er methods, including topical microbicides, to reduce HIV transmission. Although a complete understanding of virus infection of cells in the vaginal vault has yet to emerge in the microbicide ld, it is accepted that infection likely occurs inef- iently in the st moments following the introduction of the viral inoculum in the vagina. In order to further explore the biology of infection, we have evaluated the effects of seminal plasma, vaginal ids, other amlodipine mucopolysaccharide additives and vaginal pH on both the infectivity of HIV and the efacy of microbicides being developed for clinical use. Each of these additives or environmental changes might be expected to play roles alone and in combination with one another on the ef? ciency of virus transmission. The effects of these environmental conditions can be modeled and quantid in vitro. Our results would indicate that authentic seminal plasma has a major impact on the ability of HIV to transmit to target cells in assays which mimic the vaginal environment. Vaginal ids have less effect on transmission but both biological ids can affect the activity of potential microbicides in both negative and positive ways. We will present the results of studies demonstrating the impact of biological ids on virus transmission and microbicide efacy. doi: 10.1016/j.antiviral.2007.01.096 2 84 P. Rusmini et al. / Neurobiology of Disease 41 (2011) 83 ?95 demethoxygeldanamycin (17-AAG), induces ARpolyQ degradation counteracting SBMA progression in transgenic mice models of SBMA ( Tokui et al., 2009; Waza et al., 2005 ).

This mechanism occurs apparently without affecting the endogenous proteasome system ( Tokui et al., 2009 ). On this basis, we have deeply studied the possible amlodipine 88150-42-9 mechanisms of action of the 17-AAG in cellular models of SBMA, and carefully evaluated whether 17-AAG induces modi ?cations of the biochemical properties of ARpolyQ, as well as its impact on the two major intracellular degradative systems, the ubiquitin- proteasome pathway (UPP) and the autophago-lysosome pathway (APLP). We also have analyzed the possible effects of 17-AAG on two other disease mutant proteins: Cu, Zn superoxide dismutase (SOD1) and TAR DNA-binding protein (TDP-43), involved in sALS and fALS. Materials and methods All chemicals have been obtained from Sigma-Aldrich (Sigma- Aldrich, MO, USA). Plasmids The plasmids AR.Q23 and AR.Q46, routinely used in our laboratory, have been previously described ( Simeoni et al., 2000 ). The GFP-AR. Q22 and GFP-AR.Q48 were obtained by insertion of AR cDNA into the Enhanced Green Fluorescent Protein vector, expressing chimeric ?uo- rescent fusion proteins as previously described ( Stenoien et al., 1999 ). The plasmids GFPu (from Ron Kopito) ( Bence et al., 2001 ) and its yellow ?uorescent variant,

YFPu ( Rusmini et al., 2007 ), are protea- some activity reporters and code for ?uorescent proteins fused to a constitutive degron signal (CL-1). pCDNA3-wtSOD1 and pCDNA3-G93A-SOD1 express wtSOD1 and mutant G93A-SOD1 ( Tortarolo et al., 2004 ). pFLAG-FL TDP-43 and pFLAG-  C TDP-43 express FLAG-tagged wt full length human TDP-43 and a C-terminus truncated form (from E. Buratti, Italy) ( Ayala et al., 2008 ). pmRFP-LC3 expresses mRFP tagged LC3 (from Aviva Tolkovsky, UK) ( Klionsky et al., 2008 ). pDsRed-monomer-C1 encodes Ds Red Monomer, a monomeric mutant of the buy amlodipine Discosoma sp. Red ?uorescent protein DsRed (Clontech Laboratories, Inc., CA, USA). LC3 expression was silenced using shRNA constructs (four different target sequences) designed against Mus musculus Map1LC3B gene (OriGene Technolo- gies, Inc. MD, USA). The oligonucleotides inserted in pGVP-V-RS- vector were transfected as described below.

A scrambled non- effective shRNA was also utilized as control (OriGene Techn.). Cell cultures and transfection The immortalized motorneuron cell line, NSC34 ( Cashman cured et al., 1992 ), is routinely used in our laboratory ( Piccioni et al., 2001; Pozzi et al., 2003; Simeoni et al., 2000; Vismara et al.,