23 research outputs found
Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins
AbstractGiant protoplasts of Saccharomyces cerevisiae of 10–35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1–12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66±0.07 µF/cm2) and conductance (23–44 µS/cm2) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels
A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger
Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Biochimica et Biophysica Acta - Biomembranes 1788 (2009): 1255-1262, doi:10.1016/j.bbamem.2008.12.016.Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve
Na+/Ca2+ exchanger. Mass spectroscopy and Western blot analysis established that this
factor is a member of the lipocalin super family of lipid binding proteins of 132 amino
acids in length. We named it Regulatory protein of the squid nerve sodium calcium
exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far-
UV circular dichroism and infrared spectra suggest a majority of β-strand in the secondary
structure. Moreover, the predicted tertiary structure indicates ten β-sheets and two short α-
helices characteristic of most lipid binding proteins. Functional experiments showed that in
order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP
by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ
is able to stimulate the exchanger in the absence of ATP. In addition to the identification of
a new member of the lipid binding protein family, this work shows, for the first time, the
requirement of a lipid binding protein for metabolic regulation of an ion transporting
system.The work was supported by Grants from the US National Science Foundation [MCB
0444598], Fondo Nacional para Investigaciones CientĂficas y TecnolĂłgicas [PICT-05-
12397 and PICT-05-38073], Consejo Nacional de Investigfaciones CientĂficas y TĂ©cnicas
[PIP 5118 and PIP 5593] SecretarĂa de Ciencia y TĂ©cnica Universidad Nacional de
CĂłrdoba, Argentina, Fondo Nacional para Ciencia y TĂ©cnica [S1-9900009046 and G-
2001000637] and FundaciĂłn Polar, Venezuela and The Rhode Island Idea Network of
Biomedical Research Excellence (INBRE)
The Squid Axon Na<sup>+</sup>/Ca<sup>2+</sup> Exchanger Shows Ping Pong Kinetics only when the Ca<sub>i</sub>-regulatory Site is Saturated
Metronomic Photodynamic Therapy with Conjugated Polymer Nanoparticles in Glioblastoma Tumor Microenvironment
Alternative therapies such as photodynamic therapy (PDT) that combine light, oxygen and photosensitizers (PSs) have been proposed for glioblastoma (GBM) management to overcome conventional treatment issues. An important disadvantage of PDT using a high light irradiance (fluence rate) (cPDT) is the abrupt oxygen consumption that leads to resistance to the treatment. PDT metronomic regimens (mPDT) involving administering light at a low irradiation intensity over a relatively long period of time could be an alternative to circumvent the limitations of conventional PDT protocols. The main objective of the present work was to compare the effectiveness of PDT with an advanced PS based on conjugated polymer nanoparticles (CPN) developed by our group in two irradiation modalities: cPDT and mPDT. The in vitro evaluation was carried out based on cell viability, the impact on the macrophage population of the tumor microenvironment in co-culture conditions and the modulation of HIF-1α as an indirect indicator of oxygen consumption. mPDT regimens with CPNs resulted in more effective cell death, a lower activation of molecular pathways of therapeutic resistance and macrophage polarization towards an antitumoral phenotype. Additionally, mPDT was tested in a GBM heterotopic mouse model, confirming its good performance with promising tumor growth inhibition and apoptotic cell death induction.Fil: Caverzan, Matias Daniel. Universidad Nacional de RĂo Cuarto. Facultad de Ciencias Exactas FisicoquĂmicas y Naturales. Instituto de Investigaciones en TecnologĂas EnergĂ©ticas y Materiales Avanzados. - Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - CĂłrdoba. Instituto de Investigaciones en TecnologĂas EnergĂ©ticas y Materiales Avanzados; ArgentinaFil: Oliveda, Paula Martina. Universidad Nacional de RĂo Cuarto. Facultad de Ciencias Exactas FisicoquĂmicas y Naturales. Departamento de BiologĂa Molecular; ArgentinaFil: BeaugĂ©, LucĂa. Universidad Nacional de RĂo Cuarto. Facultad de Ciencias Exactas FisicoquĂmicas y Naturales. Instituto de Investigaciones en TecnologĂas EnergĂ©ticas y Materiales Avanzados. - Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - CĂłrdoba. Instituto de Investigaciones en TecnologĂas EnergĂ©ticas y Materiales Avanzados; ArgentinaFil: Palacios, Rodrigo Emiliano. Universidad Nacional de RĂo Cuarto. Facultad de Ciencias Exactas FisicoquĂmicas y Naturales. Instituto de Investigaciones en TecnologĂas EnergĂ©ticas y Materiales Avanzados. - Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - CĂłrdoba. Instituto de Investigaciones en TecnologĂas EnergĂ©ticas y Materiales Avanzados; ArgentinaFil: Chesta, Carlos Alberto. Universidad Nacional de RĂo Cuarto. Facultad de Ciencias Exactas FisicoquĂmicas y Naturales. Instituto de Investigaciones en TecnologĂas EnergĂ©ticas y Materiales Avanzados. - Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - CĂłrdoba. Instituto de Investigaciones en TecnologĂas EnergĂ©ticas y Materiales Avanzados; ArgentinaFil: Ibarra, Luis Exequiel. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentin