The rockefeller university

Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits y-tubulin, through the carboxyl terminus of its hDgt6 subunit to nucleate microtubules within spindles. However, augmin's biochemical complexity has restricted analysis of its structural organization and function. Here, we reconstitute human augmin and show that it is a Y-shaped complex that can adopt multiple conformations. Further, we find that a dimeric sub-complex retains in vitro microtubule-binding properties of octameric complexes, but not proper metaphase spindle localization. Addition of octameric augmin complexes to Xenopus egg extracts promotes microtubule aster formation, an activity enhanced by Ran-GTP. This activity requires microtubule binding, but not the characterized hDgt6 y-tubulin-recruitment domain. Tetrameric sub-complexes induce asters, but activity and microtubule bundling within asters are reduced compared with octameric complexes. Together, our findings shed light on augmin's structural organization and microtubule-binding properties, and define subunits required for its function in organizing microtubule-based structures.

Metachromatic leukodystrophy (MLD), a fatal disorder caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA), is associated with an accumulation of sulfatides, causing widespread demyelination in both central and peripheral nervous systems. On the basis of prior studies demonstrating that adeno-associated virus AAVrh.10 can mediate widespread distribution in the CNS of a secreted lysosomal transgene, and as a prelude to human trials, we comparatively assessed the optimal CNS delivery route of an AAVrh.10 vector encoding human ARSA in a large animal model for broadest distribution of ARSA enzyme. Five routes were tested (each total dose, 1.5x10(12) genome copies of AAVrh.10hARSA-FLAG): (1) delivery to white matter centrum ovale; (2) deep gray matter delivery (putamen, thalamus, and caudate) plus overlying white matter; (3) convection-enhanced delivery to same deep gray matter locations; (4) lateral cerebral ventricle; and (5) intraarterial delivery with hyperosmotic mannitol to the middle cerebral artery. After 13 weeks, the distribution of ARSA activity subsequent to each of the three direct intraparenchymal administration routes was significantly higher than in phosphate-buffered saline-administered controls, but administration by the intraventricular and intraarterial routes failed to demonstrate measurable levels above controls. Immunohistochemical staining in the cortex, white matter, deep gray matter of the striatum, thalamus, choroid plexus, and spinal cord dorsal root ganglions confirmed these results. Of the five routes studied, administration to the white matter generated the broadest distribution of ARSA, with 80% of the brain displaying more than a therapeutic (10%) increase in ARSA activity above PBS controls. No significant toxicity was observed with any delivery route as measured by safety parameters, although some inflammatory changes were seen by histopathology. We conclude that AAVrh.10-mediated delivery of ARSA via CNS administration into the white matter is likely to be safe and yields the widest distribution of ARSA, making it the most suitable route of vector delivery.

Given the complex heterogeneity of pathological changes occurring in Alzheimer's disease (AD), any therapeutic effort absolutely requires a multi-targeted approach, because attempts addressing only a single event may result ineffective. Palmitoylethanolamide (PEA), a naturally occurring lipid amide between palmitic acid and ethanolamine, seems to be a compound able to fulfill the criteria of a multi-factorial therapeutic approach. Here, we describe the anti-inflammatory and neuroprotective activities of systemic administration of PEA in adult male rats given intrahippocampal injection of beta amyloid 1-42 (A beta 1-42). Moreover, to investigate the molecular mechanisms responsible for the effects induced by PEA, we co-administered PEA with the GW6471, an antagonist of peroxisome proliferator-activated receptor-alpha (PPAR-alpha). We found that A beta 1-42 infusion results in severe changes of biochemical markers related to reactive gliosis, amyloidogenesis, and tau protein hyperphosphorylation. Interestingly, PEA was able to restore the A beta 1-42-induced alterations through PPAR-alpha involvement. In addition, results from the Morris water maze task highlighted a mild cognitive deficit during the reversal learning phase of the behavioral study. Similarly to the biochemical data, also mnestic deficits were reduced by PEA treatment. These data disclose novel findings about the therapeutic potential of PEA, and suggest novel strategies that hopefully could have the potential not just to alleviate the symptoms but also to modify disease progression.

Mycosis fungoides, the most common type of cutaneous T-cell lymphoma (CTCL), is characterized by a helper T-cell 2 (Th2) skewing with a mature CD4(+) memory T-cell phenotype. Using skin samples from patients with mycosis fungoides (n = 21), healthy volunteers (n = 17), and individuals with atopic dermatitis (n = 17) and psoriasis (n = 9), we found IL32 mRNA expression significantly higher in mycosis fungoides samples than in samples from benign inflammatory skin diseases, and its expression increases with disease progression. By IHC and immunofluorescence, we confirmed IL32 protein expression in many CD3(+)CD4(+)T cells and some epidermotropic T cells in mycosis fungoides lesions. MyLa cells (a mycosis fungoides cell line) express IL32, which, in turn, could promote cellular proliferation and viability in a dose-dependent fashion. IL32-treated MyLa and CTCL HH cells upregulated cell proliferation and survival genes. Of the major "polarizing" T-cell cytokines, only IFN gamma mRNA increases with mycosis fungoides progression and positively correlates with IL32 mRNA expression. Th2 cytokines do not positively correlate with IL32 mRNA expression or mycosis fungoides progression. Furthermore, by flow cytometry, IL32 production by circulating activated T cells in healthy individuals was found in both IFN gamma(+) and IFN gamma(-) cells but not in IL4(+)or IL13(+) cells. In conclusion, we have identified IL32(+) cells as the likely tumor cells in mycosis fungoides, and demonstrated that IL32 mRNA expression increases with mycosis fungoides progression and is significantly higher than mRNA expression in other skin diseases, and that some IL32(+) T cells are independent from the defined Th subsets. Thus, IL32 may play a unique role in mycosis fungoides progression as an autocrine cytokine. (C)2014 AACR.

It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti-HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (similar to 1:100) and potentially achievable by vaccination.

Cross-protection triggered by a mild strain of virus acts as a prophylaxis to prevent subsequent infections by related viruses in plants; however, the underling mechanisms are not fully understood. Through mutagenesis, we isolated a mutant strain of Turnip mosaic virus (TuMV), named Tu-GK, that contains an Arg182Lys substitution in helper component-proteinase (HC-Pro(K)) that confers complete cross-protection against infection by a severe strain of TuMV in Nicotiana benthamiana, Arabidopsis thaliana Col-0, and the Arabidopsis dcl2-4/dcl4-1 double mutant defective in DICER-like ribonuclease (DCL)2/DCL4-mediated silencing. Our analyses showed that HC-ProK loses the ability to interfere with microRNA pathways, although it retains a partial capability for RNA silencing suppression triggered by DCL. We further showed that Ta-GK infection triggers strong salicylic acid (SA)-dependent and SA-independent innate immunity responses. Our data suggest that DCL2/4-dependent and independent RNA silencing pathways are involved, and may crosstalk with basal innate immunity pathways, in host defense and in cross-protection.

In our prior publications we characterized a conserved acetylation motif (K(R) xxKK) of evolutionarily related nuclear receptors. Recent reports showed that peroxisome proliferator activated receptor gamma (PPAR gamma) deacetylation by SIRT1 is involved in delaying cellular senescence and maintaining the brown remodeling of white adipose tissue. However, it still remains unknown whether lysyl residues 154 and 155 (K154/155) of the conserved acetylation motif (RIHKK) in Ppar gamma 1 are acetylated. Herein, we demonstrate that Ppar gamma 1 is acetylated and regulated by both endogenous TSA-sensitive and NAD-dependent deacetylases. Acetylation of lysine 154 was identified by mass spectrometry (MS) while deacetylation of lysine 155 by SIRT1 was confirmed by in vitro deacetylation assay. An in vivo labeling assay revealed K154/K155 as bona fide acetylation sites. The conserved acetylation sites of Ppar gamma 1 and the catalytic domain of SIRT1 are both required for the interaction between Ppar gamma 1 and SIRT1. Sirt1 and Ppar gamma 1 converge to govern lipid metabolism in vivo. Acetylation-defective mutants of Ppar gamma 1 were associated with reduced lipid synthesis in ErbB2 overexpressing breast cancer cells. Together, these results suggest that the conserved lysyl residues K154/K155 of Ppar gamma 1 are acetylated and play an important role in lipid synthesis in ErbB2-positive breast cancer cells.

Broadly neutralizing antibodies (bNAbs) against HIV-1 provide both effective pre-exposure prophylaxis and treatment of HIV-1 infection in murine and nonhuman primate models, suggesting their potential use in humans. Although much is known about the role of variable domains in the neutralization breadth and potency of these bNAbs, the contribution of Fc domains to their activities is, by contrast, poorly characterized. Assessment of the in vivo activity of several bNAbs revealed that Fc gamma R-mediated effector function contributes substantially to their capacity to block viral entry, suppress viremia, and confer therapeutic activity. Enhanced in vivo potency of anti-HIV-1 bNAbs was associated with preferential engagement of activating, but not inhibitory Fc gamma Rs, and Fc domain-engineered bNAb variants with selective binding capacity for activating Fc gamma Rs displayed augmented protective activity. These findings reveal key roles for Fc effector function in the in vivo activity of anti-HIV-1 bNAbs and provide strategies for generating bNAbs with improved efficacy.

The rates of RNA synthesis and the folding of nascent RNA into biologically active structures are linked via pausing by RNA polymerase (RNAP). Structures that form within the RNA-exit channel can either increase pausing by interacting with RNAP or decrease pausing by preventing backtracking. Conversely, pausing is required for proper folding of some RNAs. Opening of the RNAP clamp domain has been proposed to mediate some effects of nascent-RNA structures. However, the connections among RNA structure formation and RNAP clamp movement and catalytic activity remain uncertain. Here, we assayed exit-channel structure formation in Escherichia coil RNAP with disulfide cross-links that favor closed- or open-clamp conformations and found that clamp position directly influences RNA structure formation and RNAP catalytic activity. We report that exit-channel RNA structures slow pause escape by favoring clamp opening through interactions with the flap that slow translocation.