Effect of botulinum neurotoxins from Mendoza of clostridium botulinum strains on cytoskeletal proteins of mammary tumor cells

The botulinum neurotoxin serotype A (BoNT A) produced by Clostridium botulinum, which causes botulism, is used for the treatment of multiple neurological diseases and its therapeutic action against cancer is currently being evaluated. In previous studies, we have shown that BoNT A from autochthonous soil strains (Su) have different properties than the reference A Hall strain. Among these, its molecular structure, its enzymatic activity against brain SNARE proteins and its greater specific toxic activity (AE) stand out. In cells from human mammary carcinoma (MCF-7) treated with BoNTs for 45 min, we found a marked effect on the expression of cytoskeletal proteins. Therefore, in this work, we delve into the study of the action of autochthonous BoNTs A and prototype A Hall on the distribution of actin and tubulin in these cells. Native forms of autochthonous BoNT (Su strains 1935 and 1891, Tupungato) and prototype A Hall were purified by saline precipitation. Their AE values (LD50 / mg protein) were established and their electrophoretic characteristics were evaluated under non-denaturing conditions. 250 LD50 of the BoNTs were incubated to MCF-7 cell cultures for 10 or 25 min. Later, the cells were fixed and processed for indirect immunofluorescence with the use of specific antibodies that recognize tubulin or actin. The samples were visualized by fluorescence microscopy. At the two times evaluated, the three types of BoNTs produced a marked redistribution of the actin cytoskeleton, patch form, on areas coinciding with the plasma membrane. Tubulin was redistributed to multiple areas with high signal density at 10 min of incubation only in the presence of BoNT 1891. At 25 min of incubation, the cells treated with BoNTs 1891 and 1935 showed this effect, while in those incubated with A Hall, the distribution of these proteins was not modified. The notable alterations in the distribution of components of the tumor cell cytoskeleton by BoNT from native strains of Mendoza soils open new perspectives for therapy against solid tumors.Fil: Chapana, Agostina Lucía. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Guarniolo, D.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Carvelli, Flavia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Sosa, E.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Fernández, R. A.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Sosa, M. A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Caballero, P. A.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaIV Reunión Conjunta de Sociedades de Biología de la República ArgentinaArgentinaSociedad de Biología de CuyoSociedad Argentina de BiologíaSociedad de Biología de RosarioSociedad Chilena de Reproducción y DesarrolloAsociación de Biología de TucumánSociedad de Biología de Córdob

Predictive modeling of the current density and radiative recombination in blue polymer-based light-emitting diodes

The results of a combined experimental and modeling study of charge transport, recombination and light emission in blue organic light-emitting diodes (OLEDs) based on a polyfluorene derivative are presented. It is shown that the measured temperature-dependent current-voltage curves and the voltage-dependent current efficiency are accurately described using an OLED device model that is based on the separately determined unipolar electron and hole mobility functions. The recombination rate is calculated using the Langevin formula, including recombination of holes with free as well as trapped electrons. The light emission is obtained from the exciton formation profile using independently determined values of the exciton radiative decay probability, the average dipole orientation, and assuming a fraction of singlet excitons ¿S¿¿ = ¿(22±3)%, close to the quantum-statistical value. No additional free parameter is used. This shows that predictive one-dimensional device modeling of OLEDs is feasible

Tetraspanin (TSP-17) Protects Dopaminergic Neurons against 6-OHDA-Induced Neurodegeneration in <i>C. elegans</i>

Parkinson's disease (PD), the second most prevalent neurodegenerative disease after Alzheimer's disease, is linked to the gradual loss of dopaminergic neurons in the substantia nigra. Disease loci causing hereditary forms of PD are known, but most cases are attributable to a combination of genetic and environmental risk factors. Increased incidence of PD is associated with rural living and pesticide exposure, and dopaminergic neurodegeneration can be triggered by neurotoxins such as 6-hydroxydopamine (6-OHDA). In C. elegans, this drug is taken up by the presynaptic dopamine reuptake transporter (DAT-1) and causes selective death of the eight dopaminergic neurons of the adult hermaphrodite. Using a forward genetic approach to find genes that protect against 6-OHDA-mediated neurodegeneration, we identified tsp-17, which encodes a member of the tetraspanin family of membrane proteins. We show that TSP-17 is expressed in dopaminergic neurons and provide genetic, pharmacological and biochemical evidence that it inhibits DAT-1, thus leading to increased 6-OHDA uptake in tsp-17 loss-of-function mutants. TSP-17 also protects against toxicity conferred by excessive intracellular dopamine. We provide genetic and biochemical evidence that TSP-17 acts partly via the DOP-2 dopamine receptor to negatively regulate DAT-1. tsp-17 mutants also have subtle behavioral phenotypes, some of which are conferred by aberrant dopamine signaling. Incubating mutant worms in liquid medium leads to swimming-induced paralysis. In the L1 larval stage, this phenotype is linked to lethality and cannot be rescued by a dop-3 null mutant. In contrast, mild paralysis occurring in the L4 larval stage is suppressed by dop-3, suggesting defects in dopaminergic signaling. In summary, we show that TSP-17 protects against neurodegeneration and has a role in modulating behaviors linked to dopamine signaling

N-Terminal Phosphorylation of the Dopamine Transporter Is Required for Amphetamine-Induced Efflux

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22—normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake

The Lantern Vol. 50, No. 2, Spring 1984

• The Storm • Je ne sais pas • The Ghetious Blastious • An Empty Cradle • The Playing Hands • Battle Hymn • A Limerick • Parting Thoughts • The River • Miss You • De la Tristeza • Two So Special • Time of the Unicorn • The Absence • Thru The Breeze • Is the World Really a Round Ball? • Brother • To Michael • Gravity • Refuge • Der Witwer • Plastic Flowers Never Die • Book on the Shelfhttps://digitalcommons.ursinus.edu/lantern/1124/thumbnail.jp

Operating organic light-emitting diodes imaged by super-resolution spectroscopy

Super-resolution stimulated emission depletion (STED) microscopy is adapted here for materials characterization that would not otherwise be possible. With the example of organic light-emitting diodes (OLEDs), spectral imaging with pixel-by-pixel wavelength discrimination allows us to resolve local-chain environment encoded in the spectral response of the semi-conducting polymer, and correlate chain packing with local electroluminescence by using externally applied current as the excitation source. We observe nanoscopic defects that would be unresolvable by traditional microscopy. They are revealed in electroluminescence maps in operating OLEDs with 50 nm spatial resolution. We find that brightest emission comes from regions with more densely packed chains. Conventional microscopy of an operating OLED would lack the resolution needed to discriminate these features, while traditional methods to resolve nanoscale features generally cannot be performed when the device is operating. This points the way towards real-time analysis of materials design principles in devices as they actually operateope