24 research outputs found
Ouabain Enhances Cell-Cell Adhesion Mediated by beta1 Subunits of the Na(+),K(+)-ATPase in CHO Fibroblasts
Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na(+),K(+)-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine beta1 subunit become adhesive, and those homotypic interactions amongst beta1 subunits of the Na(+),K(+)-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the alpha subunit of the Na(+),K(+)-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the alpha subunit. In this study, we investigated whether the adhesion between beta1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the beta1 subunit of the Na(+),K(+)-ATPase (CHO beta1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO beta1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the beta1 subunit of the Na(+),K(+)-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO beta1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO beta1 cells. Collectively, our findings suggest that the beta1 subunit adhesion is modulated by the expression levels of the Na(+),K(+)-ATPase at the plasma membrane, which is regulated by ouabain
Particle formation and surface processes on atmospheric aerosols: a review of applied quantum chemical calculations
Aerosols significantly influence atmospheric processes such as cloud nucleation, het- erogeneous chemistry, and heavy-metal transport in the troposphere. The chemical and physical complexity of atmospheric aerosols results in large uncertainties in their climate and health effects. In this article, we review recent advances in scientific understanding of aerosol processes achieved by the application of quantum chemical calculations. In particular, we emphasize recent work in two areas: new particle for- mation and heterogeneous processes. Details in quantum chemical methods are pro- vided, elaborating on computational models for prenucleation, secondary organic aerosol formation, and aerosol interface phenomena. Modeling of relative humidity effects, aerosol surfaces, and chemical kinetics of reaction pathways is discussed. Because of their relevance, quantum chemical calculations and field and laboratory experiments are compared. In addition to describing the atmospheric relevance of the computational models, this article also presents future challenges in quantum chemical calculations applied to aerosols
Differential expression of zinc transporters accompanies the differentiation of C2C12 myoblasts
Zinc transporters facilitate metal mobilization and compartmentalization, playing a key role in cellular development. Little is known about the mechanisms and pathways of Zn movement between Zn transporters and metalloproteins during myoblast differentiation. We analyzed the differential expression of ZIP and ZnT transporters during C2C12 myoblast differentiation. Zn transporters account for a transient decrease of intracellular Zn upon myogenesis induction followed by a gradual increase of Zn in myotubes. Considering the subcellular localization and function of each of the Zn transporters, our findings indicate that a fine regulation is necessary to maintain correct metal concentrations in the cytosol and subcellular compartments to avoid toxicity, maintain homeostasis, and for loading metalloproteins needed during myogenesis. This study advances our basic understanding of the complex Zn transport network during muscle differentiation
MTF1, a classic metal sensing transcription factor, promotes myogenesis in response to copper [preprint]
MTF1 is a conserved metal-binding transcription factor in eukaryotes that binds to conserved DNA sequence motifs, termed metal response elements (MREs). MTF1 responds to metal excess and deprivation, protects cells from oxidative and hypoxic stresses, and is required for embryonic development in vertebrates. We used multiple strategies to identify an unappreciated role for MTF1 and copper (Cu) in cell differentiation. Upon initiation of myogenesis from primary myoblasts, MTF1 expression increased, as did nuclear localization. Mtf1 knockdown impaired differentiation, while addition of non-toxic concentrations of Cu+ enhanced MTF1 expression and promoted myogenesis. Cu+ bound stoichiometrically to a C-terminus tetra-cysteine of MTF1. MTF1 bound to chromatin at the promoter regions of myogenic genes and binding was stimulated by copper. MTF1 formed a complex with MyoD at myogenic promoters, the master transcriptional regulator of the myogenic lineage. These studies establish novel mechanisms by which copper and MTF1 regulate gene expression in myoblast differentiation
The mitochondrial Cu+ transporter PiC2 (SLC25A3) is a target of MTF1 and contributes to the development of skeletal muscle in vitro
The loading of copper (Cu) into cytochrome c oxidase (COX) in mitochondria is essential for energy production in cells. Extensive studies have been performed to characterize mitochondrial cuproenzymes that contribute to the metallation of COX, such as Sco1, Sco2, and Cox17. However, limited information is available on the upstream mechanism of Cu transport and delivery to mitochondria, especially through Cu-impermeable membranes, in mammalian cells. The mitochondrial phosphate transporter SLC25A3, also known as PiC2, binds Cu+ and transports the ion through these membranes in eukaryotic cells, ultimately aiding in the metallation of COX. We used the well-established differentiation model of primary myoblasts derived from mouse satellite cells, wherein Cu availability is necessary for growth and maturation, and showed that PiC2 is a target of MTF1, and its expression is both induced during myogenesis and favored by Cu supplementation. PiC2 deletion using CRISPR/Cas9 showed that the transporter is required for proliferation and differentiation of primary myoblasts, as both processes are delayed upon PiC2 knock-out. The effects of PiC2 deletion were rescued by the addition of Cu to the growth medium, implying the deleterious effects of PiC2 knockout in myoblasts may be in part due to a failure to deliver sufficient Cu to the mitochondria, which can be compensated by other mitochondrial cuproproteins. Co-localization and co-immunoprecipitation of PiC2 and COX also suggest that PiC2 may participate upstream in the copper delivery chain into COX, as verified by in vitro Cu+-transfer experiments. These data indicate an important role for PiC2 in both the delivery of Cu to the mitochondria and COX, favoring the differentiation of primary myoblasts.Fil: McCann, Cat. Wesleyan University; Estados UnidosFil: Quinteros, Michael. Wesleyan University; Estados UnidosFil: Adelugba, Ifeoluwa. University of Massachussets; Estados UnidosFil: Morgada, Marcos Nicolás. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Castelblanco, Aida R.. Skidmore College; Estados UnidosFil: Davis, Emily J.. Skidmore College; Estados UnidosFil: Lanzirotti, Antonio. University of Chicago; Estados UnidosFil: Hainer, Sarah J.. University of Pittsburgh; Estados UnidosFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Navea, Juan G.. Skidmore College; Estados UnidosFil: Padilla-Benavides, Teresita. Wesleyan University; Estados Unido
Studies on the application of laser photoionization in supersonic-jets for the generation of intense ionic clusters.
Includes bibliographical references (p. 201-208).Pulsed photoionization in a supersonic expansion was investigated for the production of intense ionic cluster beams from seeded organic molecules. Neutral clusters were photoionized in a pulsed system and studied by means of an optic ion array; the molecular beam design was
optimized and is described as a mean to investigate ionic clusters. Neutral clusters were generated and directed to an acceleration region where ionization occurred via a focused 266 nm pulsed laser beam. A set of static voltage plates in the acceleration region provide the ionic
clusters with the kinetic energy required for mass separation and detection. Optimization of the parameters involved in the molecular beam apparatus are discussed as well as the calibration of the Time of
Flight-Mass Spectrometer (TOF-MS).
Low order clusters were recorded and cluster cations of (C6H5CH3)n+, (n-C6H14)n+, (ClC6H5)n+ were produced by a multiple photon photoionization process in the acceleration region. The observed cluster
mass spectra for (C6H5CH3)n+,(n-C6H14)n+, (ClC6H5)n+ presented characteristic features that are discussed. Cationic and neutral toluene clusters were computationally minimized by Density Functional Theory (DFT) and Hartree-Fock (HF) Theory. The geometry and potential energy of observed toluene clusters was calculated using Gaussian 98 system of programs by the method DFT B3LYP 6-31 (G)*. Conformational switching was found to have a
predominant role in the stability of ionic clusters as well as favored the frontier orbital interaction of the toluene ionic dimer. The photoionization potential energy surface was calculated, involving the
conformation switching for toluene dimer and trimer. Chlorobenzene dimer was also minimized and its conformational switching is presented. A perpendicular conformation structure (T-shaped) is favored for the
chlorobenzene neutral dimer, instead of the antiparallel structure observed for toluene neutral dimer.by Juan G. Navea.Ph.D
Water Adsorption Isotherms on Fly Ash from Several Sources
In
this study, horizontal attenuated total reflection (HATR) Fourier-transform
infrared (FT-IR) spectroscopy was combined with quartz crystal microbalance
(QCM) gravimetry to investigate the adsorption isotherms of water
on fly ash, a byproduct of coal combustion in power plants. Because
of composition variability with the source region, water uptake was
studied at room temperature as a function of relative humidity (RH)
on fly ash from several regions: United States, India, The Netherlands,
and Germany. The FT-IR spectra show water features growth as a function
of RH, with water absorbing on the particle surface in both an ordered
(ice-like) and a disordered (liquid-like) structure. The QCM data
was modeled using the Brunauer, Emmett, and Teller (BET) adsorption
isotherm model. The BET model was found to describe the data well
over the entire range of RH, showing that water uptake on fly ash
takes place mostly on the surface of the particle, even for poorly
combusted samples. In addition, the source region and power-plant
efficiency play important roles in the water uptake and ice nucleation
(IN) ability of fly ash. The difference in the observed water uptake
and IN behavior between the four samples and mullite (3Al<sub>2</sub>O<sub>3</sub>·2SiO<sub>2</sub>), the aluminosilicate main component
of fly ash, is attributed to differences in composition and the density
of OH binding sites on the surface of each sample. A discussion is
presented on the RH required to reach monolayer coverage on each sample
as well as a comparison between surface sites of fly ash samples and
enthalpies of adsorption of water between the samples and mullite
Ouabain Enhances Cell-Cell Adhesion Mediated by β<sub>1</sub> Subunits of the Na<sup>+</sup>,K<sup>+</sup>-ATPase in CHO Fibroblasts
Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na+,K+-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine β1 subunit become adhesive, and those homotypic interactions amongst β1 subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na+,K+-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between β1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the β1 subunit of the Na+,K+-ATPase (CHO β1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO β1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the β1 subunit of the Na+,K+-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO β1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO β1 cells. Collectively, our findings suggest that the β1 subunit adhesion is modulated by the expression levels of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain
The classic metal-sensing transcription factor MTF1 promotes myogenesis in response to copper
Metal-regulatory transcription factor 1 (MTF1) is a conserved metal-binding transcription factor in eukaryotes that binds to conserved DNA sequence motifs, termed metal response elements. MTF1 responds to both metal excess and deprivation, protects cells from oxidative and hypoxic stresses, and is required for embryonic development in vertebrates. To examine the role for MTF1 in cell differentiation, we use multiple experimental strategies [including gene knockdown (KD) mediated by small hairpin RNA and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), immunofluorescence, chromatin immunopreciptation sequencing, subcellular fractionation, and atomic absorbance spectroscopy] and report a previously unappreciated role for MTF1 and copper (Cu) in cell differentiation. Upon initiation of myogenesis from primary myoblasts, both MTF1 expression and nuclear localization increased. Mtf1 KD impaired differentiation, whereas addition of nontoxic concentrations of Cu(+)-enhanced MTF1 expression and promoted myogenesis. Furthermore, we observed that Cu(+) binds stoichiometrically to a C terminus tetra-cysteine of MTF1. MTF1 bound to chromatin at the promoter regions of myogenic genes, and Cu addition stimulated this binding. Of note, MTF1 formed a complex with myogenic differentiation (MYOD)1, the master transcriptional regulator of the myogenic lineage, at myogenic promoters. These findings uncover unexpected mechanisms by which Cu and MTF1 regulate gene expression during myoblast differentiation
Effects of Coadsorbed Water on the Heterogeneous Photochemistry of Nitrates Adsorbed on TiO<sub>2</sub>
Nitric
acid, a well-known sink of NO<sub><i>x</i></sub> gases in
the atmosphere, has been found to be photoactive while
adsorbed on tropospheric particles. When adsorbed onto semiconductive
metal oxides, nitrate’s photochemical degradation can be interpreted
as a photocatalytic process. Yet, the photolysis of nitrate ions on
the surface of aerosols can also be initiated by changes in the symmetry
of the ion upon adsorption. In this study, we use quantum chemistry
to model the vibrational spectra of adsorbed nitrate on TiO<sub>2</sub>, a semiconductor component of atmospheric aerosols, and determine
the kinetics of the heterogeneous photochemical degradation of nitrate
under simulated solar light. Frequencies and geometry calculations
suggest that the symmetry of chemisorbed nitrate ion depends strongly
on coadsorbed water, with water changing the reactive surface of TiO<sub>2</sub>. Upon irradiation, surface nitrate undergoes photolysis to
yield nitrogen-containing gaseous products including NO<sub>2</sub>, NO, HONO, and N<sub>2</sub>O, in proportions that depend on relative
humidity (RH). In addition, the heterogeneous photochemistry rate
constant decreases an order of magnitude, from (5.7 ± 0.1) ×
10<sup>–4</sup> s<sup>–1</sup> on a dry surface to (7.1
± 0.8) × 10<sup>–5</sup> s<sup>–1</sup> when
nitrate is coadsorbed with water above monolayer coverage. Little
is known about the roles of coadsorbed water on the heterogeneous
photochemistry of nitrates on TiO<sub>2</sub>, along with its impact
on the chemical balance of the atmosphere. This work discusses the
roles of water in the photolysis of surface nitrates on TiO<sub>2</sub> and the concomitant renoxification of the atmosphere