Caveolin Transfection Results in Caveolae Formation but Not Apical Sorting of Glycosylphosphatidylinositol (GPI)-anchored Proteins in Epithelial Cells

Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The “raft” hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI- anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI- anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42–53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI- anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes

Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder. MJD results from polyglutamine repeat expansion in the MJD-1 gene, conferring a toxic gain of function to the ataxin-3 protein. In this study, we aimed at overexpressing ataxin-3 in the rat brain using lentiviral vectors (LV), to generate an in vivo MJD genetic model and, to study the disorder in defined brain regions: substantia nigra, an area affected in MJD, cortex and striatum, regions not previously reported to be affected in MJD. LV encoding mutant or wild-type human ataxin-3 was injected in the brain of adult rats and the animals were tested for behavioral deficits and neuropathological abnormalities. Striatal pathology was confirmed in transgenic mice and human tissue. In substantia nigra, unilateral overexpression of mutant ataxin-3 led to: apomorphine-induced turning behavior; formation of ubiquitinated ataxin-3 aggregates; α-synuclein immunoreactivity; and loss of dopaminergic markers (TH and VMAT2). No neuropathological changes were observed upon wild-type ataxin-3 overexpression. Mutant ataxin-3 expression in striatum and cortex, resulted in accumulation of misfolded ataxin-3, and within striatum, loss of neuronal markers. Striatal pathology was confirmed by observation in MJD transgenic mice of ataxin-3 aggregates and substantial reduction of DARPP-32 immunoreactivity and, in human striata, by ataxin-3 inclusions, immunoreactive for ubiquitin and α-synuclein. This study demonstrates the use of LV encoding mutant ataxin-3 to produce a model of MJD and brings evidence of striatal pathology, suggesting that this region may contribute to dystonia and chorea observed in some MJD patients and may represent a target for therapie

Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

Upregulation of fatty acid synthase (FASN) is a common event in cancer, although its mechanistic and potential therapeutic roles are not completely understood. In this study, we establish a key role of FASN during transformation. FASN is required for eliciting the anaplerotic shift of the Krebs cycle observed in cancer cells. However, its main role is to consume acetyl-CoA, which unlocks isocitrate dehydrogenase (IDH)-dependent reductive carboxylation, producing the reductive power necessary to quench reactive oxygen species (ROS) originated during the switch from two-dimensional (2D) to three-dimensional (3D) growth (a necessary hallmark of cancer). Upregulation of FASN elicits the 2D-to-3D switch; however, FASN's synthetic product palmitate is dispensable for this process since cells satisfy their fatty acid requirements from the media. In vivo, genetic deletion or pharmacologic inhibition of FASN before oncogenic activation prevents tumor development and invasive growth. These results render FASN as a potential target for cancer prevention studies.M.Q.F. is a recipient of the following grants: FIS PI13/00430 and FIS PI16/00354 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF) and AECC Scientific Foundation (Beca de Retorno 2010). R.C. is a recipient of the following grants: FIS PI11/00832 and FIS PI14/00726 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF), II14/00009 and PIE15/00068 from the Ministerio de Sanidad, Spain. N.S.C. is a recipient of an NIH grant (5R35CA197532). O.Y.T. is a recipient of the grants BFU2014-57466 from the Ministerio de Economia y Competitividad (MINECO). J.P.B. is funded by MINECO (SAF2016-78114-R), Instituto de Salud Carlos III (RD12/0043/0021), Junta de Castilla y Leon (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigacion Biomedicina 2017 Fundacion BBVA, and Fundacion Ramon Areces. This study was partially supported by the generous donations from Fundacion CRIS Contra el Cancer and AVON Spain. We thank Drs. Erwin Wagner and Nabil Djouder for their critical review of the paper.S

Synthesis of Thiodisaccharide Sulfoxides and Sulfones – Determination of the Configuration of the Sulfur Stereocentre

The oxidation of per-O-acetyl (1,3)- and (1,4)-linked thiodisaccharides containing glucose, gulose and galactose residues, with an excess of m-chloroperbenzoic acid afforded the corresponding sulfoxides or sulfones. The latter were formed when the oxidation reaction was conducted for longer times. The sulfoxides were obtained as diastereomeric mixtures due to the chirality of the sulfur atom. Both diastereoisomers of the (1,3)-thiodisaccharides S-oxides were isolated by column chromatography, while the S-(1,4)-linked analogues could not be separated. The absolute configuration of the sulfur stereocenter of sulfoxides was assigned using NMR spectroscopy and taking into account the preferred conformations of the molecules and the shielding/deshielding of proton signals caused by anisotropy of the S=O bond and related effects. Most of the thiodisaccharide S-oxides were successfully O-deacetylated with MeOH-Et3N-H2O, whereas the sulfone underwent elimination reactions. Therefore, the oxidation was performed on the free thiodisaccharides, and the corresponding sulfones were obtained in very good yields.Fil: Colomer, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina;Fil: Manzano, Veronica Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina;Fil: Varela, Oscar Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina

Synthesis of thiodisaccharides related to 4-thiolactose: Specific structural modifications increase the inhibitory activity against E. coli β-galactosidase

In the search for new glycosidase inhibitors, a set of benzyl β-d-Gal-S-(1→4)-3-deoxy-4-thio-α-d-hexopyranosides was synthesized. Diverse configurations were installed at C-2 and C-4 of the glucose residue. The benzyl glycosidic group was kept intact or substituted by an electron-donating or electron-withdrawing group that could also participate in hydrogen bonding. All thiodisaccharides were found to be inhibitors of E. coli β-galactosidase. In general, benzyl thiodisaccharides were better inhibitors than those substituted (NO2 or NH2) on the benzyl ring. Thiodisaccharides containing a hexopyranoside, instead of a pentopyranoside, showed a weaker inhibitory activity, except for those having the α-d-xylo configuration, which exhibited inhibition constants of the same order of magnitude. These and previous results indicated that the inhibition process by thiodisaccharides is strongly dependent on the configuration of the 3-deoxy-4-thiopyranoside, as well as its substitution pattern (such as the presence of a benzyl glycoside). The enzyme-inhibitor interaction during the hydrolysis process involves a conformational selection resulting from rotation around the thioglycosidic bond and the flexibility of the terminal six-membered ring. Thus, the mentioned structural features of the inhibitor could give rise to favorable ground state conformations for the interaction with the enzyme, similar to those found for selected thiodisaccharides in the bound state. These studies demonstrated that the performance of thiodisaccharides as enzyme inhibitors could be increased by selecting the appropriate configuration and substitution of the hexopyranoside replacing the glucose moiety of 4-thiolactose.Fil: Dada, Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Colomer, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Manzano, Veronica Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Varela, Oscar Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentin

A Comparative Study Among Constraint, Robot-Aided and Standard Therapies in Upper Limb Rehabilitation of Children with Acquired Brain Injury

The functional recovery of upper limbs is one of the goals of the rehabilitation of children affected by Acquired Brain Injury. Sensorimotor stimulation exercises, particularly the Constraint-Induced Movement Therapy (CIMT) and robot-aided therapy are promising in children. However, exhaustive data about their effectiveness in the clinical practice and differences of these treatments still lack in the literature. Therefore, the aim of this study is to perform a preliminary comparative evaluation among CIMT, robot-aided therapy with Armeo®Spring and conventional physiokinesitherapy about their efficacy in upper limb rehabilitation of children after ABI. A group of 10 children was treated with two of the three abovementioned therapies, randomly chosen in the order. The evaluation included clinical and functional scales as well as 3D kinematics to objectively measure the outcome. Our results showed improvements at the elbow and shoulder after CIMT and ARMEO, whereas physiokinesitherapy frequently showed changes in the opposite direction. Further, many improvements in terms of trunk rotation and joints selectivity were conveyed by the robot-aided therapy. Future studies will be aimed at increasing the sample size and thus generalizing these results