication in primary liver cells is less robust than that in hepatoma cell lines, suggesting that innate antiviral mechanisms in primary cells may limit HCV replication or spread. Here we analyzed the expression of 47 genes associated with interferon (IFN) induction and signaling following HCV infection of primary human fetal liver cell (HFLC) cultures from 18 different donors. We report that cell culture-produced HCV (HCVcc) induced expression of Type III (lambda) IFNs and of IFN-stimulated genes (ISGs). Little expression of Type I IFNs was detected. Levels of IFN lambda and ISG induction varied among donors and, often, between adapted and nonadapted HCV chimeric constructs. Higher levels of viral replication were associated with greater induction of ISGs and of lambda IFNs. Gene induction was dependent on HCV replication, as ultraviolet light-inactivated virus was not stimulatory and an antiviral drug, 2'-C-methyladenosine, reduced induction of lambda IFNs and ISGs. The level of IFN lambda protein induced was sufficient to inhibit HCVcc infection of naive cultures. Conclusion: Together, these results indicate that despite its reported abilities to blunt the induction of an IFN response, HCV infection is capable of inducing antiviral cytokines and pathways in primary liver cell cultures. Induction of ISGs and lambda IFNs may limit the growth and spread of HCV in primary cell cultures and in the infected liver. HCV infection of HFLC may provide a useful model for the study of gene induction by HCV in vivo. (HEPATOLOGY 2011; 54: 1913-1923)